Increased production of retinoic acid by intestinal macrophages contributes to their inflammatory phenotype in patients with Crohn's disease.

BACKGROUND & AIMS: Reduced generation of all-trans retinoic acid (RA) by CD103(+) intestinal dendritic cells (DCs) is linked to intestinal inflammation in mice. However, the role of RA in intestinal inflammation in humans is unclear. We investigated which antigen-presenting cells (APCs) produce RA in the human intestine and whether generation of RA is reduced in patients with Crohn's disease (CD). METHODS: Ileal and colonic tissues were collected from patients with CD during endoscopy or surgery, and healthy tissues were collected from subjects who were undergoing follow-up because of rectal bleeding, altered bowel habits, or cancer (controls). Cells were isolated from the tissue samples, and APCs were isolated by flow cytometry. Retinaldehyde dehydrogenase (RALDH) activity was assessed by Aldefluor assay, and ALDH1A expression was measured by quantitative real-time polymerase chain reaction. Macrophages were derived by incubation of human blood monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF). RESULTS: CD103(+) and CD103(-) DCs and CD14(+) macrophages from healthy human intestine had RALDH activity. Although ALDH1A1 was not expressed by DCs, it was the predominant RALDH enzyme isoform expressed by intestinal CD14(+) macrophages and their putative precursors, CD14(+) monocytes. RALDH activity was up-regulated in all 3 populations of APCs from patients with CD; in CD14(+) macrophages, it was associated with local induction of ALDH1A1 expression. Blocking of RA receptor signaling during GM-CSF-mediated differentiation of monocytes into macrophages down-regulated CD14 and HLA-DR expression and reduced the development of tumor necrosis factor α-producing inflammatory macrophages. CONCLUSIONS: RA receptor signaling promotes differentiation of human tumor necrosis factor α-producing inflammatory macrophages in vitro. In vivo, more CD14(+) macrophages from the intestinal mucosa of patients with CD than from controls are capable of generating RA, which might increase the inflammatory phenotype of these cells. Strategies to reduce the generation of RA by CD14(+) macrophages could provide new therapeutic options for patients with CD.

A CD3-specific antibody reduces cytokine production and alters phosphoprotein profiles in intestinal tissues from patients with inflammatory bowel disease.

BACKGROUND & AIMS: T cells mediate the development of inflammation in inflammatory bowel disease (IBD). We investigated the effects of an antibody against CD3 called otelixizumab, which induces immune tolerance, in intestinal mucosa samples from patients. METHODS: Intestinal tissues were isolated from patients undergoing routine endoscopy or from patients undergoing intestinal surgery for colon cancer or IBD; healthy surrounding tissues were collected as controls. Isolated lamina propria mononuclear cells (LPMCs) and mucosal tissue explants were incubated with otelixizumab for 24 or 48 hours. Production of inflammatory cytokines was determined by enzyme-linked immunosorbent assay. Levels of 36 cytokines and chemokines and phosphorylation of 39 receptor tyrosine kinases and signaling molecules were measured using protein arrays. Immunoblot analysis was used to analyze T-cell transcription factors. RESULTS: Incubation of intestinal tissues or LPMCs with otelixizumab reduced production of interferon gamma, interleukin (IL)-17A, and other inflammatory cytokines and chemokines, simultaneously increasing production of IL-10. Mucosal biopsy specimens from patients with IBD retained inflammation-associated tyrosine phosphoprotein profiles ex vivo. Incubation of the inflamed tissue with otelixizumab reduced phosphorylation of these proteins to levels observed in control tissues. Otelixizumab also markedly reduced phosphorylation of proteins associated with T-cell receptor activation. Neutralization of IL-10 blocked the anti-inflammatory effects of otelixizumab. CONCLUSIONS: We observed anti-inflammatory effects of anti-CD3 in inflamed intestinal tissues from patients with IBD. The antibody appears to down-regulate T-cell activation via IL-10.

Proinflammatory Vδ2+ T cells populate the human intestinal mucosa and enhance IFN-γ production by colonic αß T cells.

In nonhuman primates, Vγ9Vδ2(+) (Vδ2)T cells proliferate and accumulate in mucosal tissues following microbial activation. Human Vδ2T cells produce proinflammatory cytokines in response to bacterial species that colonize the gut, but the role played by Vδ2T cells in intestinal immunity is unknown. We hypothesized that circulating Vδ2T cells can populate the human intestine and contribute to mucosal inflammation. Cell suspensions prepared from peripheral blood and intestinal biopsies were stimulated with microbial phosphoantigen (1-hydroxy-2-methyl-2-buten-4-yl 4-diphosphate [HDMAPP]) and analyzed by flow cytometry to determine Vδ2T cell phenotype, cytokine production, and proliferative potential. Circulating Vδ2T cells expressed gut-homing integrin α4ß7 (>70%), which was coexpressed with skin-associated cutaneous leukocyte Ag by up to 15% of the total population. However, Vδ2T cell activation with HDMAPP and exposure to retinoic acid (signaling via retinoic acid receptor α) increased α4ß7 expression and enhanced binding to mucosal addressin cell adhesion molecule-1 in vitro while simultaneously suppressing cutaneous leukocyte Ag, thereby generating a committed gut-tropic phenotype. Confocal microscopy and flow cytometry identified frequent Vδ2T cells that migrated out of human intestinal biopsies and comprised both CD103(+) and CD103(-) subsets that produced TNF-α and IFN-γ upon phosphoantigen exposure, with more frequent cytokine-producing cells in the CD103(-) population. Activated intestinal Vδ2T cells expressed CD70 and HLA-DR but were unable to drive the proliferation of allogeneic naive CD4(+) T cells. Instead, phosphoantigen-activated CD103(-) Vδ2T cells increased T-bet expression and enhanced IFN-γ production by autologous colonic αß T cells via an IFN-γ-dependent mechanism. These data demonstrate that circulating Vδ2T cells display enhanced gut-homing potential upon microbial activation and populate the human intestinal mucosa, generating functionally distinct CD103(+) and CD103(-) subsets that can promote inflammation by colonic αß T cells.

Intestinal Mononuclear Phagocytes in Health and Disease.

The intestine is the tissue of the body with the highest constitutive exposure to foreign antigen and is also a common entry portal for many local and systemic pathogens. Therefore, the local immune system has the unenviable task of balancing efficient responses to dangerous pathogens with tolerance toward beneficial microbiota and food antigens. As in most tissues, the decision between tolerance and immunity is critically governed by the activity of local myeloid cells. However, the unique challenges posed by the intestinal environment have necessitated the development of several specialized mononuclear phagocyte populations with distinct phenotypic and functional characteristics that have vital roles in maintaining barrier function and immune homeostasis in the intestine. Intestinal mononuclear phagocyte populations, comprising dendritic cells and macrophages, are crucial for raising appropriate active immune responses against ingested pathogens. Recent technical advances, including microsurgical approaches allowing collection of cells migrating in intestinal lymph, intravital microscopy, and novel gene-targeting approaches, have led to clearer distinctions between mononuclear phagocyte populations in intestinal tissue. In this review, we present an overview of the various subpopulations of intestinal mononuclear phagocytes and discuss their phenotypic and functional characteristics. We also outline their roles in host protection from infection and their regulatory functions in maintaining immune tolerance toward beneficial intestinal antigens.

Azathioprine therapy selectively ablates human Vδ2⁺ T cells in Crohn's disease.

Tumor-derived and bacterial phosphoantigens are recognized by unconventional lymphocytes that express a Vγ9Vδ2 T cell receptor (Vδ2 T cells) and mediate host protection against microbial infections and malignancies. Vδ2 T cells are absent in rodents but readily populate the human intestine, where their function is largely unknown. Here, we assessed Vδ2 T cell phenotype and function by flow cytometry in blood and intestinal tissue from Crohn's disease patients (CD patients) and healthy controls. Blood from CD patients included an increased percentage of gut-tropic integrin ß7-expressing Vδ2 T cells, while "Th1-committed" CD27-expressing Vδ2 T cells were selectively depleted. A corresponding population of CD27+ Vδ2 T cells was present in mucosal biopsies from CD patients and produced elevated levels of TNFα compared with controls. In colonic mucosa from CD patients, Vδ2 T cell production of TNFα was reduced by pharmacological blockade of retinoic acid receptor-α (RARα) signaling, indicating that dietary vitamin metabolites can influence Vδ2 T cell function in inflamed intestine. Vδ2 T cells were ablated in blood and tissue from CD patients receiving azathioprine (AZA) therapy, and posttreatment Vδ2 T cell recovery correlated with time since drug withdrawal and inversely correlated with patient age. These results indicate that human Vδ2 T cells exert proinflammatory effects in CD that are modified by dietary vitamin metabolites and ablated by AZA therapy, which may help resolve intestinal inflammation but could increase malignancy risk by impairing systemic tumor surveillance.

A role for gut-associated lymphoid tissue in shaping the human B cell repertoire.

We have tracked the fate of immature human B cells at a critical stage in their development when the mature B cell repertoire is shaped. We show that a major subset of bone marrow emigrant immature human B cells, the transitional 2 (T2) B cells, homes to gut-associated lymphoid tissue (GALT) and that most T2 B cells isolated from human GALT are activated. Activation in GALT is a previously unknown potential fate for immature human B cells. The process of maturation from immature transitional B cell through to mature naive B cell includes the removal of autoreactive cells from the developing repertoire, a process which is known to fail in systemic lupus erythematosus (SLE). We observe that immature B cells in SLE are poorly equipped to access the gut and that gut immune compartments are depleted in SLE. Thus, activation of immature B cells in GALT may function as a checkpoint that protects against autoimmunity. In healthy individuals, this pathway may be involved in generating the vast population of IgA plasma cells and also the enigmatic marginal zone B cell subset that is poorly understood in humans.