Dietary Yeasts Reduce Inflammation in Central Nerve System via Microflora.

OBJECTIVES: The intestinal microflora affects the pathogenesis of several autoimmune diseases by influencing immune system function. Some bacteria, such as lactic acid bacteria, have been reported to have beneficial effects on immune function. However, little is known about the effects of yeasts. Here, we aimed to investigate the effects of various dietary yeasts contained in fermented foods on experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), and to elucidate the mechanisms underlying these effects. METHODS: The effects of eight yeasts selected from 18 types of yeasts contained in fermented foods were examined using an EAE model. Of these, Candida kefyr was investigated by analyzing the intestinal microflora and its effects on intestinal and systemic immune states. RESULTS: Administration of C. kefyr ameliorated the severity of EAE. Reduced numbers of Th17 cells, suppressed interleukin (IL)-6 production by intestinal explants, and increased Tregs and CD103-positive regulatory dendritic cells in mesenteric lymph nodes (MLNs) were observed. Analysis of 16s-rDNA from feces of C. kefyr-treated mice demonstrated increased Lactobacillales and decreased Bacteroides compared to control flora. Transfer of intestinal microbiota also resulted in decreased Bacteroides and ameliorated symptoms of EAE. Thus, oral administration of C. kefyr ameliorated EAE by altering the microflora, accompanied by increased Tregs and CD103-positive regulatory dendritic cells in MLNs and decreased Th17 cells in the intestinal lamina propria. INTERPRETATION: Oral ingestion of C. kefyr may have beneficial effects on MS by modifying microflora. In addition, our findings also suggested the potential health benefits of dietary yeasts.

Gene and protein localisation of tumour necrosis factor (TNF)-α converting enzyme in gingival tissues from periodontitis patients with drug-induced gingival overgrowth.

OBJECTIVE: It is known that tumour necrosis factor (TNF)-α converting enzyme (TACE) plays a crucial role in fibrotic inflammatory diseases, and is specifically inhibited by tissue inhibitor of metalloproteinase (TIMP)-3. Fibrotic drug-induced gingival overgrowth (GO) is often combined with periodontitis. However, neither TACE nor TIMP-3 has been thoroughly examined in periodontal tissues to date. The aim of the present study was to analyse mRNA expression of TACE and TIMP-3, and protein localisation of TACE in gingival tissues removed from drug-(calcium-channel blocker) induced GO and periodontitis. METHODS: A total of 30 gingival tissue samples were taken from 15 GO and 15 periodontitis patients. The mRNA expression levels were analysed by quantitative reverse transcription polymerase chain-reaction (qRT-PCR) and the protein localisation was investigated by immunohistochemistry. Statistical analysis was performed using the Mann-Whitney U-test. RESULTS: TACE and TIMP-3 mRNA levels were significantly higher in GO compared to the periodontitis groups, as revealed by qRT-PCR (p<0.05). TACE-producing cells were immunohistochemically detected among monocytes/macrophages, plasma cells and some epithelial cells. TACE immunoreactivity was shown to be more intense in GO than in periodontitis-gingival tissue. CONCLUSIONS: We have demonstrated TACE expression in cells such as macrophages, plasma cells and epithelial cells, and its predominant expression in GO tissues. This data suggests that TACE expression in GO-gingiva could be involved in the pathogenesis of disease.

IL-7 is essential for lymphopenia-driven turnover of colitogenic CD4(+) memory T cells in chronic colitis.

We previously demonstrated that IL-7 is essential for the persistence of T-cell-mediated colitis, by showing that adoptive transfer of CD4(+)CD45RB(high) T cells into IL-7(-/-) x RAG-1(-/-) mice did not induce colitis; and that intestinal IL-7 is not essential for this colitis model, by showing that IL-7(-/-) x RAG-1(-/-) mice parabiosed with colitic CD4(+)CD45RB(high) T-cell-transferred RAG-1(-/-) mice developed colitis. Here, we investigated the role of IL-7 in the maintenance of colitogenic CD4(+) T cells by surgically separating these parabionts. Surprisingly, the separated IL-7(-/-) x RAG-1(-/-) mice were consistently diseased after separation, although no IL-7 mRNA was detected in the tissues of separated IL-7(-/-) x RAG-1(-/-) partners. CD4(+) T cells isolated from the separated RAG-1(-/-) or IL-7(-/-) x RAG-1(-/-) mice were then transferred into new RAG-1(-/-) or IL-7(-/-) x RAG-1(-/-) mice. Regardless of the source of donor cells, RAG-1(-/-) recipients developed colitis, whereas IL-7(-/-) x RAG-1(-/-) recipients did not. Collectively, these results demonstrate that IL-7 is essential for lymphopenia-driven turnover of colitogenic CD4(+) T cells rather than the maintenance of those cells in established colitic mice. They also provide a basis for the timing of IL-7/IL-7R blockade for the treatment of inflammatory bowel diseases.

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 (T(R)) cells exist as part of the normal immune population and prevent the development of intestinal inflammation. We have recently shown that CD4(+)CD25(+) T(R) 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(+) T(R) cells in the intestine. We first found that RANKL was preferentially expressed on both CD4(+)CD25(+) T(R) cells and colitogenic CD4(+) T cells, whereas RANK was expressed on dendritic cells. Although neutralizing anti-RANKL mAb did not affect T(R) activity of CD4(+)CD25(+) T(R) cells to suppress the proliferation of CD4(+) responder cells in vitro, in vivo administration of anti-RANKL mAb abrogated CD4(+)CD25(+) T(R) cell-mediated suppression of colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells into SCID mice. Interestingly, an adoptive transfer experiment using Ly5.1(+)CD4(+)CD45RB(high) cells and Ly5.2(+)CD4(+)CD25(+) T(R) cells revealed that the ratio of CD4(+)CD25(+) T(R) 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(+) T(R) 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(+) T(R) cells in colitis.

Persistent retention of colitogenic CD4+ memory T cells causes inflammatory bowel diseases to become intractable.

Despite the advent of an age when "malignant" leukemia is cured by bone marrow transplantation, "benign" inflammatory bowel diseases (IBDs) are still intractable lifelong diseases. Why is it that once an IBD develops it lasts a long time? We propose that, the same as in the response to vaccination, immune memory T cells that remember the disease are formed in IBDs and, perceiving them as "benign T-cell leukemia"-like lifelong pathology that hematogenously spreads throughout the body, we here propose that the bone marrow itself, which produces large amounts of the survival factor IL-7, is the reservoir for colitogenic CD4(+) memory T cells responsible for the intractability of IBDs.

FTY720 suppresses the development of colitis in lymphoid-null mice by modulating the trafficking of colitogenic CD4+ T cells in bone marrow.

2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride (FTY720) suppresses T-cell egress from LN, thereby preventing pathogenic T cells from migrating toward disease sites. However, little is known about whether FTY720 could control the trafficking of T cells without the presence of lymphoid tissues. Here we demonstrate that FTY720 treatment suppresses the recirculation of CD4(+) T cells in splenectomized (SPX) lymphotoxin-alpha(-/-) (LT-alpha(-/-)) mice that lack LN and spleen, as shown by peripheral blood (PB) lymphopenia in FTY720-treated SPX LT-alpha(-/-) mice. In a short-term transfer experiment, the cell number of transferred Ly5.1(+)CD4(+) T cells recovered from host FTY720-treated SPX LT-alpha(-/-) mice (Ly5.2(+)) was markedly decreased in PB, but conversely increased in BM. Notably, FTY720 treatment prevented the development of colitis that is otherwise induced in untreated SPX LT-alpha(-/-) x RAG-2(-/-) mice upon transfer of colitic lamina propria CD4(+) T cells. In such mice, the number of CD4(+) T cells in PB or lamina propria of FTY720-treated SPX LT-alpha(-/-) x RAG-2(-/-) recipients was significantly reduced, but that in the BM was significantly increased as compared with untreated control mice. Altogether, the present results indicate that FTY720 treatment may offer an additional role to direct trafficking of CD4(+) T cells in BM, resulting in the prevention of colitis.

Colitogenic CD4+ effector-memory T cells actively recirculate in chronic colitic mice.

BACKGROUND: Although the clinical usefulness of leukocytapheresis for patients with inflammatory bowel disease (IBD) has been reported as a selective removal therapy targeting pathogenic immune cells in blood circulation, it remains unclear whether colitogenic CD4(+) T cells continuously recirculate in peripheral blood during the chronic phase of colitis. METHODS: To resolve this question we conducted a series of in vivo experiments using a murine chronic colitis model induced by adoptive transfer of CD4(+)CD45RB(high) cells into SCID mice in combination with a parabiosis system. RESULTS: In colitic SCID recipients, first, almost all CD4(+) CD45RB(high) donor cells were converted to CD4(+)CD44(high)CD62L(-) IL-7Ralpha(high) effector-memory T (T(EM)) cells at 8 weeks after transfer and were distributed throughout the whole body, including colonic lamina propria, mesenteric lymph nodes, thoracic duct, peripheral blood, spleen, and bone marrow. Second, SCID mice retransferred with the colitic peripheral blood CD4(+) T cells developed colitis that is identical to the original colitis. Third, CD4(+) cells in parabionts between established colitic RAG-2(-/-) mice induced by adoptive transfer of Ly5.1(+) or Ly5.2(+) CD4(+)CD45RB(high) T cells were well mixed in almost equal proportions at various sites 2 weeks after parabiosis surgery, and the redistribution of Ly5.1(+) and Ly5.2(+) CD4(+) T cells was significantly suppressed in FTY720-treated parabionts. CONCLUSIONS: Together, these findings indicate that colitogenic CD4(+) T(EM) cells continuously recirculate in established colitic mice, suggesting that therapeutic approaches targeting systemic CD4(+) T(EM) cells, such as bone marrow transplantation, rather than those targeting only intestinal CD4(+) T cells, may be feasible for the treatment of IBD.

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 TCRalphabeta(+) 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(+)CD45RB(high) T cells, both CD4(+)CD45RB(high) donor cells and the expanding colitogenic lamina propria CD4(+)CD44(high) 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(+)CD45RB(high) 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(+)CD45RB(high) cells. When RAG-2(-/-) mice were transferred with the same number of MyD88(+/+) (Ly5.1(+)) and MyD88(-/-) (Ly5.2(+)) CD4(+)CD45RB(high) 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-x(L) molecules and less production of IFN-gamma 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.

Continuous generation of colitogenic CD4(+) T cells in persistent colitis.

Inflammatory bowel diseases take chronic courses due to the expansion of colitogenic CD4(+) cells. However, it is unclear whether the persistent disease is driven by continuous reactivation of colitogenic memory CD4(+) cells to generate effector CD4(+) cells or by continuous generation of effector CD4(+) cells from naïve cells. To clarify this issue, we performed a series of sequential adoptive transfers of Ly5.2(+) and Ly5.1(+) CD4(+)CD45RB(high) cells into RAG-2(-/-) mice at different time points. We show here that the secondarily transferred CD4(+)CD45RB(high) cells can be converted to CD4(+)CD44(high)CD62L(-)IL-7Ralpha(high) effector-memory T cells even in the presence of pre-existing effector-memory CD4(+) cells. Although the total cell numbers of CD4(+) cells in established colitic mice were consistently equivalent irrespective of the number of primarily transferred cells, the ratio of primarily and secondarily transferred cells was dependent on the ratio of the transferred cell numbers, but not on the order of the transfer. Of note, we found that primarily transferred CD4(+) cells produced significantly lower amounts of IFN-gamma and IL-17 than CD4(+) cells arising from secondary transfer. In conclusion, the continuous generation of colitogenic CD4(+) cells that compensate for exhausted CD4(+) cells may be one of the mechanisms involved in the persistence of colitis.

Immunosenescent colitogenic CD4(+) T cells convert to regulatory cells and suppress colitis.

Inflammatory bowel diseases progress steadily by the expansion of colitogenic CD4(+) cells. However, it remains unknown whether colitogenic CD4(+) cells are long-living like memory cells or exhausted like effector cells. To assess the longevity of colitogenic lamina propria (LP) CD4(+) cells, we performed sequential transfers of LP CD4(+) cells from colitic CD4(+)CD45RB(high) cell-transferred SCID mice into new SCID mice. Although SCID mice transferred with colitic LP CD4(+) cells stably developed colitis until at least the sixth transfer, the interval to the development of colitis gradually lengthened as the number of transfers increased. The incidence of colitis gradually decreased after the seventh transfer. Furthermore, non-colitic LP CD4(+) cells from mice transferred over seven times expressed significantly higher levels of PD-1 and produced significantly lower amounts of IFN-gamma, TNF-alpha, and IL-17 than colitic LP CD4(+) cells recovered after the first transfer. Most notably, we found that re-transfer of non-colitic LP CD4(+) cells recovered after multiple transfers prevented the development of colitis in SCID mice co-transferred with CD4(+)CD45RB(high) cells. Thus, colitogenic LP CD4(+) cells may be exhausted over time, become non-functional, convert to regulatory cells, and finally suppress colitis in the process of immunosenescence.

Systemic, but not intestinal, IL-7 is essential for the persistence of chronic colitis.

We previously demonstrated that IL-7 is produced by intestinal goblet cells and is essential for the persistence of colitis. It is well known, however, that goblet cells are decreased or depleted in the chronically inflamed mucosa of animal colitis models or human inflammatory bowel diseases. Thus, in this study, we assess whether intestinal IL-7 is surely required for the persistence of colitis using a RAG-1/2-/- colitis model induced by the adoptive transfer of CD4+CD45RBhigh T cells in combination with parabiosis system. Surprisingly, both IL-7-/-xRAG-1-/- and IL-7+/+xRAG-1-/- host mice developed colitis 4 wk after parabiosis to a similar extent of colitic IL-7+/+xRAG-1-/- donor mice that were previously transferred with CD4+CD45RBhigh T cells. Of note, although the number of CD4+ T cells recovered from the spleen or the bone marrow of IL-7-/-xRAG-1-/- host mice was significantly decreased compared with that of IL-7+/+xRAG-1-/- host mice, an equivalent number of CD4+ T cells was recovered from the lamina propria of both mice, indicating that the expansion of CD4+ T cells in the spleen or in the bone marrow is dependent on IL-7, but not in the lamina propria. Development of colitis was never observed in parabionts between IL-7+/+xRAG-1-/- host and noncolitic IL-7-/-xRAG-1-/- donor mice that were transferred with CD4+CD45RBhigh T cells. Collectively, systemic, but not intestinal, IL-7 is essential for the persistence of colitis, suggesting that therapeutic approaches targeting the systemic IL-7/IL-7R signaling pathway may be feasible in the treatment of inflammatory bowel diseases.