Suxia Yao

Th17 Cells Upregulate Polymeric Ig Receptor and Intestinal IgA and Contribute to Intestinal Homeostasis

Although CD4+ Th17 cells are enriched in normal intestines, their role in regulation of the host response to microbiota, and whether and how they contribute to intestinal homeostasis, is still largely unknown. It is also unclear whether Th17 cells regulate intestinal IgA production, which is also abundant in the intestinal lumen and has a crucial role as the first defense line in host response to microbiota. In this study, we found that intestinal polymeric Ig receptor (pIgR) and IgA production was impaired in T cell-deficient TCR-βxδ−/− mice. Repletion of TCR-βxδ−/− mice with Th17 cells from CBir1 flagellin TCR transgenic mice, which are specific for a commensal Ag, increased intestinal pIgR and IgA. The levels of intestinal pIgR and IgA in B6.IL-17R (IL-17R−/−) mice were lower than wild type mice. Treatment of colonic epithelial HT-29 cells with IL-17 increased pIgR expression. IL-17R−/− mice demonstrated systemic antimicroflora Ab response. Consistently, administering dextran sulfate sodium (DSS) to C57BL/6 mice after treatment with IL-17–neutralizing Ab resulted in more severe intestinal inflammation compared with control Ab. Administering DSS to IL-17R−/− mice resulted in increased weight loss and more severe intestinal inflammation compared with wild type mice, indicating a protective role of Th17 cells in intestinal inflammation. Individual mice with lower levels of pIgR and intestinal-secreted IgA correlated with increased weight loss at the end of DSS administration. Collectively, our data reveal that microbiota-specific Th17 cells contribute to intestinal homeostasis by regulating intestinal pIgR expression and IgA secretion.

Microbiota downregulates dendritic cell expression of miR-10a, which targets IL-12/IL-23p40.

Commensal flora plays important roles in the regulation of the gene expression involved in many intestinal functions and the maintenance of immune homeostasis, as well as in the pathogenesis of inflammatory bowel diseases. The microRNAs (miRNAs), a class of small, noncoding RNAs, act as key regulators in many biological processes. The miRNAs are highly conserved among species and appear to play important roles in both innate and adaptive immunity, as they can control the differentiation of various immune cells, as well as their functions. However, it is still largely unknown how microbiota regulates miRNA expression, thereby contributing to intestinal homeostasis and pathogenesis of inflammatory bowel disease. In our current study, we found that microbiota negatively regulated intestinal miR-10a expression, because the intestines, as well as intestinal epithelial cells and dendritic cells of specific pathogen-free mice, expressed much lower levels of miR-10a compared with those in germ-free mice. Commensal bacteria downregulated dendritic cell miR-10a expression via TLR–TLR ligand interactions through a MyD88-dependent pathway. We identified IL-12/IL-23p40, a key molecule for innate immune responses to commensal bacteria, as a target of miR-10a. The ectopic expression of the miR-10a precursor inhibited, whereas the miR-10a inhibitor promoted, the expression of IL-12/IL-23p40 in dendritic cells. Mice with colitis expressing higher levels of IL-12/IL-23p40 exhibited lower levels of intestinal miR-10a compared with control mice. Collectively, our data demonstrated that microbiota negatively regulates host miR-10a expression, which may contribute to the maintenance of intestinal homeostasis by targeting IL-12/IL-23p40 expression.

miR-10a inhibits dendritic cell activation and Th1/Th17 cell immune responses in IBD

Objective Although both innate and adaptive responses to microbiota have been implicated in the pathogenesis of IBD, it is still largely unknown how they are regulated during intestinal inflammation. In this report, we investigated the role of microRNA (miR)-10a, a small, non-coding RNA, in the regulation of innate and adaptive responses to microbiota in IBD. Methods miR-10a expression was analysed in the inflamed mucosa of IBD patients treated with or without antitumour necrosis factor (anti-TNF) monoclonal antibodies (mAb) (infliximab) by qRT-PCR. Human monocyte-derived dendritic cells (DC) and IBD CD4+ T cells were transfected with miR-10a precursor to define their effect on the function of DC and CD4+ T cells. Results The expression of miR-10a was markedly decreased, while NOD2 and interleukin (IL)-12/IL-23p40 were significantly increased, in the inflamed mucosa of IBD patients compared with those in healthy controls. Commensal bacteria, TNF and interferon-γ inhibited human DC miR-10a expression in vitro. Anti-TNF mAb treatment significantly promoted miR-10a expression, whereas it markedly inhibited NOD2 and IL-12/IL-23p40 in the inflamed mucosa. We further identified NOD2, in addition to IL-12/IL-23p40, as a target of miR-10a. The ectopic expression of the miR-10a precursor inhibited IL-12/IL-23p40 and NOD2 in DC. Moreover, miR-10a was found to markedly suppress IBD T helper (Th)1 and Th17 cell responses. Conclusions Our data indicate that miR-10a is decreased in the inflamed mucosa of IBD and downregulates mucosal inflammatory response through inhibition of IL-12/IL-23p40 and NOD2 expression, and blockade of Th1/Th17 cell immune responses. Thus, miR-10a could play a role in the pathogenesis and progression of IBD.

ERK differentially regulates Th17- and Treg-cell development and contributes to the pathogenesis of colitis.

Although the development of T‐cell subsets is mainly regulated by a master transcriptional regulator and phosphorylation of the STAT protein in response to distinct cytokine stimulation, accumulating data indicate that other signaling pathways are also involved in regulating or fine‐tuning T‐cell lineage commitment. In this report, we investigated the role of ERK, mitogen‐activated protein kinase (MAPK), in Th17 and Treg cell development. We demonstrate that blockade of ERK activation inhibited Th17‐cell development while upregulating Treg cells under Th17 polarization conditions. Inhibition of ERK decreased IL‐6 induction of RAR‐related orphan receptor γt but enhanced TGF‐β induction of Foxp3, and ERK inhibitor‐treated T cells under Th17 conditions possessed suppressive function in vitro because they produced more IL‐10 and TGF‐β and inhibited naïve T‐cell proliferation and IFN‐γ production at levels comparable with that of Treg cells. Furthermore, ERK inhibitor‐treated T cells under Th17 polarization conditions had a decreased potency to induce colitis in vivo. Collectively, our data demonstrated that the ERK pathway differentially regulates Th17‐ and Treg‐cell differentiation, and thus interfering with the ERK pathway could represent a therapeutic treatment for inflammatory bowel diseases and other Th17‐related autoimmune diseases.

Microbiota metabolite short-chain fatty acid acetate promotes intestinal IgA response to microbiota which is mediated by GPR43

Intestinal IgA, which is regulated by gut microbiota, has a crucial role in maintenance of intestinal homeostasis and in protecting the intestines from inflammation. However, the means by which microbiota promotes intestinal IgA responses remain unclear. Emerging evidence suggests that the host can sense gut bacterial metabolites in addition to pathogen-associated molecular patterns and that recognition of these small molecules influences host immune response in the intestines and beyond. We reported here that microbiota metabolite short-chain fatty acid acetate promoted intestinal IgA responses, which was mediated by “metabolite-sensing” GPR43. GPR43−/− mice demonstrated lower levels of intestinal IgA and IgA+ gut bacteria compared with those in wild type (WT) mice. Feeding WT but not GPR43−/− mice acetate but not butyrate promoted intestinal IgA response independent of T cells. Acetate promoted B-cell IgA class switching and IgA production in vitro in the presence of WT but not GPR43−/− dendritic cells (DCs). Mechanistically, acetate-induced DC expression of Aldh1a2, which converts Vitamin A into its metabolite retinoic acid (RA). Moreover, blockade of RA signaling inhibited the acetate induction of B-cell IgA production. Our studies thus identified a new pathway by which microbiota promotes intestinal IgA response through its metabolites.

Interleukin (IL)-21 promotes intestinal IgA response to microbiota

Commensal microbiota-specific T helper type 17 (Th17) cells are enriched in the intestines, which can convert into T follicular helper (Tfh) in Peyer’s patches, and are crucial for production of intestinal immunoglobulin A (IgA) against microbiota; however, the role of Th17 and Tfh cytokines in regulating the mucosal IgA response to enteric microbiota is still not completely known. In this study, we found that intestinal IgA was impaired in mice deficient in interleukin (IL)-17 or IL-21 signaling. IL-21, but not IL-17, is able to augment B-cell differentiation to IgA+ cells as mediated by transforming growth factor β1 (TGFβ1) and accelerate IgA class switch recombination (CSR). IL-21 and retinoic acid (RA) induce IgA+ B-cell development and IgA production and drives autocrine TGFβ1 production to initiate IgA CSR. Repletion of T-cell-deficient TCRβxδ−/− mice with Th17 cells specific for commensal bacterial antigen increased the levels of IgA+ B cells and IgA production in the intestine, which was blocked by neutralizing IL-21. Thus IL-21 functions to strongly augment IgA production under intestinal environment. Furthermore, IL-21 promotes intestinal B-cell homing through α4β7 expression, alone or with TGFβ and RA. Together, IL-21 from microbiota-specific Th17 and/or Tfh cells contributes to robust intestinal IgA levels by enhancing IgA+ CSR, IgA production and B-cell trafficking into the intestine.

TGF-β converts Th1 cells into Th17 cells through stimulation of Runx1 expression

Differentiated CD4+ T cells preserve plasticity under various conditions. However, the stability of Th1 cells is unclear, as is whether Th1 cells can convert into Th17 cells and thereby contribute to the generation of IFN‐γ+IL‐17+CD4+ T cells, the number of which correlates with severity of colitis. We investigated whether IFN‐γ+Th1 cells can convert into Th17 cells under intestinal inflammation and the mechanisms involved. IFN‐γThy1.1+ Th1 cells were generated by culturing naïve CD4+ T cells from IFN‐γThy1.1 CBir1 TCR‐Tg reporter mice, whose TCR is specific for an immunodominant microbiota antigen, CBir1 flagellin, under Th1 polarizing conditions. IFN‐γThy1.1+ Th1 cells induced colitis in Rag−/− mice after adoptive transfer and converted into IL‐17+Th17, but not Foxp3+Treg cells in the inflamed intestines. TGF‐β and IL‐6, but not IL‐1β and IL‐23, regulated Th1 conversion into Th17 cells. TGF‐β induction of transcriptional factor Runx1 is crucial for the conversion, since silencing Runx1 by siRNA inhibited Th1 conversion into Th17 cells. Furthermore, TGF‐β enhanced histone H3K9 acetylation but inhibited H3K9 trimethylation of Runx1‐ and ROR‐γt‐binding sites on il‐17 or rorc gene in Th1 cells. We conclude that Th1 cells convert into Th17 cells under inflammatory conditions in intestines, which is possibly mediated by TGF‐β induction of Runx1.

Downregulation of microRNA-107 in intestinal CD11c(+) myeloid cells in response to microbiota and proinflammatory cytokines increases IL-23p19 expression.

Commensal flora plays an important role in the development of the mucosal immune system and in maintaining intestinal homeostasis. However, the mechanisms involved in regulation of host‐microbiota interaction are still not completely understood. In this study, we examined how microbiota and intestinal inflammatory conditions regulate host microRNA expression and observed lower microRNA‐107 (miR‐107) expression in the inflamed intestines of colitic mice, compared with that in normal control mice. miR‐107 was predominantly reduced in epithelial cells and CD11c+ myeloid cells including dendritic cells and macrophages in the inflamed intestines. We demonstrate that IL‐6, IFN‐γ, and TNF‐α downregulated, whereas TGF‐β promoted, miR‐107 expression. In addition, miR‐107 expression was higher in the intestines of germ‐free mice than in mice housed under specific pathogen‐free conditions, and the presence of microbiota downregulated miR‐107 expression in DCs and macrophages in a MyD88‐ and NF‐κB‐dependent manner. We determined that the ectopic expression of miR‐107 specifically repressed the expression of IL‐23p19, a key molecule in innate immune responses to commensal bacteria. We concluded that regulation of miR‐107 by intestinal microbiota and proinflammatory cytokine serve as an important pathway for maintaining intestinal homeostasis.

mTOR Mediates IL-23 Induction of Neutrophil IL-17 and IL-22 Production.

It has been shown recently that neutrophils are able to produce IL-22 and IL-17, which differentially regulate the pathogenesis of inflammatory bowel disease. However, it is still largely unknown how the neutrophil production of IL-22 and IL-17 is regulated, and their role in the pathogenesis of inflammatory bowel disease. In this study, we found that IL-23 promoted neutrophil production of IL-17 and IL-22. IL-23 stimulated the neutrophil expression of IL-23R as well as rorc and ahr. Retinoid acid receptor–related orphan receptor γ t and aryl-hydrocarbon receptor differentially regulated IL-23 induction of neutrophil IL-17 and IL-22. In addition, IL-23 induced the activation of mTOR in neutrophils. Blockade of the mTOR pathway inhibited IL-23–induced expression of rorc and ahr, as well as IL-17 and IL-22 production. By using a microbiota Ag-specific T cell–mediated colitis model, we demonstrated that depletion of neutrophils, as well as blockade of IL-22, resulted in a significant increase in the severity of colitis, thereby indicating a protective role of neutrophils and IL-22 in chronic colitis. Collectively, our data revealed that neutrophils negatively regulate microbiota Ag-specific T cell induction of colitis, and IL-23 induces neutrophil production of IL-22 and IL-17 through induction of rorc and ahr, which is mediated by the mTOR pathway.

TLR4 regulates IFN-γ and IL-17 production by both thymic and induced Foxp3+ Tregs during intestinal inflammation

Tregs play a crucial role in the maintenance of intestinal immune homeostasis. However, significant numbers of Foxp3+ Tregs accumulate in the inflamed lesions in experimental colitis and in IBD patients. Treg production of the proinflammatory cytokines IFN‐γ and/or IL‐17 may arguably explain their ineffectiveness in suppressing intestinal inflammation. However, it remains unknown whether iTreg and tTreg produce proinflammatory cytokines and how TLR signaling regulates this process. Here, we found that Foxp3+Tregs were increased in the intestines of B6.TLR4−/− and B6.IL‐10−/− mice when compared with WT B6 mice. TLR4−/− and IL‐10−/− resulted in more Tregs within inflamed intestines. The majority of Foxp3+ Tregs in the spleen was Helios+Nrp1+, whereas most Foxp3+ Tregs in the intestinal LP were Helios−Nrp1−. More Helios+Nrp1+ Tregs expressed IFN‐γ and/or IL‐17 than did Helios−Nrp1− Tregs in the spleen and intestine, which was increased with TLR4−/−. TLR4 signaling in T cells and APCs inhibited Foxp3+ induction via MyD88‐dependent, TRIF‐independent pathways, which was negatively regulated by SOCS3. Collectively, these data demonstrate Helios+Nrp1+ tTregs and Helios−Nrp1− iTregs produce proinflammatory cytokines in the intestines during inflammation, which was regulated by TLR4 signaling.