Xinshou Ouyang

Evidence for licensing of IFN-γ-induced IFN regulatory factor 1 transcription factor by MyD88 in toll-like receptor-dependent gene induction program

The recognition of microbial components by Toll-like receptors (TLRs) initiates signal transduction pathways, which trigger the expression of a series of target genes. It has been reported that TLR signaling is enhanced by cytokines such as IFN-γ, but the mechanisms underlying this enhancement remain unclear. The MyD88 adaptor, which is essential for signaling by many TLRs, recruits members of the IFN regulatory factor (IRF) family of transcription factors, such as IRF5 and IRF7, to evoke the activation of TLR target genes. In this study we demonstrate that IRF1, which is induced by IFN-γ, also interacts with and is activated by MyD88 upon TLR activation. We provide evidence that MyD88-associated IRF1 migrates into the nucleus more efficiently than non-MyD88-associated IRF1 and that this IRF1 selectively participates in the TLR-dependent gene induction program. The critical role of MyD88-dependent “IRF1 licensing” is underscored by the observation that the induction of a specific gene subset downstream of the TLR–MyD88 pathway, such as IFN-β, inducible NO synthase, and IL-12p35, are impaired in Irf1-deficient cells. Thus, our present study places IRF1 as an additional member participating in MyD88 signaling and provides a mechanistic insight into the enhancement of the TLR-dependent gene induction program by IFN-γ.

Interleukin 10 suppresses Th17 cytokines secreted by macrophages and T cells

IL‐17 and IL‐22 are typical cytokines produced by the Th17 T cell subset, but it is unclear if Th17 cytokines can be produced by other cell types. We demonstrate that IL‐10‐deficient and IL‐10R‐deficient macrophages stimulated with lipopolysaccharide produce high levels of IL‐17 and IL‐22. Addition of exogenous IL‐10 to IL‐10‐deficient macrophages abolished IL‐17 production. When IL‐10‐deficient and IL‐10R‐deficient splenocytes were cultured under Th17 polarizing conditions, the population of IL‐17‐producing cells was increased and the cultures produced significantly higher levels of IL‐17 and IL‐22. The addition of recombinant IL‐10 to IL‐10‐deficient splenocytes significantly decreased the percentage of IL‐17‐producing CD4+ T cells. Finally, the mRNA for the Th17 transcription factor retinoic acid‐related orphan receptor (ROR)γt was significantly elevated in IL‐10‐deficient spleen cells and macrophages. These data demonstrate that Th17 cytokines and RORγt are also expressed in macrophages and that IL‐10 negatively regulates the expression of Th17 cytokines and RORγt by both macrophages and T cells.

A critical link between Toll-like receptor 3 and type II interferon signaling pathways in antiviral innate immunity

A conundrum of innate antiviral immunity is how nucleic acid-sensing Toll-like receptors (TLRs) and RIG-I/MDA5 receptors cooperate during virus infection. The conventional wisdom has been that the activation of these receptor pathways evokes type I IFN (IFN) responses. Here, we provide evidence for a critical role of a Toll-like receptor 3 (TLR3)-dependent type II IFN signaling pathway in antiviral innate immune response against Coxsackievirus group B serotype 3 (CVB3), a member of the positive-stranded RNA virus family picornaviridae and most prevalent virus associated with chronic dilated cardiomyopathy. TLR3-deficient mice show a vulnerability to CVB3, accompanied by acute myocarditis, whereas transgenic expression of TLR3 endows even type I IFN signal-deficient mice resistance to CVB3 and other types of viruses, provided that type II IFN signaling remains intact. Taken together, our results indicate a critical cooperation of the RIG-I/MDA5-type I IFN and the TLR3-type II IFN signaling axes for efficient innate antiviral immune responses.

Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9

Nonalcoholic steatohepatitis (NASH) is the most common liver disease in industrialized countries. NASH is a progressive disease that can lead to cirrhosis, cancer, and death, and there are currently no approved therapies. The development of NASH in animal models requires intact TLR9, but how the TLR9 pathway is activated in NASH is not clear. Our objectives in this study were to identify NASH-associated ligands for TLR9, establish the cellular requirement for TLR9, and evaluate the role of obesity-induced changes in TLR9 pathway activation. We demonstrated that plasma from mice and patients with NASH contains high levels of mitochondrial DNA (mtDNA) and intact mitochondria and has the ability to activate TLR9. Most of the plasma mtDNA was contained in microparticles (MPs) of hepatocyte origin, and removal of these MPs from plasma resulted in a substantial decrease in TLR9 activation capacity. In mice, NASH development in response to a high-fat diet required TLR9 on lysozyme-expressing cells, and a clinically applicable TLR9 antagonist blocked the development of NASH when given prophylactically and therapeutically. These data demonstrate that activation of the TLR9 pathway provides a link between the key metabolic and inflammatory phenotypes in NASH.

AP-1 activated by Toll-like receptors regulates expression of IL-23 p19

Interleukin (IL)-23, a new member of the IL-12 family, plays a central role in the Th17 immune response and in autoimmune diseases. It is clear that activated macrophages and dendritic cells produce IL-23, but the molecular mechanisms whereby inflammatory signals stimulate IL-23 expression are not fully understood. We demonstrate that induction of IL-23 p19 gene expression by LPS depends on the TLR4 and MyD88 pathways. All three MAPK pathways (ERK, JNK, and p38) that are activated by lipopolysaccharide (LPS) stimulation were shown to exert a positive effect on p19 expression. We cloned a 1.3-kb putative p19 promoter and defined its transcription initiation sites by the 5′-rapid amplification of cDNA ends method. By analyzing IL-23 p19 promoter mutants, we have identified a promoter region (−413 to +10) that contains several important elements, including NF-κB and AP-1. In addition to NF-κB, we have demonstrated that the proximal AP-1 site is important for p19 promoter activation. Mutation of the AP-1 site resulted in the loss of p19 promoter activation. Electrophoretic mobility shift assay (EMSA) analysis showed that c-Jun and c-Fos bind to the AP-1 site, which was confirmed by a chromatin immunoprecipitation assay. Furthermore, co-transfection of c-Jun and ATF2 synergistically induced p19 promoter activation, and c-Jun and ATF2 formed a protein complex, demonstrated by co-immunoprecipitation. Finally, LPS-stimulated peritoneal macrophages from IL-10-deficient mice expressed significantly higher IL-23 p19 than macrophages from wild type mice, and the addition of recombinant IL-10 strongly inhibited LPS-induced p19 expression. Thus, this study suggests that MyD88-dependent Toll-like receptor signaling induces IL-23 p19 gene expression through both MAPKs and NF-κB.

Epstein-Barr virus-induced gene 3 negatively regulates IL-17, IL-22 and RORγt

Epstein‐Barr virus‐induced gene 3 (EBI3) associates with p28 to form IL‐27 and with IL‐12p35 to form IL‐35. IL‐27Rα–/– mice studies indicate that IL‐27 negatively regulates Th17 cell differentiation. However, no EBI3, p28 or p35‐deficiency studies that directly address the role of EBI3, p28 or p35 on Th17 cells have been done. Here, we demonstrate that spleen cells derived from EBI3–/– mice produce significantly higher levels of IL‐17 as well as IL‐22 upon stimulation with OVA. In vitro derived EBI3–/– Th17 cells also produced significantly higher levels of IL‐17 and IL‐22 than WT cells. The frequency of IL‐17‐producing cells was also elevated when EBI3–/– cells were cultured under Th17 conditions. In addition, spleen cells from EBI3–/– mice immunized with Listeria monocytogenes produced significantly elevated levels of IL‐17 and IL‐22. Furthermore, the Th17 transcription factor RORγt was significantly enhanced in EBI3–/– cells. Finally, EBI3–/– mice exhibited a reduced bacterial load following an acute challenge with L. monocytogenes or a re‐challenge of previously immunized mice, suggesting that EBI3 negatively regulates both innate and adaptive immunity. Taken together, these data provide direct evidence that EBI3 negatively regulates the expression of IL‐17, IL‐22 and RORγt as well as protective immunity against L. monocytogenes.

Transcription factor IRF8 directs a silencing programme for TH17 cell differentiation

TH17 cells are recognized as a unique subset of T helper cells that have critical roles in the pathogenesis of autoimmunity and tissue inflammation. Although RORγt is necessary for the generation of TH17 cells, the molecular mechanisms underlying the functional diversity of TH17 cells are not fully understood. Here we show that a member of interferon regulatory factor (IRF) family of transcription factors, IRF8, has a critical role in silencing TH17-cell differentiation. Mice with a conventional knockout, as well as a T cell-specific deletion, of the Irf8 gene exhibited more efficient TH17 cells. Indeed, studies of an experimental model of colitis showed that IRF8 deficiency resulted in more severe inflammation with an enhanced TH17 phenotype. IRF8 was induced steadily and inhibited TH17-cell differentiation during TH17 lineage commitment at least in part through its physical interaction with RORγt. These findings define IRF8 as a novel intrinsic transcriptional inhibitor of TH17-cell differentiation.

Inflammasome components Asc and caspase-1 mediate biomaterial-induced inflammation and foreign body response

Implantation of biomaterials and devices into soft tissues leads to the development of the foreign body response (FBR), which can interfere with implant function and eventually lead to failure. The FBR consists of overlapping acute and persistent inflammatory phases coupled with collagenous encapsulation and currently there are no therapeutic options. Initiation of the FBR involves macrophage activation, proceeding to giant cell formation, fibroblast activation, and collagen matrix deposition. Despite the recognition of this sequence of events, the molecular pathways required for the FBR have not been elucidated. We have identified that the acute inflammatory response to biomaterials requires nucleotide-binding domain and leucine-rich repeat-containing 3 (Nlrp3), apoptosis-associated speck-like protein containing CARD (Asc), and caspase-1, as well as plasma membrane cholesterol, and Syk signaling. Full development of the FBR is dependent on Asc and caspase-1, but not Nlrp3. The common antiinflammatory drug aspirin can reduce inflammasome activation and significantly reduce the FBR. Taken together, these findings expand the role of the inflammasome from one of sensing damage associated molecular patterns (DAMPs) to sensing all particulate matter irrespective of size. In addition, implication of the inflammasome in biomaterial recognition identifies key pathways, which can be targeted to limit the FBR.

Potentiation of Th17 cytokines in aging process contributes to the development of colitis.

Th17 cells, which produce IL-17 and IL-22, promote autoimmunity in mice and have been implicated in the pathogenesis of autoimmune/inflammatory diseases in humans. However, the Th17 immune response in the aging process is still not clear. In the present study, we found that the induction of IL-17-producing CD4(+) T cells was significantly increased in aged individuals compared with young healthy ones. The mRNA expression of IL-17, IL-17F, IL-22, and RORC2 was also significantly increased in aged people. Similar to humans, Th17 cells as well as mRNAs encoding IL-17, IL-22 and RORγt were dramatically elevated in naïve T cells from aged mouse compared to young ones. In addition, CD44 positive IL-17-producing CD4(+) T cells were significantly higher in aged mice, suggesting that memory T cells are an important source of IL-17 production. Furthermore, the percentage of IL-17-producing CD4(+) T cells generated in co-culture with dendritic cells from either aged or young mice did not show significant differences, suggesting that dendritic cells do not play a primary role in the elevation of Th17 cytokines in aged mouse cells. Importantly, transfer of CD4(+)CD45Rb(hi) cells from aged mice induced more severe colitis in RAG(-/-) mice compared to cells from young mice, Taken together, these results suggest that Th17 immune responses are elevated in aging humans and mice and may contribute to the increased development of inflammatory disorders in the elderly.

Reduction of Stat3 Activity Attenuates HIV-Induced Kidney Injury

HIV-1 Nef induces podocyte proliferation and dedifferentiation by activating the Stat3 and MAPK1,2 pathways. Activation of Stat3 also occurs in human kidneys affected by HIV-associated nephropathy (HIVAN), but its contribution to the development of HIVAN is unknown. Here, we generated HIV-1 transgenic mice (Tg26) with either 75% Stat3 activity (Tg26-SA/+) or 25% Stat3 activity (Tg26-SA/-). The kidneys of Tg26-SA/+ mice, but not Tg26-SA/- mice, showed increased Stat3 phosphorylation. The Tg26-SA/+ phenotype was not different from Tg26 mice, but Tg26-SA/- mice developed significantly less proteinuria, glomerulosclerosis, and tubulointerstitial injury. Tg26-SA/+ mice exhibited reduced expression of podocyte differentiation markers and increased expression of VEGF and proliferation markers as compared to Tg26-SA/- mice. Primary podocytes isolated from Tg26-SA/+ mice showed increased Stat3 phosphorylation and reduced expression of podocyte differentiation markers. The tubulointerstitial compartment and isolated tubules of Tg26-SA/+ mice also had increased Stat3 phosphorylation and expression of Stat3 target genes. We confirmed that the expression of the HIV-1 transgene and reduction of Stat3 activity did not affect T and B cell development. In conclusion, Stat3 plays a critical role in the pathogenesis of HIVAN.