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We take a multidisciplinary approach to understand the immune-intestinal epithelial interactions during homeostasis and how alterations in these pathways predispose to the development and maintenance of chronic inflammatory diseases such as inflammatory bowel diseases (IBD), food-induced anaphylaxis and cystic fibrosis (CF).
Our projects employ in vitro and in vivo models and cell and epithelial biology, transport physiology and mucosal immunology to 1) define fundamentals of epithelial barrier function; 2) understand the role of the epithelial barrier in regulating other mucosal processes, e.g. immune responses; 3) identify immune pathways that modulate epithelial barrier function and how these pathways alter susceptibility and severity to disease; and 4) develop novel approaches to correct barrier dysfunction and restore health.
Inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC) affect more than 5 million people in North America and Europe with a 25 percent incidence rate of childhood onset. Pediatric UC presents as a more moderate to severe disease than adult UC, with delayed growth, impaired quality of life, high rate of corticosteroid (CS) dependency and frequent relapse requiring more aggressive therapy including anti-TNF therapy or surgery. Strikingly, 10 years following diagnosis, more than 40 percent of children undergo colectomy.
Incidence, family twin and phenotype concordance studies suggest that IBD is a complex genetic disease. Genome-wide association studies of pediatric and adult IBD have revealed a number of IBD risk susceptibility genes associated with innate (CARD15, ATG16L1 and IRGM) and adaptive (IL23R, IL10, IL12B and STAT-3) immunity; notably, a number of unique loci were identified to be associated with early-onset IBD susceptibility including 17q12 near the eosinophil-specific chemokines gene, CCL11.
Over the last six years, we have performed experimental- and clinical-based studies to assess the relative contribution of CCL11 and eosinophils to pediatric UC. Gene profiling on a cohort of pediatric UC patients at diagnosis identified significant upregulation of CCL11. Importantly, CCL11 levels correlated with tissue eosinophil numbers, which in turn correlated with the UC Histologic Index of Severity (UCHIS). Experimental evidence employing mice deficient in eosinophils, eosinophil peroxidase (EPO) and CCL11 revealed a critical role for eosinophils and EPO in the histopathology associated with DSS-induced colitis and that eosinophil recruitment is critically dependent on CCL11.
In pursuit of defining the cellular source of CCL11, we identified a positive correlation between recruited macrophages (MФ) (F4/80+, CD11b+, Ly6Chigh) and eosinophil numbers in DSS-colitis. Purification of recruited MФ identified this cell population to be a source of CCL11. Employing CCR2 deficient mice and performing bone marrow chimera experiments, we demonstrated a pivotal role for F4/80+, CD11b+, Ly6Chigh MФ’s in CCL11 expression colonic eosinophilic inflammation and DSS-induced histopathology. In support of these observations we identified CD68+ macrophages (MФ) as a potential source of CCL11 in pediatric UC.
In current investigations are focused on defining the stimuli and molecular signaling cascades involved in F4/80+, CD11b+, Ly6Chigh MФ-derived CCL11 expression in experimental colitis and pediatric UC.
Related Publications:Waddell A, Ahrens R, Steinbrecher K, et al. Colonic eosinophilic inflammation in experimental colitis is mediated by Ly6C(high) CCR2(+) inflammatory monocyte/macrophage-derived CCL11. J Immunol. 186:5993-6003. 2011.
Ahrens R, Waddell A, et al. Intestinal macrophage/epithelial cell-derived CCL11/eotaxin-1 mediates eosinophil recruitment and function in pediatric ulcerative colitis. J Immunol. 181(10): 7390-7399. 2008.
Forbes E, Hulett M, et al. ICAM-1-dependent pathways regulate colonic eosinophilic inflammation. J Leukoc Biol 80(2): 330-341. 2006.
Forbes E, Murase T, et al. Immunopathogenesis of experimental ulcerative colitis is mediated by eosinophil peroxidase. J Immunol. 172: 5664-5675. 2004.
Monocyte / macrophage (MØ) regulation of eosinophilic inflammation in IBD. Microbes cross the epithelium and stimulate CCR2-ligand expression, which leads to the recruitment of F4/80+ CD11b+ Ly6+ CCR2+ CX3CR1low monocytes / MØs. The monocyte / MØ upon entering the intestinal tissue are activated by unknown stimuli leading to expression of retlna, YM-1 and ARG-1 and also ccl11 (eotaxin-1). CCL11 promotes eosinophil recruitment into the colonic tissue and eosinophil degranulation and release of eosinophil peroxidase. Release of eosinophil peroxidase leads to further epithelial ulceration and damage and histopathology associated with IBD.
Triggering of the innate or adaptive immune system stimulates an activating signal leading to effective immunity. To maintain homeostasis, the vertebrate immune system possesses a counter-regulatory “inhibitory” pathway that constrains the “activating” immune signal.
The counter-regulatory pathway is mediated by immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors called immune inhibitory receptors. These receptors negatively regulate the activating signal via recruitment and activation of inhibitory signaling effector molecules; Src homology region 2 domain-containing phosphatase 1 (SHP-1) and 2 (SHP-2). Surprisingly, the importance and function of inhibitory receptors in the counter-regulation of intestinal inflammation and IBD have received limited attention.
We have recently focused on the myeloid-associated inhibitory receptor, paired immunoglobulin-like receptor B (PIR-B), in the negative-regulation of MΦ function and innate colonic inflammation. We have recently demonstrated that Pirb gene deletion causes exaggerated DSS-induced colonic injury that was critically dependent on the expression of PIR-B on macrophages. We show that direct activation of Pirb-/- macrophages by E. coli leads to exaggerated MAPK and NFkB activation as well as proinflammatory cytokine production. Finally, we demonstrate expression of PIR-B human homologues ILT-2 and ILT-3 in colonic biopsies of healthy controls and pediatric UC patients. Collectively, these studies emphasize a key role for PIR-B in the negative regulation of macrophage functions in innate intestinal immune reactions. We are currently assessing the molecular basis of PIR-B negative regulation on MΦ-derived proinflammatory cytokine production and the importance of this pathway in exacerbation of IBD.
Related Publication:Munitz A, Cole ET, et al. Paired immunoglobulin-like receptor B (PIR-B) negatively regulates macrophage activation in experimental colitis. Gastroenterology. 139: 530-541. 2010.
Figure 1. Paired immunoglobulin B (PIR-B) and immune activation. PIR-B is a tyrosine-based inhibitory motif (ITIM)-bearing receptor that negatively regulates the activating signal via recruitment and activation of inhibitory signaling effector molecules; Src homology region 2 domain-containing phosphatase 1 (SHP-1) and 2 (SHP-2). We have demonstrated that direct activation of Pirb macrophages by E. coli (TLR) leads to exaggerated MAPK and NFkB activation and proinflammatory cytokine production. We are currently assessing the molecular basis of PIR-B negative regulation on MΦ-derived proinflammatory cytokine production.
Severe food allergy-related reactions, termed food-triggered anaphylaxis, are serious, life threatening and responsible for 30,000-120,000 emergency department visits, 2,000-3,000 hospitalizations and approximately 150 deaths per year in the United States. Clinical studies indicate that food reactions account for 30-75 percent of anaphylactic cases in emergency departments in North America, Europe, Asia and Australia. The onset of symptoms from food-induced anaphylaxis is variable, occurring within seconds to a few hours following exposure to the casual food allergen, and often affects multiple organ systems including gastrointestinal (GI), cutaneous, respiratory and cardiovascular. Cutaneous symptoms (urticaria and angioedema) are the most common, occurring in approximately 80 percent of cases, whereas cardiovascular involvement occurs in 39 percent of food allergic reactions. Importantly, food anaphylactic patients do not generally present with a consistent constellation of symptoms, and thus determination of food-induced anaphylaxis susceptibility and severity cannot necessarily be predicted based on clinical history. Furthermore, there is currently no good method to distinguish between atopic individuals who do not have increased risk of anaphylaxis and atopic individuals who have increased risk of anaphylaxis and possible fatality. We have developed an experimental model of oral antigen-induced anaphylaxis in which oral antigen challenge of mice promotes an anaphylactic reaction characterized by multiorgan involvement (GI, cutaneous, respiratory and cardiovascular). We are utilizing this model to begin identifying influential effector functions (intestinal barrier function and cytokines IL-9 and IL-13) important in regulating predisposition and severity of food-induced anaphylaxis.
Figure 1. Immuno-epithelial interactions in food allergy and anaphylaxis. In nonallergic individuals, food antigens are sampled by tolerogenic antigen presenting cells (APCs) and presented to T-cells to promote a T regulatory response and oral tolerance. In food allergic individuals, APCs such as dendritic cells present food antigens to T-cells and promote a CD4+ Th2 response and production of Th2 cytokines such as IL-4, IL-5, IL-9 and IL-13. The Th2 cytokines prime the intestine (stimulates IgE synthesis, eosinophilia, intestinal epithelial secretory activity, intestinal mastocytosis and increase sensitivity of structural tissues to mast cell-derived mediators) for an anaphylactic response. We have previously demonstrated that mast cells and mast cell-derived chymase regulate steady state intestinal epithelial barrier function. Furthermore, we have shown that mast-cell dysregulation of intestinal permeability can increase food antigen-specific CD4+ Th2 responses and predisposes to food allergy. We are currently investigating the molecular pathways involved in mast cell / chymase-mediated dysregulation of intestinal epithelial barrier function in the predisposition to oral antigen sensitization and food allergy and severity of anaphylaxis.
Clinical investigations have demonstrated increased expression of IL-9 in biopsies from asthmatics, and increased expression of this cytokine correlated with disease severity. Furthermore, genetic mapping studies in both humans and mice showed linkage between the atopic phenotype and the IL-4 and IL-9 gene, identifying IL-9 as a candidate gene for asthma. These clinical investigations were supported by animal models demonstrating that IL-9 regulates the development of the allergic asthma phenotype including eosinophilic and lymphocytic inflammation, mucus accumulation, mast cell hyperplasia, subepithelial collagen deposition and airways hyper-responsiveness. The demonstration of an association between Th2-cytokines and the development of IgE-dependent systemic anaphylaxis would suggest an involvement for IL-9 in the IgE-dependent pathway. Indeed, employing a passive model of systemic anaphylaxis, investigators have revealed that constitutive expression of IL-9 in all tissues (cIL-9Tg mice) (murine pim-1 promoter containing an Em enhancer and Moloney murine leukemia virus long terminal repeat) increases susceptibility to IgE-mediated fatal systemic anaphylaxis. Given the potential importance of IL-9 in anaphylaxis, we have recently focused on the role of the Th2-cytokine interleukin (IL)-9 in oral antigen-induced allergic intestinal reactions. In preliminary analysis, employing IL-9 deficient mice, intestinal IL-9 transgenic mice and experimental models of intestinal anaphylaxis, we have demonstrated that IL-9 profoundly increases intestinal mast cells, intestinal permeability and predisposes to intestinal anaphylaxis. Remarkably, overexpression of IL-9 is able to overcome the requirement of systemic antigen sensitization prior to intra-gastric antigen-induced intestinal anaphylaxis. We are currently investigating the contribution of IL-9 to intestinal mastocytosis and intestinal permeability and how this pathway contributes to increased susceptibility and severity of oral antigen-induced anaphylaxis.
Related Publications:Forbes EE, Groschwitz K, et al. IL-9- and mast cell-mediated intestinal permeability predisposes to oral antigen hypersensitivity. J Exp Med 205(4): 897-913. 2008.
Osterfeld HN, Ahrens RE, et al. Differential roles for the IL-9/IL-9 receptor alpha-chain pathway in systemic and oral antigen-induced anaphylaxis. J Allergy Clin Immunol. 125: 469-476. 2010.
The intestinal epithelium constitutes the largest and most important barrier between our internal and external environments, forming a selectively permeable barrier that permits the absorption of nutrients, electrolytes and water, while maintaining an effective defense against intraluminal bacteria, toxins and potentially antigenic material. Disruption of the intestinal barrier is associated with bacterial, viral and parasitic infestation, as well as autoimmune and inflammatory conditions, including inflammatory bowel disease (IBD), food allergy, celiac disease and diabetes. While altered intestinal barrier function (increased intestinal epithelial permeability) can be a consequence of disease exacerbation, clinical evidence suggests it may also be a primary etiologic factor predisposing to disease development. For example, increased intestinal permeability is found not only in patients with IBD, celiac disease and type 1 diabetes, but also their healthy first-degree relatives. We are currently defining immune (cells and cytokines) and exogenous factors (foods, toxins, environmental particulates and microbes) that regulate intestinal epithelial barrier function during steady state and whether dysregulation of these pathways contributes to increased susceptibility and severity of oral antigen-induced anaphylaxis.
Related Publication:Groschwitz KR, Hogan SP. Intestinal barrier function: molecular regulation and disease pathogenesis. J Allergy Clin Immunol. 124(1): 3-20; quiz 21-22. 2009.
Interleukin (IL)-13 is a pleiotropic Th2 cytokine that has been implicated in intestinal inflammatory diseases including enteric helminth infection, food allergy and inflammatory bowel diseases (IBD) (1,2). These observations prompted us to assess the consequence of elevated IL-13 on intestinal immunity and intestinal epithelial function in vivo. To address this, we generated IL-13 transgenic mice that constitutively overexpress IL-13 in small bowel intestinal epithelial cells. We show that constitutive overexpression of IL-13 in the small bowel induces modification of intestinal epithelial architecture (villus blunting, goblet cell hyperplasia and increased epithelial proliferation) and epithelial function (altered basolateral → apical Cl- ion conductance). Pharmacological analyses in vitro and in vivo determined that elevated Cl- conductance is mediated by altered CFTR expression and activity. Generation of iIL-13Tg/Il13Ra1-/-, iIL-13Tg/Il13Ra2-/- and iIL-13Tg/STAT-6-/- mice revealed that IL-13-mediated dysregulation of epithelial architecture and Cl- conductance is dependent on IL-13Ra1 and STAT-6. These observations demonstrate a central role for the IL-13/ IL-13Ra1 pathway in the regulation of intestinal epithelial cell Cl- secretion via upregulation of CFTR, suggesting an important role for this pathway in secretory diarrhea. We are currently defining the IL-13-dependent signaling cascades involved in the regulation of intestinal epithelial CFTR expression and function and the importance of this pathway to water loss in intestinal inflammatory diseases.
Related Publication:Wu D, Ahrens R, et al. Interleukin-13 (IL-13)/IL-13 receptor alpha1 (IL-13Ralpha1) signaling regulates intestinal epithelial cystic fibrosis transmembrane conductance regulator channel-dependent Cl- secretion. J Biol Chem. 2010.
The intestinal epithelium is a single-cell layer that functions as a selectively permeable barrier permitting the absorption of nutrients, electrolytes and water, while maintaining an effective defense against intraluminal toxins, antigens and enteric flora. Clinical and experimental data suggest that dysregulation of intestinal epithelial permeability increases exposure to luminal products and subsequently leads to increased susceptibility to a spectrum of inflammatory diseases, including inflammatory bowel disease (IBD), food allergies and autoimmune diseases such as celiac disease, type 1 diabetes and multiple sclerosis. We have recently identified a pivotal role for mast cells (MC) and MC protease-4 (Mcpt-4), the murine functional homologue of human chymase, in the maintenance of small intestinal epithelial barrier homeostasis in mice. Specifically, we have found that mice deficient in MCs (KitW-sh / W-sh) or MC chymase (Mcpt-4-/-) have significantly decreased basal small intestinal permeability compared with wild-type (WT) mice. Remarkably, engraftment of KitW-sh / W-sh mice with WT but not Mcpt4-/- MC restores normal intestinal permeability.
We are currently dissecting the molecular basis of chymase-mediated regulation of homeostatic intestinal barrier function.
Related Publication:Groschwitz KR, Ahrens R, et al. Mast cells regulate homeostatic intestinal epithelial migration and barrier function by a chymase/Mcpt4-dependent mechanism. Proc Natl Acad Sci U S A 106(52): 22381-22386. 2009.
Cystic fibrosis (CF) is the second most common life-shortening, inherited disorder in the United States, affecting approximately 30,000 Americans. The underlying molecular basis of CF is a defect in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride ion (Cl-) channel that regulates the absorption and secretion of salt and water in various tissues including the lung, sweat glands, pancreas and gastrointestinal tract.
The demonstration that a majority of CF is caused by loss of surface delivery of CFTR (Δ508) and that Δ508-CFTR possesses normal cAMP-dependent electrogenic Cl- conductance has led to a concentrated effort to develop therapeutic modalities to increase CFTR delivery to the cell surface. These efforts have led to the discovery of a number of “corrector” pharmaceutics including 4-phenylbutyrate, migustat, sildenafil, SAHA, corr-4a, VRT-325, and VX-809; however, the majority of these agents are not viable as CFTR therapeutics due to their limited increase in CFTR function (15-18% of wild-type CFTR) and/or poor selectivity.
One explanation for the limited increase in Δ508-CFTR function is a reduced intracellular pool of the CFTR in patients with Δ508-CFTR, which would cap the amount of CFTR that can be chaperoned to the cell surface to stimulate Cl- secretion. This possibility has led to the concept that stimulating CFTR expression and increasing the intracellular CFTR pool may permit increased CFTR corrector-chaperoned translocation of CFTR to the cell surface and increase CFTR-mediated Cl- secretion.
We are currently studying the role of microRNAs in the regulation of CFTR expression, surface expression and function with the overall goal of therapeutically targeting microRNAs to enhance the intracellular pool of CFTR and to drive increased CFTR surface expression and Cl- secretion. If successful, the data would suggest that therapeutic antagonism of miRNAs in the presence of correctors would be a potentially viable treatment strategy for greater than 90% of individuals with CF.
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We recently demonstrated that IL-13 upregulates CFTR mRNA expression and the CFTR-dependent Cl- secretory response. In preliminary studies, we show that IL-13-induced upregulation of CFTR abundance and function is associated with dysregulation of microRNA 375 (miR-375). We are currently investigating the role of miR-375 in the regulation of CFTR expression and CFTR-dependent Cl- secretion. If our hypotheses are correct, we expect to demonstrate that antagonism of miR-375 will lead to increased CFTR abundance and function in HBE cells with cystic fibrosis (CF) and lead to improved ion secretion and that miR-375 inhibits CFTR abundance and function. Given the limitations of current CF corrector therapeutics and our striking preliminary observations, we felt compelled to determine the therapeutic potential of miR-375 antagonism as a therapeutic modality alone and in combination with CFTR correctors for the treatment of CF.
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