Regulating Th1 Differentiation Research Articles

Itaconate Inhibits Th1 Differentiation and aGVHD Via Nrf2

Background：Acute graft-versus-host disease (aGVHD) is one of the major complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT), which is caused by the differentiation of pathogenic T cells and resulted a high mortality. Development of novel therapies is critical for the prevention and treatment of aGVHD. Aims：The current study aimed to investigate the role of itaconate in the pathogenesis of aGVHD after allo-HSCT. Methods：To establish murine aGVHD model, lethally irradiated BALB/C (H-2 d) recipients were injected intravenously with 1×10 7 bone marrow (BM) cells and 5×10 6 splenocytes from C57BL/6 or Nrf2 deficiency (H-2 b) donors. Recipients were intraperitoneally injected with 30mg/kg itaconate once a day 3 days prior to BMT for a period of one week. To explore the potential mechanisms of itaconate in the regulation of aGVHD, we profiled the immune cell responses in splenocytes by flow cytometry 2 weeks post transplantation. To detect a CD4 or CD8 dependent manner, recipients were transplanted with TCD-BMs together with either CD4 or CD8 depleted splenocytes respectively. To elucidate the role of itaconate in Th1 development, naïve CD4 +T cells were sorted from WT or Nrf2 KO mice and induced in a Th1 polarization condition for 3 days. IFN-γ production was measured in the supernatant of Th1 cells by ELISA. RNA-Seq was performed to investigate the mechanism of itaconate involved in the regulation of Th1 cell differentiation. Immunofluorescence staining were performed to detect the function of itaconate on Nrf2 expression. The expression of Nrf2 downstream genes were determined by real-time PCR. Total ROS releasing in Th1 cells were detected by a DCFH-DA method. Results：The survival of receipts administrated with itaconate was significantly prolonged than control mice, companied by a lower aGVHD clinical score and decreased tissue damage. The proportions of CD3 +T, CD4 +T, CD8 +T cells were comparable between recipients treated with PBS or itaconate. T cell activations in recipients treated with itaconate were significantly suppressed compared to those of controls. However, itaconate didn't affect the percentages of Tregs and myeloid subsets, including DCs, macrophages and neutrophils. Interestingly, the population of IFN-γ producing CD4 +T, but not CD8 +T, was substantially decreased after itaconate treatment. Moreover, productions of IL-6 and IFN-γ in serum were significantly decreased. The protective effect of itaconate were diminished after depletion of donor derived CD4 +T cells or IFN-γ. RNA-seq and in vitro studies showed that itaconate could directly inhibit Th1 development by regulating oxidative stress response. Itaconate activated Nrf2 downstream genes, thus suppressed ROS production. Both in vitro and in vivo Th1 differentiation were abrogated after Nrf2 depletion. The protective role of itaconate in aGVHD was also diminished when utilizing Nrf2 deficient mice as donors. Conclusion：Itaconate significantly inhibited the activation and IFN-γ production of T cells, rather than interrupted the distribution of myeloid subsets, corresponding to mitigated aGVHD development. Itaconate directly suppressed Th1 differentiation and regulated aGVHD by a CD4 dependent manner. Mechanistically, itaconate promoted the expression of Nrf2 and its downstream genes thus suppressed ROS production, leading to a defective Th1 differentiation. Thus, our results revealed a protective role of itaconate in the pathogenesis of aGVHD by regulating Th1 differentiation.

Junctional adhesion molecule-A on dendritic cells regulates Th1 differentiation

The junctional adhesion molecule-A (JAM-A) is an adhesion molecule present in the surface of several cell types, such as endothelial cells and leukocytes as well as Dendritic Cells (DC). Given the potential relevance of JAM-A in diverse pathological conditions such as inflammatory diseases and cancer, we investigated the role of JAM-A in CD4+ T cell priming. We demonstrate that JAM-A is present in the immunological synapse formed between T cells and DC during priming. Furthermore, an antagonistic anti-JAM-A mAb could disrupt the interaction between CD4+ T cell and DC. Antagonism of JAM-A also attenuated T cell activation and proliferation with a decrease in T-bet expression and increased IL-6 and IL-17 secretion. These findings demonstrate a functional role for JAM-A in interactions between CD4+ T cells and DCs during T cell priming as a positive regulator of Th1 differentiation.

Transcription factor p73 regulates Th1 differentiation

Inter-individual differences in T helper (Th) cell responses affect susceptibility to infectious, allergic and autoimmune diseases. To identify factors contributing to these response differences, here we analyze in vitro differentiated Th1 cells from 16 inbred mouse strains. Haplotype-based computational genetic analysis indicates that the p53 family protein, p73, affects Th1 differentiation. In cells differentiated under Th1 conditions in vitro, p73 negatively regulates IFNγ production. p73 binds within, or upstream of, and modulates the expression of Th1 differentiation-related genes such as Ifng and Il12rb2. Furthermore, in mouse experimental autoimmune encephalitis, p73-deficient mice have increased IFNγ production and less disease severity, whereas in an adoptive transfer model of inflammatory bowel disease, transfer of p73-deficient naïve CD4+ T cells increases Th1 responses and augments disease severity. Our results thus identify p73 as a negative regulator of the Th1 immune response, suggesting that p73 dysregulation may contribute to susceptibility to autoimmune disease.

Tpl2 Promotes Innate Cell Recruitment and Effector T Cell Differentiation To Limit Citrobacter rodentium Burden and Dissemination.

Tumor progression locus 2 (Tpl2) is a serine-threonine kinase that regulates Th1 differentiation, secretion of the inflammatory cytokine gamma interferon (IFN-γ), and host defense against the intracellular pathogens Toxoplasma gondii, Listeria monocytogenes, and Mycobacterium tuberculosis However, relatively little is known about the contribution of Tpl2 to Th17 differentiation and immune cell function during infection with an extracellular pathogen. The goal of this study was to determine whether Tpl2 influences the immune response generated to the extracellular bacterium Citrobacter rodentium, which induces a mixed Th1 and Th17 response. During peak infection with C. rodentium, Tpl2-/- mice experienced greater bacterial burdens with evidence of dissemination to the liver and spleen but ultimately cleared the bacteria within 3 weeks postinfection, similar to the findings for wild-type mice. Tpl2-/- mice also recruited fewer neutrophils and monocytes to the colon during peak infection, which correlated with increased bacterial burdens. In mixed bone marrow chimeras, Tpl2 was shown to play a T cell-intrinsic role in promoting both IFN-γ and interleukin-17A production during infection with C. rodentium However, upon CD4 T cell transfer into Rag-/- mice, Tpl2-/- CD4 T cells were as protective as wild-type CD4 T cells against the dissemination of bacteria and mortality. These data indicate that the enhanced bacterial burdens in Tpl2-/- mice are not caused primarily by impairments in CD4 T cell function but result from defects in innate immune cell recruitment and function.

MTORC1 Promotes T-bet Phosphorylation To Regulate Th1 Differentiation.

CD4+ T cells lacking the mTORC1 activator Rheb fail to secrete IFN-γ under Th1 polarizing conditions. We hypothesized that this phenotype is due to defects in regulation of the canonical Th1 transcription factor T-bet at the level of protein phosphorylation downstream of mTORC1. To test this hypothesis, we employed targeted mass-spectrometry proteomic analysis-multiple reaction monitoring mass spectrometry. We used this method to detect and quantify predicted phosphopeptides derived from T-bet. By analyzing activated murine wild-type and Rheb-deficient CD4+ T cells, as well as murine CD4+ T cells activated in the presence of rapamycin, a pharmacologic inhibitor of mTORC1, we were able to identify six T-bet phosphorylation sites. Five of these are novel, and four sites are consistently dephosphorylated in both Rheb-deficient CD4+ T cells and T cells treated with rapamycin, suggesting mTORC1 signaling controls their phosphorylation. Alanine mutagenesis of each of the six phosphorylation sites was tested for the ability to impair IFN-γ expression. Single phosphorylation site mutants still support induction of IFN-γ expression; however, simultaneous mutation of three of the mTORC1-dependent sites results in significantly reduced IFN-γ expression. The reduced activity of the triple mutant T-bet is associated with its failure to recruit chromatin remodeling complexes to the Ifng gene promoter. These results establish a novel mechanism by which mTORC1 regulates Th1 differentiation, through control of T-bet phosphorylation.

TGF-β receptor 3 (betaglycan) regulates Th1 differentiation and T dependent B cell responses.

Abstract TGFβR3, also known as betaglycan, is a co-receptor for the TGFβ receptor and is predicted to have an important function in promoting high affinity binding of TGF-β to TGFβR1 or TGFβR2. TGFβR3 also binds inhibins with high affinity and antagonizes activin dependent activation of SMADs a function independent of TGFβR1/TGFβR2 signaling. Contrary to TGFβR1 and TGFβR2, our knowledge on the role of TGFβR3 in the immune system is very sparse. Its biology has been elusive because the knock-out of Tgfbr3 exhibit embryonic lethality. We developed the first Tgfbr3f/f mouse in which Cre mediated deletion of Tgfbr3 is very efficient. We found that deletion of TGFβR3 in thymic T cells (Tgfbr3f/f.CD4Cre) had no effect on CD4 or CD8 double positive (DP) or single positive (SP) T cell populations or peripheral T cell populations. Treg numbers in the thymus and periphery were unaltered and the mice did not develop spontaneous autoimmunity as observed when Tgfbr2 is deleted during thymic development. These observations indicated to us that TGFβR3 likely had a function independent of “classical” TGF-β signaling through TGFβR1/2 receptors. To test for this possibility we generated Tgfbr3f/f.dLckCre) mice in which is Tgfbr3 is selectively deleted in mature CD4 and CD8 T cells. We found that T dependent Ab responses, particularly of the IgG2c subclass, was greatly enhanced in mice with T-cell restricted loss of TGFβR3. We further determined that the differentiation of naïve CD4 T cells to IFN-γ expressing Th1 cells under polarizing and non-polarizing conditions was enhanced when TGFβR3 was deficient. These and other data indicate that regulation of Th1 differentiation by TGFβR3 is novel and independent of TGF-β signaling.

Tpl2 differentially regulates IFNγ and IL-17 production by CD4+ T cells in a T cell transfer model of colitis. (LYM8P.636)

Abstract Autoimmune diseases are approaching epidemic levels, estimated to affect 5-8% of the population. A number of autoimmune diseases are driven by autoreactive T cells, specifically T helper 1 (Th1) and T helper 17 (Th17) cells. One molecule gaining interest as a therapeutic target is the serine-threonine kinase, Tpl2, which promotes expression of proinflammatory mediators. We previously demonstrated that Tpl2 regulates Th1 differentiation, secretion of the inflammatory cytokine IFNγ, and host defense against the intracellular parasite Toxoplasma gondii. The goal of this study was to determine whether Tpl2 also regulates Th1 or Th17 differentiation in vivo in a T cell transfer model of colitis associated with mixed Th1/Th17 pathology. In vitro, Tpl2-/- naïve CD4+ T cells were impaired in IL-17A secretion under traditional Th17 inducing conditions of IL-6+TGF-β. Reduced IL-17A secretion correlated with increased expression of FoxP3, a transcription factor known to antagonize RORγt function. In vivo, transfer of Tpl2-/- T cells resulted in reduced proportions of CD4+ T cells expressing IFNγ, but not IL-17A, compared to that induced by wild type T cells. Further studies revealed that Th17 cells alternatively induced by IL-6+IL-23 was unaffected by Tpl2 deficiency. Collectively, these results implicate Tpl2 in TGF-β-induced FoxP3 expression. Additionally, they underscore the contribution of Tpl2 to Th1-dependent responses, including Th1-mediated immunopathology during autoimmunity.

Tumor progression locus 2 differentially regulates IFNγ and IL-17 production by effector CD4+ T cells in a T cell transfer model of colitis.

Autoimmune diseases are approaching epidemic levels, estimated to affect 5–8% of the population. A number of autoimmune diseases are believed to be driven by autoreactive T cells, specifically by T helper 1 (Th1) cells and T helper 17 (Th17) cells. One molecule gaining interest as a therapeutic target is the serine-threonine kinase, Tpl2, which promotes expression of proinflammatory mediators. We previously demonstrated that Tpl2 regulates Th1 differentiation, secretion of the inflammatory cytokine IFNγ, and host defense against the intracellular parasite Toxoplasma gondii. The goal of this study was to determine whether Tpl2 also regulates Th1 or Th17 differentiation in vivo in a model of colitis associated with mixed Th1/Th17 pathology. In vitro, Tpl2−/− naïve CD4 T cells were significantly impaired in IL-17A secretion under traditional Th17 inducing conditions. Reduced IL-17A secretion correlated with increased expression of FoxP3, a transcription factor known to antagonize RORγt function. In a murine T cell transfer model of colitis, transfer of Tpl2−/− T cells resulted in reduced proportions of CD4 T cells expressing IFNγ, but not IL-17A, compared to that induced by wild type T cells. Further studies revealed that IL-17A differentiation induced by IL-6 and IL-23, cytokines implicated in driving Th17 differentiation in vivo, was unaffected by Tpl2 deficiency. Collectively, these results implicate Tpl2 in TGF-β-induced FoxP3 expression. Additionally, they underscore the contribution of Tpl2 to Th1 immunopathology specifically, which suggests that Tpl2 inhibitors may selectively target Th1-based inflammation.

NOTCH signaling in immune-mediated bone marrow failure of aplastic anemia.

Severe aplastic anemia is a rare bone marrow failure disease with the majority of cases caused by aberrant immune destruction of blood progenitors. Although the Th1-mediated pathology of aplastic anemia is well-described, the molecular mechanisms that drive disease progression remain ill-defined. The NOTCH signaling pathway mediates Th1 differentiation in the presence of polarizing cytokines, an action requiring enzymatic processing of NOTCH receptors by γ- secretase. We used a mouse model of aplastic anemia to demonstrate that expression both of intracellular NOTCH1 (NOTCH1IC) and T-BET, a key transcription factor regulating Th1 differentiation, were increased in T cells in the spleen and bone marrow during active disease. Conditionally deleting NOTCH1 or administering γ-secretase inhibitors (GSI) in vivo, attenuated disease and rescued mice from lethal bone marrow failure. In peripheral T cells from patients with untreated aplastic anemia, NOTCH1IC was significantly elevated and was detected at the TBX21 promoter, showing NOTCH1 directly regulates the gene encoding T-BET. Treating patients’ cells ex vivo with GSI lowered NOTCH1IC levels, decreased the level of NOTCH1 detectable at the TBX21 promoter, and also decreased T-BET expression, indicating NOTCH1 signaling is responsive to GSI during active disease. Collectively, these results identify NOTCH1 signaling as a primary driver of Th1-mediated pathogenesis in aplastic anemia and may represent a novel target for therapeutic intervention.

Induction of IgG2a class switching in B cells by IL-27.

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. However, its role in B cells remains unexplored. We here show a role for IL-27 in the induction of T-bet expression and regulation of Ig class switching in B cells. Expression of WSX-1, one subunit of IL-27R, was detected at the mRNA level in primary mouse spleen B cells, and stimulation of these B cells by IL-27 rapidly activated STAT1. IL-27 then induced T-bet expression and IgG2a, but not IgG1, class switching in B cells activated with anti-CD40 or LPS. In contrast, IL-27 inhibited IgG1 class switching induced by IL-4 in activated B cells. Similar induction of STAT1 activation, T-bet expression and IgG2a class switching was observed in IFN-gamma-deficient B cells, but not in STAT1-deficient ones. The induction of IgG2a class switching was abolished in T-bet-deficient B cells activated with LPS. These results suggest that primary spleen B cells express functional IL-27R and that the stimulation of these B cells by IL-27 induces T-bet expression and IgG2a, but not IgG1, class switching in a STAT1-dependent but IFN-gamma-independent manner. The IL-27-induced IgG2a class switching is highly dependent on T-bet in response to T-independent stimuli such as LPS. Thus, IL-27 may be a novel attractive candidate as a therapeutic agent against diseases such as allergic disorders by not only regulating Th1 differentiation but also directly acting on B cells and inducing IgG2a class switching.

Up-regulation of the IL-12 receptor beta 2 chain in Crohn's disease.

Crohn' s disease (CD) is a chronic intestinal inflammatory disorder characterized by aberrant mucosal Th1 cell activation and production of IL-12, the major Th1-driving factor. The T cell response to IL-12 is dependent on the expression of a specific receptor composed of two subunits, termed IL-12Rbeta1 and IL-12Rbeta2. The content of IL-12Rbeta2, as measured at the mRNA level, is crucial in regulating Th1 differentiation. In this study we therefore investigated IL-12Rbeta2 RNA transcripts in CD. IL-12Rbeta2 expression was increased in active CD as well as Helicobacter pylori (HP)-associated gastritis and Salmonella colitis compared with that in inactive CD, ulcerative colitis, noninflammatory controls, and celiac disease. In contrast, IL-12Rbeta1 transcripts were expressed at comparable levels in all samples. In CD, IL-12Rbeta2 expression strictly correlated with tyrosine phosphorylation of STAT4, a key component of the IL-12-dependent Th1 polarization. This was associated with a pronounced expression of IFN-gamma. Transcripts for IL-12/p40 were detected in CD, HP-positive, and Salmonella colitis patients, but not in celiac disease, indicating that IL-12Rbeta2 up-regulation occurs only in IL-12-associated Th1 gastrointestinal diseases. Finally, we showed that stimulation of lamina propria mononuclear cells with IL-12 enhanced IL-12Rbeta2, suggesting that IL-12 regulates IL-12Rbeta2 expression in human gastrointestinal mucosa. The data show that the signaling pathway used by IL-12 to induce Th1 differentiation is increased at the site of disease in CD, further supporting the view that IL-12/IL-12R signals contribute to the inflammatory response in this condition.