Yingzi Cong

Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota

Summary:  There are now many experimental models of inflammatory bowel disease (IBD), most of which are due to induced mutations in mice that result in an impaired homeostasis with the intestinal microbiota. These models can be clustered into several broad categories that, in turn, define the crucial cellular and molecular mechanisms of host microbial interactions in the intestine. The first of these components is innate immunity defined broadly to include both myeloid and epithelial cell mechanisms. A second component is the effector response of the adaptive immune system, which, in most instances, comprises the CD4+ T cell and its relevant cytokines. The third component is regulation, which can involve multiple cell types, but again particularly involves CD4+ T cells. Severe impairment of a single component can result in disease, but many models demonstrate milder defects in more than one component. The same is true for both spontaneous models of IBD, C3H/HeJBir and SAMPI/Yit mice. The thesis is advanced that ‘multiple hits’ or defects in these interacting components is required for IBD to occur in both mouse and human.

Tumor Exosomes Inhibit Differentiation of Bone Marrow Dendritic Cells

The production of exosomes by tumor cells has been implicated in tumor-associated immune suppression. In this study, we show that, in mice, exosomes produced by TS/A murine mammary tumor cells target CD11b+ myeloid precursors in the bone marrow (BM) in vivo, and that this is associated with an accumulation of myeloid precursors in the spleen. Moreover, we demonstrate that TS/A exosomes block the differentiation of murine myeloid precursor cells into dendritic cells (DC) in vitro. Addition of tumor exosomes at day 0 led to a significant block of differentiation into DC, whereas addition at later time points was less effective. Similarly, exosomes produced by human breast tumor cells inhibited the differentiation of human monocytes in vitro. The levels of IL-6 and phosphorylated Stat3 were elevated 12 h after the tumor exosome stimulation of murine myeloid precursors, and tumor exosomes were less effective in inhibiting differentiation of BM cells isolated from IL-6 knockout mice. Addition of a rIL-6 to the IL-6 knockout BM cell culture restored the tumor exosome-mediated inhibition of DC differentiation. These data suggest that tumor exosome-mediated induction of IL-6 plays a role in blocking BM DC differentiation.

TGF-β Promotes Th17 Cell Development through Inhibition of SOCS3

TGF-β, together with IL-6 and IL-21, promotes Th17 cell development. IL-6 and IL-21 induce activation of STAT3, which is crucial for Th17 cell differentiation, as well as the expression of suppressor of cytokine signaling (SOCS)3, a major negative feedback regulator of STAT3-activating cytokines that negatively regulates Th17 cells. However, it is still largely unclear how TGF-β regulates Th17 cell development and which TGF-β signaling pathway is involved in Th17 cell development. In this report, we demonstrate that TGF-β inhibits IL-6- and IL-21-induced SOCS3 expression, thus enhancing as well as prolonging STAT3 activation in naive CD4+CD25− T cells. TGF-β inhibits IL-6-induced SOCS3 promoter activity in T cells. Also, SOCS3 small interfering RNA knockdown partially compensates for the action of TGF-β on Th17 cell development. In mice with a dominant-negative form of TGF-β receptor II and impaired TGF-β signaling, IL-6-induced CD4+ T cell expression of SOCS3 is higher whereas STAT3 activation is lower compared with wild-type B6 CD4+ T cells. The addition of a TGF-β receptor I kinase inhibitor that blocks Smad-dependent TGF-β signaling greatly, but not completely, abrogates the effect of TGF-β on Th17 cell differentiation. Our data indicate that inhibition of SOCS3 and, thus, enhancement of STAT3 activation is at least one of the mechanisms of TGF-β promotion of Th17 cell development.

Molecular mechanism of lipopolysaccharide-induced SOCS-3 gene expression in macrophages and microglia.

Immunological activation of macrophages/microglia within the CNS leads to the production of cytokines and chemokines that ultimately impact on glial and neuronal function. Suppressor of cytokine signaling (SOCS) proteins are negative regulators of adaptive and innate immune responses. Our previous studies demonstrated that SOCS-3 attenuates macrophage/microglial activation in vitro, suggesting that SOCS-3 may exert beneficial effects for immune-mediated CNS diseases in vivo. In this study, we describe LPS as a potent inducer of SOCS-3 transcription and expression in macrophages/microglia. An analysis of the SOCS-3 promoter indicates that AP-1 and IFN-γ activation sequence (GAS) elements are involved in LPS-induced SOCS-3 transcription. LPS-induced SOCS-3 expression was diminished in IL-10-deficient macrophages at later time points, indicating the involvement of endogenous IL-10 in this response. Blocking STAT-3 expression and activation using STAT-3 small interfering RNA reduced LPS-induced SOCS-3 gene expression. LPS activated the MAPK-ERK1/2, JNK, and p38 pathways that, in addition to STAT-3, were also involved in LPS-induced SOCS-3 expression. LPS treatment of cells led to the acetylation of histones H3 and H4 on the SOCS-3 promoter and the recruitment of STAT-3, c-Jun, c-Fos, CREB-binding protein, p300, and RNA polymerase II to the endogenous SOCS-3 promoter in a time-dependent manner. These results indicate that LPS-induced MAPK activation, the production of endogenous IL-10, and STAT-3 activation play critical roles in SOCS-3 expression, which provides for feedback attenuation of cytokine-induced immune and inflammatory responses in macrophages and microglia.

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.

Th17 Cells Induce Colitis and Promote Th1 Cell Responses through IL-17 Induction of Innate IL-12 and IL-23 Production

Both Th1 and Th17 cells have been implicated in the pathogenesis of inflammatory bowel disease and experimental colitis. However, the complex relationship between Th1 and Th17 cells and their relative contributions to the pathogenesis of inflammatory bowel disease have not been completely analyzed. Although it has been recently shown that Th17 cells can convert into Th1 cells, the underlying in vivo mechanisms and the role of Th1 cells converted from Th17 cells in the pathogenesis of colitis are still largely unknown. In this study, we report that Th17 cells from CBir1 TCR transgenic mice, which are specific for an immunodominant microbiota Ag, are more potent than Th1 cells in the induction of colitis, as Th17 cells induced severe colitis, whereas Th1 cells induced mild colitis when transferred into TCRβxδ−/− mice. High levels of IL-12 and IL-23 and substantial numbers of IFN-γ+ Th1 cells emerged in the colons of Th17 cell recipients. Administration of anti–IL-17 mAb abrogated Th17 cell-induced colitis development, blocked colonic IL-12 and IL-23 production, and inhibited IFN-γ+ Th1 cell induction. IL-17 promoted dendritic cell production of IL-12 and IL-23. Furthermore, conditioned media from colonic tissues of colitic Th17 cell recipients induced IFN-γ production by Th17 cells, which was inhibited by blockade of IL-12 and IL-23. Collectively, these data indicate that Th17 cells convert to Th1 cells through IL-17 induction of mucosal innate IL-12 and IL-23 production.

Interleukin-12 Converts Foxp3+ Regulatory T Cells to Interferon–γ-Producing Foxp3+ T Cells That Inhibit Colitis

BACKGROUND & AIMS Regulatory T (Treg) cells are plastic, but the in vivo mechanisms by which they are converted into foxhead box p3 (Foxp3+) interferon (IFN)-γ+ T cells and whether these converted cells retain the ability to inhibit colitis are not clear. METHODS Foxp3+ Treg cells were generated by culture of naïve CD4+ T cells from Foxp3GFP CBir1 T-cell receptor (TCR) transgenic (Tg) (CBir1-Tg) mice, which are specific for CBir1 flagellin (an immunodominant microbiota antigen), with transforming growth factor-β. Foxp3GFP+ CBir1-Tg Treg cells were isolated by fluorescence-activated cell sorting and transferred into TCRβxδ-/- mice. Colitis was induced by transfer of naïve CBir1-Tg CD4+ T cells into immunodeficient mice. RESULTS Microbiota antigen-specific Foxp3+ Treg cells were converted, in the intestine, to IFN-γ+ T-helper (Th)1 cells, interleukin (IL)-17+ Th17 cells, and Foxp3+ T cells that coexpress IFN-γ and/or IL-17. Conversion of Treg cells into IFN-γ-producing Th1 cells and Foxp3+IFN-γ+ T cells required innate cell production of IL-12 in the intestine; blocking IL-12 with an antibody inhibited their conversion to Th1 and Foxp3+IFN-γ+ T cells in the intestines of mice that were recipients of Treg cells. Addition of IL-12, but not IL-23, promoted conversion of Treg cells into Th1 and Foxp3+IFN-γ+ T cells, in vitro. Foxp3+IFN-γ+ T cells had regulatory activity because they suppressed proliferation of naïve T cells, in vitro, and inhibited induction of colitis by microbiota antigen-specific T cells. IFN-γ+ Th1 cells were not converted into Treg cells; Foxp3+IFN-γ+ T cells differentiated into IFN-γ+ but not Foxp3+ T cells. CONCLUSIONS IL-12 promotes conversion of Treg cells into IFN-γ-expressing cells; Foxp3+IFN-γ+ T cells retain their regulatory functions and develop during the transition of Foxp3+ Treg cells into IFN-γ+ Th1 cells.

Experimental Models to Study Molecular Mechanisms Underlying Intestinal Inflammation

ABSTRACT: Experimental animal models, particularly the newer mouse models, have convincingly demonstrated that CD+ T cells play a central role in chronic intestinal inflammation. Such CD4+ effector T cells are induced by the bacterial flora. In at least one model, it is conventional protein antigens that are stimulating these pathogenic T cells. The antigens driving disease seem to be a selective subset of immunodominant proteins, likely derived from a subset of organisms. Multiple genes contribute to colitis susceptibility and a number of these genes are being localized.

Colitis Induced by Enteric Bacterial Antigen-Specific CD4+ T Cells Requires CD40-CD40 Ligand Interactions for a Sustained Increase in Mucosal IL-12

C3H/HeJBir is a mouse substrain that is highly susceptible to colitis. Their CD4+ T cells react to Ags of the commensal enteric bacteria, and the latter can mediate colitis when activated by these Ags and transferred to histocompatible scid recipients. In this study, multiple long-term C3H/HeJBir CD4+ T cell (Bir) lines reactive to commensal enteric bacterial Ags have been generated. All these were Ag specific, pauciclonal, and Th1 predominant; most induced colitis uniformly after transfer to scid recipients. Lesions were focal and marked by increased expression of IL-12p40 and IFN-γ mRNA and protein. Pathogenic Bir T cell lines expressed CD40 ligand (CD40L) when cultured with Ag-pulsed APCs in vitro. Production of IL-12 was also increased in such cultures, an effect that was Ag- and T cell-dependent and required costimulation by CD40, but not by B7. The two Bir T cell lines that did not induce lesions after transfer failed to significantly express CD40L or increase IL-12 when cultured with Ag-pulsed APCs. Administration of anti-CD40L blocked disease expression induced by pathogenic T cells. We conclude that interactions in the colon mucosa between CD40L-expressing Bir Th1 cells with APCs endogenously loaded with commensal bacterial Ags are critical for sustained increases in local IL-12 production and progression to colitis.

Isolation of Flagellated Bacteria Implicated in Crohn's Disease

Background: Serologic expression cloning has identified flagellins of the intestinal microbiota as immunodominant antigens in experimental colitis in mice and in individuals with Crohns disease (CD). The present study was done to identify the microbial source of such flagellins. Methods: Using a variety of isolation and culture approaches, a number of previously unknown flagellated bacteria were isolated. Based on 16S ribosomal DNA sequences, these bacteria fall into the family Lachnospiraceae of the phylum Firmicutes. Results: Serum IgG from patients with CD and from mice with colitis reacted to the flagellins of these bacteria, and only their flagellins, whereas serum IgG from controls did not. The sequence of these flagellins demonstrate conserved amino‐ and carboxy‐terminal domains that cluster phylogenetically and have a predicted 3D structure similar to Salmonella fliC, including an intact TLR5 binding site. The flagellin of 1 of these bacteria was likely O‐glycosylated. Conclusions: The conserved immune response in both mouse and human to these previously unknown flagellins of the microbiota indicate that they play an important role in host–microbe interactions in the intestine. (Inflamm Bowel Dis 2007)