Calcitriol attenuates TLR2/IL-33 signaling pathway to repress Th9 cell differentiation and potentially limits the pathophysiology of rheumatoid arthritis

There is limited information regarding the TLR2 signaling pathway involved in Th9 cell differentiation. The role of calcitriol in regulating TLR2-mediated Th9 cell development is unknown. Thus, we aimed to unravel the TLR2 signaling pathway in Th9 cells and its regulation by calcitriol. We have used n?=?5–6 animals for each murine experiment. Human studies involved five healthy volunteers. Moreover, ten healthy individuals and ten RA patients were included in the study. Murine and human Th9 cells were treated with Calcitriol (100 nM) and Pam₃CSK₄ (2 µg/mL). The number of IL-9+ve cells was determined by flow cytometry. Real-time PCR was used to assess the gene expression. Serum 25(OH)D₃ levels were determined by HPLC. We observed that TLR2 signals via IL-33/ST2 in Th9 cells. Increased TLR2 expression associated with increased IL9 expression and augmented disease severity in RA patients. Calcitriol attenuated TLR2 signaling in murine and human Th9 cells. Low serum vitamin D3 level negatively associated with increased IL-9 and TLR2 expression and disease severity in RA patients. Our data suggest a potential role of calcitriol to ameliorate the disease severity of RA patients.

     

Differentiation and recruitment of Th9 cells stimulated by pleural mesothelial cells in human Mycobacterium tuberculosis infection

Newly discovered IL-9–producing CD4⁺ helper T cells (Th9 cells) have been reported to contribute to tissue inflammation and immune responses, however, differentiation and immune regulation of Th9 cells in tuberculosis remain unknown. In the present study, our data showed that increased Th9 cells with the phenotype of effector memory cells were found to be in tuberculous pleural effusion as compared with blood. TGF-β was essential for Th9 cell differentiation from naïve CD4⁺ T cells stimulated with PMA and ionomycin in vitro for 5 h, and addition of IL-1β, IL-4 or IL-6 further augmented Th9 cell differentiation. Tuberculous pleural effusion and supernatants of cultured pleural mesothelial cells were chemotactic for Th9 cells, and this activity was partly blocked by anti-CCL20 antibody. IL-9 promoted the pleural mesothelial cell repairing and inhibited IFN-γ-induced pleural mesothelial cell apoptosis. Moreover, pleural mesothelial cells promoted Th9 cell differentiation by presenting antigen. Collectively, these data provide new information concerning Th9 cells, in particular the collaborative immune regulation between Th9 cells and pleural mesothelial cells in human M. tuberculosis infection. In particular, pleural mesothelial cells were able to function as antigen-presenting cells to stimulate Th9 cell differentiation.     

NFκB attenuates IL-5 production and upregulates T-box transcription factors in Th2-like T cells

IL-5 plays important roles in eosinophil differentiation, expansion, and recruitment. The regulation of IL-5 seems critical for the treatment of eosinophil-mediated allergic reactions. However, the precise mechanisms for IL-5 regulation remain unknown. In this study, we investigated how IL-5 production is regulated. The transduction of GATA-3 into a murine T cell hybridoma resulted in acquiring the ability to produce IL-5 in response to an antigenic stimulus like Th2 cells. This production was dependent on the cAMP-PKA pathway, but not on p38 activation. Transduction of NIK largely impaired IL-5 production. RelA and RelB similarly impaired IL-5 production. RelA decreased not only IL-5 protein amount but mRNA. RelA also inhibited Il5-luciferase reporter activity. The transduction of GATA-3 decreased the expression of Tbx21 and Eomes, but the additional transduction of RelA abrogated the decreased expression of GATA-3-induced Tbx21 and Eomes. Furthermore, the transduction of T-bet or Eomes into the GATA-3-transduced T cell hybridoma impaired IL-5 production. These results suggested that strong enhancement of the NFκB pathway downregulates IL-5 production and upregulates T-box protein expression to shift an immune response from Th2 to inflammatory Th1.     

Development of a unique epigenetic signature during in vivo Th17 differentiation

Activated naive CD4⁺ T cells are highly plastic cells that can differentiate into various T helper (Th) cell fates characterized by the expression of effector cytokines like IFN-γ (Th1), IL-4 (Th2) or IL-17A (Th17). Although previous studies have demonstrated that epigenetic mechanisms including DNA demethylation can stabilize effector cytokine expression, a comprehensive analysis of the changes in the DNA methylation pattern during differentiation of naive T cells into Th cell subsets is lacking. Hence, we here performed a genome-wide methylome analysis of ex vivo isolated naive CD4⁺ T cells, Th1 and Th17 cells. We could demonstrate that naive CD4⁺ T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions, findings which are in line with the previously reported plasticity of Th17 cells. We could identify seven regions located in Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 showing pronounced demethylation selectively in ex vivo isolated Th17 cells when compared to other ex vivo isolated Th cell subsets and in vitro generated Th17 cells, suggesting that this unique epigenetic signature allows identifying and functionally characterizing in vivo generated Th17 cells.     

Th17 cell plasticity towards a T-bet-dependent Th1 phenotype is required for bacterial control in Staphylococcus aureus infection

Staphylococcus aureus is frequently detected in patients with sepsis and thus represents a major health burden worldwide. CD4⁺ T helper cells are involved in the immune response to S. aureus by supporting antibody production and phagocytosis. In particular, Th1 and Th17 cells secreting IFN-γ and IL-17A, are involved in the control of systemic S. aureus infections in humans and mice.To investigate the role of T cells in severe S. aureus infections, we established a mouse sepsis model in which the kidney was identified to be the organ with the highest bacterial load and abundance of Th17 cells. In this model, IL-17A but not IFN-γ was required for bacterial control. Using Il17aCre × R26YFP mice we could show that Th17 fate cells produce Th17 and Th1 cytokines, indicating a high degree of Th17 cell plasticity. Single cell RNA-sequencing of renal Th17 fate cells uncovered their heterogeneity and identified a cluster with a Th1 expression profile within the Th17 cell population, which was absent in mice with T-bet/Tbx21-deficiency in Th17 cells (Il17aCre x R26eYFP x Tbx21-flox). Blocking Th17 to Th1 transdifferentiation in Th17 fate cells in these mice resulted in increased S. aureus tissue loads.In summary, we highlight the impact of Th17 cells in controlling systemic S. aureus infections and show that T-bet expression by Th17 cells is required for bacterial clearance. While targeting the Th17 cell immune response is an important therapeutic option in autoimmunity, silencing Th17 cells might have detrimental effects in bacterial infections.     

Galectin-9 and IL-21 Mediate Cross-regulation between Th17 and Treg Cells during Acute Hepatitis C

Loss of CD4 T cell help correlates with virus persistence during acute hepatitis C virus (HCV) infection, but the underlying mechanism(s) remain unknown. We developed a combined proliferation/intracellular cytokine staining assay to monitor expansion of HCV-specific CD4 T cells and helper cytokines expression patterns during acute infections with different outcomes. We demonstrate that acute resolving HCV is characterized by strong Th1/Th17 responses with specific expansion of IL-21-producing CD4 T cells and increased IL-21 levels in plasma. In contrast, viral persistence was associated with lower frequencies of IL-21-producing CD4 T cells, reduced proliferation and increased expression of the inhibitory receptors T cell immunoglobulin and mucin-domain-containing-molecule-3 (Tim-3), programmed death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) on HCV-specific CD8 T cells. Progression to persistent infection was accompanied by increased plasma levels of the Tim-3 ligand Galectin-9 (Gal-9) and expansion of Gal-9 expressing regulatory T cells (Tregs). In vitro supplementation of Tim-3^high HCV-specific CD8 T cells with IL-21 enhanced their proliferation and prevented Gal-9 induced apoptosis. siRNA-mediated knockdown of Gal-9 in Treg cells rescued IL-21 production by HCV-specific CD4 T cells. We propose that failure of CD4 T cell help during acute HCV is partially due to an imbalance between Th17 and Treg cells whereby exhaustion of both CD4 and CD8 T cells through the Tim-3/Gal-9 pathway may be limited by IL-21 producing Th17 cells or enhanced by Gal-9 producing Tregs.     

Disruption of IL-21 Signaling Affects T Cell-B Cell Interactions and Abrogates Protective Humoral Immunity to Malaria

Interleukin-21 signaling is important for germinal center B-cell responses, isotype switching and generation of memory B cells. However, a role for IL-21 in antibody-mediated protection against pathogens has not been demonstrated. Here we show that IL-21 is produced by T follicular helper cells and co-expressed with IFN-γ during an erythrocytic-stage malaria infection of Plasmodium chabaudi in mice. Mice deficient either in IL-21 or the IL-21 receptor fail to resolve the chronic phase of P. chabaudi infection and P. yoelii infection resulting in sustained high parasitemias, and are not immune to re-infection. This is associated with abrogated P. chabaudi-specific IgG responses, including memory B cells. Mixed bone marrow chimeric mice, with T cells carrying a targeted disruption of the Il21 gene, or B cells with a targeted disruption of the Il21r gene, demonstrate that IL-21 from T cells signaling through the IL-21 receptor on B cells is necessary to control chronic P. chabaudi infection. Our data uncover a mechanism by which CD4+ T cells and B cells control parasitemia during chronic erythrocytic-stage malaria through a single gene, Il21, and demonstrate the importance of this cytokine in the control of pathogens by humoral immune responses. These data are highly pertinent for designing malaria vaccines requiring long-lasting protective B-cell responses.     

Stat4 limits DNA methyltransferase recruitment and DNA methylation of the IL-18Ralpha gene during Th1 differentiation

Stat4 is required for Th1 development, although how a transiently activated factor generates heritable patterns of gene expression is still unclear. We examined the regulation of IL-18Ralpha expression to define a mechanism for Stat4-dependent genetic programming of a Th1-associated gene. Although Stat4 binds the Il18r1 promoter following IL-12 stimulation and transiently increases acetylated histones H3 and H4, patterns of histone acetylation alone in Th1 cells may not be sufficient to explain cell-type-specific patterns of gene expression. The level of DNA methylation and recruitment of Dnmt3a to Il18r1 inversely correlate with IL-18Ralpha expression, and blocking DNA methylation increases IL-18Ralpha expression. Moreover, there was decreased Il18r1-Dnmt3a association and DNA methylation following transient trichostatin A-induced histone hyperacetylation in Stat4-/-Th1 cultures. Increased association of Dnmt3a and the Dnmt3a cofactor Dnmt3L with the promoters of several Stat4-dependent genes was found in Stat4-/- Th1 cultures, providing a general mechanism for Stat4-dependent gene programming. These data support a mechanism wherein the transient hyperacetylation induced by Stat4 prevents the recruitment of DNA methyltransferases and the subsequent repression of the Il18r1 locus.