Theodore J. Sanders

Use of Methylation Patterns to Determine Expansion of Stem Cell Clones in Human Colon Tissue

BACKGROUND & AIMS It is a challenge to determine the dynamics of stem cells within human epithelial tissues such as colonic crypts. By tracking methylation patterns of nonexpressed genes, we have been able to determine how rapidly individual stem cells became dominant within a human colonic crypt. We also analyzed methylation patterns to study clonal expansion of entire crypts via crypt fission. METHODS Colonic mucosa was obtained from 9 patients who received surgery for colorectal cancer. The methylation patterns of Cardiac-specific homeobox, Myoblast determination protein 1, and Biglycan were examined within clonal cell populations, comprising either part of, or multiple adjacent, normal human colonic crypts. Clonality was demonstrated by following cytochrome c oxidase-deficient (CCO⁻) cells that shared an identical somatic point mutation in mitochondrial DNA. RESULTS Methylation pattern diversity among CCO⁻ clones that occupied only part of a crypt was proportional to clone size; this allowed us to determine rates of clonal expansion. Analysis indicated a slow rate of niche succession within the crypt. The 2 arms of bifurcating crypts had distinct methylation patterns, indicating that fission can disrupt epigenetic records of crypt ancestry. Adjacent clonal CCO⁻ crypts usually had methylation patterns as dissimilar to one another as methylation patterns of 2 unrelated crypts. Mathematical models indicated that stem cell dynamics and epigenetic drift could account for observed dissimilarities in methylation patterns. CONCLUSIONS Methylation patterns can be analyzed to determine the rates of recent clonal expansion of stem cells, but determination of clonality over many decades is restricted by epigenetic drift. We developed a technique to follow changes in intestinal stem cell dynamics in human epithelial tissues that might be used to study premalignant disease.

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

Transitional 2 B cells home to gut-associated lymphoid tissue and present an activated phenotype in healthy subjects, but gut immune compartments are depleted in SLE.

Proinflammatory Vδ2+ T Cells Populate the Human Intestinal Mucosa and Enhance IFN-γ Production by Colonic αβ 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 α4β7 (>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 α4β7 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 αβ 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 αβ T cells.

Regulation of human intestinal T-cell responses by type 1 interferon-STAT1 signaling is disrupted in inflammatory bowel disease

Type 1 interferon (IFN-1) promotes regulatory T-cell function to suppress inflammation in the mouse intestine, but little is known about IFN-1 in the human gut. We therefore assessed the influence of IFN-1 on CD4+ T-cells isolated from human colon tissue obtained from healthy controls or patients with inflammatory bowel disease (IBD). Immunofluorescent imaging revealed constitutive expression of IFNβ in human intestinal tissue, and colonic T-cells were responsive to exogenous IFN-1 as assessed by phosphorylation of signal transduction and activator of transcription 1 (pSTAT1) and induction of interferon stimulated genes (ISGs). Unlike their blood counterparts, intestinal T-cells from non-inflamed regions of IBD colon displayed enhanced responsiveness to IFN-1, increased frequency of pSTAT1+ cells, and greater induction of ISGs upon IFN-1 exposure in vitro. In healthy tissue, antibody neutralization of IFNβ selectively reduced T-cell production of the pro-regulatory cytokine interleukin-10 (IL-10) and increased IFNγ synthesis. In contrast, neutralization of IFNβ in IBD tissue cultures increased the frequency of T-cells producing inflammatory cytokines but did not alter IL-10 expression. These data support a role for endogenous IFN-1 as a context-dependent modulator of T-cell function that promotes regulatory activity in healthy human intestine, but indicate that the IFN-1/STAT1 pathway is dysregulated in inflammatory bowel disease.

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.

OC-009 Human anti-microbial vδ2+ t-cells are novel intestinal lymphocytes with functional relevance in crohn's disease

Introduction Vγ9Vδ2+ “unconventional” (Vδ2) T-cells are a population of circulating anti-microbial lymphocytes found only in higher primates and whose role in human intestinal immunity is unknown. In macaques, microbe-activated Vδ2T-cells expand and accumulate in mucosal tissues, and human Vδ2T-cells can produce key mediators of intestinal inflammation such as IFNγ, TNFα and IL-17A in response to bacterial species present among the gut microbiota. We therefore hypothesised that Vδ2T-cells might contribute to the pathogenesis of Crohns disease (CD). Methods Disaggregated intestinal biopsies and peripheral blood were analysed by flow-cytometry in CD patients (n=22), and healthy controls (n=36). Blood and biopsy-derived cell suspensions were stimulated with microbial phosphoantigen (HDMAPP) and IL-2 in vitro to determine Vδ2T-cell phenotype, cytokine production and proliferative potential in the presence or absence of azathioprine. Results Blood Vδ2T-cells proliferated, expressed “gut-homing” integrin β7, and produced IFNγ and TNFα upon activation with HDMAPP and IL-2 in vitro. Vδ2T-cells were also identified by confocal microscopy in both healthy and inflamed colonic lamina propria. In contrast to their blood counterparts, mucosal Vδ2T-cells expressed high levels of CD103 integrin, which is implicated in interactions with the intestinal epithelium. Although the frequency of mucosal Vδ2T-cells was low, these cells proliferated rapidly and up-regulated CD70 co-stimulatory molecule upon exposure to HDMAPP and IL-2 in vitro, consistent with responsiveness to the gut microbiota. In CD patients receiving azathioprine therapy, Vδ2T-cells were selectively lost from the blood and were markedly depleted from the lamina propria. Accordingly, physiological concentrations of azathioprine were sufficient to block HDMAPP activation of Vδ2T-cells in vitro. Conclusion Human Vδ2T-cells primarily reside in the blood but display gut-homing potential upon microbial activation and can be detected in the intestinal mucosa. Intestinal Vδ2T-cells may contribute to local pro-inflammatory responses against the gut microbiota but are depleted by azathioprine therapy in CD. Competing interests None declared.

Hydrogen Sulfide Reduces Myeloid-Derived Suppressor Cell-Mediated Inflammatory Response in a Model of Helicobacter hepaticus-Induced Colitis

Chronic inflammation contributes to tumor initiation in colitis-associated colorectal cancer (CRC). Indeed, inflammatory bowel disease (IBD) patients show an increased risk of developing CRC. Cancer immune evasion is a major issue in CRC and preclinical and clinical evidence has defined a critical role for myeloid-derived suppressor cells (MDSCs) that contribute to tumor growth and progression by suppressing T-cells and modulating innate immune responses. MDSCs comprise a heterogeneous population of immature myeloid cells that can be distinct in two subtypes: CD11b+Ly6G+Ly6Clow with granulocytic phenotype (G-MDSCs) and CD11b+Ly6G−Ly6Chigh with monocytic phenotype (M-MDSCs). Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule that regulates various physiological and pathophysiological functions. In particular, several studies support its anti-inflammatory activity in experimental colitis and ulcer. However, the role of the H2S pathway in innate immune-mediated IBD has not yet been elucidated. To better define a possible link between MDSCs and H2S pathway in colitis-associated CRC development, we used an innate immune-mediated IBD model induced by infection with the bacterium Helicobacter hepaticus (Hh), closely resembling human IBD. Here, we demonstrated an involvement of MDSCs in colitis development. A significant time-dependent increase of both G-MDSCs and M-MDSCs was observed in the colon and in the spleen of Hh-infected mice. Following, we observed that chronic oral administration of the H2S donor DATS reduced colon inflammation by limiting the recruitment of G-MDSCs in the colon of Hh-infected mice. Thus, we identify the metabolic pathway l-cysteine/H2S as a possible new player in the immunosuppressive mechanism responsible for the MDSCs-promoted colitis-associated cancer development.

Sa1778 Constitutive Type I Interferon, via STAT1 Activation, Selectively Promotes Regulatory T Cell Function in the Healthy Human Intestine, but Not in Inflammatory Bowel Disease

WT Balb/C mice (p<0.001 for each cytokine btw uninfected control and helminth-infected; N=at least 3 independent experiments), while no regulation of IFNγ and modest induction of IL4 or IL10 was seen in STAT6-/mice. Helminths did not induce TGFβ production in STAT6-/animals. In an acute GVHD and colitis model, helminths regulated WT donor T cell Th1 cytokine generation, serum Th1 cytokines, colitis and survival in WT and not STAT6-/recipients (p<0.05 for each parameter btw. helminth infected WT and helminthinfected STAT6-/recipients, multiple experiments). Helminth infection was associated with the induction of donor and recipient FoxP3+ regulatory T cells (Tregs) in WT and not STAT6-/bone marrow transplanted mice (p<0.05 btw. helminth infected WT and helminthinfected STAT6-/recipients, multiple experiments). Addition of TGFβ to STAT6-/T cell cultures restored T cell IL10 production and Treg generation that regulate Th1 inflammation. Conclusions: STAT6 is a critical transcription factor directing mucosal TGFβ producing Th3 cell generation. Helminths utilize STAT6 pathway to induce mucosal Tregs, trigger mucosal T cell IL10 secretion and regulate alloreactive colitis in a TGFβ dependent manner.

PWE-256 Intestinal inflammation regulates retinoic acid dependent imprinting of gut tropism by dendritic cells independently of RALDH expression

Introduction In the mouse, tissue-specific expression of retinaldehyde dehydrogenase (RALDH) enzymes by CD103+ intestinal dendritic cells (DC) enables them to generate all-trans retinoic acid (RA) and thereby imprint a gut-tropic phenotype on T cells via induction of homing receptors including α4β7 integrin. In health, RA from CD103+ also enhances their generation of Treg, contributing to intestinal homeostasis. In murine models of inflammatory bowel disease (IBD) RALDH expression by CD103+ DC is reduced but little is known about the function of RA in the human intestine. The aim of this study was to determine whether factors present in the healthy and inflamed human intestine regulate RA generation and activity. Methods Conditioned media (CM) were generated by culture of intestinal biopsies from healthy individuals and IBD patients (inflamed/non-inflamed regions). DC were differentiated from monocytes using GM-CSF and IL-4 in the presence or absence of CM. Expression of RA-generating enzymes was assessed by qRT-PCR and RALDH activity determined using the Aldefluor assay. Induction of α4β7 following activation of naïve allogeneic CD4+ T cells was determined by flow cytometry. Results Activation of naïve CD4+ T cells by human monocyte-derived DC resulted in RA-dependent upregulation of α4β7. These DC possessed retinal-inhibitable Aldefluor activity and expressed both alcohol dehydrogenase (RDH10) and RALDH (RALDH1,2,3) enzymes required for the generation of RA from retinol via retinal. Aldefluor activity was regulated by GM-CSF and RA, and reflected predominately the activity of RALDH2 as suggested by qRT-PCR analysis of sorted Aldefluor+ DC. CM significantly suppressed Aldefluor activity (p<0.0001) irrespective of whether generated from healthy or IBD tissue (inflamed or non-inflamed). The inhibitory effect of CM generated from healthy tissue could be partially reversed with the prostaglandin E2 (PGE2) EP-2 receptor antagonist AH6809 but this effect was less marked with CM from IBD tissue suggesting the involvement of distinct RALDH regulators. Although the effects of inflamed and non-inflamed CM on Aldefluor activity were similar, DC differentiated in the presence of inflamed CM induced significantly higher (p<0.05) levels of CD4 T cell α4β7 expression. Conclusion Factors generated in the human intestinal mucosa limit RALDH activity in DC and may thereby impact upon their generation of RA. Factors other than PGE2 are involved particularly in inflamed tissue. Intestinal mediators influence the imprinting of gut tropism independently of effects on RA-generating enzymes. Manipulation of RA availability may offer new therapeutic options in IBD. Competing interests None declared.