Teruji Totsuka

CD4+CD25bright T Cells in Human Intestinal Lamina Propria as Regulatory Cells

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only active suppression by regulatory T cells plays an important role in the normal intestinal homeostasis, but also its dysregulation leads to the development of inflammatory bowel disease. In this study, we demonstrate that the CD4+CD25bright T cells reside in the human intestinal lamina propria (LP) and functionally retain regulatory activities. All human LP CD4+ T cells regardless of CD25 expression constitutively expressed CTLA-4, glucocorticoid-induced TNFR family-related protein, and Foxp3 and proliferate poorly. Although LP CD4+CD25− T cells showed an activated and anergic/memory phenotype, they did not retain regulatory activity. In LP CD4+CD25+ T cells, however, cells expressing CD25 at high levels (CD4+CD25bright) suppressed the proliferation and various cytokine productions of CD4+CD25− T cells. LP CD4+CD25bright T cells by themselves produced fewer amounts of IL-2, IFN-γ, and IL-10. Interestingly, LP CD4+CD25bright T cells with regulatory T activity were significantly increased in patients with active inflammatory bowel disease. These results suggest that CD4+CD25bright T cells found in the normal and inflamed intestinal mucosa selectively inhibit the host immune response and therefore may contribute to the intestinal immune homeostasis.

MyD88-deficient mice develop severe intestinal inflammation in dextran sodium sulfate colitis.

BackgroundGut commensal microbes affect the development and activation of the mucosal and systemic immune systems. However, the exact molecular mechanism of these microbes that is involved in the development of colitis remains unclear.MethodsThe present study was conducted to determine the distinct role of the innate immune system in the development of a dextran sulfate sodium (DSS) colitis model in MyD88−/− mice, because myeloid differentiation protein (MyD88) is a major adaptor molecule essential for signaling via Toll-like receptors (TLRs). To this end, MyD88−/− and wild-type (WT) mice received sterile distilled water containing 1.2% DSS for 8 days. The survival rate, total clinical score (body weight loss, stool consistency, and rectal bleeding), colon length, and histological score were assessed. The expression of surface markers (F4/80 and CD4) on infiltrating lamina propria mononuclear cells was analyzed immunohistochemistrically.ResultsMyD88−/− mice exhibited increased susceptibility to DSS-induced colitis, as reflected by significantly higher lethality and higher clinical and histological scores, and more severe colonic shortening compared to WT mice. Immunohistochemical analysis revealed a significant increase of both F4/80+ macrophages and CD4+ T cells in the inflamed mucosa in DSS-fed MyD88−/− mice compared to DSS-fed WT mice.ConclusionsThese findings suggest that, via MyD88 signaling, the innate immune system in the gut plays an important protective role in colitis.

Blockade of B7-H1 Suppresses the Development of Chronic Intestinal Inflammation

A newly identified costimulatory molecule, programmed death-1 (PD-1), provides a negative signal that is essential for immune homeostasis. However, it has been suggested that its ligands, B7-H1 (PD-L1) and B7-dendritic cells (B7-DC; PD-L2), could also costimulate T cell proliferation and cytokine secretion. Here we demonstrate the involvement of PD-1/B7-H1 and B7-DC interaction in the development of colitis. We first examined the expression profiles of PD-1 and its ligands in both human inflammatory bowel disease and a murine chronic colitis model induced by adoptive transfer of CD4+CD45RBhigh T cells to SCID mice. Second, we assessed the therapeutic potential of neutralizing anti-B7-H1 and/or B7-DC mAbs using this colitis model. We found significantly increased expression of PD-1 on T cells and of B7-H1 on T, B, and macrophage/DCs in inflamed colon from both inflammatory bowel disease patients and colitic mice. Unexpectedly, the administration of anti-B7-H1, but not anti-B7-DC, mAb after transfer of CD4+CD45RBhigh T cells suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced the production of IFN-γ, IL-2, and TNF-α, but not IL-4 or IL-10, by lamina propria CD4+ T cells. These data suggest that the interaction of PD-1/B7-H1, but not PD-1/B7-DC, might be involved in intestinal mucosal inflammation and also show a possible role of interaction between B7-H1 and an as yet unidentified receptor for B7-H1 in inducing T cell activation.

Regulation of Murine Inflammatory Bowel Disease by CD25+ and CD25−CD4+ Glucocorticoid-Induced TNF Receptor Family-Related Gene Regulatory T Cells

CD4+CD25+ regulatory T cells in normal animals are engaged in the maintenance of immunological self-tolerance and prevention of autoimmune disease. However, accumulating evidence suggests that a fraction of the peripheral CD4+CD25− T cell population also possesses regulatory activity in vivo. Recently, it has been shown glucocorticoid-induced TNFR family-related gene (GITR) is predominantly expressed on CD4+CD25+ regulatory T cells. In this study, we show evidence that CD4+GITR+ T cells, regardless of the CD25 expression, regulate the mucosal immune responses and intestinal inflammation. SCID mice restored with the CD4+GITR− T cell population developed wasting disease and severe chronic colitis. Cotransfer of CD4+GITR+ population prevented the development of CD4+CD45RBhigh T cell-transferred colitis. Administration of anti-GITR mAb-induced chronic colitis in mice restored both CD45RBhigh and CD45RBlow CD4+ T cells. Interestingly, both CD4+CD25+ and CD4+CD25− GITR+ T cells prevented wasting disease and colitis. Furthermore, in vitro studies revealed that CD4+CD25−GITR+ T cells as well as CD4+CD25+GITR+ T cells expressed CTLA-4 intracellularly, showed anergic, suppressed T cell proliferation, and produced IL-10 and TGF-β. These data suggest that GITR can be used as a specific marker for regulatory T cells controlling mucosal inflammation and also as a target for treatment of inflammatory bowel disease.

Signaling pathway via TNF-α/NF-κB in intestinal epithelial cells may be directly involved in colitis-associated carcinogenesis

Treatment with anti-TNF-alpha MAb has been accepted as a successful maintenance therapy for patients with inflammatory bowel diseases (IBD). Moreover, it has been recently reported that blockade of TNF receptor (TNFR) 1 signaling in infiltrating hematopoietic cells may prevent the development of colitis-associated cancer (CAC). However, it remains unclear whether the TNF-alpha signaling in epithelial cells is involved in the development of CAC. To investigate this, we studied the effects of anti-TNF-alpha MAb in an animal model of CAC by administration of azoxymethane (AOM) followed by sequential dextran sodium sulfate (DSS) ingestion. We observed that the NF-kappaB pathway is activated in colonic epithelia from DSS-administered mice in association with upregulation of TNFR2 rather than TNFR1. Immunoblot analysis also revealed that the TNFR2 upregulation accompanied by the NF-kappaB activation is further complicated in CAC tissues induced in AOM/DSS-administered mice compared with the nontumor area. Such NF-kappaB activity in the epithelial cells is significantly suppressed by the treatment of MP6-XT22, an anti-TNF-alpha MAb. Despite inability to reduce the severity of colitis, sequential administration of MP6-XT22 reduced the numbers and size of tumors in association with the NF-kappaB inactivation. Taken together, present studies suggest that the TNFR2 signaling in intestinal epithelial cells may be directly involved in the development of CAC with persistent colitis and imply that the maintenance therapy with anti-TNF-alpha MAb may prevent the development of CAC in patients with long-standing IBD.

Intestinal Lamina Propria Retaining CD4+CD25+ Regulatory T Cells Is A Suppressive Site of Intestinal Inflammation

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only does active suppression by regulatory T (TREG) cells play an important role in the normal intestinal homeostasis, but also that its dysregulation of immune response leads to the development of inflammatory bowel disease. In this study, we demonstrate that murine CD4+CD25+ T cells residing in the intestinal lamina propria (LP) constitutively express CTLA-4, glucocorticoid-induced TNFR, and Foxp3 and suppress proliferation of responder CD4+ T cells in vitro. Furthermore, cotransfer of intestinal LP CD4+CD25+ T cells prevents the development of chronic colitis induced by adoptive transfer of CD4+CD45RBhigh T cells into SCID mice. When lymphotoxin (LT)α-deficient intercrossed Rag2 double knockout mice (LTα−/− × Rag2−/−), which lack mesenteric lymph nodes and Peyer’s patches, are transferred with CD4+CD45RBhigh T cells, they develop severe wasting disease and chronic colitis despite the delayed kinetics as compared with the control LTα+/+ × Rag2−/− mice transferred with CD4+CD45RBhigh T cells. Of note, when a mixture of splenic CD4+CD25+ TREG cells and CD4+CD45RBhigh T cells are transferred into LTα−/− × Rag2−/− recipients, CD4+CD25+ TREG cells migrate into the colon and prevent the development of colitis in LTα−/− × Rag2−/− recipients as well as in the control LTα+/+ × Rag2−/− recipients. These results suggest that the intestinal LP harboring CD4+CD25+ TREG cells contributes to the intestinal immune suppression.

IL-7 Is Essential for the Development and the Persistence of Chronic Colitis

Although IL-7 has recently emerged as a key cytokine involved in controlling the homeostatic turnover and the survival of peripheral resting memory CD4+ T cells, its potential to be sustained pathogenic CD4+ T cells in chronic immune diseases, such as inflammatory bowel diseases, still remains unclear. In this study, we demonstrate that IL-7 is essential for the development and the persistence of chronic colitis induced by adoptive transfer of normal CD4+CD45RBhigh T cells or colitogenic lamina propria (LP) CD4+ memory T cells into immunodeficient IL-7+/+ × RAG-1−/− and IL-7−/− × RAG-1−/− mice. Although IL-7+/+ × RAG-1−/− recipients transferred with CD4+CD45RBhigh splenocytes developed massive inflammation of the large intestinal mucosa concurrent with massive expansion of Th1 cells, IL-7−/− × RAG-1−/− recipients did not. Furthermore, IL-7−/− × RAG-1−/−, but not IL-7+/+ × RAG-1−/−, mice transferred with LP CD4+CD44highCD62L−IL-7Rαhigh effector-memory T cells (TEM) isolated from colitic CD4+CD45RBhigh-transferred mice did not develop colitis. Although rapid proliferation of transferred colitogenic LP CD4+ TEM cells was observed in the in IL-7−/− × RAG-1−/− mice to a similar extent of those in IL-7+/+ × RAG-1−/− mice, Bcl-2 expression was significantly down-modulated in the transferred CD4+ T cells in IL-7−/− × RAG-1−/− mice compared with those in IL-7+/+ × RAG-1−/− mice. Taken together, IL-7 is essential for the development and the persistence of chronic colitis as a critical survival factor for colitogenic CD4+ TEM cells, suggesting that therapeutic approaches targeting IL-7/IL-7R signaling pathway may be feasible in the treatment of inflammatory bowel diseases.

MyD88-Dependent Pathway in T Cells Directly Modulates the Expansion of Colitogenic CD4+ T Cells in Chronic Colitis

TLRs that mediate the recognition of pathogen-associated molecular patterns are widely expressed on/in cells of the innate immune system. However, recent findings demonstrate that certain TLRs are also expressed in conventional TCRαβ+ T cells that are critically involved in the acquired immune system, suggesting that TLR ligands can directly modulate T cell function in addition to various innate immune cells. In this study, we report that in a murine model of chronic colitis induced in RAG-2−/− mice by adoptive transfer of CD4+CD45RBhigh T cells, both CD4+CD45RBhigh donor cells and the expanding colitogenic lamina propria CD4+CD44high memory cells expresses a wide variety of TLRs along with MyD88, a key adaptor molecule required for signal transduction through TLRs. Although RAG-2−/− mice transferred with MyD88−/−CD4+CD45RBhigh cells developed colitis, the severity was reduced with the delayed kinetics of clinical course, and the expansion of colitogenic CD4+ T cells was significantly impaired as compared with control mice transferred with MyD88+/+CD4+CD45RBhigh cells. When RAG-2−/− mice were transferred with the same number of MyD88+/+ (Ly5.1+) and MyD88−/− (Ly5.2+) CD4+CD45RBhigh cells, MyD88−/−CD4+ T cells showed significantly lower proliferative responses assessed by in vivo CFSE division assay, and also lower expression of antiapoptotic Bcl-2/Bcl-xL molecules and less production of IFN-γ and IL-17, compared with the paired MyD88+/+CD4+ T cells. Collectively, the MyD88-dependent pathway that controls TLR signaling in T cells may directly promote the proliferation and survival of colitogenic CD4+ T cells to sustain chronic colitis.

Interleukin-18 overproduction exacerbates the development of colitis with markedly infiltrated macrophages in interleukin-18 transgenic mice

Background and Aim:  The authors have previously shown that production of interleukin (IL)‐18 was increased in the inflamed mucosa of patients with Crohns disease (CD) and blockade of IL‐18 ameliorated the murine model of CD. This demonstrated that IL‐18 plays a significant role during intestinal inflammation. However, the initial role of IL‐18 during intestinal inflammation was unclear; therefore the susceptibility of IL‐18 transgenic (Tg) mice to acute dextran sulfate sodium (DSS)‐induced colitis was examined.

Rank-rankl signaling pathway is critically involved in the function of CD4+CD25+ regulatory T cells in chronic colitis

It is now clear that functional CD4+CD25+ regulatory T (TR) cells exist as part of the normal immune population and prevent the development of intestinal inflammation. We have recently shown that CD4+CD25+ TR cells reside in the intestine and control intestinal homeostasis in humans and mice. In this study, we demonstrate that the TNF family molecule RANKL and its receptor RANK are critically involved in controlling the function of CD4+CD25+ TR cells in the intestine. We first found that RANKL was preferentially expressed on both CD4+CD25+ TR cells and colitogenic CD4+ T cells, whereas RANK was expressed on dendritic cells. Although neutralizing anti-RANKL mAb did not affect TR activity of CD4+CD25+ TR cells to suppress the proliferation of CD4+ responder cells in vitro, in vivo administration of anti-RANKL mAb abrogated CD4+CD25+ TR cell-mediated suppression of colitis induced by adoptive transfer of CD4+CD45RBhigh T cells into SCID mice. Interestingly, an adoptive transfer experiment using Ly5.1+CD4+CD45RBhigh cells and Ly5.2+CD4+CD25+ TR cells revealed that the ratio of CD4+CD25+ TR cells in total CD4+ T cells in inflamed mucosa was significantly decreased by anti-RANKL mAb treatment. Consistent with this, the expression of RANK on lamina propria CD11c+ cells from colitic mice was significantly increased as compared with that from normal mice, and in vitro treatment with anti-RANKL mAb suppressed the expansion of CD4+Foxp3+ TR cells in culture with colitic lamina propria CD11c+ cells. Together, these results suggest that the RANK-RANKL signaling pathway is critically involved in regulating the function of CD4+CD25+ TR cells in colitis.