| 2021 | Scaffold compound L971 exhibits anti-inflammatory activities through inhibition of JAK/STAT and NFκB signalling pathways. | J Cell Mol Med | JAK/STAT and NFκB signalling pathways play essential roles in regulating inflammatory responses, which are important pathogenic factors of various serious immune-related diseases, and function individually or synergistically. To find prodrugs that can treat inflammation, we performed a preliminary high-throughput screening of 18 840 small molecular compounds and identified scaffold compound L971 which significantly inhibited JAK/STAT and NFκB driven luciferase activities. L971 could inhibit the constitutive and stimuli-dependent activation of STAT1, STAT3 and IκBα and could significantly down-regulate the proinflammatory gene expression in mouse peritoneal macrophages stimulated by LPS. Gene expression profiles upon L971 treatment were determined using high-throughput RNA sequencing, and significant differentially up-regulated and down-regulated genes were identified by DESeq analysis. The bioinformatic studies confirmed the anti-inflammatory effects of L971. Finally, L971 anti-inflammatory character was further verified in LPS-induced sepsis shock mouse model in vivo. Taken together, these data indicated that L971 could down-regulate both JAK/STAT and NFκB signalling activities and has the potential to treat inflammatory diseases such as sepsis shock. |
| 2021 | Transcriptional profiling of Microtus fortis responses to S. japonicum: New sight into Mf-Hsp90α resistance mechanism. | Parasite Immunol | Schistosomiasis is a parasitic disease with a chronic debilitating character caused by parasitic flatworms of the genus Schistosoma. The main disease-causing species of Schistosoma in China is S. japonicum. M fortis has been proved to be a nonpermissive host of S. japonicum. Mf-HSP90α (Microtus fortis heat shock protein 90alpha), the homologue of HSP90α, display anti-schistosome effect in vitro and in vivo. In the current study, in order to investigate the mechanism of anti-schistosome effect of Mf-HSP90α, we conducted RNA-Seq to obtain the transcriptome profile of M. fortis liver infected with S. japonicum at different time points.By mapping the differential expressed genes (DEGs) to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we found that the JAK2/STAT1 pathway was highly enriched with an elevated level of IL-10 and HSP90α. We then checked the IL-10-JAK2/STAT1-HSP90α pathway, and found that this pathway was activated in the infected mice with S. japonicum. The expression of the molecules in this pathway was elevated on the 10 day after infection and gradually decreased on the 20 day.The IL-10-JAK2/STAT1-HSP90α axis was associated with the anti-schistosome effect of Mf-HSP90α, and targeting IL-10-JAK2/STAT1-HSP90α axis might be a novel therapeutic strategy for developing resistance to S. japonicum infection. |
| 2021 | The Endothelium is in Shock and Interferon-α/STAT1 Signaling is to Blame. | Am J Respir Cell Mol Biol | |
| 2021 | A Translational Investigation of Interferon-α and STAT1 Signaling in Endothelial Cells during Septic Shock Provides Therapeutic Perspectives. | Am J Respir Cell Mol Biol | Septic shock and disseminated intravascular coagulation (DIC) are knowingly characterized by an endothelial cell dysfunction. The molecular mechanisms underlying this relationship are, however, poorly understood. In this work, me aimed at investigating human circulating interferon-α (IFN-α) in septic shock-induced DIC patients and tested the potential role of endothelial Stat1 as a therapeutic target in a mouse model of sepsis. For this, circulating type I, II and III IFNs and procoagulant microvesicles were quantified in a prospective cohort of septic shock patients. Next, we used a septic shock model induced by cecal ligation and puncture (CLP) in wild-type (WT) mice, in Ifnar1 (type I IFN receptor subunit 1)-knockout (KO) mice, as well as in Stat1 (Signal transducer and activator of transcription 1) conditional KO mice. In humans samples, we observed higher levels of circulating IFN-α and IFN-α1 in DIC compared to non-DIC patients, while levels of IFN-β, IFN-γ, IFN-λ1, IFN-λ2, IFN-λ3 were not different. IFN-α level was positively correlated with CD105-microvesicle levels, reflecting endothelial injury. In Ifnar1-/- mice, CLP did not induce septic shock and was characterized by lesser endothelial cell injury, with lower aortic inflammatory cytokine expression, endothelial inflammatory-related genes expression and fibrinolysis. In mice in which Stat1 was specifically ablated in endothelial cells, a marked protection against sepsis was also observed, suggesting the relevance of an endothelium-targeted strategy. Our work highlights the key roles of type I interferons as pathogenic players in septic shock-induced DIC and the potential pertinence of endothelial STAT1 as a therapeutic target. |
| 2021 | Analyzing the potential therapeutic mechanism of Huashi Baidu Decoction on severe COVID-19 through integrating network pharmacological methods. | J Tradit Complement Med | Huashi Baidu Decoction (HSBD) is a novel complex prescription which has positive effects on severe COVID-19. This study was aimed to discover key Chinese materia medica, main active compounds, hub therapeutic target proteins and core signal pathways in the potential therapeutic mechanism of HSBD on severe COVID-19 through integrating network pharmacological methods.TCMSP, TCMID and STITCH databases were used to screen out active compounds and target proteins of HSBD. GeneCards database was used to screen out disease genes of severe COVID-19. The potential therapeutic targets of HSBD on severe COVID-19 were used to construct protein-protein interaction network through STRING database and the hub target proteins were discovered. Next, GO and KEGG enrichment analysis were carried out to discover core signal pathways. Finally, the network diagram of "Chinese materia medica-active compounds-therapeutic target proteins" was built, then key Chinese materia medica and main active compounds were selected.HSBD might treat severe COVID-19 through 45 potential target genes, among them, there were 13 hub target genes: RELA, TNF, IL6, IL1B, MAPK14, TP53, CXCL8, MAPK3, MAPK1, IL4, MAPK8, CASP8, STAT1. Meanswhile, GO_BiologicalProcess and KEGG signaling pathways analysis results showed that the core signal pathways were inflammation and immune regulation pathways. Finally, 4 key Chinese materia medica and 11 main active compounds were discovered in the HSBD. In conclusion, the therapeutic mechanism of HSBD on severe COVID-19 might involve its pharmacological effects of anti-inflammation and immune regulation via acting on 45 disease-related proteins of severe COVID-19.Viral Pneumonia, COVID-19, Acute Respiratory Distress Syndrome, Septic Shock, Chinese Herbal Medicine. |
| 2021 | Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes. | Cell | COVID-19 is characterized by excessive production of pro-inflammatory cytokines and acute lung damage associated with patient mortality. While multiple inflammatory cytokines are produced by innate immune cells during SARS-CoV-2 infection, we found that only the combination of TNF-α and IFN-γ induced inflammatory cell death characterized by inflammatory cell death, PANoptosis. Mechanistically, TNF-α and IFN-γ co-treatment activated the JAK/STAT1/IRF1 axis, inducing nitric oxide production and driving caspase-8/FADD-mediated PANoptosis. TNF-α and IFN-γ caused a lethal cytokine shock in mice that mirrors the tissue damage and inflammation of COVID-19, and inhibiting PANoptosis protected mice from this pathology and death. Furthermore, treating with neutralizing antibodies against TNF-α and IFN-γ protected mice from mortality during SARS-CoV-2 infection, sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other infectious and autoinflammatory diseases by limiting tissue damage/inflammation. |
| 2020 | COVID-19 cytokines and the hyperactive immune response: Synergism of TNF-α and IFN-γ in triggering inflammation, tissue damage, and death. | bioRxiv | The COVID-19 pandemic has caused significant morbidity and mortality. Currently, there is a critical shortage of proven treatment options and an urgent need to understand the pathogenesis of multi-organ failure and lung damage. Cytokine storm is associated with severe inflammation and organ damage during COVID-19. However, a detailed molecular pathway defining this cytokine storm is lacking, and gaining mechanistic understanding of how SARS-CoV-2 elicits a hyperactive inflammatory response is critical to develop effective therapeutics. Of the multiple inflammatory cytokines produced by innate immune cells during SARS-CoV-2 infection, we found that the combined production of TNF-α and IFN-γ specifically induced inflammatory cell death, PANoptosis, characterized by gasdermin-mediated pyroptosis, caspase-8-mediated apoptosis, and MLKL-mediated necroptosis. Deletion of pyroptosis, apoptosis, or necroptosis mediators individually was not sufficient to protect against cell death. However, cells deficient in both RIPK3 and caspase-8 or RIPK3 and FADD were resistant to this cell death. Mechanistically, the STAT1/IRF1 axis activated by TNF-α and IFN-γ co-treatment induced iNOS for the production of nitric oxide. Pharmacological and genetic deletion of this pathway inhibited pyroptosis, apoptosis, and necroptosis in macrophages. Moreover, inhibition of PANoptosis protected mice from TNF-α and IFN-γ-induced lethal cytokine shock that mirrors the pathological symptoms of COVID-19. In vivo neutralization of both TNF-α and IFN-γ in multiple disease models associated with cytokine storm showed that this treatment provided substantial protection against not only SARS-CoV-2 infection, but also sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock models, demonstrating the broad physiological relevance of this mechanism. Collectively, our findings reveal that blocking the COVID-19 cytokine-mediated inflammatory cell death signaling pathway identified in this study may benefit patients with COVID-19 or other cytokine storm-driven syndromes by limiting inflammation and tissue damage. The findings also provide a molecular and mechanistic description for the term cytokine storm. Additionally, these results open new avenues for the treatment of other infectious and autoinflammatory diseases and cancers where TNF-α and IFN-γ synergism play key pathological roles. |
| 2021 | Oleanolic acid protects against mast cell-mediated allergic responses by suppressing Akt/NF-κB and STAT1 activation. | Phytomedicine | Oleanolic acid (OA) is an active compound found in a variety of medicinal herbs and plants. Though OA has been widely attributed with a variety of biological activities, studies focused on its anti-allergic inflammation properties are insufficient.Given the rapid increase in allergic diseases and the lack of fundamental treatment options, this study aimed to find a safe and effective therapy for allergic disorders.We evaluated the inhibitory effect of OA on allergic inflammatory response and the possible mechanisms underlying the effect using phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated human mast cell (HMC)-1, and a mouse model of compound 48/80-induced anaphylactic shock.OA suppressed pro-inflammatory cytokine expressions in PMACI-induced HMC-1 cells by inhibiting activation of the Akt, p38 mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and signal transducer and activator of transcription (STAT) 1 signaling pathways. Moreover, OA showed a protective effect against compound 48/80-induced anaphylactic shock through inhibition of histamine release and immunoglobulin E level via regulation of NF-κB and STAT1 activation.The results showed that OA suppressed mast cell-mediated allergic response by transcriptional regulation. We suggest that OA has potential effect against allergic inflammatory disorders, including anaphylaxis, and might be a useful therapeutic agent for allergic disease. |
| 2020 | HDAC6 Mediates Macrophage iNOS Expression and Excessive Nitric Oxide Production in the Blood During Endotoxemia. | Front Immunol | Excessive nitric oxide (NO) production and NO-mediated nitrative stress contribute to vascular dysfunction, inflammation, and tissue injury in septic shock. New therapeutic targets are urgently needed to provide better control of NO level during septic shock. In the present study, we investigated the role of HDAC6 in the regulation of NO production and nitrative stress in a mouse model of endotoxin-induced septic shock. HDAC6 deficient mice and a specific HDAC6 inhibitor were utilized in our studies. Our data clearly indicate that HDAC6 is an important mediator of NO production in macrophages. HDAC6 mediates NO production through the regulation of iNOS expression in macrophages. HDAC6 up-regulates iNOS expression in macrophages by modulating STAT1 activation and IRF-1 expression. HDAC6 inhibition potently blocked endotoxin-induced STAT1 activation and iNOS expression in macrophages. Furthermore, HDAC6 contributes to excessive NO production and nitrotyrosine level in the blood and promotes iNOS expression in the lung tissues during septic shock. Our data reveal a novel HDAC6/STAT1/iNOS pathway that mediates excessive NO production and nitrative stress in septic shock. |
| 2020 | Zinc inhibits lethal inflammatory shock by preventing microbe-induced interferon signature in intestinal epithelium. | EMBO Mol Med | The cytokine TNF drives inflammatory diseases, e.g., Crohn's disease. In a mouse model of TNF-induced systemic inflammatory response syndrome (SIRS), severe impact on intestinal epithelial cells (IECs) is observed. Zinc confers complete protection in this model. We found that zinc no longer protects in animals which lack glucocorticoids (GCs), or express mutant versions of their receptor GR in IECs, nor in mice which lack gut microbiota. RNA-seq studies in IECs showed that zinc caused reduction in expression of constitutive (STAT1-induced) interferon-stimulated response (ISRE) genes and interferon regulatory factor (IRF) genes. Since some of these genes are involved in TNF-induced cell death in intestinal crypt Paneth cells, and since zinc has direct effects on the composition of the gut microbiota (such as several Staphylococcus species) and on TNF-induced Paneth cell death, we postulate a new zinc-related anti-inflammatory mechanism. Zinc modulates the gut microbiota, causing less induction of ISRE/IRF genes in crypt cells, less TNF-induced necroptosis in Paneth cells, and less fatal evasion of gut bacteria into the system. |
| 2020 | Strategies to reduce the risk of platinum containing antineoplastic drug-induced ototoxicity. | Expert Opin Drug Metab Toxicol | Cisplatin is a highly effective chemotherapeutic agent against a variety of solid tumors in adults and in children. Unfortunately, a large percentage of patients suffer permanent sensorineural hearing loss. Up to 60% of children and at least 50% of adults suffer this complication that seriously compromises their quality of life. Hearing loss is due to damage to the sensory cells in the inner ear. The mechanisms of cochlear damage are still being investigated. However, it appears that inner ear damage is triggered by reactive oxygen species (ROS) formation and inflammation 34.We discuss a number of potential therapeutic targets that can be addressed to provide hearing protection. These strategies include enhancing the endogenous antioxidant pathways, heat shock proteins, G protein coupled receptors and counteracting ROS and reactive nitrogen species, and blocking pathways that produce inflammation, including TRPV1 and STAT1 36.Numerous potential protective agents show promise in animal models by systemic or local administration. However, clinical trials have not shown much efficacy to date with the exception of sodium thiosulfate. There is an urgent need to discover safe and effective protective agents that do not interfere with the efficacy of cisplatin against tumors yet preserve hearing 151. |
| 2020 | "Small" Intestinal Immunopathology Plays a "Big" Role in Lethal Cytokine Release Syndrome, and Its Modulation by Interferon-γ, IL-17A, and a Janus Kinase Inhibitor. | Front Immunol | Chimeric antigen receptor T cell (CART) therapy, administration of certain T cell-agonistic antibodies, immune check point inhibitors, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) and Toxic shock syndrome (TSS) caused by streptococcal as well as staphylococcal superantigens share one common complication, that is T cell-driven cytokine release syndrome (CRS) accompanied by multiple organ dysfunction (MOD). It is not understood whether the failure of a particular organ contributes more significantly to the severity of CRS. Also not known is whether a specific cytokine or signaling pathway plays a more pathogenic role in precipitating MOD compared to others. As a result, there is no specific treatment available to date for CRS, and it is managed only symptomatically to support the deteriorating organ functions and maintain the blood pressure. Therefore, we used the superantigen-induced CRS model in HLA-DR3 transgenic mice, that closely mimics human CRS, to delineate the immunopathogenesis of CRS as well as to validate a novel treatment for CRS. Using this model, we demonstrate that (i) CRS is characterized by a rapid rise in systemic levels of several Th1/Th2/Th17/Th22 type cytokines within a few hours, followed by a quick decline. (ii) Even though multiple organs are affected, small intestinal immunopathology is the major contributor to mortality in CRS. (iii) IFN-γ deficiency significantly protected from lethal CRS by attenuating small bowel pathology, whereas IL-17A deficiency significantly increased mortality by augmenting small bowel pathology. (iv) RNA sequencing of small intestinal tissues indicated that IFN-γ-STAT1-driven inflammatory pathways combined with enhanced expression of pro-apoptotic molecules as well as extracellular matrix degradation contributed to small bowel pathology in CRS. These pathways were further enhanced by IL-17A deficiency and significantly down-regulated in mice lacking IFN-γ. (v) Ruxolitinib, a selective JAK-1/2 inhibitor, attenuated SAg-induced T cell activation, cytokine production, and small bowel pathology, thereby completely protecting from lethal CRS in both WT and IL-17A deficient HLA-DR3 mice. Overall, IFN-γ-JAK-STAT-driven pathways contribute to lethal small intestinal immunopathology in T cell-driven CRS. |
| 2020 | An Essential Role for Perforin-2 in Type I IFN Signaling. | J Immunol | Type I IFNs play a complex role in determining the fate of microbial pathogens and may also be deleterious to the host during bacterial and viral infections. Upon ligand binding, a receptor proximal complex consisting of IFN-α and -β receptors 1 and 2 (IFNAR1, IFNAR2, respectively), tyrosine kinase 2 (Tyk2), Jak1, and STAT2 are assembled and promote the phosphorylation of STAT1 and STAT2. However, how the IFNARs proximal complex is assembled upon binding to IFN is poorly understood. In this study, we show that the membrane-associated pore-forming protein Perforin-2 (P2) is critical for LPS-induced endotoxic shock in wild-type mice. Type I IFN-mediated JAK-STAT signaling is severely impaired, and activation of MAPKs and PI3K signaling pathways are delayed in P2-deficient mouse bone marrow-derived macrophages, mouse embryonic fibroblasts (MEFs), and human HeLa cells upon IFN stimulation. The P2 -glycosylated extracellular membrane attack complex/perforin domain and the P2 domain independently associate with the extracellular regions of IFNAR1 and IFNAR2, respectively, in resting MEFs. In addition, the P2 cytoplasmic tail domain mediated the constitutive interaction between STAT2 and IFNAR2 in resting MEFs, an interaction that is dependent on the association of the extracellular regions of P2 and IFNAR2. Finally, the constitutive association of P2 with both receptors and STAT2 is critical for the receptor proximal complex assembly and reciprocal transphosphorylation of Jak1 and Tyk2 as well as the phosphorylation and activation of STAT1 and STAT2 upon IFN-β stimulation. |
| 2020 | TRAF1 suppresses antifungal immunity through CXCL1-mediated neutrophil recruitment during Candida albicans intradermal infection. | Cell Commun Signal | Candida albicans is the most common opportunistic human fungal pathogen. The chemokine ligand CXCL1 plays a protective role in fungal infection through the recruitment of neutrophils. TRAF1 (tumor necrosis factor-associated factor 1) can be highly induced by proinflammatory stimuli such as LPS and TNF and has been implicated in septic shock. However, the role of TRAF1 in infection, especially fungal infection, remains elusive. Herein, we reveal that TRAF1 suppresses the antifungal immune response to Candida albicans intradermal infection through the regulation of CXCL1 induction and neutrophil recruitment.A mouse model of C. albicans intradermal infection was established. The Traf1 mice and Traf1 immortalized human keratinocytes were generated. The p65 inhibitor triptolide, STAT1 inhibitor fludarabine, neutrophil-depletion antibody Ly6G, and neutralizing antibody for CXCL1 were utilized. The expression of proinflammatory cytokines and chemokines was assessed by real-time PCR and ELISA, and the activation of signaling molecules was analyzed by Western blotting. Hematoxylin and eosin staining and periodic acid Schiff staining were used for histology or fungal detection, respectively. The immunofluorescence and flow cytometry analyses were employed in the assessment of immune cell infiltration. Bone marrow transplantation and adoptive transfer experiments were conducted to establish a role for TRAF1 in the macrophage compartment in fungal skin infection.TRAF1-deficient mice demonstrated improved control of Candida albicans intradermal infection, and concomitant increase in neutrophil recruitment and reduction in fungal burden. The chemokine CXCL1 was upregulated in the TRAF1-deficient macrophages treated with heat-killed C. albicans. Mechanistically, TRAF1-deficient macrophages showed increased activation of transcription factor NFκB p65. The human CXCL8 was also highly induced in the TRAF1-deficient human keratinocytes upon TNF stimulation through decreasing the activation of transcription factor STAT1. TRAF1-deficient macrophages played a critical role in containing the C. albicans skin infection in vivo.TRAF1-deficient mice can better control fungal infection in the skin, a process attributable to the CXCL-neutrophil axis. Mechanistically, TRAF1 likely regulates CXCL1 expression in both macrophages and keratinocytes through the transcriptional factor NFκB and STAT1, respectively. Our finding offers new insight into the understanding of the immune regulatory mechanisms in host defense against C. albicans infection. |
| 2020 | In-vitro effect of heat stress on bovine monocytes lifespan and polarization. | Immunobiology | Heat stress (HS) has a negative impact on dairy cows' health, milk production, reproductive performance and immune defenses. Cellular and molecular responses to high temperatures in bovine polymorphonuclear cells and peripheral blood mononuclear cells (PBMCs) have been investigated so far. On the contrary, the effects of high temperatures on isolated monocytes remain almost undisclosed. The aim of this study was to unravel the in vitro effects of high temperatures, simulating a severe HS related body hyperthermia, on bovine lifespan and M1/M2 polarisation. The PBMCs were isolated from whole blood of 9 healthy dairy cattle. Monocytes were sorted by magnetic activated cell sorting and cultured over night at 39 °C (normothermia) or 41 °C (HS). Apoptotic rate and viability were assessed and mRNA abundance for heat shock proteins (HSPs), heat transcription factors (HSFs) and genes involved in monocyte/macrophage polarization (STAT1, STAT2, STAT3, STAT6, IL1β, TGF1β, IL-10, COX2) were quantified by qPCR. We found that apoptosis increased in monocytes exposed to 41 °C, as compared to control, while viability conversely decreased. HS increased the abundance of HSF1 and HSP70. The concomitant decrease of STAT1 and STAT2 and the increase of STAT6 genes abundance at 41 °C suggest, at transcriptional factors level, a polarization of monocytes from a classical activated M1 to a non-classically activated M2 monocytes. In conclusion, the exposure of bovine monocytes to high temperatures affects their lifespan as well as the abundance of genes involved in HS response and in monocyte/macrophages polarization phenotype, confirming that bovine immune response may be significantly affected by hyperthermia. |
| 2019 | Novel pharmacological inhibition of EZH2 attenuates septic shock by altering innate inflammatory responses to sepsis. | Int Immunopharmacol | The function of histone methyltransferase enhancer of zeste homolog 2 (EZH2) in sepsis remains unknown. We reported here that the expression of EZH2 and H3K27me3 was significantly upregulated in the circulation of septic patients, whereas patients who survived presented downregulated the expression of EZH2 on CD14+ monocytes. We further identified increased expression of EZH2 in the circulation, peritoneal fluid, and septic lungs from CLP mice. 3-DZNeP treated CLP mice improved mortality and protected from organ injury. EZH2 inhibition not only suppressed the activation of inflammatory cells and release of cytokines in the circulation and infectious sites, but also promoted bacteria clearance and replenished the circulating monocyte and neutrophil pool from bone marrow. Blockage of EZH2 also suppressed the progression of lung injury and alleviated inflammation by decreasing the pulmonary cell apoptosis, reducing inflammatory cells infiltration and cytokines release through inhibition of the STAT3 signaling pathway and recovery of PPARγ activation. In addition, EZH2 inhibitor blunted macrophage M1 polarization by SOCS3/STAT1 pathway. Overall, these data suggest that EZH2 could be a potential biomarker predicting clinical outcome and a new target for therapeutic interference in sepsis. |
| 2018 | Lipopolysaccharide shock reveals the immune function of indoleamine 2,3-dioxygenase 2 through the regulation of IL-6/stat3 signalling. | Sci Rep | Indoleamine 2,3-dioxygenase 2 (Ido2) is a recently identified catalytic enzyme in the tryptophan-kynurenine pathway that is expressed primarily in monocytes and dendritic cells. To elucidate the biological role of Ido2 in immune function, we introduced lipopolysaccharide (LPS) endotoxin shock to Ido2 knockout (Ido2 KO) mice, which led to higher mortality than that in the wild type (WT) mice. LPS-treated Ido2 KO mice had increased production of inflammatory cytokines (including interleukin-6; IL-6) in serum and signal transducer and activator of transcription 3 (stat3) phosphorylation in the spleen. Moreover, the peritoneal macrophages of LPS-treated Ido2 KO mice produced more cytokines than did the WT mice. By contrast, the overexpression of Ido2 in the murine macrophage cell line (RAW) suppressed cytokine production and decreased stat3 expression. Finally, RAW cells overexpressing Ido2 did not alter nuclear factor κB (NF-κB) or stat1 expression, but IL-6 and stat3 expression decreased relative to the control cell line. These results reveal that Ido2 modulates IL-6/stat3 signalling and is induced by LPS, providing novel options for the treatment of immune disorders. |
| 2018 | Phospho-proteomic analysis of primary human colon epithelial cells during the early Trypanosoma cruzi infection phase. | PLoS Negl Trop Dis | The protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, causes severe morbidity and mortality in afflicted individuals. About 30% of T. cruzi-infected individuals present with cardiac, gastrointestinal tract, and/or neurological disorders. Megacolon, one of the major pathologies of Chagas disease, is accompanied by gastrointestinal motility disorders. The molecular mechanism of T. cruzi-mediated megacolon in Chagas disease is currently unknown. To decipher the molecular mechanism of T. cruzi-induced alteration in the colon during the early infection phase, we exposed primary human colonic epithelial cells (HCoEpiC) to invasive T. cruzi trypomastigotes at multiple time points to determine changes in the phosphoprotein networks in the cells following infection using proteome profiler Human phospho-kinase arrays. We found significant changes in the phosphorylation pattern that can mediate cellular deregulations in colonic epithelial cells after infection. We detected a significant increase in the levels of phosphorylated heat shock protein (p-HSP) 27 and transcription factors that regulate various cellular functions, including c-Jun and CREB. Our study confirmed significant upregulation of phospho (p-) Akt S473, p-JNK, which may directly or indirectly modulate CREB and c-Jun phosphorylation, respectively. We also observed increased levels of phosphorylated CREB and c-Jun in the nucleus. Furthermore, we found that p-c-Jun and p-CREB co-localized in the nucleus at 180 minutes post infection, with a maximum Pearson correlation coefficient of 0.76±0.02. Increased p-c-Jun and p-CREB have been linked to inflammatory and profibrotic responses. T. cruzi infection of HCoEpiC induces an increased expression of thrombospondin-1 (TSP-1), which is fibrogenic at elevated levels. We also found that T. cruzi infection modulates the expression of NF-kB and JAK2-STAT1 signaling molecules which can increase pro-inflammatory flux. Bioinformatics analysis of the phosphoprotein networks derived using the phospho-protein data serves as a blueprint for T. cruzi-mediated cellular transformation of primary human colonic cells during the early phase of T. cruzi infection. |
| 2018 | The Circadian Clock Controls Immune Checkpoint Pathway in Sepsis. | Cell Rep | Sepsis and septic shock are associated with life-threatening organ dysfunction caused by an impaired host response to infections. Although circadian clock disturbance impairs the early inflammatory response, its impact on post-septic immunosuppression remains poorly elucidated. Here, we show that Bmal1, a core circadian clock gene, plays a role in the regulation of host immune responses in experimental sepsis. Mechanistically, Bmal1 deficiency in macrophages increases PKM2 expression and lactate production, which is required for expression of the immune checkpoint protein PD-L1 in a STAT1-dependent manner. Consequently, targeted ablation of Pkm2 in myeloid cells or administration of anti-PD-L1-neutralizing antibody or supplementation with recombinant interleukin-7 (IL-7) facilitates microbial clearance, inhibits T cell apoptosis, reduces multiple organ dysfunction, and reduces septic death in Bmal1-deficient mice. Collectively, these findings suggest that the circadian clock controls the immune checkpoint pathway in macrophages and therefore represents a potential therapeutic target for lethal infection. |
| 2018 | Overall Downregulation of mRNAs and Enrichment of H3K4me3 Change Near Genome-Wide Association Study Signals in Systemic Lupus Erythematosus: Cell-Specific Effects. | Front Immunol | This study was designed to define gene expression and H3K4me3 histone modifications in T cells, B cells, and monocytes in systemic lupus erythematosus (SLE). Array studies of total peripheral blood mononuclear cells have demonstrated gene expression signatures related to neutrophils, interferon, and other inflammatory pathways. It is not clear how consistent these effects are across different cell types. In this study, RNA-seq and chromatin immunoprecipitation-seq were utilized to identify gene expression patterns and H3K4me3 histone modifications related to promoter activation in SLE. Across the three cell types, there was 55% concordance for gene expression changes related to SLE. Key conserved pathways were ribosome biogenesis among upregulated genes and heat shock response among downregulated genes. ETS family transcription factors (TFs) and STAT1 were revealed as common regulators by position weight matrices. When epigenetic changes were leveraged with gene expression, the pivotal TFs ATF3 and FOS were defined with ATF3 also cross-referencing with gene expression-identified TFs. Genome-wide association study (GWAS) single nucleotide polymorphisms associated with SLE were cross-referenced with both mRNA and H3K4me3 changes in SLE. Baseline mRNA expression and H3K4me3 peak height was higher at sites that cross-referenced with GWAS signals, however, all three cell types exhibited an overall decrease in expression of GWAS-associated RNAs differentially expressed in SLE. H3K4me3 changes in SLE were also enriched in GWAS-associated sites. In summary, the SLE disease process is associated with both shared and cell-specific changes in gene expression and epigenetics. Surprisingly, GWAS-associated RNAs were overall markedly decreased across all three cell types. TF analysis identified ATF3, FOS, STAT1, and ETS family members as critical, all pathways with a recognized relationship to the SLE disease process. GWAS signals clearly mark both cell-type specific changes in SLE as well as concordant changes across all three cell types. Interpretation of single nucleotide polymorphism effects in SLE will require tissue-specific mechanistic studies and therapeutics will require mechanistic studies in multiple cell types. |
| 2017 | Quaking Deficiency Amplifies Inflammation in Experimental Endotoxemia the Aryl Hydrocarbon Receptor/Signal Transducer and Activator of Transcription 1-NF-κB Pathway. | Front Immunol | Macrophages, characterized by considerable diversity and plasticity, play a crucial role in a broad spectrum of biological processes, including inflammation. However, the molecular mechanisms underlying the diverse phenotypes of macrophages are not well defined. Here, we show that the RNA-binding protein, quaking (QKI), dynamically modulates macrophage polarization states. After lipopolysaccharide (LPS) stimulation, QKI-silenced RAW 264.7 cells displayed a pro-inflammatory M1 phenotype characterized by increased expression of iNOS, TNF-α, and IL-6 and decreased expression of anti-inflammatory factors, such as IL-10, found in inflammatory zone (Fizz1), and chitinase-like 3 (Chil3 or Ym1). By contrast, QKI5 overexpression led to a suppressive phenotype resembling M2 macrophages, even under M1 differentiation conditions. Moreover, myeloid-specific QKI-deficient mice tended to be more susceptible to LPS-induced endotoxic shock, while the exogenous transfer of macrophages overexpressing QKI5 exerted a significant improving effect. This improvement corresponded to a higher proportion of M2 macrophages, in line with elevated levels of IL-10, and a decrease in levels of pro-inflammatory mediators, such as IL-6, TNF-α, and IL-1β. Further mechanistic studies disclosed that QKI was a potent inhibitor of the nuclear factor-kappa B (NF-κB) pathway, suppressing p65 expression and phosphorylation. Strikingly, reduced expression of the aryl hydrocarbon receptor (Ahr) and reduced phosphorylation of signal transducer and activator of transcription 1 in QKI-deficient cells failed to restrain the transcriptional activity of NF-κB and NRL pyrin domain containing 3 (NLRP3) activation, while restoring QKI expression skewed the above M1-like response toward an anti-inflammatory M2 state. Taken together, these findings suggest a role for QKI in restraining overt innate immune responses by regulating the Ahr/STAT1-NF-κB pathway. |
| 2018 | Signalling Profiles of Blood Leucocytes in Sepsis and in Acute Pancreatitis in Relation to Disease Severity. | Scand J Immunol | Intracellular signalling in blood leucocytes shows multiple aberrations in acute pancreatitis (AP) complicated by organ dysfunction (OD). We studied whether the aberrations associate with severity of AP and occur in sepsis complicated by OD. The study comprises 14 sepsis patients (11 with shock), 18 AP patients (nine mild; six moderately severe; three severe) and 28 healthy volunteers. Within 48 h after admission to hospital, phosphorylation of nuclear factor-ĸB (NF-ĸB), signal transducers and activators of transcription (STATs) 1,3, and extracellular signal-regulated kinases 1/2 were measured from stimulated or non-stimulated leucocytes using phosphospecific whole blood flow cytometry. In sepsis, as compared with healthy subjects, phosphorylated NF-ĸB levels of monocytes promoted by bacterial lipopolysaccharides, tumour necrosis factor or Escherichia coli cells were lower (P < 0.001 for all), pSTAT1 levels of monocytes promoted by IL-6 were lower (P < 0.05 for all), and STAT3 was constitutively phosphorylated in monocytes, neutrophils and lymphocytes (P < 0.001 for all). In AP, severity was associated with proportions of pSTAT1-positive monocytes and lymphocytes promoted by IL-6 (P < 0.01 for both), constitutive STAT3 phosphorylation in neutrophils (P < 0.05), but not with any of the pNF-ĸB levels. Monocyte pSTAT3 fluorescence intensity, promoted by IL-6, was lower in sepsis and AP patients with OD than in AP patients without OD (P < 0.001). Collectively, signalling aberrations in sepsis with OD mimic those described previously in AP with OD. Possibility that aberrations in STAT1 and STAT3 pathways provide novel markers predicting evolution of OD warrants studies including patients presenting without OD but developing it during follow-up. |
| 2018 | Rotavirus Degrades Multiple Interferon (IFN) Type Receptors To Inhibit IFN Signaling and Protects against Mortality from Endotoxin in Suckling Mice. | J Virol | STAT1 phosphorylation in response to exogenous interferon (IFN) administration can be inhibited by rotaviral replication both and In addition many rotavirus strains are resistant to the actions of different IFN types. The regulation by rotaviruses (RVs) of antiviral pathways mediated by multiple IFN types is not well understood. In this study, we find that during infection and , RVs significantly deplete IFN type I, II, and III receptors (IFNRs). Regulation of IFNRs occurred exclusively within RV-infected cells and could be abrogated by inhibiting the lysosomal-endosomal degradation pathway. , IFNR degradation was conserved across multiple RV strains that differ in their modes of regulating IFN induction. In suckling mice, exogenously administered type I, II, or III IFN induced phosphorylation of STAT1-Y701 within intestinal epithelial cells (IECs) of suckling mice. Murine EW strain RV infection transiently activated intestinal STAT1 at 1 day postinfection (dpi) but not subsequently at 2 to 3 dpi. In response to injection of purified IFN-α/β or -λ, IECs in EW-infected mice exhibited impaired STAT1-Y701 phosphorylation, correlating with depletion of different intestinal IFNRs and impaired IFN-mediated transcription. The ability of EW murine RV to inhibit multiple IFN types led us to test protection of suckling mice from endotoxin-mediated shock, an outcome that is dependent on the host IFN response. Compared to mortality in controls, mice infected with EW murine RV were substantially protected against mortality following parenteral endotoxin administration. These studies identify a novel mechanism of IFN subversion by RV and reveal an unexpected protective effect of RV infection on endotoxin-mediated shock in suckling mice. Antiviral functions of types I, II, and III IFNs are mediated by receptor-dependent activation of STAT1. Here, we find that RV degrades the types I, II, and III IFN receptors (IFNRs) In a suckling mouse model, RV effectively blocked STAT1 activation and transcription following injection of different purified IFNs. This correlated with significantly decreased protein expression of intestinal types I and II IFNRs. Recent studies demonstrate that in mice lipopolysaccharide (LPS)-induced lethality is prevented by genetic ablation of IFN signaling genes such as IFNAR1 and STAT1. When suckling mice were infected with RV, they were substantially protected from lethal exposure to endotoxin. These findings provide novel insights into the mechanisms underlying rotavirus regulation of different interferons and are likely to stimulate new research into both rotavirus pathogenesis and endotoxemia. |
| 2017 | Therapeutic Effects of Monoclonal Antibody against Dengue Virus NS1 in a STAT1 Knockout Mouse Model of Dengue Infection. | J Immunol | Dengue virus (DENV) is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome and is endemic to tropical and subtropical regions of the world. Our previous studies showed the existence of epitopes in the C-terminal region of DENV nonstructural protein 1 (NS1) which are cross-reactive with host Ags and trigger anti-DENV NS1 Ab-mediated endothelial cell damage and platelet dysfunction. To circumvent these potentially harmful events, we replaced the C-terminal region of DENV NS1 with the corresponding region from Japanese encephalitis virus NS1 to create chimeric DJ NS1 protein. Passive immunization of DENV-infected mice with polyclonal anti-DJ NS1 Abs reduced viral Ag expression at skin inoculation sites and shortened DENV-induced prolonged bleeding time. We also investigated the therapeutic effects of anti-NS1 mAb. One mAb designated 2E8 does not recognize the C-terminal region of DENV NS1 in which host-cross-reactive epitopes reside. Moreover, mAb 2E8 recognizes NS1 of all four DENV serotypes. We also found that mAb 2E8 caused complement-mediated lysis in DENV-infected cells. In mouse model studies, treatment with mAb 2E8 shortened DENV-induced prolonged bleeding time and reduced viral Ag expression in the skin. Importantly, mAb 2E8 provided therapeutic effects against all four serotypes of DENV. We further found that mAb administration to mice as late as 1 d prior to severe bleeding still reduced prolonged bleeding time and hemorrhage. Therefore, administration with a single dose of mAb 2E8 can protect mice against DENV infection and pathological effects, suggesting that NS1-specific mAb may be a therapeutic option against dengue disease. |
| 2017 | HSP90 is necessary for the ACK1-dependent phosphorylation of STAT1 and STAT3. | Cell Signal | Signal transducers and activators of transcription (STATs) are latent, cytoplasmic transcription factors. Janus kinases (JAKs) and activated CDC42-associated kinase-1 (ACK1/TNK2) catalyse the phosphorylation of STAT1 and the expression of its target genes. Here we demonstrate that catalytically active ACK1 promotes the phosphorylation and nuclear accumulation of STAT1 in transformed kidney cells. These processes are associated with STAT1-dependent gene expression and an interaction between endogenous STAT1 and ACK1. Moreover, the E3 ubiquitin ligase seven-in-absentia homolog-2 (SIAH2), which targets ACK1 through valine-909 for proteasomal degradation, attenuates the ACK1-STAT1 signalling node. We further show that ACK1 promotes the phosphorylation and nuclear accumulation of STAT3 in cultured cells and that the levels of ACK1 correlate positively with the levels of tyrosine phosphorylated STAT3 in primary lung adenocarcinoma (ADC) cells. Global analysis of ACK1 interaction partners validated the interaction of ACK1 with heat shock protein 90 (HSP90α/β). Inhibition of this chaperone with the novel drug Onalespib (AT13387) demonstrates that HSP90 is an upstream regulator of the ACK1-dependent phosphorylation of STAT1 and STAT3. In addition to these molecular insights, our data offer a pharmacological strategy to control the ACK1-STAT signalling axis. |
| 2017 | Stat proteins as intracellular regulators of resistance to myocardial injury in the context of cardiac remodeling and targeting for therapy. | Adv Clin Exp Med | The roles of STAT (signal transducers and activators of transcription) proteins are widely discussed in relation to other agents like IFN-γ that are involved in cardiovascular diseases. STAT3 protects cardiomyocytes during endotoxic shock and ischemia and prolongs survival of these cells by activation of antiapoptotic genes like Bcl-2 and c-Fos. Moreover, IL-6 dependent expression of STAT3 is probably responsible for hypertrophy of cardiomyocytes. On the contrary, STAT1 mediates cell death by induction of caspase-1. STAT6 probably enhances cellular damage in myocardial infraction, which is significantly reduced in mice with the knockout STAT6 gene. Considering these facts, we attempted to review in this paper the role of STAT proteins in myocardial remodeling, highlighting STAT3 as a potent mediator of cardioprotection. Our review also aims to acquaint a broad audience of internal medicine practitioners with the STAT3-related molecular mechanisms that underlie the therapeutic properties of such widely administered drugs as angiotensin II type 1 (AT1) receptor antagonists and HMG-CoA reductase inhibitors, such as losartan and lovastatin. |
| 2017 | Schisandrin A inhibits dengue viral replication via upregulating antiviral interferon responses through STAT signaling pathway. | Sci Rep | Dengue virus (DENV) infects 400 million people worldwide annually. Infection of more than one serotype of DENV highly corresponds to dengue hemorrhagic fever and dengue shock syndrome, which are the leading causes of high mortality. Due to lack of effective vaccines and unavailable therapies against DENV, discovery of anti-DENV agents is urgently needed. We first characterize that Schisandrin A can inhibit the replication of four serotypes of DENV in a concentration- and time-dependent manner, with an effective half-maximal effective concentration 50% (EC) value of 28.1 ± 0.42 μM against DENV serotype type 2 without significant cytotoxicity. Furthermore, schisandrin A can effectively protect mice from DENV infection by reducing disease symptoms and mortality of DENV-infected mice. We demonstrate that STAT1/2-mediated antiviral interferon responses contribute to the action of schisandrin A against DENV replication. Schisandrin A represents a potential antiviral agent to block DENV replication in vitro and in vivo. In conclusion, stimulation of STAT1/2-mediated antiviral interferon responses is a promising strategy to develop antiviral drug. |
| 2017 | Regulatory Mechanisms of Hsp90. | Biochem Mol Biol J | The ability of Hsp90 to activate a disparate clientele implicates this chaperone in diverse biological processes. To accommodate such varied roles, Hsp90 requires a variety of regulatory mechanisms that are coordinated in order to modulate its activity appropriately. Amongst these, the master-regulator heat shock factor 1 (HSF1) is critically important in upregulating Hsp90 during stress, but is also responsible, through interaction with specific transcription factors (such as STAT1 and Strap/p300) for the integration of a variety of biological signals that ultimately modulate Hsp90 expression. Additionally, transcription factors, such as STAT1, STAT3 (including STAT1-STAT3 oligomers), NF-IL6, and NF-kB, are known to influence Hsp90 expression directly. Co-chaperones offer another mechanism for Hsp90 regulation, and these can modulate the chaperone cycle appropriately for specific clientele. Co-chaperones include those that deliver specific clients to Hsp90, and others that regulate the chaperone cycle for specific Hsp90-client complexes by modulating Hsp90s ATPase activity. Finally, post-translational modification (PTM) of Hsp90 and its co-chaperones helps too further regulate the variety of different Hsp90 complexes found in cells. |
| 2017 | Downregulates IP-10 Production and Prevents Th1 Cell Recruitment. | J Immunol | Staphylococcal superantigens cause toxic shock syndrome, which is characterized by massive T cell activation and a predominant Th1 profile of cytokine production. However, superantigen-producing strains are often part of the human nasal microbiome, and this carrier state has often been associated with some type 2 immune responses such as chronic sinusitis with polyps and atopic dermatitis. We have previously reported that the cell wall downregulates the human T cell response to superantigens through a TLR2-dependent, IL-10-mediated mechanism. In this study, we show that also regulates the profile of superantigen-induced T cell recruitment. The staphylococcal superantigen SEE induced the production of Th1 cell-recruiting chemokines, including IP-10, through an IFN-γ-dependent mechanism. Such an induction was suppressed by the concomitant presence of The downregulation of IP-10 by was mediated by components of its cell wall, but was not due to peptidoglycan-induced IL-10 production. Instead, triggered activation of MAPKs p38 and ERK, as well as inhibition of STAT1 signaling in monocytes, altogether contributing to the downregulation of IP-10 and other Th1 cell-recruiting chemokines (e.g., CXCL9 and CXCL11). These effects translated into inhibition of superantigen-induced Th1 cell recruitment. Taken together, our data may explain why colonization of superantigen-producing can induce, under some circumstances, mucosal type 2 immune responses. |
| 2018 | HSP70L1-mediated intracellular priming of dendritic cell vaccination induces more potent CTL response against cancer. | Cell Mol Immunol | Heat-shock protein (HSP)-based immunotherapy is established on its adjuvant effects when applied via an extracellular approach to pulse and activate dendritic cells (DCs). Our previous studies indicate that DCs pulsed with recombinant fusion proteins of antigenic fragment and HSP70-like protein 1 (HSP70L1) are potent in stimulating antigen-specific Th1 responses. We herein evaluated the cytotoxic T cell (CTL) response by an intracellular approach of priming DCs with transfection of recombinant adenovirus-expressing the fusion gene of the 576-699 fragment of carcinoembryonic antigen (CEA) and HSP70L1. As compared with DCs pulsed with extracellular fusion protein, the DCs transfected with recombinant adenovirus expressing the fusion gene displayed equivalent mature phenotypes but less inflammatory appearance. However, the transfected DCs were superior to the pulsed DCs in inducing CEA-specific CTLs. Consistently, immunization of HLA-A2.1/H-2K transgene mice with the transfected DCs could induce more quantities of HLA-A2.1-restricted CEA-specific CTLs, protecting nude mice more significantly from human CEA-expressing colon tumor challenge when adoptively transferred. Mechanistic investigation indicated that intracellular expression of the fusion protein empowered the transfected DCs by activation of STAT1 possibly via inducing IFN-β and ERK pathways. Therefore, the more potent ability to induce anti-CEA CTL responses enables the DCs, which transfected with recombinant adenovirus expressing the fusion gene of antigenic CEA fragment and Th1 adjuvant, as an alternative promising approach for the immunotherapy of CEA-positive tumors. |
| 2016 | K45A mutation of RIPK1 results in poor necroptosis and cytokine signaling in macrophages, which impacts inflammatory responses in vivo. | Cell Death Differ | Receptor interacting protein kinase 1 (RIPK1) participates in several cell signaling complexes that promote cell activation and cell death. Stimulation of RIPK1 in the absence of caspase signaling induces regulated necrosis (necroptosis), which promotes an inflammatory response. Understanding of the mechanisms through which RIPK1 promotes inflammation has been unclear. Herein we have evaluated the impact of a K45A mutation of RIPK1 on necroptosis of macrophages and the activation of inflammatory response. We show that K45A mutation of RIPK1 results in attenuated necroptosis of macrophages in response to stimulation with LPS, TNFα and IFNβ in the absence of caspase signaling. Impairment in necroptosis correlated with poor phosphorylation of RIPK1, RIPK3 and reduced trimerization of MLKL. Furthermore, K45A mutation of RIPK1 resulted in poor STAT1 phosphorylation (at S727) and expression of RANTES and MIP-1α following TNF-R engagement in the absence of caspase activation. Our results further indicate that in the absence of stimulation by pathogen-associated molecular patterns (PAMPs), cellular inhibitors of apoptotic proteins (cIAPs) prevent the K45-dependent auto-phosphorylation of RIPK1, leading to resistance against necroptosis. Finally, RIPK1(K45A) mice displayed attenuated inflammatory response in vivo as they were significantly resistant against endotoxin shock, but highly susceptible against a challenge with Salmonella typhimurium. This correlated with reduced expression of IL-1β and ROS, and poor processing of caspase 8 by RIPK1(K45A) macrophages. Overall, these results indicate that K45 mediated kinase activity of RIPK1 is not only important for necroptosis but it also has a key role in promoting cytokine signaling and host response to inflammatory stimuli. |
| 2016 | Biflorin, Isolated from the Flower Buds of Syzygium aromaticum L., Suppresses LPS-Induced Inflammatory Mediators via STAT1 Inactivation in Macrophages and Protects Mice from Endotoxin Shock. | J Nat Prod | Two chromone C-glucosides, biflorin (1) and isobiflorin (2), were isolated from the flower buds of Syzygium aromaticum L. (Myrtaceae). Here, inhibitory effects of 1 and 2 on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophages were evaluated, and 1 (IC50 = 51.7 and 37.1 μM, respectively) was more potent than 2 (IC50 > 60 and 46.0 μM). The suppression of NO and PGE2 production by 1 correlated with inhibition of iNOS and COX-2 protein expression. Compound 1 reduced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression via inhibition of their promoter activities. Compound 1 inhibited the LPS-induced production and mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6. Furthermore, 1 reduced p-STAT1 and p-p38 expression but did not affect the activity of nuclear factor κ light-chain enhancer of activated B cells (NF-κB) or activator protein 1 (AP-1). In a mouse model of LPS-induced endotoxemia, 1 reduced the mRNA levels of iNOS, COX-2, and TNF-α, and the phosphorylation-mediated activation of the signal transducer and activator of transcription 1 (STAT1), consequently improving the survival rates of mice. Compound 1 showed a significant anti-inflammatory effect on carrageenan-induced paw edema and croton-oil-induced ear edema in rats. The collective data indicate that the suppression of pro-inflammatory gene expression via p38 mitogen-activated protein kinase and STAT1 inactivation may be a mechanism for the anti-inflammatory activity of 1. |
| 2015 | Antagonism of toll-like receptor 2 attenuates the formation and progression of abdominal aortic aneurysm. | Acta Pharm Sin B | Abdominal aortic aneurysm (AAA) is an inflammatory vascular disorder with high mortality. Accumulating evidence shows that toll-like receptor 2 (TLR2) plays a critical role in the regulation of wound-repairing process after tissue injury. We wondered if TLR2 signaling contributed to the pathogenesis of AAA and that targeting TLR2 would attenuate AAA development and progression. In this study, enhanced expression of TLR2 and its ligands were observed in human AAA tissue. Neutralization of TLR2 protected against AAA development and caused established AAA to regress in mouse models of AAA. In addition, TLR2-deficient mice also failed to develop AAA. The prophylactic and therapeutic effects of blocking TLR2 were accompanied by a significant resolution of inflammation and vascular remodeling, as indicated by the decreased expression or activity of MMP-2/9, α-SMA, inflammatory cytokines, and transcription factors NF-κB, AP-1 and STAT1/3 in AAA tissue. Mechanistically, blocking TLR2 decreased the expression and interaction of TLR2 and several endogenous ligands, which diminished chronic inflammation and vascular remodeling in the vascular tissue of AAA. Our studies indicate that the interactions between TLR2 and its endogenous ligands contribute to the pathogenesis of AAA and that targeting TLR2 offers great potential toward the development of therapeutic agents against AAA. |
| 2015 | Requirement for endogenous heat shock factor 1 in inducible nitric oxide synthase induction in murine microglia. | J Neuroinflammation | Inducible nitric oxide synthase (iNOS) makes a great contribution to host defense and inflammation. In many settings, lipopolysaccharide (LPS) induces iNOS expression through activation of the inhibitor of κB-α (IκB-α)-nuclear factor-κB (NF-κB) cascade, whereas interferon-γ (IFN-γ) acts through Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1) signals. Heat shock factor 1 (HSF1), a major regulator of heat shock protein transcription, has been shown to regulate the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but it remains obscure whether and how HSF1 affects iNOS induction.Western blot was used to measure the protein expression. The mRNA level was measured by real-time PCR. Silence of HSF1 was achieved by small interfering RNA. Nitric oxide (NO) content and NF-κB binding activity were assayed by commercial kits. Chromatin immunoprecipitation (ChIP) was used to measure the binding activity of NF-κB and STAT1 to iNOS promoters.HSF1 inhibition or knockdown prevented the LPS- and/or IFN-γ-stimulated iNOS protein expression in cultured microglia. HSF1 inhibition blocked iNOS mRNA transcription. These inhibitory effects of HSF1 inhibition on iNOS expression were confirmed in brain tissues from endotoxemic mice. Further analysis showed that HSF1 inhibition had no effect on IκB-α degradation and NF-κB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-κB or STAT1 was also not affected by HSF1 inhibition, but HSF1 inhibition reduced the binding of NF-κB and STAT1 to their DNA elements. In addition, HSF1 inhibition reduced NF-κB and STAT1 bindings to iNOS promoter inside the LPS/IFN-γ-stimulated cells.This preventing effect of HSF1 inhibition on iNOS mRNA transcription presents the necessary role of HSF1 in iNOS induction. |
| 2016 | HSP90 inhibition leads to degradation of the TYK2 kinase and apoptotic cell death in T-cell acute lymphoblastic leukemia. | Leukemia | We previously found that tyrosine kinase 2 (TYK2) signaling through its downstream effector phospho-STAT1 acts to upregulate BCL2, which in turn mediates aberrant survival of T-cell acute lymphoblastic leukemia (T-ALL) cells. Here we show that pharmacologic inhibition of heat shock protein 90 (HSP90) with a small-molecule inhibitor, NVP-AUY922 (AUY922), leads to rapid degradation of TYK2 and apoptosis in T-ALL cells. STAT1 protein levels were not affected by AUY922 treatment, but phospho-STAT1 (Tyr-701) levels rapidly became undetectable, consistent with a block in signaling downstream of TYK2. BCL2 expression was downregulated after AUY922 treatment, and although this effect was necessary for AUY922-induced apoptosis, it was not sufficient because many T-ALL cell lines were resistant to ABT-199, a specific inhibitor of BCL2. Unlike ABT-199, AUY922 also upregulated the proapoptotic proteins BIM and BAD, whose increased expression was required for AUY922-induced apoptosis. Thus, the potent cytotoxicity of AUY922 involves the synergistic combination of BCL2 downregulation coupled with upregulation of the proapoptotic proteins BIM and BAD. This two-pronged assault on the mitochondrial apoptotic machinery identifies HSP90 inhibitors as promising drugs for targeting the TYK2-mediated prosurvival signaling axis in T-ALL cells. |
| 2015 | DLK1 is a novel inflammatory inhibitor which interferes with NOTCH1 signaling in TLR-activated murine macrophages. | Eur J Immunol | Delta-like protein 1 (DLK1) is a noncanonical ligand that inhibits NOTCH1 receptor activity and regulates multiple differentiation processes. In macrophages, NOTCH signaling increases TLR-induced expression of key pro-inflammatory mediators. We have investigated the role of DLK1 in macrophage activation and inflammation using Dlk1-deficient mice and Raw 264.7 cells overexpressing Dlk1. In the absence of Dlk1, NOTCH1 expression is increased and the activation of macrophages with TLR3 or TLR4 agonists leads to higher production of IFN-β and other pro-inflammatory cytokines, including TNF-α, IL-12, and IL-23. The expression of key proteins involved in IFN-β signaling, such as IRF3, IRF7, IRF1, or STAT1, as well as cRel, or RelB, which are responsible for the generation of IL-12 and IL-23, is enhanced in Dlk1 KO macrophages. Consistently, Dlk1 KO mice are more sensitive to LPS-induced endotoxic shock. These effects seem to be mediated through the modulation of NOTCH1 signaling. TLR4 activation reduces DLK1 expression, whereas increases NOTCH1 levels. In addition, DLK1 expression diminishes during differentiation of human U937 cells to macrophages. Overall, these results reveal a novel role for DLK1 as a regulator of NOTCH-mediated, pro-inflammatory macrophage activation, which could help to ensure a baseline level preventing constant tissue inflammation. |
| 2016 | SUMO modification of Akt regulates global SUMOylation and substrate SUMOylation specificity through Akt phosphorylation of Ubc9 and SUMO1. | Oncogene | SUMOylation is an important post-translational modification, and Akt SUMOylation was found to regulate cell proliferation, tumorigenesis and cell cycle, but the molecular mechanism of Akt SUMOylation is less well known. Here, we show both endogenous and ectopic Akt SUMOylation and Lys276 is the major SUMO acceptor on Akt. Further, Akt SUMOylation is Akt phosphorylation dependent and Akt SUMOylation increases Akt kinase activity without affecting the phosphorylation level of Akt. Moreover, endogenous Akt SUMOylation is enhanced by insulin treatment and this is Akt activity dependent. Heat-shock stimulus also increases Akt SUMOylation and it is also Akt activity dependent. Endogenous Akt SUMOylation is also found in the rat brain and it is enhanced by insulin-like growth factor-1 stimulation. In addition, Akt directly phosphorylates Ubc9 at Thr35 and phosphorylates SUMO1 at Thr76. Ubc9 phosphorylation at Thr35 promotes Ubc9 thioester bond formation and SUMO1 phosphorylation at Thr76 stabilizes the SUMO1 protein. Through these distinct mechanisms, Akt SUMOylation regulates global SUMOylation, including Akt and Ubc9 SUMOylation, and substrate SUMOylation specificity, including STAT1 and CREB SUMOylation, in different manners. Akt SUMOylation also enhances phosphatase and tensin homolog (PTEN) SUMOylation through Akt phosphorylation of Ubc9 and SUMO1, which serves as an endogenous mechanism to stop the positive feedback loop resulted from Akt activation. Further, Akt SUMOylation increases cyclin D1 expression and cell proliferation, and these effects are also mediated through Ubc9 phosphorylation at Thr35 and SUMO1 phosphorylation at Thr76. Here, we have identified a novel mechanism for SUMOylation regulation. Because of the important role Akt plays in tumorigenesis, this mechanism may also be involved in Akt-regulated tumorigenesis. |
| 2015 | Dengue Virus Control of Type I IFN Responses: A History of Manipulation and Control. | J Interferon Cytokine Res | The arthropod-borne diseases caused by dengue virus (DENV) are a major and emerging problem of public health worldwide. Infection with DENV causes a series of clinical manifestations ranging from mild flu syndrome to severe diseases that include hemorrhage and shock. It has been demonstrated that the innate immune response plays a key role in DENV pathogenesis. However, in recent years, it was shown that DENV evades the innate immune response by blocking type I interferon (IFN-I). It has been demonstrated that DENV can inhibit both the production and the signaling of IFN-I. The viral proteins, NS2A and NS3, inhibit IFN-I production by degrading cellular signaling molecules. In addition, the viral proteins, NS2A, NS4A, NS4B, and NS5, can inhibit IFN-I signaling by blocking the phosphorylation of the STAT1 and STAT2 molecules. Finally, NS5 mediates the degradation of STAT2 using the proteasome machinery. In this study, we briefly review the most recent insights regarding the IFN-I response to DENV infection and its implication for pathogenesis. |
| 2014 | Specific phosphorylation of histone demethylase KDM3A determines target gene expression in response to heat shock. | PLoS Biol | Histone lysine (K) residues, which are modified by methyl- and acetyl-transferases, diversely regulate RNA synthesis. Unlike the ubiquitously activating effect of histone K acetylation, the effects of histone K methylation vary with the number of methyl groups added and with the position of these groups in the histone tails. Histone K demethylases (KDMs) counteract the activity of methyl-transferases and remove methyl group(s) from specific K residues in histones. KDM3A (also known as JHDM2A or JMJD1A) is an H3K9me2/1 demethylase. KDM3A performs diverse functions via the regulation of its associated genes, which are involved in spermatogenesis, metabolism, and cell differentiation. However, the mechanism by which the activity of KDM3A is regulated is largely unknown. Here, we demonstrated that mitogen- and stress-activated protein kinase 1 (MSK1) specifically phosphorylates KDM3A at Ser264 (p-KDM3A), which is enriched in the regulatory regions of gene loci in the human genome. p-KDM3A directly interacts with and is recruited by the transcription factor Stat1 to activate p-KDM3A target genes under heat shock conditions. The demethylation of H3K9me2 at the Stat1 binding site specifically depends on the co-expression of p-KDM3A in the heat-shocked cells. In contrast to heat shock, IFN-γ treatment does not phosphorylate KDM3A via MSK1, thereby abrogating its downstream effects. To our knowledge, this is the first evidence that a KDM can be modified via phosphorylation to determine its specific binding to target genes in response to thermal stress. |
| 2014 | Antibody-validated proteins in inflamed islets of fulminant type 1 diabetes profiled by laser-capture microdissection followed by mass spectrometry. | PLoS One | There are no reports of proteomic analyses of inflamed islets in type 1 diabetes.Proteins expressed in the islets of enterovirus-associated fulminant type 1 diabetes (FT1DM) with extensive insulitis were identified by laser-capture microdissection mass spectrometry using formalin-fixed paraffin-embedded pancreatic tissues.Thirty-eight proteins were identified solely in FT1DM islets, most of which have not been previously linked to type 1 diabetes. Five protein-protein interacting clusters were identified, and the cellular localization of selected proteins was validated immunohistochemically. Migratory activity-related proteins, including plastin-2 (LCP1), moesin (MSN), lamin-B1 (LMNB1), Ras GTPase-activating-like protein (IQGAP1) and others, were identified in CD8+ T cells and CD68+ macrophages infiltrated to inflamed FT1DM islets. Proteins involved in successive signaling in innate/adaptive immunity were identified, including SAM domain and HD domain-containing protein 1 (SAMHD1), Ras GTPase-activating-like protein (IQGAP1), proteasome activator complex subunit 1 (PSME1), HLA class I histocompatibility antigen (HLA-C), and signal transducer and activator of transcription 1-alpha/beta (STAT1). Angiogenic (thymidine phosphorylase (TYMP)) and anti-angiogenic (tryptophan-tRNA ligase (WARS)) factors were identified in migrating CD8+ T cells and CD68+ macrophages. Proteins related to virus replication and cell proliferation, including probable ATP-dependent RNA helicase DEAD box helicase 5 (DDX5) and heterogeneous nuclear ribonucleoprotein H (HNRNPH1), were identified. The anti-apoptotic protein T-complex protein 1 subunit epsilon (CCT5), the anti-oxidative enzyme 6-phosphogluconate dehydrogenase (PDG), and the anti-viral and anti-apoptotic proteins serpin B6 (SERPINB6) and heat shock 70 kDa protein1-like (HSPA1L), were identified in FT1DM-affected islet cells.The identified FT1DM-characterizing proteins include those involved in aggressive beta cell destruction through massive immune cell migration and proteins involved in angiogenesis and islet vasculature bleeding, cell repair, and anti-inflammatory processes. Several target proteins for future type 1 diabetes interventions were identified. |
| 2014 | Soluble IFN receptor potentiates in vivo type I IFN signaling and exacerbates TLR4-mediated septic shock. | J Immunol | Circulating levels of a soluble type I IFNR are elevated in diseases, such as chronic inflammation, infections, and cancer, but whether it functions as an antagonist, agonist, or transporter is unknown. In this study, we elucidate the in vivo importance of the soluble type I IFNAR, soluble (s)IFNAR2a, which is generated by alternative splicing of the Ifnar2 gene. A transgenic mouse model was established to mimic the 10-15-fold elevated expression of sIFNAR2a observed in some human diseases. We generated transgenic mouse lines, designated SolOX, in which the transgene mRNA and protein-expression patterns mirrored the expression patterns of the endogenous gene. SolOX were demonstrated to be more susceptible to LPS-mediated septic shock, a disease model in which type I IFN plays a crucial role. This effect was independent of "classical" proinflammatory cytokines, such as TNF-α and IL-6, whose levels were unchanged. Because the increased levels of sIFNAR2a did not affect the kinetics of the increased interferonemia, this soluble receptor does not potentiate its ligand signaling by improving IFN pharmacokinetics. Mechanistically, increased levels of sIFNAR2a are likely to facilitate IFN signaling, as demonstrated in spleen cells overexpressing sIFNAR2a, which displayed quicker, higher, and more sustained activation of STAT1 and STAT3. Thus, the soluble IFNR is an important agonist of endogenous IFN actions in pathophysiological processes and also is likely to modulate the therapeutic efficacy of clinically administered IFNs. |
| 2014 | Interferon-γ induces senescence in normal human melanocytes. | PLoS One | Interferon-γ (IFN-γ) plays an important role in the proceedings of vitiligo through recruiting lymphocytes to the lesional skin. However, the potential effects of IFN-γ on skin melanocytes and the subsequent contribution to the vitiligo pathogenesis are still unclear.To investigate the effects of IFN-γ on viability and cellular functions of melanocytes.Primary human melanocytes were treated with IFN-γ. Cell viability, apoptosis, cell cycle melanin content and intracellular reactive oxygen species (ROS) level were measured. mRNA expression was examined by real-time PCR. The release of interleukin 6 (IL-6) and heat shock protein 70 (HSP-70) was monitored by ELISA. β-galactosidase staining was utilized to evaluate melanocyte senescence.Persistent IFN-γ treatment induced viability loss, apoptosis, cell cycle arrest and senescence in melanocytes. Melanocyte senescence was characterized as the changes in pigmentation and morphology, as well as the increase of β-galactosidase activity. Increase of p21Cip1/Waf1 protein was evident in melanocytes after IFN-γ treatment. IFN-γ induction of senescence was attenuated by siRNAs against p21, Janus kinase 2 (JAK2) or signal transducer and activator of transcription 1 (STAT1), but not by JAK1 siRNA nor by p53 inhibitor pifithrin-α. IFN-γ treatment increased the accumulation of intracellular ROS in melanocytes, while ROS scavenger N-acetyl cysteine (NAC) effectively inhibited IFN-γ induced p21 expression and melanocyte senescence. IL-6 and HSP-70 release was significantly induced by IFN-γ treatment, which was largely inhibited by NAC. The increase of IL-6 and HSP-70 release could also be observed in senescent melanocytes.IFN-γ can induce senescence in melanocytes and consequently enhance their immuno-competency, leading to a vitiligo-prone milieu. |
| 2013 | 5,6,7-trimethoxyflavone suppresses pro-inflammatory mediators in lipopolysaccharide-induced RAW 264.7 macrophages and protects mice from lethal endotoxin shock. | Food Chem Toxicol | 5,6,7-Trimethoxyflavone (TMF), methylations of the hydroxyl groups of oroxylin A or baicalein, was found to significantly inhibit the productions of nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. However, no report has been issued on the anti-inflammatory potential of TMF and the underlying molecular mechanism. In the present study, we investigated the anti-inflammatory effects of TMF in LPS-induced RAW 264.7 macrophages and LPS-induced septic shock in mice. TMF dose-dependently inhibits iNOS and COX-2 at the protein, mRNA, and promoter binding levels and that these inhibitions cause attendant decreases in the productions of NO and PGE2. TMF inhibits the productions and mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 induced by LPS. Furthermore, TMF suppress the transcriptional activity of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1), and nuclear translocations of NF-κB, AP-1, and signal transducer and activator of transcription 1/3 (STAT1/3). Pretreatment with TMF increase the survival rate of mice with LPS-induced endotoxemia and reduced the serum levels of cytokines. Taken together, these findings suggest that TMF down-regulates the expressions of the pro-inflammatory iNOS, COX-2, TNF-α, IL-1β, and IL-6 genes in macrophages by interfering with the activation of NF-κB, AP-1, and STAT1/3. |
| 2013 | Transcriptomic profiling of progesterone in the male fathead minnow (Pimephales promelas) testis. | Gen Comp Endocrinol | P4 is a hormone with diverse functions that include roles in reproduction, growth, and development. The objectives of this study were to examine the effects of P4 on androgen production in the mature teleost testis and to identify molecular signaling cascades regulated by P4 to improve understanding of its role in male reproduction. Fathead minnow (FHM) testis explants were treated in vitro with two concentrations of P4 (10(-8) and 10(-6) M) for 6 and 12 h. P4 significantly increased testosterone (T) production in the FHM testis but did not affect 11-ketotestosterone. Gene network analysis revealed that insulin growth factor (Igf1) and tumor necrosis factor receptor (Tnfr) signaling was significantly depressed with P4 treatment after 12h. There was also a 20% increase in a gene network for follicle-stimulating hormone secretion and an 18% decrease in genes involved in vasopressin signaling. Genes in steroid metabolism (e.g. star, cyp19a, 11bhsd) were not significantly affected by P4 treatments in this study, and it is hypothesized that pre-existing molecular machinery may be more involved in the increased production of T rather than the de novo expression of steroid-related transcripts and receptors. There was a significant decrease in prostaglandin E synthase 3b (cytosolic) (ptges3b) after treatment with P4, suggesting that there is cross talk between P4 and prostaglandin pathways in the reproductive testis. P4 has a role in regulating steroid production in the male testis and may do so by modulating gene networks related to endocrine pathways, such as Igf1, Tnfr, and vasopressin. |
| 2013 | Phosphorylation of signal transducer and activator of transcription 1 reduces bortezomib-mediated apoptosis in cancer cells. | Cell Death Dis | The potent and selective proteasome inhibitor bortezomib has shown remarkable antitumor activity and is now entering clinical trials for several cancers. However, the molecular mechanisms by which bortezomib induces cytotoxicity in ovarian cancer cells still remain unclear. In this study, we show that bortezomib induced apoptosis, which was demonstrated by the downregulation of antiapoptotic molecules (Bcl-2, Bcl-XL, p-Bad, and p-AKT) and the upregulation of proapoptotic proteins (p21, p27, and cleaved-Bid) in ovarian cancer cell lines. Moreover, bortezomib stimulates Janus kinase (JAK) phosphorylation and activates heat-shock transcription factor-1 (HSF-1) and heat-shock protein 70 (HSP70), ultimately leading to signal transducer and activator of transcription 1 (STAT1) phosphorylation. Phosphorylated STAT1 partially counteracted apoptosis induced by bortezomib in cancer cells. These findings suggest that the antitumor activity of bortezomib in ovarian cancer can be improved by inhibiting bortezomib-induced STAT1 phosphorylation. This effect can be achieved by STAT1 knockdown, HSP70 knockdown, JAK inhibition, or the addition of cisplatin, one of the most commonly used anticancer drugs. These results provide the first evidence that STAT1 phosphorylation can play a role in bortezomib resistance by exerting antiapoptotic effects. They also suggest the possibility to abolish or reduce bortezomib chemoresistance in ovarian cancer by the addition of cisplatin or JAK inhibitors. |
| 2013 | Requirement of heat shock protein 70 for inducible nitric oxide synthase induction. | Cell Signal | Inducible nitric oxide synthase (iNOS) contributes critically to inflammation and host defense. While normally undetectable, iNOS expression is induced by endotoxins and cytokines via discrete signaling pathways. Lipopolysaccharide (LPS) triggers iNOS gene transactivation by the IKK-NF-κB cascade, whereas interferon-γ (IFN-γ) acts through transcriptional factor STAT1 and IRF-1. Previous studies showed that heat shock protein 90 is essential for iNOS gene transactivation. But the role of the closely related heat shock protein 70 (Hsp70) in iNOS induction remains unknown. We address this issue in cultured cells and endotoxemic mice. With mouse macrophages, Hsp70 inhibition or knockdown prevented LPS/IFN-γ-stimulated iNOS protein expression. RT-PCR experiments showed that both LPS- and IFN-γ-induced iNOS mRNA transcriptions were blocked by Hsp70 inhibitor. The preventing effect of Hsp70 inhibition on iNOS gene transcription was confirmed in vivo in endotoxemic mice. Further studies revealed that Hsp70 inhibition disabled IKK activation in LPS-stimulated cells, hence precluding NF-κB-initiated iNOS gene transcription. Intriguingly, Hsp70 inhibition had little effect on IFN-γ-elicited STAT1 activation or IRF-1 upregulation. But ChIP assays showed that both STAT1 and IRF-1 bindings to iNOS promoters were markedly reduced in Hsp70-inhibited cells. Hsp70 inhibition had no significant effect on iNOS mRNA stability. These studies uncover the necessity of Hsp70 for iNOS induction. Hsp70 is required for IKK activation and STAT1/IRF-1 promoter binding amid iNOS gene transactivation. Selectively targeting Hsp70 may be a new approach to intervene iNOS expression in diseases. |
| 2013 | Fatal Mycobacterium colombiense/cytomegalovirus coinfection associated with acquired immunodeficiency due to autoantibodies against interferon gamma: a case report. | BMC Infect Dis | Reports of acquired immunodeficiency due to autoantibodies against interferon gamma in the adult population are increasing. The interleukin-12-dependent interferon-gamma axis is a major regulatory pathway of cell-mediated immunity and is critical for protection against a few intracellular organisms, including non-tuberculous mycobacteria and Salmonella spp. We report the first case of a fatal disseminated Mycobacterium colombiense/cytomegalovirus coinfection in an adult woman associated with the acquisition of autoantibodies against interferon-gamma.A 49-year-old woman, born to nonconsanguineous parents in Laos, but who had lived in Canada for the past 30 years, presented with a 1-month history of weight loss, fatigue, cough, and intermittent low-grade fever. A thoracic computed tomography scan revealed an 8 × 7 cm irregular mass impacting the right superior lobar bronchus along with multiple mediastinal and hilar adenopathies. On the fourth day of admission, the patient developed fever with purulent expectorations. Treatment for a post-obstructive bacterial pneumonia was initiated while other investigations were being pursued. Almost every culture performed during the patient's hospitalization was positive for M. colombiense. Given the late presentation of symptoms - at the age of 49 years - and the absence of significant family or personal medical history, we suspected an acquired immunodeficiency due to the presence of anti-interferon-gamma autoantibodies. This was confirmed by their detection at high levels in the plasma and a STAT1 phosphorylation assay on human monocytes. The final diagnosis was immunodeficiency secondary to the production of autoantibodies against interferon-gamma, which resulted in a post-obstructive pneumonia and disseminated infection of M. colombiense. The clinical course was complicated by the presence of a multiresistant Pseudomonas aeruginosa post-endobronchial ultrasound mediastinitis, cytomegalovirus pneumonitis with dissemination, and finally, susceptible P. aeruginosa ventilator-associated pneumonia with septic shock and multiple organ failure, leading to death despite appropriate antibacterial and anti-mycobacterial treatment.Although rare, acquired immunodeficiency syndromes should be considered in the differential diagnosis of patients with severe, persistent, or recurrent infections. Specifically, severe non-tuberculous mycobacteria or Salmonella infections in adults without any other known risk factors may warrant examination of autoantibodies against interferon-gamma because of their increasing recognition in the literature. |
| 2013 | The aerial part of Taraxacum coreanum extract has an anti-inflammatory effect on peritoneal macrophages in vitro and increases survival in a mouse model of septic shock. | J Ethnopharmacol | Taraxacum coreanum Nakaiis a dandelion native to Korea and is widely consumed as an edible and medicinal herb. The aerial part of Taraxacum coreanum (TC) has been used therapeutically as a diuretic and anti-inflammatory agent, but its mechanism of action has not yet been evaluated.To investigate the anti-inflammatory potential of a Taraxacum coreanum chloroform fraction(TCC) and its mechanisms of action in vitro and in vivo.Isolated mouse peritoneal macrophages were stimulated in vitro with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) in the presence or absence of TCC. The anti-inflammatory effects of TCC were assessed by measuring nitric oxide (NO) and prostaglandin E2 (PGE2) production, as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), IκBα, phospho-IKK, mitogen-activated protein kinases (MAPKs), and signal transducer and activator of transcription (STAT1). The effects of TCC were tested in vivo by measuring cytokine production and survival in a mouse model of lethal septic shock. And the standard compounds of Taraxacum coreanum were analyzed by HPLC using a C18 column.Treatment of primary macrophages with TCC in vitro significantly inhibited all of the inflammatory parameters measured, including LPS-induced NO and PGE2 production, iNOS and COX-2 expression, IκBα degradation, IKK phosphorylation, and MAPK and STAT1 activation. In a mouse model of LPS-induced septic shock, TCC inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, and increased survival by 83%.Standard compounds (gallic acid, syringic acid) of Taraxacum coreanum were qualified by HPLC analysis.TCC possesses potent anti-inflammatory activity in vitro and in vivo, which occurs at least partly through inhibition of proinflammatory signaling and mediator release. These results strongly support the therapeutic potential of TCC as an anti-inflammatory agent in vivo. |
| 2013 | Suppressors of cytokine signaling 2 and 3 diametrically control macrophage polarization. | Immunity | Suppressors of cytokine signaling (SOCS) are important regulators of lipopolysaccharide (LPS) and cytokine responses but their role in macrophage polarization is unknown. We have shown here that myeloid-restricted Socs3 deletion (Socs3(Lyz2cre)) resulted in resistance to LPS-induced endotoxic shock, whereas Socs2(-/-) mice were highly susceptible. We observed striking bias toward M2-like macrophages in Socs3(Lyz2cre) mice, whereas the M1-like population was enriched in Socs2(-/-) mice. Adoptive transfer experiments showed that responses to endotoxic shock and polymicrobial sepsis were transferable and macrophage dependent. Critically, this dichotomous response was associated with enhanced regulatory T (Treg) cell recruitment by Socs3(Lyz2cre) cells, whereas Treg cell recruitment was absent in the presence of Socs2(-/-) macrophages. In addition, altered polarization coincided with enhanced interferon-gamma (IFN-γ)-induced signal transducer and activator of transcription-1 (STAT1) activation in Socs2(-/-) macrophages and enhanced interleukin-4 (IL-4) plus IL-13-induced STAT6 phosphorylation in Socs3(Lyz2cre) macrophages. SOCS, therefore, are essential controllers of macrophage polarization, regulating inflammatory responses. |
| 2012 | STAT1-deficient mice are resistant to cecal ligation and puncture-induced septic shock. | Shock | STAT1 (signal transducer and activator of transcription 1) is a member of the JAK-STAT signaling family and plays a key role in facilitating gene transcription in response to activation of the types I and II interferon (IFN) receptors. TYK2 is essential for type I, but not type II, IFN-induced STAT1 activation. Previous studies show that STAT1-deficient mice are resistant to endotoxin-induced shock. The goal of the present study was to assess the response of STAT1- and TYK2-deficient mice to septic shock caused by cecal ligation and puncture (CLP). End points included survival, core temperature, organ injury, systemic cytokine production, and bacterial clearance. Results showed that survival rates were significantly higher in STAT1 knockout (STAT1KO) mice compared with wild-type controls (80% vs. 10%). The improved survival of STAT1KO mice was associated with less hypothermia, metabolic acidosis, hypoglycemia, and hepatocellular injury. Plasma interleukin 6, MIP-2, CXCL10, and IFN-α concentrations were significantly lower in STAT1KO mice than in wild-type mice. In the absence of antibiotic treatment, blood and lung bacterial counts were significantly lower in STAT1KO mice than in controls. However, treatment with antibiotics ablated that difference. A survival advantage was not observed in TYK2-deficient mice compared with control. However, CLP-induced hypothermia and systemic interleukin 6 and CXCL10 production were significantly attenuated in TYK2-deficient mice. These results indicate that STAT1 activation is an important factor in the pathogenesis of CLP-induced septic shock and is associated with the development of systemic inflammation and organ injury. TYK2 activation also appears to contribute to CLP-induced inflammation, but to a lesser extent than STAT1. |
| 2012 | CLEC5A regulates Japanese encephalitis virus-induced neuroinflammation and lethality. | PLoS Pathog | CLEC5A/MDL-1, a member of the myeloid C-type lectin family expressed on macrophages and neutrophils, is critical for dengue virus (DV)-induced hemorrhagic fever and shock syndrome in Stat1⁻/⁻ mice and ConA-treated wild type mice. However, whether CLEC5A is involved in the pathogenesis of viral encephalitis has not yet been investigated. To investigate the role of CLEC5A to regulate JEV-induced neuroinflammation, antagonistic anti-CLEC5A mAb and CLEC5A-deficient mice were generated. We find that Japanese encephalitis virus (JEV) directly interacts with CLEC5A and induces DAP12 phosphorylation in macrophages. In addition, JEV activates macrophages to secrete proinflammatory cytokines and chemokines, which are dramatically reduced in JEV-infected Clec5a⁻/⁻ macrophages. Although blockade of CLEC5A cannot inhibit JEV infection of neurons and astrocytes, anti-CLEC5A mAb inhibits JEV-induced proinflammatory cytokine release from microglia and prevents bystander damage to neuronal cells. Moreover, JEV causes blood-brain barrier (BBB) disintegrity and lethality in STAT1-deficient (Stat1⁻/⁻) mice, whereas peripheral administration of anti-CLEC5A mAb reduces infiltration of virus-harboring leukocytes into the central nervous system (CNS), restores BBB integrity, attenuates neuroinflammation, and protects mice from JEV-induced lethality. Moreover, all surviving mice develop protective humoral and cellular immunity against JEV infection. These observations demonstrate the critical role of CLEC5A in the pathogenesis of Japanese encephalitis, and identify CLEC5A as a target for the development of new treatments to reduce virus-induced brain damage. |
| 2012 | Suppression of antigen-specific CD4+ T cell activation by SRA/CD204 through reducing the immunostimulatory capability of antigen-presenting cell. | J Mol Med (Berl) | Pattern recognition scavenger receptor SRA/CD204, primarily expressed on specialized antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages, has been implicated in multiple physiological and pathological processes, including atherosclerosis, Alzheimer's disease, endotoxic shock, host defense, and cancer development. SRA/CD204 was also recently shown to function as an attenuator of vaccine response and antitumor immunity. Here, we, for the first time, report that SRA/CD204 knockout (SRA(-/-)) mice developed a more robust CD4(+) T cell response than wild-type mice after ovalbumin immunization. Splenic DCs from the immunized SRA(-/-) mice were much more efficient than those from WT mice in stimulating naïve OT-II cells, indicating that the suppressive activity of SRA/CD204 is mediated by DCs. Strikingly, antigen-exposed SRA(-/-) DCs with or without lipopolysaccharide treatment exhibited increased T-cell-stimulating activity in vitro, which was independent of the classical endocytic property of the SRA/CD204. Additionally, absence of SRA/CD204 resulted in significantly elevated IL12p35 expression in DCs upon CD40 ligation plus interferon gamma (IFN-γ) stimulation. Molecular studies reveal that SRA/CD204 inhibited the activation of STAT1, mitogen activated protein kinase p38, and nuclear factor-kappa B signaling activation in DCs treated with anti-CD40 antibodies and IFN-γ. Furthermore, splenocytes from the generated SRA(-/-) OT-II mice showed heightened proliferation upon stimulation with OVA protein or MHC-II-restricted OVA(323-339) peptide compared with cells from the SRA(+/+) OT-II mice. These results not only establish a new role of SRA/CD204 in limiting the intrinsic immunogenicity of APCs and CD4(+) T cell activation but also provide additional insights into the molecular mechanisms involved in the immune suppression by this molecule. |
| 2011 | Obligatory role of heat shock protein 90 in iNOS induction. | Am J Physiol Cell Physiol | Inducible nitric oxide (NO) synthase (iNOS) plays an important role in cell injury and host defense. While undetectable in normal tissues, iNOS expression is induced by endotoxins and inflammatory cytokines primarily via the IκB kinase/nuclear factor-κB (IKK-NF-κB) and Janus kinase (JAK)-signal transducers and activators of transcription 1 (STAT1) pathways. Our previous studies found that heat shock protein 90 (Hsp90) associates with iNOS, and this association enhances iNOS activity. Here we show that Hsp90 is also essential for iNOS induction. With mouse macrophages, Hsp90 inhibition by geldanamycin or knockdown with small interfering RNA (siRNA) prevented lipopolysaccharide (LPS) or interferon-γ (IFN-γ)-stimulated iNOS protein expression. RT-PCR experiments showed that iNOS mRNA transcription was blocked by Hsp90 inhibition. Radicicol, another Hsp90 inhibitor whose structure is different from that of geldanamycin, also blocked iNOS mRNA transcription. These cell biology findings were confirmed in infarcted myocardium where iNOS expression was markedly attenuated by Hsp90 inhibition in vivo. Intriguingly, further analyses showed that inhibiting Hsp90 had no significant effect on the activation of either IKK-NF-κB or JAK-STAT1 in LPS/IFN-γ-stimulated cells. Neither was the nuclear transport of active NF-κB or STAT1 affected by Hsp90 inhibition. But Hsp90 inhibition markedly reduced the binding of active NF-κB and STAT1 to their DNA elements. Chromatin immunoprecipitation assays confirmed that Hsp90 was essential for NF-κB and STAT1 bindings to iNOS promoters inside cells. These studies reveal that besides acting as an allosteric enhancer, Hsp90 is also required for transcriptional factor binding amid iNOS mRNA transcription. In view of the essential role of Hsp90 in iNOS gene transactivation, targeting Hsp90 may represent a new approach to intervene iNOS expression in diseases. |
| 2011 | STAT2 mediates innate immunity to Dengue virus in the absence of STAT1 via the type I interferon receptor. | PLoS Pathog | Dengue virus (DENV) is a mosquito-borne flavivirus, and symptoms of infection range from asymptomatic to the severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). High viral loads correlate with disease severity, and both type I & II interferons (IFNs) are crucial for controlling viral replication. We have previously reported that signal transducer and activator of transcription (STAT) 1-deficient mice are resistant to DENV-induced disease, but little is known about this STAT1-independent mechanism of protection. To determine the molecular basis of the STAT1-independent pathway, mice lacking STAT1, STAT2, or both STAT1 and STAT2 were infected with a virulent mouse-adapted strain of DENV2. In the first 72 hours of infection, the single-deficient mice lacking STAT1 or STAT2 possessed 50-100 fold higher levels of viral RNA than wild type mice in the serum, spleen, and other visceral tissues, but remained resistant to DENV-induced death. In contrast, the double-deficient mice exhibited the early death phenotype previously observed in type I and II IFN receptor knockout mice (AG129), indicating that STAT2 is the mediator of the STAT1-independent host defense mechanism. Further studies demonstrated that this STAT2-dependent STAT1-independent mechanism requires the type I IFN receptor, and contributes to the autocrine amplification of type I IFN expression. Examination of gene expression in the spleen and bone marrow-derived macrophages following DENV infection revealed STAT2-dependent pathways can induce the transcription of a subset of interferon stimulated genes even in the absence of STAT1. Collectively, these results help elucidate the nature of the poorly understood STAT1-independent host defense mechanism against viruses by identifying a functional type I IFN/STAT2 signaling pathway following DENV infection in vivo. |
| 2010 | A switch from hBrm to Brg1 at IFNγ-activated sequences mediates the activation of human genes. | Cell Res | The SWI/SNF chromatin-remodeling complexes utilize energy from ATP hydrolysis to reposition nucleosomes and regulate the expression of human genes. Here, we studied the roles of human Brahma (hBrm) and Brahma-related gene 1 (Brg1), the ATPase subunits of the SWI/SNF complexes, in regulating human genes. Our results indicate that both hBrm and Brg1 interact with Signal transducer and activator of transcription (Stat) 1 in vitro. However, Stat1 in its native form only recruits hBrm to IFNγ-activated sequences (GAS) of individual genes; by contrast, in a stress-induced phosphorylated form, Stat1 mainly binds to Brg1. Under basal conditions, hBrm is recruited by native Stat1 to the GAS and exists in a mSin3/HDAC co-repressor complex on the hsp90α gene, which shows a compact chromatin structure. Upon heat-shock, hBrm is acetylated by p300 and dissociates from the co-repressor complex, which the phosphorylated Stat1 is increased, and binds and recruits Brg1 to the GAS, leading to elevated induction of the gene. This hBrm/Brg1 switch also occurs at the GAS of all of the three examined immune genes in heat-shocked cells; however, this switch only occurs in specific cell types upon exposure to IFNγ. Regardless of the stimulus, the hBrm/Brg1 switch at the GAS elicits an increase in gene activity. Our data are consistent with the hypothesis that the hBrm/Brg1 switch is an indicator of the responsiveness of a gene to heat-shock or IFNγ stimulation and may represent an "on-off switch" of gene expression in vivo. |
| 2010 | The early whole-blood transcriptional signature of dengue virus and features associated with progression to dengue shock syndrome in Vietnamese children and young adults. | J Virol | Dengue is a pantropic public health problem. In children, dengue shock syndrome (DSS) is the most common life-threatening complication. The ability to predict which patients may develop DSS may improve triage and treatment. To this end, we conducted a nested case-control comparison of the early host transcriptional features in 24 DSS patients and 56 sex-, age-, and virus serotype-matched uncomplicated (UC) dengue patients. In the first instance, we defined the "early dengue" profile. The transcriptional signature in acute rather than convalescent samples (≤72 h post-illness onset) was defined by an overabundance of interferon-inducible transcripts (31% of the 551 overabundant transcripts) and canonical gene ontology terms that included the following: response to virus, immune response, innate immune response, and inflammatory response. Pathway and network analyses identified STAT1, STAT2, STAT3, IRF7, IRF9, IRF1, CEBPB, and SP1 as key transcriptional factors mediating the early response. Strikingly, the only difference in the transcriptional signatures of early DSS and UC dengue cases was the greater abundance of several neutrophil-associated transcripts in patients who progressed to DSS, a finding supported by higher plasma concentrations of several canonical proteins associated with neutrophil degranulation (bactericidal/permeability-increasing protein [BPI], elastase 2 [ELA2], and defensin 1 alpha [DEF1A]). Elevated levels of neutrophil-associated transcripts were independent of the neutrophil count and also of the genotype of the infecting virus, as genome-length sequences of dengue virus serotype 1 (DENV-1) (n = 15) and DENV-2 (n = 3) sampled from DSS patients were phylogenetically indistinguishable from those sampled from uncomplicated dengue patients (32 DENV-1 and 9 DENV-2 sequences). Collectively, these data suggest a hitherto unrecognized association between neutrophil activation, pathogenesis, and the development of DSS and point to future strategies for guiding prognosis. |
| 2010 | N-ethyl-N-nitrosourea-induced mutation in ubiquitin-specific peptidase 18 causes hyperactivation of IFN-αß signaling and suppresses STAT4-induced IFN-γ production, resulting in increased susceptibility to Salmonella typhimurium. | J Immunol | To deepen our knowledge of the natural host response to pathogens, our team undertook an in vivo screen of mutagenized 129S1 mice with Salmonella Typhimurium. One mutation affecting Salmonella susceptibility was mapped to a region of 1.3 Mb on chromosome 6 that contains 15 protein-coding genes. A missense mutation was identified in the Usp18 (ubiquitin-specific peptidase 18) gene. This mutation results in an increased inflammatory response (IL-6, type 1 IFN) to Salmonella and LPS challenge while paradoxically reducing IFN-gamma production during bacterial infection. Increased STAT1 phosphorylation correlated with impaired STAT4 phosphorylation, resulting in overwhelming IL-6 secretion but reduced IFN-gamma production during infection. The reduced IFN-gamma levels, along with the increased inflammation, rationalize the S. Typhimurium susceptibility in terms of increased bacterial load in target organs and cytokine-induced septic shock and death. |
| 2010 | Activation of the stress protein response inhibits the STAT1 signalling pathway and iNOS function in alveolar macrophages: role of Hsp90 and Hsp70. | Thorax | Alveolar fluid clearance is impaired by inducible nitric oxide synthase (iNOS)/nitric oxide (NO)-dependent mechanisms in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The activation of the stress protein response (SPR) in alveolar macrophages on iNOS-dependent NO production in response to interferon gamma (IFNgamma), a major cytokine present in the airspace of patients with ALI, was investigated.The SPR was activated in murine and primary human alveolar macrophages prior to analysis of signal transducer and activator of transcription factor 1 (STAT1) activation, iNOS mRNA and protein synthesis, and NO production.SPR activation resulted in inhibition of IFNgamma-mediated NO production (p=0.001) with >95% detergent insolubilisation of the STAT1 protein. Its subsequent proteasomal degradation was partially reversed with pretreatment of cells with the chemical chaperone glycerol. This early effect of the SPR was caused by the complete disruption of heat shock protein 90 (Hsp90)-STAT1 binding, as shown by immunoprecipitation. Recovery of STAT1 activation and recovery of iNOS synthesis occurred within 12 h after SPR activation (p=0.02). NO production (as compared with non-SPR controls) did not occur until 48 h later (p=0.02). SPR-induced Hsp70 (Hsp70i) expression caused a late inhibition of NO production (p=0.02). Inhibiting >50% Hsp70i expression recovered NO production to control levels whereas overexpressing Hsp70i in the absence of the SPR inhibited NO production (p=0.02).Early inhibition of STAT1 following its dissociation from Hsp90, and later inhibition of iNOS activity by Hsp70i, represent novel mechanisms by which SPR activation modulates the IFNgamma signalling in alveolar macrophages. These results highlight a potential clinical application for Hsp90 inhibitors in modulating NO signalling during the early phase of acute lung injury. |
| 2010 | Stat1 mediates an auto-regulation of hsp90beta gene in heat shock response. | Cell Signal | We have reported earlier that a heat shock element in the first intron of human hsp90beta gene (iHSE) acts as an intronic enhancer to bind the heat shock factor (HSF1) and activates hsp90beta gene under heat shock. Here, we show that, in addition to the HSF1, Stat1 phosphorylation is indispensable in the event. We show that Jak2, a Janus kinase specifically associated with the beta subunit of IFNgamma receptor, and PKCepsilon an isoform of the atypical PKC family, are the two dominant kinases responsible for the heat shock induced phosphorylation on Y701 and S727 of Stat1. However, the activation of these kinases under heat shock requires the association of chaperone proteins of the Hsp90 family, in particular, the Hsp90beta under heat shock. Furthermore, Brg1, an ATPase subunit of the SWI/SNF chromatin remodeling complex is likely recruited by HSF1 and Stat1 at the iHSE under heat shock. Brg1 further confers an open chromatin conformation at the promoter region that is pivotal to the heat shock induced fully activation of the hsp90beta gene in Jurkat cells. This is a novel example of how multiple activation steps occur under heat shock, first on the kinases and then the Stat1 and the SWI/SNF chromatin remodeling complex that follows to conduct an auto-regulation based fully activation of the gene. |
| 2009 | Therapeutic applications and mechanisms underlying the activity of immunosuppressive oligonucleotides. | Ann N Y Acad Sci | Synthetic oligodeoxynucleotides (ODN) capable of "neutralizing" or "inhibiting" immune responses have been described. This review will focus on the properties of phosphorothioate ODN that mimic the immunosuppressive activity of the repetitive TTAGGG motifs present in mammalian telomeres. These TTAGGG multimers block the production of pro-inflammatory and T helper type 1 cytokines elicited when immune cells are activated by a wide variety of Toll-like receptor ligands, polyclonal activators, and antigens. Several mechanisms contribute to the suppressive activity of such ODN. Ongoing microarray studies indicate that suppressive ODN interfere with the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT4, thereby blocking the inflammation mediated by STAT-associated signaling cascades. In animal models, suppressive ODN can be used to prevent or treat diseases characterized by persistent immune activation, including collagen-induced arthritis, inflammatory arthritis, systemic lupus erythematosus, silicosis, and toxic shock. These findings suggest that TTAGGG multimers may find broad use in the treatment of diseases characterized by over-exuberant/persistent immune activation. |
| 2009 | Aryl hydrocarbon receptor in combination with Stat1 regulates LPS-induced inflammatory responses. | J Exp Med | Toll-like receptor (TLR) signals perform a crucial role in innate immune responses to pathogens. In this study, we found that the aryl hydrocarbon receptor (Ahr) negatively regulates inflammatory responses mediated by lipopolysaccharide (LPS) in macrophages. Ahr was induced in macrophages stimulated by LPS, but not by transforming growth factor (TGF)-beta plus interleukin (IL)-6, which can induce Ahr in naive T cells. The production of IL-6 and tumor necrosis factor (TNF)-alpha by LPS was significantly elevated in Ahr-deficient macrophages compared with that in wild-type (WT) cells. Ahr-deficient mice were more highly sensitive to LPS-induced lethal shock than WT mice. Signal transducer and activator of transcription 1 (Stat1) deficiency, as well as Ahr deficiency, augmented LPS-induced IL-6 production. We found that Ahr forms a complex with Stat1 and nuclear factor-kappa B (NF-kappaB) in macrophages stimulated by LPS, which leads to inhibition of the promoter activity of IL-6. Ahr thus plays an essential role in the negative regulation of the LPS signaling pathway through interaction with Stat1. |
| 2009 | HSP90 is essential for Jak-STAT signaling in classical Hodgkin lymphoma cells. | Cell Commun Signal | In classical Hodgkin lymphoma (cHL) chemotherapeutic regimens are associated with stagnant rates of secondary malignancies requiring the development of new therapeutic strategies. We and others have shown that permanently activated Signal Transducer and Activator of Transcription (STAT) molecules are essential for cHL cells. Recently an overexpression of heat-shock protein 90 (HSP90) in cHL cells has been shown and inhibition of HSP90 seems to affect cHL cell survival. Here we analysed the effects of HSP90 inhibition by geldanamycin derivative 17-AAG or RNA interference (RNAi) on aberrant Jak-STAT signaling in cHL cells. Treatment of cHL cell lines with 17-AAG led to reduced cell proliferation and a complete inhibition of STAT1, -3, -5 and -6 tyrosine phosphorylation probably as a result of reduced protein expression of Janus kinases (Jaks). RNAi-mediated inhibition of HSP90 showed similar effects on Jak-STAT signaling in L428 cHL cells. These results suggest a central role of HSP90 in permanently activated Jak-STAT signaling in cHL cells. Therapeutics targeting HSP90 may be a promising strategy in cHL and other cancer entities associated with deregulated Jak-STAT pathway activation. |
| 2009 | Inhibition of zymosan-induced kidney dysfunction by tyrphostin AG-490. | J Inflamm (Lond) | Zymosan-induced shock has been associated with an increased production of pro-inflammatory cytokines and mediators, causing a generalized dysfunction of liver, lung and kidneys. Herein, we investigate the effects of tyrphostin AG-490 on the early inflammation and on the late renal injury provoked by zymosan injection.Shock was induced by intraperitoneal injection of zymosan in a dose of 0.8-1.0 mg/g body weight in BALB/c mice and 0.8 mg/g body weight in SCID mice. Tyrphostin AG-490 was administered intraperitoneally in a dose of 5 mg/kg immediately after shock induction. Blood, peritoneal lavage and kidneys were collected at certain time points after zymosan injection. The levels of MIP-1alpha, RANTES, IL-6, IL-10, alpha1-antitrypsin and C5a in plasma were determined by ELISA. The number of IL-10-secreting cells in peritoneum was assayed by ELISPOT. Kidney function was monitored by measurement of urine/plasma creatinine levels and proteinuria. Histological assessment of renal injury was performed in a blinded fashion after hematoxylin/eosin staining. Immunohistochemistry analyses were used to evaluate the expression of C5aR, STAT1, STAT3 and the binding ability of IgGs in kidneys.Tyrphostin AG-490 attenuated the early phase of zymosan-induced shock via inhibition of MIP-1alpha, RANTES and C5a plasma levels and via elevation of IL-10 in plasma. The drug increased IL-10 production in peritoneum and the number of IL-10-secreting peritoneal cells. AG-490 was able to retain the time of coagulation and the level of alpha1-antitrypsin to normal values. At the late stage of shock, AG-490 decreased scores of tubular injury, cell infiltration and glomerular lesions in parallel with diminished creatinine plasma level and protein excretion. These beneficial effects of AG-490 were related to lowered levels of circulating IL-6, MIP-1alpha and C5a, and to inhibited expression of STAT1, STAT3 and C5aR in kidneys. The drug diminished the production of zymosan-specific IgG antibodies and hindered the glomeruli from IgGs recognition.Tyrphostin AG-490 reduced the magnitude of the initial inflammatory response in zymosan-induced shock and prevented the development of severe kidney dysfunction. Our data suggest that the drug might be used as a therapeutic approach in cases where shock is combined with acute renal injury. |
| 2009 | Identification of novel SHPS-1-associated proteins and their roles in regulation of insulin-like growth factor-dependent responses in vascular smooth muscle cells. | Mol Cell Proteomics | Tyrosine phosphatase non-receptor type substrate-1 (SHPS-1), a transmembrane protein, plays a vital role in cell migration and proliferation. Our previous studies have shown that insulin-like growth factor-I (IGF-I) stimulates SHPS-1 phosphorylation, leading to recruitment of SHP-2, c-Src, Shc, and Grb2.p85 to phosphorylated SHPS-1. Assembly of this signaling complex is required for optimal stimulation of both mitogen-activated protein and phosphatidylinositol 3-kinase pathways. The main aim of the present study was to identify novel proteins that interacted with the cytoplasmic domain of SHPS-1 (SHPS-1/CD) in response to IGF-I stimulation and define the role of these interactions in mediating specific biological functions. We performed a functional proteomic screening to identify SHPS-1 binding partners using combination of mRNA display and the tandem affinity purification-tag methods. Screening identified a number of proteins not previously known to interact with phosphorylated SHPS-1/CD. These novel SHPS-1 binding partners represent several functional categories including heat shock proteins, protein kinases and phosphatases, and proteins that regulate transcription or translation. In Vivo and in vitro studies suggested that most of the proteins bound to SHPS-1 via binding to one of the four SH2 domain containing proteins, SHP-2, CTK, SUPT6H, and STAT1, that directly bound to SHPS-1. Although the binding of most of these proteins to SHPS-1 was positively regulated by IGF-I, a few were negatively regulated, suggesting differential regulation of protein complexes assembled on SHPS-1/CD in response to IGF-I. Further studies showed that truncation of SHPS-1/CD significantly impaired IGF-I-dependent AKT signal transduction and subsequent biological functions including cell survival, protein synthesis, protein aggregation, and prevention of apoptosis. The results emphasize the importance of formation of SHPS-1 signaling complex induced by IGF-I and provide novel insights into our knowledge of the role of this molecular scaffold in regulation of IGF-I-stimulated signal transduction and biological actions. |
| 2008 | Effects of levo- and dextrosimendan on NF-kappaB-mediated transcription, iNOS expression and NO production in response to inflammatory stimuli. | Br J Pharmacol | Levosimendan is used in the treatment of decompensated heart failure. It increases the contractility of the myocardium by sensitizing troponin C to calcium. In addition, levosimendan has been reported to have beneficial effects in experimental models of septic shock. Because heart failure and sepsis have been associated with excessive nitric oxide (NO) production through inducible NOS (iNOS), we investigated the effects of the simendans on NO production and iNOS expression and on generation of pro-inflammatory cytokines.Macrophages and fibroblasts were exposed to inflammatory stimuli to induce iNOS expression. Proteins were measured by western blot and mRNA expression was determined by quantitative RT-PCR. Promoter activity and nuclear factor-kappaB (NF-kappaB) and the gamma-activated site (GAS; binding site for signal transducer and activator of transcription 1; STAT1)-mediated transcription were investigated using luciferase reporter constructs. Cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were measured by ELISA.Levosimendan and dextrosimendan decreased NO production in a dose-dependent manner in cells exposed to inflammatory stimuli. The simendans decreased iNOS protein and mRNA expression but did not affect iNOS mRNA decay. These compounds decreased iNOS promoter activity and inhibited NF-kappaB-mediated transcription but not that mediated by STAT1/GAS. The simendans reduced IL-6 production slightly but they had no effect on TNF-alpha synthesis.The simendans downregulated NF-kappaB-dependent transcription and decreased iNOS promoter activity, iNOS expression and NO production. These mechanisms may contribute to their beneficial clinical effects. |
| 2008 | Tyrphostin AG-490 inhibited the acute phase of zymosan-induced inflammation. | Int Immunopharmacol | Tyrphostins, derivatives of benzylidene malononitrile are recognized as tyrosine kinase inhibitors that have been applied in some models of acute inflammatory conditions, like LPS and zymosan-induced shock. In the present study, we have investigated the effects of tyrphostin AG-490, on the development of multiple organ failure induced by i.p. injection of zymosan (1 mg/g body weight) in mice. Organ dysfunction and systemic inflammation was estimated 24 h after zymosan administration. Treatment of mice with AG-490 (dose, 5 mg/kg i.p. simultaneously with zymosan) decreased the number of cells and the level of NO in the peritoneal lavage. The substance attenuated the elevation of creatinine (indicator of renal failure), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and bilirubin (indicators for liver dysfunction) and prevented the accelerated coagulation time. The injection of zymosan resulted in a substantial increase in the serum level of TNF-alpha and IL-6, which was strongly inhibited by AG-490. Tyrphostin abolished the expression of iNOS and TNF-alphaR in the liver. Moreover, immunohistochemistry of liver showed decreased phosphorylation of Stat1 and Stat3. In conclusion, the administration of tyrphostin AG-490 in zymosan-induced nonseptic shock significantly improved the rate of survival and lead to less exerted signs of multiple organ failure. |
| 2008 | CLEC5A is critical for dengue-virus-induced lethal disease. | Nature | Dengue haemorrhagic fever and dengue shock syndrome, the most severe responses to dengue virus (DV) infection, are characterized by plasma leakage (due to increased vascular permeability) and low platelet counts. CLEC5A (C-type lectin domain family 5, member A; also known as myeloid DAP12-associating lectin (MDL-1)) contains a C-type lectin-like fold similar to the natural-killer T-cell C-type lectin domains and associates with a 12-kDa DNAX-activating protein (DAP12) on myeloid cells. Here we show that CLEC5A interacts with the dengue virion directly and thereby brings about DAP12 phosphorylation. The CLEC5A-DV interaction does not result in viral entry but stimulates the release of proinflammatory cytokines. Blockade of CLEC5A-DV interaction suppresses the secretion of proinflammatory cytokines without affecting the release of interferon-alpha, supporting the notion that CLEC5A acts as a signalling receptor for proinflammatory cytokine release. Moreover, anti-CLEC5A monoclonal antibodies inhibit DV-induced plasma leakage, as well as subcutaneous and vital-organ haemorrhaging, and reduce the mortality of DV infection by about 50% in STAT1-deficient mice. Our observation that blockade of CLEC5A-mediated signalling attenuates the production of proinflammatory cytokines by macrophages infected with DV (either alone or complexed with an enhancing antibody) offers a promising strategy for alleviating tissue damage and increasing the survival of patients suffering from dengue haemorrhagic fever and dengue shock syndrome, and possibly even other virus-induced inflammatory diseases. |
| 2008 | Dipyrithione inhibits lipopolysaccharide-induced iNOS and COX-2 up-regulation in macrophages and protects against endotoxic shock in mice. | FEBS Lett | Dipyrithione (PTS2) possesses anti-bacterial and anti-fungal activity. In the present study, we found that PTS2 dose-dependently inhibited the LPS-induced up-regulation of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein level in RAW264.7 cells. RT-PCR experiments showed that PTS2 suppressed LPS-induced iNOS but not COX-2 expression at the mRNA level. As expected, PTS2 prevented NO secretion in RAW264.7 cells. Furthermore, PTS2 administration significantly decreased LPS-induced mortality in mice. Mechanistically, PTS2 decreased expression and phosphorylation of STAT1, but did not interfere with the MAPK and NF-kappaB pathways. In conclusion, PTS2 protects mice against endotoxic shock and inhibits LPS-induced production of pro-inflammatory mediators, suggesting that PTS2 could play an anti-inflammatory role in response to LPS. |
| 2007 | Altered gene expression in rat colonic adenocarcinomas induced in an azoxymethane plus 2-amino-1-methyl-6-phenylimidazo[4,5-b]- pyridine initiation-promotion model. | Oncology | 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant food-derived mutagenic/carcinogenic heterocyclic amine (HCA), has attracted particular attention as a probable human colon carcinogen. Some studies have shown that PhIP administered in the post-initiation phase is able to enhance rat colon carcinogenesis remarkably. To determine whether this genotoxicant leaves a DNA footprint in colon carcinogenesis, 6-week-old male F344 rats were first subcutaneously injected with azoxymethane (AOM) and then continuously treated with various doses (0-200 ppm) of PhIP added to their diet. Animals were killed at week 36 for histopathological examination, and colonic adenocarcinomas derived from animals receiving 0, 50 and 200 ppm PhIP were subjected to a novel three-dimensional (3D)-microarray and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. A total of five candidate genes were identified in adenocarcinomas following 200 ppm of PhIP and AOM initiation, with a dose-dependent increment. Among them, Stat1 (signal transducer and activator of transcription 1) and VEGFc (vascular endothelial growth factor c) demonstrated statistically significant upregulation by real-time RT-PCR. In addition, HSP90 (heat shock protein 90) and VEGFa showed a non-significant tendency to increase. In summary, overexpression of Stat1, VEGF and other genes could be involved in PhIP-enhanced colon tumorigenesis in the post-initiation phase. |
| 2007 | Changes in gene expression in experimental glaucoma and optic nerve transection: the equilibrium between protective and detrimental mechanisms. | Invest Ophthalmol Vis Sci | The authors studied retinal gene expression changes in rats after experimental intraocular pressure elevation and optic nerve transection to elucidate molecular mechanisms of retinal ganglion cell (RGC) death.Translimbal laser photocoagulation was used to induce unilateral IOP elevation in 41 albino Wistar rats. In 38 additional animals, unilateral transection of the optic nerve was performed. Retinas were harvested 1 day, 3 days, 1 week, 2 weeks, 4 weeks, and 8 weeks after each treatment, and total RNA was isolated. Pooled RNA from each time point was analyzed with rat genome arrays. Array results were confirmed by real-time PCR, and localization studies were performed using in situ hybridization for select genes.Genes that were upregulated in glaucoma, but not after transection, included Cyclin D2, Stat1, Stat3, c-Fos, Junb, Anxa1, Anxa 3, and CCAAT/enhancer binding protein (Cebp-delta). In glaucoma and transection models, the upregulation of c-Jun, Activating transcription factor 3, Heat shock protein 27, and Timp1 were observed. Comparisons among microarray databases were performed between our data and reports of retinal and optic nerve injury models in mice, rats, and monkeys.Gene expression changes specific to experimental glaucoma injury were identified. The present analysis supports the importance of neuroinflammation and the participation of the tumor necrosis factor alpha signaling pathway in glaucoma injury. The alterations observed include processes that are both protective of and detrimental to the survival of RGCs. |
| 2007 | STAT proteins in innate immunity during sepsis: lessons from gene knockout mice. | Acta Med Okayama | The innate immune system provides immediate defense against infection and serves as the first line of host defense during infection. In innate immunity, leukocytes such as neutrophils and macrophages recognize and respond to pathogens in a non-specific manner. Therefore, the recruitment and activation of leukocytes are essential in innate immunity, and are governed by a variety of chemical mediators including cytokines. Cytokines are generally divided into 2 types, termed type-1 and type-2 cytokines. Type-1 cytokines are important in local host defense, while type-2 cytokines play a protective role when inflammatory response spreads to the body. These cytokines exert their biological functions through the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. STAT1/3/4/6 are transcription factors that mediate IFNgamma/IL-10/IL-12/IL-13 cytokine signaling, respectively. Evidence indicates that STAT proteins have a significant impact on innate immunity during sepsis. This review focuses on recent understandings in the regulation of innate immunity by STAT proteins during sepsis and septic shock. The suppressor of cytokine signaling (SOCS) proteins are a family of SH2 domain-containing cytoplasmic proteins that complete a negative feedback loop to attenuate signal transduction from cytokines that act through the JAK/STAT pathway. The participation of SOCS proteins in sepsis is also discussed. |
| 2008 | Tyk2 and signal transducer and activator of transcription 1 contribute to intestinal I/R injury. | Shock | Previously, we have shown that the Jak-signal transducer and activator of transcription signaling constituents Tyk2 and STAT1 play a role in the development of multiple organ failure during endotoxin shock. Here, we report that Tyk2 and STAT1 contribute to death caused by intestinal I/R injury. Tyk2- and STAT1-deficient mice showed increased survival to I/R because their intestines were protected from gross histomorphological tissue destruction and neutrophil infiltration. On the molecular level, the reduced ischemia induced inflammatory response in mutant versus wild-type mice was accompanied by an impaired up-regulation of the adhesion molecules P-selectin and intercellular adhesion molecule 1 and of the matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MMP-14 in the reperfused intestine. In conclusion, this study demonstrates for the first time that Tyk2 or STAT1 promote intestinal I/R-induced shock based on a deregulated local inflammatory response and a destruction of the gut intestinal barrier. |
| 2007 | Control of specificity and magnitude of NF-kappa B and STAT1-mediated gene activation through PIASy and PIAS1 cooperation. | Proc Natl Acad Sci U S A | NF-kappaB and STATs regulate multiple cellular processes through the transcriptional activation of genes with diversified functions. Although the molecular mechanisms that can turn on/off the overall NF-kappaB/STAT signaling have been extensively studied, how NF-kappaB/STAT-target genes can be differentially regulated is poorly understood. Here we report that PIASy, a member of the PIAS (for protein inhibitor of activated STAT) protein family, is a physiologically important transcriptional repressor of NF-kappaB and STAT1. Piasy deletion in dendritic cells resulted in enhanced expression of a subset of NF-kappaB and STAT1-dependent genes in response to LPS or IFN-gamma treatment, respectively. Consistently, Piasy null mice are hypersensitive to the LPS-induced endotoxic shock. Furthermore, PIASy and PIAS1 display specific as well as redundant effects on the regulation of NF-kappaB/STAT1 signaling. Pias1-/-Piasy-/- embryos died before day 11.5. The disruption of one allele of Pias1 in the Piasy-/- background significantly enhanced the effect of Piasy deletion on the transcriptional induction of NF-kappaB/STAT1-dependent genes, and vice versa. Our results demonstrate that PIASy cooperates with PIAS1 to regulate the specificity and magnitude of NF-kappaB/STAT1-mediated gene activation. |
| 2007 | Heme oxygenase-1 mediated cytoprotection against liver ischemia and reperfusion injury: inhibition of type-1 interferon signaling. | Transplantation | Toll-like receptor (TLR)-4 signaling plays a key role in initiating exogenous antigen-independent innate immunity-dominated liver ischemia/reperfusion injury (IRI). Heme oxygenase (HO)-1, a heat-shock protein 32, exerts potent adaptive anti-oxidant and anti-inflammatory functions. Signal transducers and activator of transcription (STAT)-1 activation triggers interferon (IFN)-inducible protein 10 (CXCL-10), one of major products of type-1 IFN pathway downstream of TLR4. This study focuses on the role of type-1 IFN pathway in the mechanism of HO-1 cytoprotection during liver IRI.Cobalt protoporphyrin (CoPP)-induced HO-1 overexpression ameliorated liver damage in a well-defined mouse model of liver warm IRI, as evidenced by improved hepatic function (serum alanine aminotransferase levels) and liver histology (Suzuki's scores). HO-1 downregulated phospho-STAT-1 and its key product, CXCL-10. In contrast, TLR4 expression remained elevated regardless of the IRI status. To dissect the mechanism of HO-1 upon CXCL-10, we cultured RW 264.7 (macrophage) cells with exogenous rIFN-beta to stimulate CXCL-10 production via TLR4 pathway in vitro. Indeed, CoPP-induced HO-1 suppressed otherwise highly upregulated rIFN-beta-triggered CXCL-10. Moreover, consistent with our in vitro data, CoPP pretreatment diminished rIFN-beta-induced CXCL-10 production in normal mouse livers.Hepatic IRI activates TLR4 signaling in vivo to elaborate CXCL-10. HO-1 overexpression downregulates activation of STAT1 via type-1 IFN pathway downstream of TLR4, which in turn decreases CXCL-10 production. This study provides evidence for a novel mechanism by which HO-1 exerts adaptive cytoprotective and anti-inflammatory functions in the context of innate TLR4 activation. |
| 2007 | Advanced glycation end-product-induced mitogenesis is dependent on Janus kinase 2-induced heat shock protein 70 in normal rat kidney interstitial fibroblast cells. | Transl Res | Kidney interstitial fibroblast proliferation is important in the pathogenesis of diabetic renal fibrosis. In this regard, advanced glycation end-product (AGE)-induced proliferation in normal rat kidney interstitial fibroblast (NRK-49F) cells is dependent on the Janus kinase 2 (JAK2) signal transducers and activators of transcription (STAT) pathway. Heat shock protein (Hsp) is a molecular target of JAK/STAT. Thus, the role of Hsp70 in AGE-induced mitogenesis in NRK-49F cells was studied. The AGE dose (100-200 microg/mL) and time (16-72 h) dependently increased Hsp70 protein expression. AGE-induced Hsp70 was attenuated by AG-490 (a JAK2 inhibitor) and N-acetylcysteine. AGE also increased tyrosine phosphorylation of Hsp70, cyclin E, and cyclin D1 (to a lesser extent) while increasing Hsp70 protein interactions with STAT1, STAT3, STAT5b, cyclin D1, and cyclin E. AGE-induced tyrosine phosphorylation of Hsp70 and cyclin E (but not cyclin D1) was attenuated by AG-490. AGE-induced mitogenesis, cyclin D1, and cyclin E were attenuated by Hsp70 antisense oligodeoxynucleotide and 2-aminopurine (an Hsp70 inhibitor). AGE-induced Hsp70 and mitogenesis were also attenuated by N-acetylcysteine. It was concluded that AGE-induced Hsp70 protein expression and tyrosine phosphorylation are dependent on JAK2 in NRK-49F cells. AGE increased protein-protein interactions among Hsp70, STAT1, STAT3, STAT5b, cyclin D1, and cyclin E. Moreover, AGE-induced mitogenesis is dependent on Hsp70 and oxidative stress. |
| 2007 | Diverse effects of Stat1 on the regulation of hsp90alpha gene under heat shock. | J Cell Biochem | Stat1 has been known as a regulator of gene expression and a mediator of IFNgamma signaling in mammalian cells, while its effect in a heat shock response remains unclear. We used RNAi knockdown, point mutations, ChIP and promoter activity assays to study the effect of Stat1 on the heat-shock induction of the hsp90alpha gene under heat shock conditions. We found that Stat1 regulates the heat shock induction of its target genes, the hsp90alpha gene in a heat shock response while the constitutive activity of the gene remains unaffected. The result of Stat1 in complex with Stat3 and HSF1 that bound at the GAS to lead a moderate heat shock induction was designated as an "intrinsic" induction of the hsp90alpha gene. Additionally a reduced or an elevated level of heat shock induction was also controlled by the Stat1 on hsp90alpha. These diverse effects on the hsp90alpha gene were a "reduced" induction with over-expressed Stat1 elicited by transfection of wild-type Stat1 or IFNgamma treatment, bound at the GAS as homodimer; and an "enhanced" heat shock induction with a mutation-mediated prohibition of Stat1/GAS binding. In conclusion, the status and efficacy of Stat1 bound at the GAS of its target gene are pivotal in determining the impact of Stat1 under heat shock. The results provided the first evidence on the tumor suppressor Stat1 that it could play diverse roles on its target genes under heat shock that also shed lights on patients with fever or under thermotherapy. |
| 2006 | STAT1 and STAT3 phosphorylation by porins are independent of JAKs but are dependent on MAPK pathway and plays a role in U937 cells production of interleukin-6. | Cytokine | A group of transcription factors, termed signal transducers and activators of transcription (STATs), appears to orchestrate the downstream events propagated by cytokine/growth factor interactions with their cognate receptors. Similarly, cytoplasmic Janus kinases (JAKs) seem to play a critical role in diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. In this work, we analysed the effects of the Salmonella enterica serovar Typhimurium porins on signaling by the JAK/STAT pathway and IL-6 release in U937 cells. Porins and LPS of membrane from Gram-negative bacteria are factors implicated in septic shock. In our assays porins induce interleukin-6 (IL-6) release (110+/-2.6pg/ml) 24h after stimulation and STAT1/STAT3 tyrosine (Tyr701/Tyr705) and serine (Ser727) phosphorylation after 15min. By using several selective inhibitors we demonstrate that porins modulate the activation of STAT1/STAT3 through mitogen activated protein kinases (MAPKs) and not JAKs. Furthermore, we demonstrated that STAT1 and STAT3 are not involved in the modulation of IL-6 release in U937 cells stimulated with porins. Inhibition of tyrosine/serine phosphorylation mediated by MAPKs of STAT1 and STAT3 decrease the IL-6 secretion following porin stimulation. Therefore, suggesting a key role of this pathway in phosphorylation of Ser 727 in STAT1 and STAT3. These results are confirmed by porin or LPS-induced nuclear translocation of STAT1 and STAT3 in U937 cells. |
| 2007 | Heat shock stress ameliorates cytokine mixture-induced permeability by downregulating the nitric oxide and signal transducer and activator of transcription pathways in Caco-2 cells. | Shock | Proinflammatory cytokines are known to impair intestinal barrier function and to activate signaling pathways, whereas heat shock responses prevent cytokine-induced mucosal damage. We hypothesized that heat shock response blocks the effects of proinflammatory cytokines by regulating nitric oxide (NO) production and the activities of the Janus kinase/signal transducer and activator of transcription (STAT) pathway. A monolayer of Caco-2 cells were pretreated with sodium arsenite (SA, 500 micromol/L) for 1 h, followed by a 1-h recovery, and then stimulated with a cytokine mixture (cytomix: tumor necrosis factor alpha [10 ng/mL], interferon beta [1000 U/mL], and interleukin [IL] 1beta [1 ng/mL]) for 24 h. The permeability of horseradish peroxidase and fluorescein isothiocyanate-conjugated Dextran and transepithelial resistance and potential difference were measured in Ussing chambers. Interleukin-6, IL-8, NO, inducible NO synthase mRNA, STAT activity, and suppressor of cytokine signaling (SOCS) expression were measured in medium or cell lysates. Cytomix resulted in increased epithelial permeability of both fluorescein isothiocyanate-conjugated Dextran and horseradish peroxidase; whereas treatment of Caco-2 cells with SA 500 micromol/L blocked the cytomix-induced permeability changes. In addition, SA treatment decreased cytomix-induced NO production and inducible NO synthase mRNA expression and decreased the levels of STAT1, STAT3, SOCS1, and SOCS3. The SA treatment also decreased cytomix-induced IL-6 and IL-8 production in a dose-dependent manner. In conclusion, cytomix increased epithelial permeability, which is associated with increased NO and STAT activities. The SA treatment ameliorated cytomix-induced permeability, possibly through the downregulation of the NO and Janus kinase/STAT pathways. |
| 2007 | Staphylococcal enterotoxin B in vivo modulates both gamma interferon receptor expression and ligand-induced activation of signal transducer and activator of transcription 1 in T cells. | Infect Immun | Superantigens (SAg) are bacterial exotoxins that provoke extreme responses in the immune system; for example, the acute hyperactivation of SAg-reactive T cells that leads to toxic shock syndrome is followed within days by strong immunosuppression. The gamma interferon (IFN-gamma) response is deeply affected in both extremes. The implication of IFN-gamma in the pathophysiology of lethal shock induced in mice after a secondary challenge with the SAg staphylococcal enterotoxin B (SEB) prompted us to study the regulation of IFN-gamma secretion and the intracellular response. We demonstrate in this study that a rechallenge with SEB becomes lethal only when given inside a critical time window after SEB priming and is associated with an increase of IFN-gamma serum release 72 h after priming. However, at this time, a selective blockade of IFN-gamma/STAT1 signaling develops in spleen cells, correlating with a lack of expression of the IFN-gamma receptor beta subunit and STAT1 in the T-cell population. Selective blockade of the STAT1 signaling pathway--while simultaneously maintaining STAT3 signaling and expression--may be a protective mechanism that shortens IFN-gamma production during the Th1 effector response. This blockade may also have consequences on switching towards a suppressor phenotype with chronic exposure to the superantigen. |
| 2006 | Serine-phosphorylated STAT1 is a prosurvival factor in Wilms' tumor pathogenesis. | Oncogene | Wilms' tumor (WT), one of the most common pediatric solid cancers, arises in the developing kidney as a result of genetic and epigenetic changes that lead to the abnormal proliferation and differentiation of the metanephric blastema. As activation of signal transducers and activators of transcription (STATs) plays an important role in the maintenance/growth and differentiation of the metanephric blastema, and constitutively activated STATs facilitate neoplastic behaviors of a variety of cancers, we hypothesized that dysregulation of STAT signaling may also contribute to WT pathogenesis. Accordingly, we evaluated STAT phosphorylation patterns in tumors and found that STAT1 was constitutively phosphorylated on serine 727 (S727) in 19 of 21 primary WT samples and two WT cell lines. An inactivating mutation of S727 to alanine reduced colony formation of WT cells in soft agar by more than 80% and induced apoptosis under conditions of growth stress. S727-phosphorylated STAT1 provided apoptotic resistance for WT cells via upregulation of expression of the heat-shock protein (HSP)27 and antiapoptotic protein myeloid cell leukemia (MCL)-1. The kinase responsible for STAT1 S727 phosphorylation in WT cells was identified based upon the use of selective inhibitors as protein kinase CK2, not p38, MAP-kinase kinase (MEK)1/2, phosphatidylinositol 3'-kinase, protein kinase C or Ca/calmodulin-dependent protein kinase II (CaMKII). The inhibition of CK2 blocked the anchorage-independent growth of WT cells and induced apoptosis under conditions of growth stress. Our findings suggest that serine-phosphorylated STAT1, as a downstream target of protein kinase CK2, plays a critical role in the pathogenesis of WT and possibly other neoplasms with similar STAT1 phosphorylation patterns. |
| 2006 | A new tumor suppressor DnaJ-like heat shock protein, HLJ1, and survival of patients with non-small-cell lung carcinoma. | J Natl Cancer Inst | We previously identified DnaJ-like heat shock protein (HLJ1) as a gene associated with tumor invasion. Here, we investigated the clinical significance of HLJ1 expression in non-small-cell lung cancer (NSCLC) patients and its role in cancer progression.We induced HLJ1 overexpression or knockdown in human lung adenocarcinoma CL1-5 cells and analyzed cell proliferation, anchorage-independent growth, in vivo tumorigenesis, cell motility, invasion, and cell cycle progression. Expression of genes that act downstream of HLJ1 was examined by DNA microarray analysis, pathway analysis, and western blotting. We measured HLJ1 expression in tumors and adjacent normal tissues of 71 NSCLC patients by quantitative reverse transcription-polymerase chain reaction. Associations between HLJ1 expression and disease-free and overall survival were determined using the log-rank test and multivariable Cox proportional hazards regression analysis. Validation was performed in an independent cohort of 56 NSCLC patients. Loss of heterozygosity (LOH) mapping of the HLJ1 locus was analyzed in 48 paired microdissected NSCLC tumors. All statistical tests were two-sided.HLJ1 expression inhibited lung cancer cell proliferation, anchorage-independent growth, tumorigenesis, cell motility, and invasion, and slowed cell cycle progression through a novel STAT1/P21(WAF1) pathway that is independent of P53 and interferon. HLJ1 expression was lower in tumors than in adjacent normal tissue in 55 of 71 patients studied. NSCLC patients with high HLJI expressing tumors had reduced cancer recurrence (hazard ratio [HR] = 0.47; 95% confidence interval [CI] = 0.23 to 0.93; P = .03) and longer overall survival (HR = 0.38; 95% CI = 0.16 to 0.89; P = .03) than those with low-expressing tumors. Validation in the independent patient cohort confirmed the association between HLJ1 expression and patient outcome. LOH mapping revealed high frequencies (66.7% and 70.8%) of allelic loss and microsatellite instability (87.5% and 95.2%) of the HLJ1 locus at chromosome 1p31.1.HLJ1 is a novel tumor suppressor in NSCLC, and high HLJ1 expression is associated with reduced cancer recurrence and prolonged survival of NSCLC patients. |
| 2006 | Gene expression profiling of the host response to HIV-1 B, C, or A/E infection in monocyte-derived dendritic cells. | Virology | Dendritic cells (DC) are among the first targets of human immunodeficiency virus type-1 (HIV-1) infection and in turn play a crucial role in viral transmission to T cells and in the regulation of the immune response. The major group of HIV-1 has diversified genetically based on variation in env sequences and comprise at least 11 subtypes. Because little is known about the host response elicited against different HIV-1 clade isolates in vivo, we sought to use gene expression profiling to identify genes regulated by HIV-1 subtypes B, C, and A/E upon de novo infection of primary immature monocyte-derived DC (iMDDCs). A total of 3700 immune-related genes were subjected to a significance analysis of microarrays (SAM); 656 genes were selected as significant and were further divided into 8 functional categories. Regardless of the time of infection, 20% of the genes affected by HIV-1 were involved in signal transduction, followed by 14% of the genes identified as transcription-related genes, and 7% were classified as playing a role in cell proliferation and cell cycle. Furthermore, 7% of the genes were immune response genes. By 72 h postinfection, genes upregulated by subtype B included the inhibitor of the matrix metalloproteinase TIMP2 and the heat shock protein 40 homolog (Hsp40) DNAJB1, whereas the IFN inducible gene STAT1, the MAPK1/ERK2 kinase regulator ST5, and the chemokine CXCL3 and SHC1 genes were induced by subtypes C and A/E. These analyses distinguish a temporally regulated host response to de novo HIV-1 infection in primary dendritic cells. |
| 2005 | Therapeutic potential of oligonucleotides expressing immunosuppressive TTAGGG motifs. | Ann N Y Acad Sci | Synthetic oligodeoxynucleotides (ODNs) expressing immunosuppressive TTAGGG motifs downregulate the production of proinflammatory and Th1 cytokines. The ability of these "suppressive ODNs" to slow or prevent the development of diseases characterized by over-exuberant immune stimulation was examined. Suppressive ODNs significantly reduced disease severity in murine models of arthritis, lupus, and LPS-induced toxic shock. These beneficial effects were accompanied by a significant reduction in serum autoantibody and cytokine levels. Underlying these protective effects was the ability of suppressive ODNs to bind to and prevent the phosphorylation of STAT1 and STAT4, thereby blocking the signaling cascade central to the initiation and/or perpetuation of these disease states. These findings suggest that suppressive ODNs might find use in the treatment of acute and chronic diseases characterized by excessive immune stimulation. |
| 2005 | Janus kinase-2 signaling mediates apoptosis in rat cardiomyocytes. | Vascul Pharmacol | We tested the hypothesis that activation Jak2, which is prominently involved in the up-regulation of the renin-angiotensin system (RAS), constitutes a focal point in relaying signals triggered by a Angiotensin II (Ang II) and hypoxia/reoxygenation separately to cause an enhanced susceptibility of cardiac myocyte to apoptotic cell death. Ang II-treated adult cardiomyocytes in culture exhibited an increased level of apoptosis that accompanied activation of pro-apoptotic as well as anti-apoptotic signaling pathways. We observed increased phosphorylation of Jak2 kinase, Stat1, JNK, with increased expression of Bax protein, followed by an increase in caspase-1 and caspase-3 activity. Activation of these pro-apoptotic pathways was blocked by the Jak2 pharmacological inhibitor, Tyrphostin AG490. We also observed an increase in phosphorylation of cardioprotective pathway components, namely S6 ribosomal protein, and heat shock protein 27 (HSP27). Likewise, the oxidative stress, via the hypoxia/reoxygenation treatment of rat adult cardiomyocytes, produced apoptosis that was dependent upon activation of Jak2. The apoptotic response was not only reduced by Losartan, an inverse agonist of the AT1, receptor, but by treatment with AG490 as well. Taken together, these observations provide clear evidence in favor of Jak2 signaling as mediator of the apoptotic response in cardiomyocytes. However, there was a concomitant induction of cytoprotective signaling that presumably provides a negative feed-back to the deleterious effects of the agonist. |
| 2005 | Human serum from patients with septic shock activates transcription factors STAT1, IRF1, and NF-kappaB and induces apoptosis in human cardiac myocytes. | J Biol Chem | Proinflammatory cytokines have been linked to depression of myocardial contractility in vivo in patients with acute septic shock and in vitro models employing isolated myocytes exposed to serum from such patients. The key pathways involved in mediating this septic organ dysfunction (cell adhesion molecule expression, inducible nitric-oxide synthase induction, and apoptosis) are known to be regulated by transcription factors STAT1, IRF1, and NF-kappaB. Utilizing a model that mimics human disease, we have demonstrated activation of the transcription factors STAT1, IRF1, and NF-kappaB in human fetal myocytes exposed to human septic serum. Both reporter and electrophoretic mobility shift assays demonstrated a 5-19-fold increase in activation of transcription factors STAT1, IRF1, and NF-kappaB in response to incubation with human septic serum. The addition of human septic serum to human fetal myocytes induced apoptosis in human fetal myocytes and activation of the mitogen-activated protein kinase c-Jun NH -terminal kinase and caspase 1 as measured by Western blot. These data suggest that transcription factor activation and early myocyte apoptosis play a mechanistic role in septic myocardial depression and sepsis-induced organ dysfunction. |
| 2005 | Critical roles for both STAT1-dependent and STAT1-independent pathways in the control of primary dengue virus infection in mice. | J Immunol | Dengue virus (DEN), a flavivirus, causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome, the most common mosquito-borne viral illnesses in humans worldwide. In this study, using STAT1(-/-) mice bearing two different mutant stat1 alleles in the 129/Sv/Ev background, we demonstrate that IFNR-dependent control of primary DEN infection involves both STAT1-dependent and STAT1-independent mechanisms. The STAT1 pathway is necessary for clearing the initial viral load, whereas the STAT1-independent pathway controls later viral burden and prevents DEN disease in mice. The STAT1-independent responses in mice with primary DEN infection included the early activation of B and NK cells as well as the up-regulation of MHC class I molecules on macrophages and dendritic cells. Infection of bone marrow-derived dendritic cell cultures with either DEN or Sindbis virus, another positive-strand RNA virus, confirmed the early vs late natures of the STAT1-dependent and STAT1-independent pathways. Collectively, these data begin to define the nature of the STAT1-dependent vs the STAT1-independent pathway in vivo. |
| 2005 | Mechanism and effect of thrombospondin-4 polymorphisms on neutrophil function. | Blood | High-throughput genomic technology identified an association between a single nucleotide polymorphism (SNP), a proline (P387) rather than the predominant alanine (A387) at position 387 in thrombospondin-4 (TSP-4) and premature myocardial infarction. The inflammatory hypothesis of atherosclerosis invokes a prominent role of leukocytes and cytokines in pathogenesis. As the expression of TSP-4 by vascular cells permits its exposure to circulating leukocytes, the interactions of human neutrophils (polymorphonuclear leukocytes [PMNs]) with both TSP-4 variants were investigated. Phorbol 12-myristate 13-acetate (PMA)-stimulated PMNs adhered and migrated well and equally on the TSP-4 variants. Integrin alpha(M)beta2 was identified as the TSP-4 receptor mediating these responses, and the 3 epidermal growth factor (EGF)-like domains of TSP-4 harboring the SNPs interacted with the alpha(M)I-domain. Despite the similarity in these responses, the P387 variant induced more robust tyrosine phosphorylation of the stress-related mitogen-activated protein kinases (MAPKs): p38MAPK and c-Jun NH2-terminal kinase (JNK), as well as signal transducer and activator of transcription-1 (STAT1) and heat shock protein 27 (HSP27) than the A387 variant. Additionally, cells adherent to P387 TSP-4 variant released 4-fold more H2O2 and secreted 2-fold more interleukin 8 (IL-8) as compared with the A387. H2O2 release and p38MAPK activation were totally inhibited by blockade of alpha(M)beta2. Thus, alpha(M)beta2 plays a central role in proinflammatory activities of TSP-4 (P387) and may contribute to the prothrombotic phenotype associated with this variant. |
| 2005 | Suppressive oligodeoxynucleotides protect mice from lethal endotoxic shock. | J Immunol | Endotoxic shock is a life-threatening condition caused by exposure to bacterial LPS. LPS triggers the release of acute phase, proinflammatory, and Th1 cytokines that facilitate the development of endotoxic shock. Synthetic oligodeoxynucleotides (ODN) expressing suppressive TTAGGG motifs effectively down-regulate the production of proinflammatory and Th1 cytokines elicited by a variety of immune stimuli. The current results demonstrate that suppressive ODN protect mice from LPS-induced endotoxic shock. Underlying this protective effect is the ability of suppressive ODN to bind to and prevent the phosphorylation of STAT1 and STAT4, thereby blocking the signaling cascade mediated by LPS-induced IFN-beta and IL-12. These findings suggest that suppressive ODN might be of use in the treatment of endotoxic shock. |
| 2004 | The role of Tyk2, Stat1 and Stat4 in LPS-induced endotoxin signals. | Int Immunol | Mice lacking Tyk2, Stat1 or Stat4, which are members of the Jak-Stat signaling cascade, were resistant to LPS-induced endotoxin shock. Interestingly, Tyk2-deficient mice had higher resistance to LPS challenge than mice lacking either Stat1 or Stat4. The activation of MAPK and NF-kappaB by LPS, and the production of TNF-alpha and IL-12 after LPS injection, were not abrogated by the absence of Tyk2, Stat1 or Stat4. In Stat1-deficient mice, the induction of IFN-beta by LPS in macrophages was severely reduced, although the serum level of IFN-gamma was elevated after LPS injection. In contrast, in Stat-4 deficient mice, the induction of IFN-beta by LPS was normal, but the serum level of IFN-gamma remained low after LPS injection. Interestingly, the induction of both IFN-beta and IFN-gamma by LPS was severely reduced in Tyk2-deficient mice. Therefore, Stat1 and Stat4 independently play substantial roles in the susceptibility to LPS. Tyk2 is essential for LPS-induced endotoxin shock, and this signaling pathway is transduced by the activation of Stat1 and Stat4. |
| 2004 | Hypoxia/re-oxygenation-induced, redox-dependent activation of STAT1 (signal transducer and activator of transcription 1) confers resistance to apoptotic cell death via hsp70 induction. | Biochem J | STAT1 (signal transducer and activator of transcription 1) is potentially involved in cell survival, as well as cell death, in different types of cells. The present study was designed to examine the effects of STAT1 on hypoxia/re-oxygenation (H/R)-induced cell death and/or survival, and the underlying mechanisms of any such effects. H/R was shown to induce apoptotic cell death of rat hepatocytes. The addition of a STAT1-specific inhibitor, fludarabine, significantly increased the fraction of apoptotic cells after H/R. Following H/R, STAT1 was activated and sequential phosphorylation of Tyr701 and Ser727 was observed, which could be inhibited by the antioxidant N-acetyl-L-cysteine. Tyrosine and serine phosphorylation of STAT1 was mediated by Janus kinase 2 and phosphoinositide 3-kinase/Akt kinase respectively in a redox-dependent manner following H/R. STAT1-induced HSP70 (heat-shock protein 70) expression and the suppression of apoptosis occurred concomitantly. In conclusion, STAT1 activation, in a redox-dependent manner, following H/R may play crucial roles in cell survival, at least partly via HSP70 induction. |
| 2004 | Implication of STAT1 and STAT3 transcription factors in the response to superantigens. | Cytokine | The activation of STAT1 and STAT3 in response to SEB was analyzed in spleen of Balb/c mice. The intraperitoneal injection of the superantigen SEB activated STAT1 and STAT3 in spleen. Activated STAT1 almost completely disappeared in 24 h even though activated STAT3 was present for more than 48 h after SEB injection. Cyclosporine A was able to block the initial STAT1 activation, but STAT3 activation was only partially affected. SEB also increased the mRNA levels for STAT1, STAT3 and SOCS1. When a second injection with SEB was given 72 h after the first stimulus, STAT1 activation was much lower than that observed after the first stimulation with SEB and no increase in the STAT1 mRNA level was observed. Nevertheless, after this second injection, STAT3 was again activated without any significant interference from the first stimulus and the STAT3 and SOCS1 mRNA levels again increased. These data indicate that a first stimulation with superantigen re-programs cells so that they respond to a second stimulation in a different way. Understanding the mechanisms implicated in this re-programming is basic for designing therapeutic strategies in processes such as septic shock. |
| 2003 | Phosphorylation of the Stat1 transactivation domain is required for full-fledged IFN-gamma-dependent innate immunity. | Immunity | Stat1 is phosphorylated on serine 727 within its transactivating domain (TAD) in response to interferons or other immunological signals. We generated gene-targeted mutant mice expressing a serine727-alanine mutant of Stat1. These animals showed increased mortality upon infection with Listeria monocytogenes and impaired clearance of the bacteria from spleen and liver. The Stat1S727A mice were more resistant to the LPS-induced septic shock syndrome, suggesting that Stat1 serine phosphorylation promotes inflammatory responses. Expression of IFN-gamma-induced genes was strongly reduced in macrophages expressing Stat1(S727A). While mutation of Stat1 at S727 did not reduce its binding to chromatin, association with the coactivator CBP and histone acetylation at the interferon-responsive GBP promoter was strongly reduced, suggesting defective recruitment of histone acetylases as the mechanism underlying IFN-gamma hyporesponsiveness. Our data demonstrate that the increase in transcription factor activity caused by Stat serine phosphorylation contributes to macrophage activation and to IFN-gamma-dependent immune responses in vivo. |
| 2003 | Plasma membrane rafts and chaperones in cytokine/STAT signaling. | Acta Biochim Pol | We and others have recently obtained data suggesting that cytokine-STAT signaling in many different cell-types is a chaperoned pathway initiated at the level of specialized plasma membrane microdomains called "rafts" (the "raft-STAT signaling hypothesis"). These findings are of broad significance in that all cytokines and growth factors initiate signaling in target cells by interacting with respective cell-surface receptors. The new data suggest that raft microdomains represent the units of function at the cell-surface through which ligand-stimulated STAT signaling is initiated. Moreover, recent evidence shows the involvement of chaperone proteins in regulating the STAT signaling pathway. These chaperones include the human homolog of the tumorous imaginal disc 1 protein (hTid1) which associates with Janus kinase 2 (JAK2) at the level of the plasma membrane, heat shock protein 90 (HSP90) which associates with STAT3 and STAT1 proteins in caveolin-1-containing raft and cytoplasmic complexes, and glucose regulated protein 58 (GRP58/ER-60/ERp57), a thiol dependent protein-disulfide isomerase, found in association with STAT3 "statosome" complexes in the cytosol and in the raft fraction. We suggest a function of the HSP90 chaperone system in preserving IL-6/STAT3 signaling in liver cells in the context of fever. The identification and function of protein partners associated with specific STAT species in rafts and in cytosolic complexes, and in the efficient departure of cytokine-activated STATs from the cytosolic face of rafts towards the cell nucleus are now areas of active investigation. |
| 2001 | [Role of STAT1 on the regulation the human hsp90 alpha gene expression]. | Zhongguo Yi Xue Ke Xue Yuan Xue Bao | To investigate the role of STAT1 on the regulation of human hsp90 alpha gene expression.We first transfected Jurkat cells with the STAT1 expression construct and analyzed the expression of hsp90 alpha gene expression via quantitative RT-PCR system. Then we co-transfected the STAT1 expression construct and the CAT reporter gene driven by different length of 5' flanking sequence of hsp90 alpha gene. Western blot was carried out to detect the level of tyrosine phosphorylation in Jurkat cells with and without heat shock treatment (42 degrees C 1 h). By electrophoretic mobility shift assays (EMSA), we evaluated the DNA binding activity of a STAT1 responsible element located in the regulatory region of hsp90 alpha gene in Jurkat cell nuclear extracts.The mRNA level of hsp90 alpha gene in Jurkat cells was decreased when transfected by STAT1 expression construct, over-expression of STAT1 down-regulates the expression of CAT reporter gene with the present of a distal fragment from -1756 to -1463 within the 5' flanking regulatory sequences of hsp90 alpha gene. The tyrosine phosphorylation of STAT1 was detectable in Jurkat cells and increased when subjected to heat shock. Electrophoretic mobility shift assays (EMSA) results showed that STAT1 could bind to its responsible element in the regulatory region of hsp90 alpha gene.STAT1 could negatively regulate the human hsp90 alpha gene expression. |
| 2003 | Immune selection in murine tumors. Ph.d thesis. | APMIS Suppl | It must be assumed that all tumor cells produce proteins which do not belong to a normal cell. These are called tumor-associated or tumor-specific antigens. In the classic immune surveillance theory it is believed that the cellular immune defense (the T-cell system) continuously discovers and eliminates newly arisen tumor cells which express such tumor-specific antigens. Since then it has been shown that one of the preconditions for the T-cell system to be able to recognize antigens is that they are presented by MHC class I histocompatibility antigens. There is a continual processing and presentation of all intracellular proteins in a cell. Thus, a tumor cell which produces an abnormal protein will also present this and thereby expose itself to being killed by cytotoxic T cells. The antigens are presented in the form of short peptides (8-9 aminoacids), which arise as a result of controlled degradation of the original proteins. The peptides thus formed are transported by specialised molecules in the so-called endogenous antigen processing and presentation pathway, and are eventually bound to and presented by MHC class I molecules. It has been shown that many tumors express less MHC class I on their surface compared to the normal tissue from which they have arisen, and also that patients with reduced immune function have an increased incidence of certain forms of cancer. It is therefore widely believed that a low MHC class I level contributes to the ability of tumor cells to avoid the T-cell-mediated immune defense. The aim of the present research project was to confirm the existence of a T-cell-mediated immune selection in primary tumors. Another of its goals was to elucidate the extent to which tumor cells with low MHC class I expression showed poor ability to present antigen, and whether the reason for this could be found in one or more of the molecular systems which participate in antigen processing and presentation. By using the chemical carcinogen 3-methylcholanthrene a total of 144 tumors were induced in immunologically normal and T-cell defective mice, respectively. It was assumed that tumors induced in normal mice would be immune selected, whilst this would not be the case for tumors from T-cell defective mice. This enabled us to work with a tumor-material where the two populations only differed in that the one part had undergone selection by a T-cell system and the other had not. Tumor induction time turned out to be shorter in immune defective than in normal mice, and the tumor frequency was higher, which might be due to the fact that in normal mice tumor growth was inhibited and in certain cases stopped by the T cells. On transplantation of the uncloned cell lines which were established from the primary tumors to immunologically normal congenic recipients, we were able to show that most of the tumors which originated from mice with a functional T-cell system, and which must therefore be assumed to have undergone selection in the primary tumor host, were not immunogenic and were therefore accepted. On the other hand, most tumors which originated from T-cell-defective mice were rejected as a sign that immunogenic tumor cells, assumed to have expressed tumor antigen, had not been eliminated in the primary tumor host. Still, we found that the ability of tumor cells to induce an immune response on transplantation was not reflected in their MHC class I expression. Both tumor lines from immunodeficient and normal mice had highly varying MHC class I levels, and contrary to expectations the highest levels were seen in tumor lines from immunologically normal mice. At the same time we found that the expression levels for the three different MHC class I molecules were the same in the individual tumor lines, which might indicate that the three genes are syn-regulated. The MHC class I mRNA content in tumors from normal mice was generally concordant with the surface level of MHC protein. Among the tumor lines from immunodeficient mice, on the other hand, we found several where there was no such agreement, which was taken to indicate that tumor cells with deviant MHC class I gene transcription had not been eliminated, in contrast to in the immunocompetent tumor hosts. The ability of tumor cells to present antigen was investigated by infecting cells with virus and thereafter assessing their ability to function as target cells for virus-specific T cells in a cytotoxic test. Their ability to do this varied considerably, but showed a correlation with their MHC class I expression. Among the transplanted tumor lines that were not able to present viral antigen, the majority were accepted, while most of the tumor lines which were rejected on transplantation possessed the ability to present virus. Closer analysis of the composition of proteasomes, heat shock protein content and TAP molecule function, which are all involved in the antigen processing system, did not immediately reveal any defects. Treatment with interferon gamma, which is known to upregulate the transcription of MHC class I and a number of other proteins which are involved in antigen presentation, showed that by far the majority of the tumor lines were able to respond normally. This was also true for the tumor lines which had deviant MHC class I gene transcription and the cells which showed poor ability to present viral antigen. We did find, however, three cell lines which did not respond to interferon gamma, and they all had defective interferon gamma-signaling, not because they did not express the interferon-receptor on the surface, but possibly on account of their lacking phosphorylation of an intracellular signal molecule, Stat1. |
| 2003 | Central role for type I interferons and Tyk2 in lipopolysaccharide-induced endotoxin shock. | Nat Immunol | Toll-like receptor-4 activation by lipopolysaccharide (LPS) induces the expression of interferon-beta (IFN-beta) in a MyD88-independent manner. Here we report that mice devoid of the JAK protein tyrosine kinase family member, Tyk2, were resistant to shock induced by high doses of LPS. Basal and LPS-induced expression of IFN-beta and IFN-alpha4 mRNA in Tyk2-null macrophages were diminished. However, Tyk2-null mice showed normal systemic production of nitric oxide and proinflammatory cytokines and the in vivo response to tumor necrosis factor (TNF) was unperturbed. IFN-beta-null but not STAT1-null mice were also resistant to high dose LPS treatment. Together, these data suggest that Tyk2 and IFN-beta are essential effectors in LPS induced lethality. |
| 2003 | Inhibition of IFN-gamma -induced STAT1 activation by 15- deoxy-Delta 12,14-prostaglandin J2. | Am J Physiol Endocrinol Metab | The inhibitory actions of 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)) on inflammatory gene expression have been attributed to the ability of this prostaglandin to inhibit the activation of NF-kappaB. In this study, we have identified an additional signaling pathway sensitive to inhibition by PGJ(2). We show that PGJ(2) inhibits interferon (IFN)-gamma-stimulated phosphorylation and DNA-binding activity of STAT1. The inhibitory actions on STAT1 phosphorylation are first apparent after a 1- to 2-h incubation and are maximal after a 6-h incubation with PGJ(2), and they correlate with the expression of heat shock protein (HSP)70 in islets. In previous studies, we have correlated the inhibitory actions of PGJ(2) on inducible nitric oxide synthase (iNOS) expression and NF-kappaB activation in response to IL-1 with the increased expression of HSP70. Using overexpression and antisense depletion, we provide evidence that HSP70 does not mediate the inhibitory actions of PGJ(2) on IL-1-induced NF-kappaB or IFN-gamma-induced STAT1 activation or cytokine-stimulated iNOS expression by beta-cells. Last, we show that the inhibitory actions of a short 6-h pulse with PGJ(2) on IL-1 plus IFN-gamma-stimulated iNOS expression and NO production by beta-cells are persistent for extended periods (< or =48 h). These findings suggest that PGJ(2) inhibits multiple cytokine-signaling pathways (IL-1 and IFN-gamma), that the inhibitory actions are persistent for extended periods, and that increased HSP70 expression correlates with, but does not appear to mediate, the inhibitory actions of PGJ(2) on IL-1 and IFN-gamma signaling in beta-cells. |
| 2002 | SOCS-1 participates in negative regulation of LPS responses. | Immunity | SOCS-1 is a negative regulatory molecule of the JAK-STAT signal cascade. Here, we demonstrate that SOCS-1 is a critical downregulating factor for LPS signal pathways. SOCS-1 expression was promptly induced in macrophages upon LPS stimulation. SOCS-1-deficient mice were highly sensitive to LPS-induced shock and produced increased levels of inflammatory cytokines. Introduction of SOCS-1 inhibited LPS-induced NF-kappaB and STAT1 activation in macrophages. Furthermore, LPS tolerance, a refractory state to second LPS stimulation, was not observed in SOCS-1-deficient mice. These results suggest SOCS-1 as an essential, negative regulator in LPS responses that protects the host from harmful overresponses to LPS and may provide new insight into the endotoxin-induced fatal syndrome that occasionally occurs following infection. |
| Administration of two macrophage-derived interferon-gamma-inducing factors (IL-12 and IL-15) induces a lethal systemic inflammatory response in mice that is dependent on natural killer cells but does not require interferon-gamma. | Cell Immunol | Activation of macrophages by microbes results in the rapid production of monokines (e.g., interleukin-12 (IL-12), IL-15, and IL-18), which induce production of interferon-gamma (IFN-gamma) by natural killer (NK) cells. We examined the effects of administering IL-15 in combination with IL-12 in a murine toxicity model to determine how these two cytokines might contribute to the inflammatory state that accompanies infectious processes. The daily, simultaneous administration of IL-15 (3 x 10(5)U) and IL-12 (1 microg) to normal mice resulted in shock and 100% mortality within 3-7 days, whereas minimal toxicity was observed following the administration of IL-15 or IL-12 alone. Mice treated with IL-15 plus IL-12 exhibited lesions of the gastrointestinal tract, elevated serum levels of acute phase reactants and pro-inflammatory cytokines, and NK cell apoptosis. Neutralization of IFN-gamma, TNF-alpha, and IL-1beta was not protective in cytokine-treated mice, however, toxicity and death could be completely abrogated by depletion of NK cells. Mice deficient in the STAT4 transcription factor also exhibited complete protection while mice deficient in IFN-gamma or its downstream mediator, STAT1, did not. These findings suggest that cytokine- stimulated NK cells are able to promote systemic inflammation via the induction of STAT4-responsive genes other than IFN-gamma or TNF-alpha. |
| 2002 | Lipopeptide adjuvants: monitoring and comparison of P3CSK4- and LPS-induced gene transcription. | Int Immunopharmacol | Bacteria-derived synthetic lipoproteins constitute potent macrophage activators in vivo and are effective stimuli, enhancing the immune response especially with respect to low or non-immunogenic compounds. N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4), exhibiting one of the most effective lipopeptide derivatives, represents a highly efficient immunoadjuvant in parenteral, oral, nasal and genetic immunization either in combination with or after covalent linkage to antigen. In order to further elucidate its molecular mode of action with respect to the transcriptional level, we focused our investigations on the P3CSK4-induced modulation of gene transcription. We could show that P3CSK4 activates/represses an array of at least 140 genes partly involved in signal transduction and regulation of the immune response. P3CSK4 activates the expression of tumor suppressor protein p53 (p53), c-rel, inhibitor of nuclear factor kappa B (NFkappaB) alpha (IkappaB alpha), type 2 (inducible) nitric oxide (NO) synthase (iNOS), CD40-LR, intercellular adhesion molecule-1 (ICAM-1) and interleukin 1/6/15 (IL-1/6/15). We detected no activation of heat shock protein (HSP) 27, 60, 84 and 86, osmotic stress protein 94 (Osp 94), IL-12, extracellular signal-regulated protein kinase 1 (ERK1), p38 mitogen activated protein (MAP)-kinase (p38), c-Jun NH2-terminal kinase (JNK), signal transducer and activator of transcription 1 (STAT1), CD14 and caspase genes. Furthermore, we monitored inhibition of STAT6, Janus kinase 3 (Jak3) and cyclin D1/D3 gene transcription after stimulating bone marrow-derived macrophages (BMDM) with lipopeptide. In addition, we monitored significant differences after lipopeptide and lipopolysaccharide (LPS) stimulation of bone marrow-derived murine macrophages. Our findings are of importance for further optimizing both conventional and genetic immunization, and for the development of novel synthetic vaccines. |
| 2002 | Natural killer cells contribute to the lethality of a murine model of Escherichia coli infection. | Surgery | Cooperative interactions between natural killer (NK) cells and macrophages occur normally during the course of the early immune response to bacterial, protozoal, and viral pathogens, with each cellular compartment providing the other with critical stimulatory factors. We conducted the present study to determine whether NK cells contribute to the dysregulated immune response that accompanies septic shock.An analysis of the lethality of Escherichia coli CP9 was conducted in mice that had been depleted of NK cells via the injection of an anti-asialo GM1 antibody and in CD epsilon transgenic mice that are deficient in both NK cells and T cells. The 2 groups of mice were analyzed for serum levels of interferon-gamma, tumor necrosis factor-alpha, and interleukin-1beta as well as activation of NFkappaB and STAT1, 2 proinflammatory transcription factors.NK cell-depleted and NK cell-deficient mice exhibited 80% survival in the face of an intraperitoneal bacterial challenge, whereas control mice all died within 12 hours. Serum levels of proinflammatory cytokines were markedly reduced in NK-depleted mice. NF kappaB and STAT1 activation were also reduced. NK-depleted mice exhibited less inflammation within multiple organs on histologic analysis.These results show that NK cells may contribute to the lethality of bacterial infections via effects on cytokine production. |
| 2001 | [The role of SRC kinase in activation of transcription factor STAT1]. | Tsitologiia | Transcription factor STAT1 (Signal Transducers and Activators of Transcription) takes part in signal transduction from receptors of growth factors and many cytokines, including interferons. In this paper, the role of tyrosinkinases Src and JAK2 was estimated in activation of STAT1 by epidermal growth factor (EGF) and hyperosmotic shock. Using a pharmacological inhibitor of Src kinases CGP77675 and cells with knockout c-src, it was shown that Src activated STAT1 upon stimulation by both epidermal growth factor and hypersomatic shock. In contrast, JAK2 activity exerted no influence on these processes. |
| 2001 | Thrombin regulates vascular smooth muscle cell growth and heat shock proteins via the JAK-STAT pathway. | J Biol Chem | The growth-stimulating effects of thrombin are mediated primarily via activation of a G protein-coupled receptor, PAR-1. Because PAR-1 has no intrinsic tyrosine kinase activity, yet requires tyrosine phosphorylation events to induce mitogenesis, we investigated the role of the Janus tyrosine kinases (JAKs) in thrombin-mediated signaling. JAK2 was activated rapidly in rat vascular smooth muscle cells (VSMC) treated with thrombin, and signal transducers and activators of transcription (STAT1 and STAT3) were phosphorylated and translocated to the nucleus in a JAK2-dependent manner. AG-490, a JAK2-specific inhibitor, and a dominant negative JAK2 mutant inhibited thrombin-induced ERK2 activity and VSMC proliferation suggesting that JAK2 is upstream of the Ras/Raf/MEK/ERK pathway. To elucidate the functional significance of JAK-STAT activation, we studied the effect of thrombin on heat shock protein (Hsp) expression, based upon the following: 1) reports that thrombin stimulates reactive oxygen species production in VSMC; 2) the putative role of Hsps in modulating cellular responses to reactive oxygen species; and 3) the presence of functional STAT1/3-binding sites in Hsp70 and Hsp90beta promoters. Indeed, thrombin up-regulated Hsp70 and Hsp90 protein expression via enhanced binding of STATs to cognate binding sites in the Hsp70 and Hsp90 promoters. Together, these results suggest that JAK-STAT pathway activation is necessary for thrombin-induced VSMC growth and Hsp gene expression. |
| 2001 | Reactive oxygen species regulate heat-shock protein 70 via the JAK/STAT pathway. | Arterioscler Thromb Vasc Biol | Reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) activate intracellular signal transduction pathways implicated in the pathogenesis of cardiovascular disease. H(2)O(2) is a mitogen for rat vascular smooth muscle cells (VSMCs), and protein tyrosine phosphorylation is a critical event in VSMC mitogenesis. Therefore, we investigated whether the mitogenic effects of H(2)O(2), such as stimulation of extracellular signal-regulated kinase (ERK)2, are mediated via activation of cytoplasmic Janus tyrosine kinases (JAKs). JAK2 was activated rapidly in VSMCs treated with H(2)O(2), and signal transducers and activators of transcription (STAT) STAT1 and STAT3 were tyrosine-phosphorylated and translocated to the nucleus in a JAK2-dependent manner. Inhibition of JAK2 activity with AG-490 partially inhibited H(2)O(2)-induced ERK2 activity, suggesting that JAK2 is upstream of the Ras/Raf/mitogen-activated protein kinase-ERK/ERK mitogenic pathway. Because heat-shock proteins (HSPs) can protect cells from ROS, we investigated the effect of H(2)O(2) on HSP expression. H(2)O(2) stimulated HSP70 expression in a time-dependent manner, and AG-490 abolished H(2)O(2)-induced HSP70 expression. H(2)O(2) activated the HSP70 promoter via enhanced binding of STATs to cognate binding sites in the promoter. Regulation of chaperones such as HSP70 via activation of the JAK/STAT pathway suggests that in addition to its growth-promoting effects, this pathway may help VSMCs adapt to oxidative stress. |
| 2000 | Requirement of heat shock protein 90 in mesangial cell mitogenesis. | Kidney Int | Hyperplasia of mesangial cells (MCs) is a frequent finding in glomerulonephritis. Heat shock protein 90 (HSP90) is a major cellular chaperone that assists protein folding under physiological and stress conditions.To identify genes that are potentially involved in the pathogenesis of glomerulonephritis, we analyzed glomerular gene expression in mesangioproliferative rat anti-Thy1.1 nephritis by representational difference analysis (RDA). Expression of HSP90beta in anti-Thy1.1 nephritis was studied by Northern and Western blot analyses and immunohistochemistry. In cultured rat MCs, the requirement of HSP90 for mitogenic signaling steps and MC replication was studied by incubation with the specific HSP90 inhibitor geldanamycin.By RDA, a cDNA fragment homologous to HSP90beta was identified. Glomerular mRNA and protein expression of HSP90beta was markedly and transiently up-regulated during the course of anti-Thy1.1 nephritis, with a maximum at day 6, coinciding with the peak of MC proliferation. By immunohistochemistry, HSP90beta expression in normal glomeruli was detected in podocytes. However, in anti-Thy1.1 nephritis, glomerular HSP90beta protein expression was strongly and transiently increased in mesangial localization. In vitro, mitogenic stimulation of rat MCs led to the induction of HSP90beta mRNA and protein. Incubation of MCs with geldanamycin dose-dependently inhibited DNA synthesis and replication. Moreover, geldanamycin interfered with mitogen-induced phosphorylation of extracellular signal-regulated kinase and transcription of c-fos and Egr-1, but not with transactivation of STAT1 transcription factor. Cell cycle analysis of serum-stimulated MCs revealed that geldanamycin inhibited kinase activity of cyclin D1/CDK4 complexes and blocked progression in the G0/G1 phase and at the S/G2 phase transition.The up-regulation of HSP90beta in anti-Thy1.1 nephritis may reflect its functional involvement in phenotypical alterations of MCs in mesangioproliferative glomerulonephritis. Our in vitro studies indicate that HSP90 governs the capacity of MCs to respond to proliferative stimuli by regulating critical mitogenic signaling steps necessary for G1 entry and S-phase progression. |
| 1999 | Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-gamma uses a different signaling pathway. | Proc Natl Acad Sci U S A | STAT1 is an essential transcription factor for macrophage activation by IFN-gamma and requires phosphorylation of the C-terminal Ser727 for transcriptional activity. In macrophages, Ser727 phosphorylation in response to bacterial lipopolysaccharide (LPS), UV irradiation, or TNF-alpha occurred through a signaling path sensitive to the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 whereas IFN-gamma-mediated Ser727 phosphorylation was not inhibited by the drug. Consistently, SB203580 did not affect IFN-gamma-mediated, Stat1-dependent transcription but inhibited its enhancement by LPS. Furthermore, LPS, UV irradiation, and TNF-alpha caused activation of p38 MAPK whereas IFN-gamma did not. An essential role for p38 MAPK activity in STAT1 Ser727 phosphorylation was confirmed by using cells expressing an SB203580-resistant p38 MAPK. In such cells, STAT1 Ser727 phosphorylation in response to UV irradiation was found to be SB203580 insensitive. Targeted disruption of the mapkap-k2 gene, encoding a kinase downstream of p38 MAPK with a key role in LPS-stimulated TNF-alpha production and stress-induced heat shock protein 25 phosphorylation, was without a significant effect on UV-mediated Ser727 phosphorylation. The recombinant Stat1 C terminus was phosphorylated in vitro by p38MAPKalpha and beta but not by MAPK-activated protein kinase 2. Janus kinase 2 activity, previously reported to be required for IFN-gamma-mediated Ser727 phosphorylation, was not needed for LPS-mediated Ser727 phosphorylation, and activation of Janus kinase 2 did not cause the appearance of STAT1 Ser727 kinase activity. Our data suggest that STAT1 is phosphorylated at Ser727 by a stress-activated signaling pathway either through p38 MAPK directly or through an unidentified kinase downstream of p38MAPK. |
| 1999 | The mitogen-activated protein (MAP) kinase p38 and its upstream activator MAP kinase kinase 6 are involved in the activation of signal transducer and activator of transcription by hyperosmolarity. | J Biol Chem | Environmental stress (e.g. aniso-osmolarity and UV light), hypoxia/reoxygenation, and reactive oxygen species activate intracellular signaling cascades such as the "stress-responsive" mitogen-activated protein kinases and nuclear factor kappaB. We have recently shown that the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway is ligand-independently activated by hyperosmotic shock. In the present study, we show that besides STAT1 also the tyrosine phosphatase SHP2 became tyrosine-phosphorylated upon hyperosmolarity. SB 202190 and SB 203580 (specific inhibitors of p38) inhibited both STAT activation and tyrosine phosphorylation of SHP2 induced by hyperosmotic stress. Overexpression of wild-type p38 mitogen-activated protein kinase and its upstream activator mitogen-activated protein kinase kinase 6 (MKK6) resulted in an enhanced STAT1 tyrosine phosphorylation upon osmotic shock. Accordingly, overexpression of dominant negative mutants of p38 and MKK6 largely decreased hyperosmotic STAT1 activation and tyrosine phosphorylation of SHP2. Furthermore, we provide evidence that a genistein-sensitive tyrosine kinase different from Jak1 is involved in stress-activation of STAT1 and tyrosine phosphorylation of SHP2. These results strongly suggest that hyperosmotic shock activates STAT1 and SHP2 via p38 and its upstream activator MKK6. |
| 1999 | A fatal cytokine-induced systemic inflammatory response reveals a critical role for NK cells. | J Immunol | The mechanism of cytokine-induced shock remains poorly understood. The combination of IL-2 and IL-12 has synergistic antitumor activity in vivo, yet has been associated with significant toxicity. We examined the effects of IL-2 plus IL-12 in a murine model and found that the daily, simultaneous administration of IL-2 and IL-12 resulted in shock and 100% mortality within 4 to 12 days depending on the strain employed. Mice treated with IL-2 plus IL-12 exhibited NK cell apoptosis, pulmonary edema, degenerative lesions of the gastrointestinal tract, and elevated serum levels of proinflammatory cytokines and acute phase reactants. The actions of TNF-alpha, IFN-gamma, macrophage-inflammatory protein-1alpha, IL-1, IL-1-converting enzyme, Fas, perforin, inducible nitric oxide synthase, and STAT1 did not contribute to the observed toxicity, nor did B or T cells. However, toxicity and death from treatment with IL-2 plus IL-12 could be completely abrogated by elimination of NK cells. These results suggest that the fatal systemic inflammatory response induced by this cytokine treatment is critically dependent upon NK cells, but does not appear to be mediated by the known effector molecules of this cellular compartment. These data may provide insight into the pathogenesis of cytokine-induced shock in humans. |
| 1998 | Molecular cloning of the promoter of the gene encoding the Rhesus monkey beta-amyloid precursor protein: structural characterization and a comparative study with other species. | Gene | Abnormal regulation of transcription of the beta-amyloid precursor protein (betaAPP) gene is implicated in the pathogenesis of Alzheimer's disease (AD). We have examined 17- kb genomic region which contains the 5'-flanking region (promoter), first exon and on of the betaAPP gene of the Rhesus monkey (rhbetaAPP). A predominant scription start site was tified 146 bp upstream of the translation initiation codon. Sequencing 5848 bp of 5'-flanking revealed the presence of multiple near consensus sequences for binding potential transcriptional regulatory factors, such as activator proteins (AP-1, AP-2), an apolipoprotein E-B1 element, estrogen-responsive element, heat shock element and NF-kappaB. The sequence of the rhbetaAPP promoter also contains several sites for the binding of proteins that serve as signal transducers and activators of transcription (STAT1) (GAS). The rhbetaAPP promoter is highly homologous to the human promoter, but less homologous to the rodents. The homology between human and Rhesus monkey of the further upstream region gradually decreased over its length. A region of 270 bp of the human betaAPP promoter is missing from the Rhesus monkey promoter. Structural analysis of the promoter suggests that it contains characteristics of inducible genes and sites for regulated activity by various transcription factors. |
| 1998 | Activation of the Janus kinase/signal transducer and activator of transcription pathway by osmotic shock. | J Biol Chem | Numerous cytokines, growth, and differentiation factors elicit their intracellular responses via Janus tyrosine kinases (Jaks) and transcription factors of the STAT (signal transducer and activator of transcription) family. Additionally, environmental stress (UV light, heat, aniso-osmolarity, and radicals) has recently been shown to activate intracellular signaling cascades such as the stress-activated protein kinases and nuclear factor-kappaB. In this study, we demonstrate that in different cell lines a particular stress, namely hyperosmolarity, results in tyrosine phosphorylation of the Janus kinases Jak1, Jak2, and Tyk2 and in the activation of STAT1 and/or STAT3. Both transcription factors are phosphorylated at a specific tyrosine residue and translocation to the nucleus was demonstrated by the use of a STAT3/green fluorescent protein fusion protein. A prominent role for Jak1 in the activation of STATs by hypertonicity was demonstrated by the use of Jak-deficient cell lines. Stress-activated STAT1 and STAT3 transactivate a reporter gene containing the acute-phase response element of the rat alpha2-macroglobulin promoter. Experiments using a diffusible solute suggest that not the increase in intracellular osmolarity but the resultant cell shrinkage is the trigger for Jak/STAT activation. |
| 1998 | Trauma-hemorrhage activates signal transduction pathways in mouse splenic T cells. | Shock | Severe impairment in the functions of immune-competent cells has been observed following trauma and hemorrhage. Inappropriate release of cytokines during trauma and hemorrhagic shock disrupt T lymphocyte functions and enable cells to activate genes whose products are detrimental for maintaining a much-needed humoral and cell-mediated immunity. The intracellular events for gene activation are mediated by cytoplasmic transcription factors present as nascent (signal transducer and activator of transcription 1 (STAT 1)) or as a complex (nuclear factor kappaB (NF-kappaB)). Receptor-initiated phosphorylation activates these transcription factors prior to their nuclear translocation and binding to cognate DNA sequences. Because T cell functions are critical to efficient functioning of the immune system, we investigated whether expression of transcription factors, STAT1 and NF-kappaB, is perturbed in splenic T cells following trauma and hemorrhage. To study this, enriched T cells harvested from spleens (pooled from three or four mice per group) of sham, trauma (consisting of midline laparotomy), sham+trauma, hemorrhage (blood pressure maintained at approximately 30 mmHg for 90 min followed by adequate fluid resuscitation), and trauma+hemorrhage groups at 16-18 h after surgical procedure were probed for signal expressions in the presence and absence of interferon-gamma using electrophoretic mobility shift and Western immunoblot assay procedures. Hemorrhage with or without trauma induced activation of Janus kinase 1, STAT1, and NF-kappaB in T cells. Stimulation of T cells with interferon-gamma led to activation of all these signals in all groups including experimental controls. STAT1 activation was accompanied by Janus kinase 1 phosphorylation, whereas NF-kappaB activation was mediated by phosphorylation and rapid degradation of IkappaBalpha. These studies demonstrate that hemorrhagic shock, with or without laparotomy, is sufficient to induce activation of transcription factors in splenic T cells. Thus, attempts to prevent the activation of transcription factors following hemorrhage by pharmacologic means might be helpful for maintaining cell-mediated immunity under these conditions. |