Richard Ahrens

IL-9– and mast cell–mediated intestinal permeability predisposes to oral antigen hypersensitivity

Previous mouse and clinical studies demonstrate a link between Th2 intestinal inflammation and induction of the effector phase of food allergy. However, the mechanism by which sensitization and mast cell responses occurs is largely unknown. We demonstrate that interleukin (IL)-9 has an important role in this process. IL-9–deficient mice fail to develop experimental oral antigen–induced intestinal anaphylaxis, and intestinal IL-9 overexpression induces an intestinal anaphylaxis phenotype (intestinal mastocytosis, intestinal permeability, and intravascular leakage). In addition, intestinal IL-9 overexpression predisposes to oral antigen sensitization, which requires mast cells and increased intestinal permeability. These observations demonstrate a central role for IL-9 and mast cells in experimental intestinal permeability in oral antigen sensitization and suggest that IL-9–mediated mast cell responses have an important role in food allergy.

Mast cells regulate homeostatic intestinal epithelial migration and barrier function by a chymase/Mcpt4-dependent mechanism.

Altered intestinal barrier function is postulated to be a central predisposing factor to intestinal diseases, including inflammatory bowel diseases and food allergies. However, the mechanisms involved in maintaining homeostatic intestinal barrier integrity remain undefined. In this study, we demonstrate that mice deficient in mast cells (KitW-sh/W-sh [Wsh]) or mast cell chymase (Mcpt4−/−) have significantly decreased basal small intestinal permeability compared with wild-type (WT) mice. Altered intestinal barrier function was linked to decreased intestinal epithelial cell migration along the villus/crypt axis, altered intestinal morphology, and dysregulated claudin-3 crypt expression. Remarkably, engraftment of Wsh mice with WT but not Mcpt4−/− mast cells restored intestinal epithelial cell migration, morphology, and intestinal epithelial barrier function. Collectively, these findings identify a mechanism by which mast cells regulate homeostatic intestinal epithelial migration and barrier function.

Intestinal Macrophage/Epithelial Cell-Derived CCL11/Eotaxin-1 Mediates Eosinophil Recruitment and Function in Pediatric Ulcerative Colitis

Clinical studies have demonstrated a link between the eosinophil-selective chemokines, eotaxins (eotaxin-1/CCL11 and eotaxin-2/CCL24), eosinophils, and the inflammatory bowel diseases, Crohn’s disease and ulcerative colitis (UC). However, the cellular source and individual contribution of the eotaxins to colonic eosinophilic accumulation in inflammatory bowel diseases remain unclear. In this study we demonstrate, by gene array and quantitative PCR, elevated levels of eotaxin-1 mRNA in the rectosigmoid colon of pediatric UC patients. We show that elevated levels of eotaxin-1 mRNA positively correlated with rectosigmoid eosinophil numbers. Further, colonic eosinophils appeared to be degranulating, and the levels positively correlated with disease severity. Using the dextran sodium sulfate (DSS)-induced intestinal epithelial injury model, we show that DSS treatment of mice strongly induced colonic eotaxin-1 and eotaxin-2 expression and eosinophil levels. Analysis of eosinophil-deficient mice defined an effector role for eosinophils in disease pathology. DSS treatment of eotaxin-2−/− and eotaxin-1/2−/− mice demonstrated that eosinophil recruitment was dependent on eotaxin-1. In situ and immunofluorescence analysis-identified eotaxin-1 expression was restricted to intestinal F4/80+CD11b+ macrophages in DSS-induced epithelial injury and to CD68+ intestinal macrophages and the basolateral compartment of intestinal epithelial cells in pediatric UC. These data demonstrate that intestinal macrophage and epithelial cell-derived eotaxin-1 plays a critical role in the regulation of eosinophil recruitment in colonic eosinophilic disease such as pediatric UC and provides a basis for targeting the eosinophil/eotaxin-1 axis in UC.

Differential roles for the IL-9/IL-9 receptor α-chain pathway in systemic and oral antigen-induced anaphylaxis

BACKGROUND The cytokine IL-9 has been implicated in allergic reactions, including food allergy, but its contribution to parenteral versus oral antigen-induced anaphylaxis remains unclear. OBJECTIVES We sought to delineate the contribution of the IL-9/IL-9 receptor alpha-chain (IL-9R) pathway to parenteral and oral antigen-induced anaphylaxis. METHODS Wild-type, IL-9-deficient (Il9(-/-)), and IL-9R-deficient (Il9R(-/-)) mice were subjected to passive and active parenteral and oral antigen (ovalbumin [OVA])-induced anaphylaxis. Severity of systemic anaphylaxis was gauged by decreased body temperature; intestinal anaphylaxis was assessed based on secretory diarrhea, intestinal mastocytosis, and serum murine mast cell protease 1 level. Specific immunoglobulin isotypes or immunoglobulin receptor-blocking antibodies were administered before challenge to define the role of the IgE and IgG pathways. RESULTS Repeated oral antigen challenge of OVA-sensitized wild-type mice induced anaphylaxis with both systemic and intestinal involvement; both were IgE dependent and attenuated in Il9(-/-) and Il9R(-/-) mice. In contrast, parenteral OVA challenge of OVA-sensitized wild-type mice induced systemic anaphylaxis, which was independent of the IL-9/IL-9R pathway. Strikingly, the IL-9/IL-9R pathway had no role in either the IgG or IgE component of parenteral antigen-induced or anti-IgE and anti-FcgammaRII/III mAb-induced systemic anaphylaxis. CONCLUSIONS Parenteral antigen-induced murine systemic anaphylaxis is mediated by both IgG- and IgE-dependent pathways, and both can occur independently of IL-9/IL-9R signaling. In contrast, oral antigen-induced intestinal and systemic anaphylaxis is strictly IgE mediated and requires IL-9/IL-9R signaling. These studies indicate differential involvement of the IL-9/IL-9R pathway in systemic and oral antigen-induced anaphylaxis.

Colonic Eosinophilic Inflammation in Experimental Colitis Is Mediated by Ly6Chigh CCR2+ Inflammatory Monocyte/Macrophage-Derived CCL11

Recent genome-wide association studies of pediatric inflammatory bowel disease have implicated the 17q12 loci, which contains the eosinophil-specific chemokine gene CCL11, with early-onset inflammatory bowel disease susceptibility. In the current study, we employed a murine model of experimental colitis to define the molecular pathways that regulate CCL11 expression in the chronic intestinal inflammation and pathophysiology of experimental colitis. Bone marrow chimera experiments showed that hematopoietic cell-derived CCL11 is sufficient for CCL11-mediated colonic eosinophilic inflammation. We show that dextran sodium sulfate (DSS) treatment promotes the recruitment of F4/80+CD11b+CCR2+Ly6Chigh inflammatory monocytes into the colon. F4/80+CD11b+CCR2+Ly6Chigh monocytes express CCL11, and their recruitment positively correlated with colonic eosinophilic inflammation. Phenotypic analysis of purified Ly6Chigh intestinal inflammatory macrophages revealed that these cells express both M1- and M2-associated genes, including Il6, Ccl4, Cxcl2, Arg1, Chi3l3, Ccl11, and Il10, respectively. Attenuation of DSS-induced F4/80+CD11b+CCR2+Ly6Chigh monocyte recruitment to the colon in CCR2−/− mice was associated with decreased colonic CCL11 expression, eosinophilic inflammation, and DSS-induced histopathology. These studies identify a mechanism for DSS-induced colonic eosinophilia mediated by Ly6ChighCCR2+ inflammatory monocyte/macrophage-derived CCL11.

Resistin-like molecule α enhances myeloid cell activation and promotes colitis

BACKGROUND Resistin-like molecule (Relm) alpha is a secreted protein and a hallmark signature gene for alternatively activated macrophages. Relm-alpha is highly induced by allergic inflammatory triggers and perceived to promote tissue repair. Yet the function of Relm-alpha remains unknown. OBJECTIVE We sough to determine the role of Relm-alpha in dextran sodium sulfate (DSS)-induced colonic injury. METHODS The cellular source of Relm-alpha was determined after oral DSS-induced colitis. Retnla(-/-) mice were generated, subjected to DSS treatment, and monitored for disease progression (clinical and histopathologic features). Cytokine production in the supernatants of ex vivo colon cultures, and of LPS-stimulated macrophages incubated with Relm-alpha was assessed. Relm-alpha was administered intraperitoneally, and the cellular recruitment to the peritoneum was assessed. RESULTS After innate intestinal stimulation with DSS, Relm-alpha was highly expressed by eosinophils and epithelial cells. Retnla gene-targeted mice were protected from DSS-induced colitis (eg, decreased diarrhea, rectal bleeding, colon shortening, disease score, and histopathologic changes). Relm-alpha coactivated IL-6 and TNF-alpha release and inhibited IL-10 release from LPS-activated bone marrow-derived macrophages. Consistent with these finding, colon cultures of DSS-treated Retnla(-/-) mice produced decreased IL-6 and increased IL-10 ex vivo. Furthermore, Retnla(-/-) mice had substantially decreased c-Jun N-terminal kinase phosphorylation in vivo. In vivo administration of Relm-alpha initiated cellular recruitment to the peritoneum, and Relm-alpha was able to induce eosinophil chemotaxis in vitro. CONCLUSIONS These findings demonstrate a central proinflammatory role for Relm-alpha in colonic innate immune responses, identifying a novel pathway for regulation of macrophage activation.

ICAM‐1‐dependent pathways regulate colonic eosinophilic inflammation

Eosinophilic inflammation is a common feature of numerous eosinophil‐associated gastrointestinal (EGID) diseases. Central to eosinophil migration into the gastrointestinal tract are the integrin‐mediated interactions with adhesion molecules. Although the mechanisms regulating eosinophil homing into the small intestine have begun to be elucidated, the adhesion pathways responsible for eosinophil trafficking into the large intestine are unknown. We investigated the role of adhesion pathways in eosinophil recruitment into the large intestine during homeostasis and disease. First, using a hapten‐induced colonic injury model, we demonstrate that in contrast to the small intestine, eosinophil recruitment into the colon is regulated by a β7‐integrin addressin cell adhesion molecule‐1‐independent pathway. Characterization of integrin expression on colonic eosinophils by flow cytometry analysis revealed that colonic CC chemokine receptor 3+ eosinophils express the intercellular adhesion molecule‐1 (ICAM‐1) counter‐receptor integrins αL, αM, and β2. Using ICAM‐1‐deficient mice and anti‐ICAM‐1 neutralizing antibodies, we show that hapten‐induced colonic eosinophilic inflammation is critically dependent on ICAM‐1. These studies demonstrate that β2‐integrin/ICAM‐1‐dependent pathways are integral to eosinophil recruitment into the colon during GI inflammation associated with colonic injury.

Intestinal Mast Cell Levels Control Severity of Oral Antigen-Induced Anaphylaxis in Mice

Food-triggered anaphylaxis can encompass a variety of symptoms that affect multiple organ systems and can be life threatening. The molecular distinction between non-life-threatening and life-threatening modes of such anaphylaxis has not yet been delineated. In this study, we sought to identify the specific immune functions that regulate the severity of oral antigen-induced anaphylaxis. We thus developed an experimental mouse model in which repeated oral challenge of ovalbumin-primed mice induced an FcεRI- and IgE-dependent oral antigen-triggered anaphylaxis that involved multiple organ systems. Strikingly, the severity of the systemic symptoms of anaphylaxis (eg, hypothermia) positively correlated with the levels of intestinal mast cells (r = -0.53; P < 0.009). In addition, transgenic mice with both increased intestinal and normal systemic levels of mast cells showed increased severity of both intestinal and extra-intestinal symptoms of IgE-mediated passive as well as oral antigen- and IgE-triggered anaphylaxis. In conclusion, these observations indicate that the density of intestinal mast cells controls the severity of oral antigen-induced anaphylaxis. Thus, an awareness of intestinal mast cell levels in patients with food allergies may aid in determining their susceptibility to life-threatening anaphylaxis and may eventually aid in the treatment of food-triggered anaphylaxis.

Chymase-mediated intestinal epithelial permeability is regulated by a protease-activating receptor/matrix metalloproteinase-2-dependent mechanism.

Mast cells regulate intestinal barrier function during disease and homeostasis. Secretion of the mast cell-specific serine protease chymase regulates homeostasis. In the present study, we employ in vitro model systems to delineate the molecular pathways involved in chymase-mediated intestinal epithelial barrier dysfunction. Chymase stimulation of intestinal epithelial (Caco-2 BBe) cell monolayers induced a significant reduction in transepithelial resistance, indicating decreased intestinal epithelial barrier function. The chymase-induced intestinal epithelial barrier dysfunction was characterized by chymase-induced protease-activated receptor (PAR)-2 activation and matrix metalloproteinase (MMP)-2 expression and activation. Consistent with this observation, in vitro analysis revealed chymase-induced PAR-2 activation and increased MAPK activity and MMP-2 expression. Pharmacological and small interfering RNA-mediated antagonism of PAR-2 and MMP-2 significantly attenuated chymase-stimulated barrier dysfunction. Additionally, the chymase/MMP-2-mediated intestinal epithelial dysfunction was associated with a significant reduction in the tight junction protein claudin-5, which was partially restored by MMP-2 inhibition. Finally, incubation of Caco-2 BBe cells with chymase-sufficient, but not chymase-deficient, bone marrow-derived mast cells decreased barrier function, which was attenuated by the chymase inhibitor chymostatin. Collectively, these results suggest that mast cell/chymase-mediated intestinal epithelial barrier function is mediated by PAR-2/MMP-2-dependent pathways.

Paired Immunoglobulin-Like Receptor B (PIR-B) Negatively Regulates Macrophage Activation in Experimental Colitis

BACKGROUND & AIMS Innate and adaptive immune responses are regulated by cross talk between activation and inhibitory signals. Dysregulation of the inhibitory signal can lead to aberrant chronic inflammatory diseases such as the inflammatory bowel diseases (IBD). Little is known about negative regulation of innate intestinal immune activation. We examined the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of macrophage function in innate intestinal immunity. METHODS We examined the susceptibility of Pirb-/- and wild-type (WT) mice to dextran sodium sulfate (DSS)-induced colitis. We assessed proinflammatory cytokine release and mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB) activation in Pirb-/- and WT macrophages following Escherichia coli stimulation. Macrophage transfer experiments were performed to define the role of PIR-B in the negative regulation of macrophage function in DSS-induced colitis. We also assessed expression of PIR-B human homologues (immunoglobulin-like transcript [ILT]-2 and ILT-3) in colon biopsy samples from healthy individuals (controls) and patients with IBD. RESULTS Pirb-/- mice had increased susceptibility to DSS-induced colitis. In vitro analysis showed increased production of proinflammatory cytokines (interleukin-6, interleukin-1beta, and tumor necrosis factor alpha) and activation of MAPK and NF-kappaB in Pirb-/- macrophages following bacterial activation. Adoptive transfer of bone marrow-derived Pirb-/- macrophages into WT mice was sufficient to increase disease susceptibility. ILT-2 and ILT-3 were expressed on CD68+ and CD68- mononuclear cells and intestinal epithelium in colon biopsy samples from patients and controls. CONCLUSIONS PIR-B negatively regulates macrophage functions in response to pathogenic bacteria and chronic intestinal inflammatory responses. Inhibitory receptors such as PIR-B might be used as therapeutic targets for treatment of patients with IBD.