Yohei Mikami

Monocyte Chemoattractant Protein-1 Contributes to Gut Homeostasis and Intestinal Inflammation by Composition of IL-10-Producing Regulatory Macrophage Subset

Lamina propria macrophages (LPMϕs) spontaneously produce large amounts of anti-inflammatory IL-10 and play a central role in regulation of immune responses against commensal bacteria. MCP-1 is a chemokine that plays an important role in recruitment of monocytes and macrophages to inflamed tissues. We demonstrated that, in addition to IL-10, LPMϕs produced large amounts of MCP-1, even in a steady state. MCP-1 deficiency caused impaired IL-10 production by LPMϕs and led to exacerbation of dextran sulfate sodium-induced acute colitis. As an explanation of this impaired IL-10 production by LPMϕs, we found that LPMϕs could be separated into two subsets with distinct side-scattered properties, namely LPMϕ1 (CD11b+F4/80+CD11c–SSChi) and LPMϕ2 (CD11b+F4/80+CD11c–SSClo). Unlike LPMϕ1, the LPMϕ2 subset migrated in response to MCP-1 and produced a larger amount of IL-10 in response to commensal bacteria. LPMϕs isolated from MCP-1–deficient mice produced less IL-10 as a consequence of the lack of the MCP-1–dependent LPMϕ2 population. This imbalanced composition in LPMϕ population may be involved in the susceptibility to DSS-induced colitis in MCP-1–deficient mice. Our results suggest that endogenous MCP-1 contributes to the composition of resident LPMϕ subsets in the intestine. Moreover, MCP-1–dependent LPMϕ2 subset may play an important role in maintenance of gut homeostasis in the steady state, and in the termination of excess inflammatory responses in the intestine, by producing IL-10.

Regulatory T Cells Suppress Development of Colitis, Blocking Differentiation of T-Helper 17 Into Alternative T-Helper 1 Cells

BACKGROUND & AIMS Although T-helper (Th) 17 and Th1 cells are involved in pathogenesis of intestinal inflammation, their developmental pathways and sufficiency to promote disease are not known; nor are the roles of CD4⁺CD25⁺ regulatory T (T(R)) cells in their development. METHODS We performed adoptive transfer experiments to investigate the induction and suppression of colitis using naïve CD4⁺CD45RB(high) T cells and/or CD4⁺CD25⁺ T(R) cells that were obtained from retinoid-related orphan receptor gamma t (RORγt) gfp/⁺ or Ly5.1/Ly5.2 congenic mice. RESULTS We observed 3 types of colitogenic CD4⁺ Th1 cells (interleukin [IL]-17A⁻interferon [IFN]-γ⁺): RORγt⁻ classical Th1 cells that differentiated directly from naïve T cells; RORγt⁺ Th1-like cells; and RORγt⁻ alternative Th1 cells that were terminally differentiated from RORγt⁺ cells via Th17 (IL-17A⁺IFN-γ⁻), Th17/Th1 (IL-17A⁺IFN-γ⁺), or Th1-like (IL-17A⁻IFN-γ⁺) cells. In this pathway, CD4⁺CD25⁺ T(R) cells suppress the development of not only classical Th1 cells, but also alternative Th1 cells at the transition of Th17/Th1 into alternative Th1 cells, resulting in accumulation of Th17 and Th17/Th1 cells in mice in which the development of colitis was suppressed. Furthermore, T(R) cells regulated the established balance of Th17 and Th1 cells under colitic conditions to yield a high ratio of Th17 and Th17/Th1 cells to Th1 cells in noncolitic conditions. CONCLUSIONS Th17 and Th17/Th1 cells become colitogenic alternative Th1 cells via Th17, Th17/Th1, and Th1-like cells, independently of classical Th1 cells. T(R) cells suppress this pathway, resulting in accumulation of Th17 and Th17/Th1 cells.

Immune aspects of the pathogenesis of inflammatory bowel disease

Although the precise etiologies of inflammatory bowel disease (IBD) (ulcerative colitis and Crohns disease) remain obscure, several reports have indicated that dysfunction of the mucosal immune system plays an important role in its pathogenesis. Recent progress with genome-wide association studies has identified many IBD susceptibility genes. In individuals with genetic risk, abnormal interactions between the host immune system and gut flora, and dysregulation of cellular responses such as autophagy and ER stress, induce an abnormal host immune response in the gut resulting in intestinal inflammation. Research progress animal models in IBD, and in human IBD, has identified several key molecules in IBD pathogenesis such as TNFα and adhesion molecules, and molecular targeting therapies based on these molecules have been developed. Here, we review immunological aspects in IBD pathogenesis and the development of immunoregulatory therapy.

CCR9 Macrophages Are Required for Acute Liver Inflammation in Mouse Models of Hepatitis

BACKGROUND & AIMS Antigen-presenting cells (APCs) are involved in the induction of liver inflammation. We investigated the roles of specific APCs in the pathogenesis of acute liver injury in mice. METHODS We used concanavalin A (con A) or carbon tetrachloride to induce acute liver inflammation in mice and studied the roles of macrophages that express CCR9. RESULTS After injection of con A, we detected CCR9(+)CD11b(+)CD11c(-) macrophages that express tumor necrosis factor (TNF)-α in livers of mice, whereas CCR9(+)Siglec-H(+)CD11b(-)CD11c(low) plasmacytoid DCs (pDCs), which are abundant in normal livers, disappeared. The CCR9(+) macrophages were also detected in the livers of RAG-2(-/-) mice, which lack lymphocytes and natural killer T cells, after injection of con A. Under inflammatory conditions, CCR9(+) macrophages induced naive CD4(+) T cells to become interferon gamma-producing Th1 cells in vivo and in vitro. CCR9(-/-) mice injected with con A did not develop hepatitis unless they also received CCR9(+) macrophages from mice that received con A; more CCR9(+) macrophages accumulated in their inflamed livers than CCR9(+) pDCs, CCR9(-) pDCs, or CCR9(-) macrophages isolated from mice that had received injections of con A. Levels of CCL25 messenger RNA increased in livers after injection of con A; neutralizing antibodies against CCL25 reduced the induction of hepatitis by con A by blocking the migration of CCR9(+) macrophages and their production of TNF-α. Peripheral blood samples from patients with acute hepatitis had greater numbers of TNF-α-producing CCR9(+)CD14(+)CD16(high) monocytes than controls. CONCLUSIONS CCR9(+) macrophages contribute to the induction of acute liver inflammation in mouse models of hepatitis.

RORγt-dependent IL-17A-producing cells in the pathogenesis of intestinal inflammation

The hypothesis of helper T (Th)1/Th2 cytokine balance proposed by Mosmann and Coffman is often invoked to explain the development of inflammatory diseases, including inflammatory bowel diseases (IBD). Recently, however, a newly identified class of Th cells—Th17 cells, which produce Th17 family cytokines—has been recognized as an essential subpopulation in the development of almost all kinds of human and animal inflammatory diseases, rather than Th1 and Th2 cells. A representative Th17 family cytokine, interleukin (IL)-17A, is produced by not only Th17 cells, but also by other types of cells, such as T-cell receptor γδ T cells, natural killer (NK) T cells, NK cells, myeloid cells, and innate lymphoid cells, which may also be critically involved in the initiation and persistence of IBD. Here we review recent advances in the study of such IL-17A-producing cells in the pathogenesis of IBD.

C-C motif chemokine receptor 9 positive macrophages activate hepatic stellate cells and promote liver fibrosis in mice†‡

Chemokine receptors mediate migration of immune cells into the liver, thereby promoting liver inflammation. C‐C motif chemokine receptor (CCR) 9+ macrophages are crucial in the pathogenesis of acute liver inflammation, but the role and underlying mechanisms of this macrophage subset in chronic liver injury and subsequent liver fibrosis are not fully understood. We confirmed that tumor necrosis factor alpha (TNF‐α)‐producing CCR9+ macrophages accumulated during the initiation of carbon tetrachloride (CCl4)‐induced liver injury, and CCR9 deficiency attenuated the degree of liver damage. Accumulation of CCR9+ macrophages persisted prominently during the process of liver fibrosis induced by repetitive CCl4 or thioacetamide (TAA)/leptin administration. Increased CCR9 expression was also found on activated hepatic stellate cells (HSCs). Importantly, experimental liver fibrosis was significantly ameliorated in CCR9−/− mice compared with wild‐type (WT) mice, assessed by α‐smooth muscle actin (α‐SMA) immunostain, Sirius red staining, and messenger RNA (mRNA) expression levels of α‐SMA, collagen 1α1, transforming growth factor (TGF)‐β1, and tissue inhibitor of metalloproteinase (TIMP)‐1. Accumulated CD11b+ macrophages in CCl4‐treated WT mice showed marked increases in TNF, NO synthase‐2, and TGF‐β1 mRNA expression compared with CCR9−/− mice, implying proinflammatory and profibrogenic properties. Hepatic CD11b+ macrophages from CCl4‐treated WT mice (i.e., CCR9+ macrophages), but not CD8+ T lymphocytes or non‐CD11b+ cells, significantly activated HSCs in vitro compared with those from CCR9−/− mice. TNF‐α or TGF‐β1 antagonism attenuated CCR9+ macrophage‐induced HSC activation. Furthermore, C‐C motif chemokine ligand (CCL) 25 mediated migration and, to a lesser extent, activation of HSCs in vitro. Conclusion: Accumulated CD11b+ macrophages are critical for activating HSCs through the CCR9/CCL25 axis and therefore promote liver fibrosis. CCR9 antagonism might be a novel therapeutic target for liver fibrosis. (HEPATOLOGY 2013;)

Cross-talk between RORγt+ innate lymphoid cells and intestinal macrophages induces mucosal IL-22 production In Crohn's disease

Background:Interleukin (IL)-22–producing ROR&ggr;t+ innate lymphoid cells (ILCs) play a pivotal role in intestinal immunity. Recent reports demonstrated that ILCs contribute to mucosal protection and intestinal inflammation in mice. In humans, numbers of ROR&ggr;t+ ILCs are significantly increased in the intestine of patients with Crohns disease (CD), suggesting that ILCs may be associated with intestinal inflammation in CD. However, the mechanism by which ILCs are regulated in the intestine of patients with CD is poorly understood. This study aimed to determine the activation mechanism of intestinal ILCs in patients with CD. Methods:CD45+ lineage marker- ILCs were isolated from intestinal lamina propria of patients with CD. ILCs were then subdivided into 4 distinct populations based on the expression of CD56 and CD127. Purified ILC subsets were cocultured with intestinal CD14+ macrophages, and IL-22 production was evaluated. Results:CD127+CD56− and CD127+CD56+ ILC, but not CD127−CD56+ or CD127−CD56− ILC, subsets expressed ROR&ggr;t and produced IL-22. IL-22 production by these ILC subsets was enhanced when ILCs were cocultured with intestinal macrophages. IL-23 or cell-to-cell contact was required for macrophage-mediated activation of ILCs. IL-22 production by ILCs was perturbed in inflamed mucosa compared with noninflamed mucosa. IL-22 induced the expression of Reg1&agr; and Claudin-1 in human intestinal epithelial organoids. Conclusions:ROR&ggr;t+ ILCs might enhance mucosal barrier function through the upregulation of Reg1&agr; through production of IL-22. Although CD14+ macrophages augment intestinal inflammation in patients with CD, macrophages also promote a negative feedback pathway through the activation of IL-22 production by ROR&ggr;t+ ILCs.

Competition between colitogenic Th1 and Th17 cells contributes to the amelioration of colitis.

Th17 cells and Th1 cells coordinate to play a critical role in the formation of inflammatory bowel diseases. To examine how Th17 and Th1 cells are regulated at inflammatory sites, we used Th1‐dominant CD4+CD45RBhigh T cell‐transferred RAG‐2−/− and Th1/Th17‐mixed IL‐10−/− mice. Interestingly, not only did colitic RAG‐2−/− mice that were parabiosed with WT mice show significant amelioration of colitis, but amelioration of disease was also observed in those parabiosed with colitic IL‐10−/− mice. To assess the interference between Th1 and Th17 colitogenic T cells, we co‐transferred colitogenic CD4+ T cells from the lamina propria (LP) of CD4+CD45RBhigh T cell‐transferred RAG‐2−/− mice and IL‐10−/− mice into RAG‐2−/− mice. Surprisingly, the co‐transferred RAG‐2−/− mice showed a vast cellular infiltration of LP CD4+ T cells similar to that seen in RAG‐2−/− mice re‐transferred with the cells from colitic RAG‐2−/− mice alone, but the co‐transferred RAG‐2−/− mice did not have the wasting symptoms, which are also absent in RAG‐2−/− mice transferred with cells from colitic IL‐10−/− mice alone. Furthermore, the percentages of Th1 and Th17 cells originating from IL‐10−/− mice and those of Th1 cells originating from colitic RAG‐2−/− mice were all significantly decreased in the co‐transferred mice as compared with the singly‐transferred paired RAG‐2−/− mice, suggesting that Th1 and Th17 cells are in competition, and that their orchestration results in a merged clinical phenotype of the two types of murine colitis.

A breakthrough in probiotics: Clostridium butyricum regulates gut homeostasis and anti-inflammatory response in inflammatory bowel disease

Intestinal immune homeostasis is regulated by gut microbiota, including beneficial and pathogenic microorganisms. Imbalance in gut bacterial constituents provokes host proinflammatory responses causing diseases such as inflammatory bowel disease (IBD). The development of next-generation sequencing technology allows the identification of microbiota alterations in IBD. Several studies have shown reduced diversity in the gut microbiota of patients with IBD. Advances in gnotobiotic technology have made possible analysis of the role of specific bacterial strains in immune cells in the intestine. Using these techniques, we have shown that Clostridium butyricum as a probiotic induces interleukin-10-producing macrophages in inflamed mucosa via the Toll-like receptor 2/myeloid differentiation primary response gene 88 pathway to prevent acute experimental colitis. In this review, we focus on the new approaches for the role of specific bacterial strains in immunological responses, as well as the potential of bacterial therapy for IBD treatments.

T-helper 17 and interleukin-17-producing lymphoid tissue inducer-like cells make different contributions to colitis in mice.

BACKGROUND & AIMS T helper (Th) 17 cells that express the retinoid-related orphan receptor (ROR) γt contribute to the development of colitis in mice, yet are found in normal and inflamed intestine. We investigated their development and functions in intestines of mice. METHODS We analyzed intestinal Th17 cells in healthy and inflamed intestinal tissues of mice. We analyzed expression of lymphotoxin (LT)α by Th17 cells and lymphoid tissue inducer-like cells. RESULTS LTα(-/-) and RORγt(-/-) mice had significantly lower percentages of naturally occurring Th17 cells in the small intestine than wild-type mice. Numbers of CD3(-)CD4(+/-)interleukin-7Rα(+)c-kit(+)CCR6(+)NKp46(-) lymphoid tissue inducer-like cells that produce interleukin-17A were increased in LTα(-/-) and LTα(-/-) × recombination activating gene (RAG)-2(-/-) mice, compared with wild-type mice, but were absent from RORγt(-/-) mice. Parabiosis of wild-type and LTα(-/-) mice and bone marrow transplant experiments revealed that LTα-dependent gut-associated lymphoid tissue structures are required for generation of naturally occurring Th17 cells. However, when wild-type or LTα(-/-) CD4(+)CD45RB(high) T cells were transferred to RAG-2(-/-) or LTα(-/-)×RAG-2(-/-) mice, all groups, irrespective of the presence or absence of LTα on the donor or recipient cells, developed colitis and generated Th1, Th17, and Th17/Th1 cells. RAG-2(-/-) mice that received a second round of transplantation, with colitogenic but not naturally occurring Th17 cells, developed intestinal inflammation. The presence of naturally occurring Th17 cells in the colons of mice inhibited development of colitis after transfer of CD4(+)CD45RB(high) T cells and increased the numbers of Foxp3(+) cells derived from CD4(+)CD45RB(high) T cells. CONCLUSIONS Gut-associated lymphoid tissue structures are required to generate naturally occurring Th17 cells that have regulatory activities in normal intestines of mice, but not for colitogenic Th17 and Th17/Th1 cells during inflammation.