Joanne C. Masterson

Interleukin 37 expression protects mice from colitis

IL-37, a newly described member of the IL-1 family, functions as a fundamental inhibitor of innate inflammation and immunity. In the present study, we examined a role for IL-37 during experimental colitis. A transgenic mouse strain was generated to express human IL-37 (hIL-37tg), and these mice were subjected to dextran sulfate sodium (DSS)-induced colitis. Despite the presence of a CMV promoter to drive expression of IL-37, mRNA transcripts were not present in colons at the resting state. Expression was observed only upon disruption of the epithelial barrier, with a six- to sevenfold increase (P = 0.02) on days 3 and 5 after continuous exposure to DSS. During the development of colitis, clinical disease scores were reduced by 50% (P < 0.001), and histological indices of colitis were one-third less in hIL-37tg mice compared with WT counterparts (P < 0.001). Reduced inflammation was associated with decreased leukocyte recruitment into the colonic lamina propria. In addition, release of IL-1β and TNFα from ex vivo colonic explant tissue was decreased 5- and 13-fold, respectively, compared with WT (P ≤ 0.005), whereas IL-10 was increased sixfold (P < 0.001). However, IL-10 was not required for the anti-inflammatory effects of IL-37 because IL-10-receptor antibody blockade did not reverse IL-37-mediated protection. Mechanistically, IL-37 originating from hematopoietic cells was sufficient to exert anti-inflammatory effects because WT mice reconstituted with hIL-37tg bone marrow were protected from colitis. Thus, IL-37 emerges as key modulator of intestinal inflammation.

The oesophageal string test: a novel, minimally invasive method measures mucosal inflammation in eosinophilic oesophagitis

Objective Eosinophil predominant inflammation characterises histological features of eosinophilic oesophagitis (EoE). Endoscopy with biopsy is currently the only method to assess oesophageal mucosal inflammation in EoE. We hypothesised that measurements of luminal eosinophil-derived proteins would correlate with oesophageal mucosal inflammation in children with EoE. Design The Enterotest diagnostic device was used to develop an oesophageal string test (EST) as a minimally invasive clinical device. EST samples and oesophageal mucosal biopsies were obtained from children undergoing upper endoscopy for clinically defined indications. Eosinophil-derived proteins including eosinophil secondary granule proteins (major basic protein-1, eosinophil-derived neurotoxin, eosinophil cationic protein, eosinophil peroxidase) and Charcot–Leyden crystal protein/galectin-10 were measured by ELISA in luminal effluents eluted from ESTs and extracts of mucosal biopsies. Results ESTs were performed in 41 children with active EoE (n=14), EoE in remission (n=8), gastro-oesophageal reflux disease (n=4) and controls with normal oesophagus (n=15). EST measurement of eosinophil-derived protein biomarkers significantly distinguished between children with active EoE, treated EoE in remission, gastro-oesophageal reflux disease and normal oesophagus. Levels of luminal eosinophil-derived proteins in EST samples significantly correlated with peak and mean oesophageal eosinophils/high power field (HPF), eosinophil peroxidase indices and levels of the same eosinophil-derived proteins in extracts of oesophageal biopsies. Conclusions The presence of eosinophil-derived proteins in luminal secretions is reflective of mucosal inflammation in children with EoE. The EST is a novel, minimally invasive device for measuring oesophageal eosinophilic inflammation in children with EoE.

Eosinophilic Esophagitis: Epithelial Mesenchymal Transition Contributes to Esophageal Remodeling and Reverses with Treatment

BACKGROUND Mechanisms underlying esophageal remodeling with subepithelial fibrosis in subjects with eosinophilic esophagitis (EoE) have not been delineated. OBJECTIVES We sought to explore a role for epithelial mesenchymal transition (EMT) in subjects with EoE and determine whether EMT resolves with treatment. METHODS Esophageal biopsy specimens from 60 children were immunostained for epithelial (cytokeratin) and mesenchymal (vimentin) EMT biomarkers, and EMT was quantified. Subjects studied had EoE (n = 17), indeterminate EoE (n = 15), gastroesophageal reflux disease (n = 7), or normal esophagus (n = 21). EMT was analyzed for relationships to diagnosis, eosinophil counts, and indices of subepithelial fibrosis, eosinophil peroxidase, and TGF-β immunostaining. EMT was assessed in pretreatment and posttreatment biopsy specimens from 18 subjects with EoE treated with an elemental diet, 6-food elimination diet, or topical corticosteroids (n = 6 per group). RESULTS TGF-β1 treatment of esophageal epithelial cells in vitro for 24 hours induced upregulation of mesenchymal genes characteristic of EMT, including N-cadherin (3.3-fold), vimentin (2.1-fold), and fibronectin (7.5-fold). EMT in esophageal biopsy specimens was associated with EoE (or indeterminate EoE) but not gastroesophageal reflux disease or normal esophagus and was correlated to eosinophil counts (r = 0.691), eosinophil peroxidase (r = 0.738), and TGF-β (r = 0.520) immunostaining and fibrosis (r = 0.644) indices. EMT resolved with EoE treatments that induced clinicopathologic remission with reduced eosinophil counts. EMT decreased significantly after treatment by 74.1% overall in the 18 treated subjects with EoE; pretreatment versus posttreatment EMT scores were 3.17 ± 0.82 versus 0.82 ± 0.39 (P < .001), with similar decreases within treatment groups. Pretreatment/posttreatment EMT was strongly correlated with eosinophil counts for combined (r = 0.804, P < .001) and individual treatment groups. CONCLUSIONS EMT likely contributes to subepithelial fibrosis in subjects with EoE and resolves with treatments that decrease esophageal inflammation, and its resolution correlates with decreased numbers of esophageal eosinophils.

Neuronal guidance molecule netrin-1 attenuates inflammatory cell trafficking during acute experimental colitis

Background Inflammatory bowel diseases, encompassing Crohns disease and ulcerative colitis, are characterised by persistent leucocyte tissue infiltration leading to perpetuation of an inappropriate inflammatory cascade. The neuronal guidance molecule netrin-1 has recently been implicated in the orchestration of leucocyte trafficking during acute inflammation. We therefore hypothesised that netrin-1 could modulate leucocyte infiltration and disease activity in a model of inflammatory bowel disease. Design DSS-colitis was performed in mice with partial genetic netrin-1 deficiency (Ntn-1+/− mice) or wild-type mice treated with exogenous netrin-1 via osmotic pump to examine the role of endogenous and therapeutically administered netrin-1. These studies were supported by in vitro models of transepithelial migration and intestinal epithelial barrier function. Results Consistent with our hypothesis, we observed induction of netrin-1 during intestinal inflammation in vitro or in mice exposed to experimental colitis. Moreover, mice with partial netrin-1 deficiency demonstrated an exacerbated course of DSS-colitis compared to littermate controls, with enhanced weight loss and colonic shortening. Conversely, mice treated with exogenous mouse netrin-1 experienced attenuated disease severity. Importantly, permeability studies and quantitative assessment of apoptosis reveal that netrin-1 signalling events do not alter mucosal permeability or intestinal epithelial cell apoptosis. In vivo studies of leucocyte transmigration demonstrate suppression of neutrophil trafficking as a key function mediated by endogenous or exogenously administered netrin-1. Finally, genetic studies implicate the A2B adenosine receptor in netrin-1-mediated protection during DSS-colitis. Conclusions The present study identifies a previously unrecognised role for netrin-1 in attenuating experimental colitis through limitation of neutrophil trafficking.

Effect of proton pump inhibitor on esophageal eosinophilia.

Objective: Differentiation between the common etiologies of dense esophageal eosinophilia such as gastroesophageal reflux disease (GERD) and eosinophilic esophagitis can be difficult. We hypothesized that histologic features may provide diagnostic clues concerning the etiology of esophageal eosinophilia. Methods: We performed a retrospective chart review of 204 children with the diagnosis of esophagitis characterized by ≥15 eosinophils (eos) per high-power field (HPF) in at least 1 biopsy. We then restricted our analysis to subjects who had received at least 8 weeks of only proton pump inhibitors (PPIs) followed by endoscopy and who had a clinicopathologic response to this treatment. Symptoms, endoscopic findings, and pathologic descriptions were reviewed and an eosinophil peroxidase (EPX) index was determined to assess for degranulation/eosinophil activation. Results: Of the 204 identified charts, 7 subjects identified met the inclusion criteria. Five of these 7 patients showed a clinicopathologic response to PPIs after their follow-up endoscopy, (mean peak eosinophil count: 92 vs 5 eos/HPF, and EPX index: 39.2 vs 14.6, pre- and posttreatment, respectively). Two patients experienced initial resolution of symptoms and esophageal eosinophilia with PPI therapy; however, within 17–23 months they redeveloped symptoms and esophageal eosinophilia while receiving PPI therapy at the time of a third endoscopy (mean peak eosinophil count: 40 vs 11 vs 36 eos/HPF, and EPX index: 44 vs 21 vs 36.5, pre-, post- and posttreatment, respectively). No clinicopathologic features or degranulation patterns differentiated subjects with GERD/PPI responsive esophageal eosinophilia from those who had transient response to PPI treatment. Conclusions: No clinicopathologic features differentiated subjects who responded to PPI treatment. PPI treatment can be helpful to exclude GERD and PPI responsive esophageal eosinophilia but long-term follow-up is critical in the management of esophagitis.

Myeloid-derived miR-223 regulates intestinal inflammation via repression of the NLRP3 inflammasome

MicroRNA (miRNA)-mediated RNA interference regulates many immune processes, but how miRNA circuits orchestrate aberrant intestinal inflammation during inflammatory bowel disease (IBD) is poorly defined. Here, we report that miR-223 limits intestinal inflammation by constraining the nlrp3 inflammasome. miR-223 was increased in intestinal biopsies from patients with active IBD and in preclinical models of intestinal inflammation. miR-223-/y mice presented with exacerbated myeloid-driven experimental colitis with heightened clinical, histopathological, and cytokine readouts. Mechanistically, enhanced NLRP3 inflammasome expression with elevated IL-1&bgr; was a predominant feature during the initiation of colitis with miR-223 deficiency. Depletion of CCR2+ inflammatory monocytes and pharmacologic blockade of IL-1&bgr; or NLRP3 abrogated this phenotype. Generation of a novel mouse line, with deletion of the miR-223 binding site in the NLRP3 3′ untranslated region, phenocopied the characteristics of miR-223-/y mice. Finally, nanoparticle-mediated overexpression of miR-223 attenuated experimental colitis, NLRP3 levels, and IL-1&bgr; release. Collectively, our data reveal a previously unappreciated role for miR-223 in regulating the innate immune response during intestinal inflammation.

Esophageal microbiome in eosinophilic esophagitis

Objective The microbiome has been implicated in the pathogenesis of a number of allergic and inflammatory diseases. The mucosa affected by eosinophilic esophagitis (EoE) is composed of a stratified squamous epithelia and contains intraepithelial eosinophils. To date, no studies have identified the esophageal microbiome in patients with EoE or the impact of treatment on these organisms. The aim of this study was to identify the esophageal microbiome in EoE and determine whether treatments change this profile. We hypothesized that clinically relevant alterations in bacterial populations are present in different forms of esophagitis. Design In this prospective study, secretions from the esophageal mucosa were collected from children and adults with EoE, Gastroesophageal Reflux Disease (GERD) and normal mucosa using the Esophageal String Test (EST). Bacterial load was determined using quantitative PCR. Bacterial communities, determined by 16S rRNA gene amplification and 454 pyrosequencing, were compared between health and disease. Results Samples from a total of 70 children and adult subjects were examined. Bacterial load was increased in both EoE and GERD relative to normal subjects. In subjects with EoE, load was increased regardless of treatment status or degree of mucosal eosinophilia compared with normal. Haemophilus was significantly increased in untreated EoE subjects as compared with normal subjects. Streptococcus was decreased in GERD subjects on proton pump inhibition as compared with normal subjects. Conclusions Diseases associated with mucosal eosinophilia are characterized by a different microbiome from that found in the normal mucosa. Microbiota may contribute to esophageal inflammation in EoE and GERD.

Role of eosinophils in inflammatory bowel and gastrointestinal diseases.

Inflammatory bowel diseases (IBD) are characterized by the invasion of leukocytes into the intestinal mucosa. However, a mixed inflammatory picture is observed that includes neutrophils, lymphocytes, monocytes, and eosinophils. To this day, the role of eosinophils in health and in disease remains unclear. Investigations into their function stem primarily from allergic diseases, asthma, and parasitic infections. This makes it even more difficult to discern a role for the fascinating eosinophil in IBDs because, unlike the lung or the skin, eosinophils reside in normal intestinal mucosa and increase in disease states; consequently, an intricate system must regulate their migration and numbers. These granulocytes are equipped with the machinery to participate in gastrointestinal (GI) inflammation and in the susceptible microenvironment, they may initiate or perpetuate an inflammatory response. A significant body of literature characterizes eosinophils present in the GI microenvironment where they have the potential to interact with other resident cells, thus promoting intestinal remodeling, mucus production, epithelial barrier, cytokine production, angiogenesis, and neuropeptide release. A number of lines of evidence support both potential beneficial and deleterious roles of eosinophils in the gut. Although studies from the gut and other mucosal organs suggest eosinophils affect mucosal GI inflammation, definitive roles for eosinophils in IBDs await discovery.

Eosinophil-mediated signalling attenuates inflammatory responses in experimental colitis

Objective Eosinophils reside in the colonic mucosa and increase significantly during disease. Although a number of studies have suggested that eosinophils contribute to the pathogenesis of GI inflammation, the expanding scope of eosinophil-mediated activities indicate that they also regulate local immune responses and modulate tissue inflammation. We sought to define the impact of eosinophils that respond to acute phases of colitis in mice. Design Acute colitis was induced in mice by administration of dextran sulfate sodium, 2,4,6-trinitrobenzenesulfonic acid or oxazolone to C57BL/6J (control) or eosinophil deficient (PHIL) mice. Eosinophils were also depleted from mice using antibodies against interleukin (IL)-5 or by grafting bone marrow from PHIL mice into control mice. Colon tissues were collected and analysed by immunohistochemistry, flow cytometry and reverse transcription PCR; lipids were analysed by mass spectroscopy. Results Eosinophil-deficient mice developed significantly more severe colitis, and their colon tissues contained a greater number of neutrophils, than controls. This compensatory increase in neutrophils was accompanied by increased levels of the chemokines CXCL1 and CXCL2, which attract neutrophils. Lipidomic analyses of colonic tissue from eosinophil-deficient mice identified a deficiency in the docosahexaenoic acid-derived anti-inflammatory mediator 10, 17- dihydroxydocosahexaenoic acid (diHDoHE), namely protectin D1 (PD1). Administration of an exogenous PD1-isomer (10S, 17S-DiHDoHE) reduced the severity of colitis in eosinophil-deficient mice. The PD1-isomer also attenuated neutrophil infiltration and reduced levels of tumour necrosis factor-α, IL-1β, IL-6 and inducible NO-synthase in colons of mice. Finally, in vitro assays identified a direct inhibitory effect of PD1-isomer on neutrophil transepithelial migration. Conclusions Eosinophils exert a protective effect in acute mouse colitis, via production of anti-inflammatory lipid mediators.

Novel Device to Sample the Esophageal Microbiome—The Esophageal String Test

A growing number of studies implicate the microbiome in the pathogenesis of intestinal inflammation. Previous work has shown that adults with esophagitis related to gastroesophageal reflux disease have altered esophageal microbiota compared to those who do not have esophagitis. In these studies, sampling of the esophageal microbiome was accomplished by isolating DNA from esophageal biopsies obtained at the time of upper endoscopy. The aim of the current study was to identify the esophageal microbiome in pediatric individuals with normal esophageal mucosa using a minimally invasive, capsule-based string technology, the Enterotest™. We used the proximal segment of the Enterotest string to sample the esophagus, and term this the “Esophageal String Test” (EST). We hypothesized that the less invasive EST would capture mucosal adherent bacteria present in the esophagus in a similar fashion as mucosal biopsy. EST samples and mucosal biopsies were collected from children with no esophageal inflammation (n = 15) and their microbiome composition determined by 16S rRNA gene sequencing. Microbiota from esophageal biopsies and ESTs produced nearly identical profiles of bacterial genera and were different from the bacterial contents of samples collected from the nasal and oral cavity. We conclude that the minimally invasive EST can serve as a useful device for study of the esophageal microbiome.