Search

The epithelial lining of the intestine, particularly the stem cell compartment, is affected by harsh conditions in the luminal environment and also is susceptible to genotoxic agents such as radiation and chemotherapy. Therefore, the ability for intestinal epithelial cells to revert to a stem cell state is an important physiological damage response to regenerate the intestinal epithelium at sites of mucosal injury. Many signaling networks involved in maintaining the stem cell niche are activated as part of the damage response to promote cellular plasticity and regeneration. The relative contribution of each cell type and signaling pathway is a critical area of ongoing research, likely dependent on the nature of injury as well as the regional specification within the intestine. Here, we review the current understanding of the multicellular cooperation to restore the intestinal epithelium after damage.

Background & Aims CD4+ T cells are regulated by activating and inhibitory cues, and dysregulation of these proper regulatory inputs predisposes these cells to aberrant inflammation and exacerbation of disease. We investigated the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of the CD4+ T-cell inflammatory response and exacerbation of the colitic phenotype. Methods We used Il10-/- spontaneous and CD4+CD45RBhi T-cell transfer models of colitis with PIR-B-deficient (Pirb-/-) mice. Flow cytometry, Western blot, and RNA sequencing analysis was performed on wild-type and Pirb-/- CD4+ T cells. In silico analyses were performed on RNA sequencing data set of ileal biopsy samples from pediatric CD and non–inflammatory bowel disease patients and sorted human memory CD4+ T cells. Results We identified PIR-B expression on memory CD4+ interleukin (IL)17a+ cells. We show that PIR-B regulates CD4+ T-helper 17 cell (Th17)-dependent chronic intestinal inflammatory responses and the development of colitis. Mechanistically, we show that the PIR-B– Src-homology region 2 domain-containing phosphatase-1/2 axis tempers mammalian target of rapamycin complex 1 signaling and mammalian target of rapamycin complex 1–dependent caspase-3/7 apoptosis, resulting in CD4+ IL17a+ cell survival. In silico analyses showed enrichment of transcriptional signatures for Th17 cells (RORC, RORA, and IL17A) and tissue resident memory (HOBIT, IL7R, and BLIMP1) networks in PIR-B+ murine CD4+ T cells and human CD4+ T cells that express the human homologue leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3). High levels of LILRB3 expression were associated strongly with mucosal injury and a proinflammatory Th17 signature, and this signature was restricted to a treatment-naïve, severe pediatric CD population. Conclusions Our findings show an intrinsic role for PIR-B/LILRB3 in the regulation of CD4+ IL17a+ T-cell pathogenic memory responses., Graphical abstract

Background & Aims Alterations in intestinal MUC2 mucin and microbial diversity are closely linked with important intestinal pathologies; however, their impact on each other and on intestinal pathogenesis has been vaguely characterized. Therefore, it was of interest in this study to delineate distinct and cooperative function of commensal microbiota and the Muc2 mucus barrier in maintaining intestinal epithelial barrier function. Methods Muc2 mucin deficient (Muc2–/–) and sufficient (Muc2+/+) littermates were used as a model for assessing the role of Muc2. To quantify the role of the microbiota in disease pathogenesis, Muc2+/+ and Muc2–/– littermates were treated with a cocktail of antibiotics that reduced indigenous bacteria, and then fecal transplanted with littermate stool and susceptibility to dextran sulphate sodium (DSS) quantified. Results Although, Muc2+/+ and Muc2–/– littermates share similar phyla distribution as evidenced by 16S sequencing they maintain their distinctive gastrointestinal phenotypes. Basally, Muc2–/– showed low-grade colonic inflammation with high populations of inflammatory and tolerogenic immune cells that became comparable to Muc2+/+ littermates following antibiotic treatment. Antibiotics treatment rendered Muc2+/+ but not Muc2–/– littermates highly susceptibility to DSS-induced colitis that was ILC3 dependent. Muc2–/– microbiota was colitogenic to Muc2+/+ as it worsened DSS-induced colitis. Microbiota dependent inflammation was confirmed by bone-marrow chimera studies, as Muc2–/– receiving Muc2+/+ bone marrow showed no difference in their susceptibility toward DSS induced colitis. Muc2–/– microbiota exhibited presence of characteristic OTUs of specific bacterial populations that were transferrable to Muc2+/+ littermates. Conclusions These results highlight a distinct role for Muc2 mucin in maintenance of healthy microbiota critical in shaping innate host defenses to promote intestinal homeostasis., Graphical abstract

Background and aims The trigger hypothesis opens the possibility of anti-flare initiation therapies by stating that ulcerative colitis (UC) flares originate from inadequate responses to acute mucosal injuries. However, experimental evidence is restricted by a limited use of suitable human models. We thus aimed to investigate the acute mucosal barrier injury responses in humans with and without UC using an experimental injury model. Methods A standardized mucosal break was inflicted in the sigmoid colon of 19 patients with UC in endoscopic and histological remission and 20 control subjects. Postinjury responses were assessed repeatedly by high-resolution imaging and sampling to perform Geboes scoring, RNA sequencing, and injury niche microbiota 16S ribosomal RNA gene sequencing. Results UC patients had more severe endoscopic postinjury inflammation than did control subjects (P < .01), an elevated modified Geboes score (P < .05), a rapid induction of innate response gene sets (P < .05) and antimicrobial peptides (P < .01), and engagement of neutrophils (P < .01). Innate lymphoid cell type 3 (ILC3) markers were increased preinjury (P < .01), and ILC3 activating cytokines were highly induced postinjury, resulting in an increase in ILC3-type cytokine interleukin-17A. Across groups, the postinjury mucosal microbiome had higher bacterial load (P < .0001) and lower α-diversity (P < .05). Conclusions UC patients in remission respond to mucosal breaks by an innate hyperresponse engaging resident regulatory ILC3s and a subsequent adaptive activation. The postinjury inflammatory bowel disease–like microbiota diversity decrease is irrespective of diagnosis, suggesting that the dysbiosis is secondary to host injury responses. We provide a model for the study of flare initiation in the search for antitrigger-directed therapies., Graphical abstract

Background & Aims How benign liver steatosis progresses to nonalcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC) remains elusive. NASH progression entails diverse pathogenic mechanisms and relies on complex cross-talk between multiple tissues such as the gut, adipose tissues, liver, and the brain. Using a hyperphagic mouse fed with a Western diet (WD), we aimed to elucidate the cross-talk and kinetics of hepatic and extrahepatic alterations during NASH–HCC progression, as well as regression. Methods Hyperphagic mice lacking a functional Alms1 gene (Foz/Foz) and wild-type littermates were fed WD or standard chow for 12 weeks for NASH/fibrosis and for 24 weeks for HCC development. NASH regression was modeled by switching back to normal chow after NASH development. Results Foz+WD mice were steatotic within 1 to 2 weeks, developed NASH by 4 weeks, and grade 3 fibrosis by 12 weeks, accompanied by chronic kidney injury. Foz+WD mice that continued on WD progressed to cirrhosis and HCC within 24 weeks and had reduced survival as a result of cardiac dysfunction. However, NASH mice that were switched to normal chow showed NASH regression, improved survival, and did not develop HCC. Transcriptomic and histologic analyses of Foz/Foz NASH liver showed strong concordance with human NASH. NASH was preceded by an early disruption of gut barrier, microbial dysbiosis, lipopolysaccharide leakage, and intestinal inflammation. This led to acute-phase liver inflammation in Foz+WD mice, characterized by neutrophil infiltration and increased levels of several chemokines/cytokines. The liver cytokine/chemokine profile evolved as NASH progressed, with subsequent predominance by monocyte recruitment. Conclusions The Foz+WD model closely mimics the pathobiology and gene signature of human NASH with fibrosis and subsequent HCC. Foz+WD mice provide a robust and relevant preclinical model of NASH, NASH-associated HCC, chronic kidney injury, and heart failure., Supplemental Graphical Summary

Celiac disease (CD) is a common autoimmune disorder triggered by the ingestion of gluten in genetically susceptible individuals. Although the mechanisms underlying gliadin-mediated activation of adaptive immunity in CD have been well-characterized, regulation of innate immune responses and the functions of certain immune cell populations within the epithelium and lamina propria are not well-understood at present. Innate lymphoid cells (ILCs) are types of innate immune cells that have lymphoid morphology, lack antigen-specific receptors, and play important roles in tissue homeostasis, inflammation, and protective immune responses against pathogens. Information regarding the diversity and functions of ILCs in lymphoid organs and at mucosal sites has grown over the past decade, and roles of different ILC subsets in the pathogenesis of some inflammatory intestinal diseases have been proposed. However, our understanding of the contribution of ILCs toward the initiation and progression of CD is still limited. In this review, we discuss current pathophysiological aspects of ILCs within the gastrointestinal tract, findings of recent investigations characterizing ILC alterations in CD and refractory CD, and suggest avenues for future research.

Background & Aims We previously showed that abundance of Candida tropicalis is significantly greater in Crohn’s disease patients compared with first-degree relatives without Crohn’s disease. The aim of this study was to determine the effects and mechanisms of action of C tropicalis infection on intestinal inflammation and injury in mice. Methods C57BL/6 mice were inoculated with C tropicalis, and colitis was induced by administration of dextran sodium sulfate in drinking water. Disease severity and intestinal permeability subsequently were evaluated by endoscopy, histology, quantitative reverse-transcription polymerase chain reaction, as well as 16S ribosomal RNA and NanoString analyses (NanoString Technologies, Seattle, WA). Results Infected mice showed more severe colitis, with alterations in gut mucosal helper T cells (Th)1 and Th17 cytokine expression, and an increased frequency of mesenteric lymph node–derived group 2 innate lymphoid cells compared with uninfected controls. Gut microbiome composition, including changes in the mucin-degrading bacteria, Akkermansia muciniphila and Ruminococcus gnavus, was altered significantly, as was expression of several genes affecting intestinal epithelial homeostasis in isolated colonoids, after C tropicalis infection compared with uninfected controls. In line with these findings, fecal microbiome transplantation of germ-free recipient mice using infected vs uninfected donors showed altered expression of several tight-junction proteins and increased susceptibility to dextran sodium sulfate–induced colitis. Conclusions C tropicalis induces dysbiosis that involves changes in the presence of mucin-degrading bacteria, leading to altered tight junction protein expression with increased intestinal permeability and followed by induction of robust Th1/Th17 responses, which ultimately lead to an accelerated proinflammatory phenotype in experimental colitic mice., Graphical abstract

The increasing application of single-cell RNA sequencing (scRNA-seq) technology in life science and biomedical research has significantly increased our understanding of the cellular heterogeneities in immunology, oncology and developmental biology. This review will summarize the development of various scRNA-seq technologies; primarily discussing the application of scRNA-seq on infectious diseases, and exploring the current development, challenges, and potential applications of scRNA-seq technology in the future.

Detection of individual cytokines in routine biopsies from patients with inflammatory skin diseases has the potential to personalize diagnosis and treatment selection, but this approach has been limited by technical feasibility. We evaluate whether a chromogen-based RNA in situ hybridization approach can be used to detect druggable cytokines in psoriasis and atopic dermatitis. A series of psoriasis (n = 20) and atopic dermatitis (n = 26) biopsies were stained using RNA in situ hybridization for IL4, IL12B (IL-12/23 p40), IL13, IL17A, IL17F, IL22, IL23A (IL-23 p19), IL31, and TNF (TNF-α). NOS2 and IFNG, canonical psoriasis biomarkers, were also included. All 20 of the psoriasis cases were positive for IL17A, which tended to be the predominant cytokine, although some cases had relatively higher levels of IL12B, IL17F, or IL23A. The majority of cytokine expression in psoriasis was epidermal. A total of 22 of 26 atopic dermatitis cases were positive for IL13, also at varying levels; a subset of cases had significant IL4, IL22, or IL31 expression. Patterns were validated in independent bulk RNA-sequencing and single-cell RNA-sequencing datasets. Overall, RNA in situ hybridization for cytokines appears highly specific with virtually no background staining and may allow for individualized evaluation of treatment-relevant cytokine targets in biopsies from patients with inflammatory skin disorders., Graphical abstract

Group 2 innate lymphoid cells (ILCs) are thought to contribute to the pathogenesis of atopic dermatitis (AD). IL-4 stimulates T helper type 2 (Th2) cells and ILC2s to proliferate and produce cytokines. Dupilumab, an antibody against the IL-4 receptor, is used in AD therapy. We speculated that its efficacy might involve blocking the activation of Th2 cells and ILC2s via IL-4. Here, we examined circulating Th2 cells and ILC2s in 27 Japanese patients with AD before and after the administration of dupilumab. Between 0 and 4 months after dupilumab administration, the percentages of Th2 cells and ILC2s were decreased. Notably, ILC2/3 ratio was decreased after dupilumab treatment. Interestingly, ILC2/3 ratio before dupilumab treatment were significantly higher in high responders than in low responders to dupilumab. To resolve the molecular signatures of the Th2 and ILC2s in AD, we sorted CD4+ T cells and ILCs from peripheral blood and analyzed their transcriptomes using the BD Rhapsody Single-cell RNA sequencing system. Between 0 and 4 months after dupilumab administration, the Th2 and ILC2 cluster gene signatures were downregulated. Thus, dupilumab might improve dermatitis by suppressing the Th2 cell and ILC2 populations and altering the Th2 and ILC2 repertoire in patients with AD., Graphical abstract

Background & Aims Hirschsprung’s disease (HSCR) is a congenital intestinal motility disorder defined by the absence of enteric neuronal cells (ganglia) in the distal gut. The development of HSCR-associated enterocolitis remains a life-threatening complication. Absence of enteric ganglia implicates innervation of acetylcholine-secreting (cholinergic) nerve fibers. Cholinergic signals have been reported to control excessive inflammation, but the impact on HSCR-associated enterocolitis is unknown. Methods We enrolled 44 HSCR patients in a prospective multicenter study and grouped them according to their degree of colonic mucosal acetylcholinesterase-positive innervation into low-fiber and high-fiber patient groups. The fiber phenotype was correlated with the tissue cytokine profile as well as immune cell frequencies using Luminex analysis and fluorescence-activated cell sorting analysis of colonic tissue and immune cells. Using confocal immunofluorescence microscopy, macrophages were identified in close proximity to nerve fibers and characterized by RNA-seq analysis. Microbial dysbiosis was analyzed in colonic tissue using 16S-rDNA gene sequencing. Finally, the fiber phenotype was correlated with postoperative enterocolitis manifestation. Results The presence of mucosal nerve fiber innervation correlated with reduced T-helper 17 cytokines and cell frequencies. In high-fiber tissue, macrophages co-localized with nerve fibers and expressed significantly less interleukin 23 than macrophages from low-fiber tissue. HSCR patients lacking mucosal nerve fibers showed microbial dysbiosis and had a higher incidence of postoperative enterocolitis. Conclusions The mucosal fiber phenotype might serve as a prognostic marker for enterocolitis development in HSCR patients and may offer an approach to personalized patient care and new therapeutic options., Graphical abstract

Background & Aims Psoriasis and inflammatory bowel disease (IBD) are both chronic inflammatory diseases occurring in the skin and gut, respectively. It is well established that psoriasis and IBD have high concordance rates, and similar changes in immune cells and microbiome composition have been reported in both conditions. To study this connection, we used a combination murine model of psoriatic dermatitis and colitis in which mice were treated topically with the Toll-like receptor 7 agonist imiquimod (IMQ) and fed dextran sulfate sodium (DSS). Methods We applied IMQ topically to B6 mice (IMQ mice) and subsequently fed them 2% DSS in their drinking water. Disease activity and immune cell phenotypes were analyzed, and the microbial composition of fecal samples was investigated using 16S ribosomal RNA sequencing. We transplanted feces from IMQ mice to germ-free IQI/Jic (IQI) mice and fed them DSS to assess the effect of the gut microbiome on disease. Results We first confirmed that IMQ mice showed accelerated DSS colitis. IMQ mice had decreased numbers of IgD+ and IgM+ B cells and increased numbers of non–cytokine-producing macrophages in the gut. Moreover, the gut microbiomes of IMQ mice were perturbed, with significant reductions of Lactobacillus johnsonii and Lactobacillus reuteri populations. Germ-free mice transplanted with feces from IMQ mice, but not with feces from untreated mice, also developed exacerbated DSS colitis. Conclusions These results suggest that skin inflammation may contribute to pathogenic conditions in the gut via immunologic and microbiological changes. Our finding of a novel potential skin–gut interaction provides new insights into the coincidence of psoriasis and IBD., Graphical abstract

Background & Aims Crohn’s disease is an inflammatory bowel disease that affects the ileum and is associated with increased cytokines. Although interleukin (IL)6, IL17, IL21, and IL22 are increased in Crohn’s disease and are associated with disrupted epithelial regeneration, little is known about their effects on the intestinal stem cells (ISCs) that mediate tissue repair. We hypothesized that ILs may target ISCs and reduce ISC-driven epithelial renewal. Methods A screen of IL6, IL17, IL21, or IL22 was performed on ileal mouse organoids. Computational modeling was used to predict microenvironment cytokine concentrations. Organoid size, survival, proliferation, and differentiation were characterized by morphometrics, quantitative reverse-transcription polymerase chain reaction, and immunostaining on whole organoids or isolated ISCs. ISC function was assayed using serial passaging to single cells followed by organoid quantification. Single-cell RNA sequencing was used to assess Il22ra1 expression patterns in ISCs and transit-amplifying (TA) progenitors. An IL22-transgenic mouse was used to confirm the impact of increased IL22 on proliferative cells in vivo. Results High IL22 levels caused decreased ileal organoid survival, however, resistant organoids grew larger and showed increased proliferation over controls. Il22ra1 was expressed on only a subset of ISCs and TA progenitors. IL22-treated ISCs did not show appreciable differentiation defects, but ISC biomarker expression and self-renewal–associated pathway activity was reduced and accompanied by an inhibition of ISC expansion. In vivo, chronically increased IL22 levels, similar to predicted microenvironment levels, showed increases in proliferative cells in the TA zone with no increase in ISCs. Conclusions Increased IL22 limits ISC expansion in favor of increased TA progenitor cell expansion., Graphical abstract

Allergic contact dermatitis and its animal model, contact hypersensitivity, are T-cell-mediated inflammatory skin diseases that require activation of the innate immune system. Here we investigate the role of innate lymphoid cells (ILCs) during the elicitation phase of 2,4,6-trinitrochlorobenzene-induced contact hypersensitivity using EomesGfp/+ x Rorc(γt)-CreTg x Rosa26RYfp/+ reporter mice. Ear swelling responses, cutaneous ILC numbers, and cytokine production were determined at different time points. Functional analyses were performed in a CD90.1/.2 congenic adoptive transfer model that allowed selective antibody-mediated depletion of ILCs before hapten challenge, and in Rorasg/floxIl7rCre/+ mice, which lack ILC2. Hapten challenge induced early increases of natural killer cells in skin and ear draining lymph nodes corresponding to the peak ear swelling response. In contrast, ILC1, 2, and 3 showed a delayed increase in numbers corresponding to the contact hypersensitivity resolution phase. Hapten challenge induced increased marker cytokines in all ILC subtypes and an activated phenotype in ILC2. Depletion of all ILC resulted in a significantly enhanced ear swelling response. Similarly, ILC2-deficient mice (Rorasg/floxIl7rCre/+) displayed increased ear swelling responses on hapten challenge, suggesting that ILC2 act as negative regulators in the type 1-dominated immune response of contact hypersensitivity.

Background Atopic dermatitis skin lesions demonstrate increased expression of IL-25 by keratinocytes and increased numbers of type 2 innate lymphoid cells (ILC2s) that express high levels of IL-25 receptor (IL-25R). IL-13 is expressed in atopic dermatitis skin lesions and plays an important role in pathogenesis of the disease. Objective Our aim was to determine the role of IL-25 and ILC2s in a mouse model of antigen-driven allergic skin inflammation. Methods Wild-type mice; mice that express an Il13-driven enhanced green fluorescent protein; and mice that lack IL-25R, IL-25 in keratinocytes, or IL-13 or IL-25R in ILC2s were subjected to acute or chronic epicutaneous sensitization with ovalbumin. Sensitized skin was examined by histology for epidermal thickening. Cellular infiltrates were analyzed for surface markers and intracellular expression of enhanced green fluorescent protein by flow cytometry. Gene expression was quantitated by RT quantitative PCR. Result In both acute and chronic antigen-driven allergic skin inflammation, signaling by keratinocyte-derived IL-25 in ILC2s is important for epidermal hyperplasia, dermal infiltration by CD4+ T cells, and cutaneous expression of Il13 and the IL-13–dependent TH2-cell–attracting chemokines Cc17 and Ccl22. ILCs are the major source of IL-13 in acutely sensitized mouse skin, whereas T cells are its major source in chronically sensitized mouse skin. Conclusion ILC2 activation by IL-25 is essential for IL-13 expression at sites of allergic skin inflammation., Graphical abstract

Graphical abstract, Inflammatory bowel disease (IBD) is a debilitating chronic inflammatory disease of the gastrointestinal (GI) tract. It affects more than 3.5 million people in the western world and places a huge financial burden on healthcare systems. IBD is highly heterogeneous; disease severity and outcomes in IBD are highly variable, and patients may experience episodes of relapse and remission. However, treatment often follows a step-up model whereby the patients start with anti-inflammatory agents (corticosteroids or immunosuppressants) and step-up to monoclonal anti-tumour necrosis factor-α (TNFα) antibodies and then other biologics if the initial drugs cannot control disease. Unfortunately, many patients do not respond to the costly biologics, and thus often still require gut-resective surgery, which decreases quality of life. In order to decrease rates of surgery and ineffective treatments, it is important to identify markers that accurately predict disease progression and treatment responses, to inform decisions about the best choice of therapeutics. Here we examine molecular approaches to patient stratification that aim to increase the effectiveness of treatments and potentially reduce healthcare costs. In the future, it may become possible to stratify patients based on their suitability for specific molecular-targeted therapeutic agents, and eventually use molecular stratification for personalised medicine in IBD.

Background & Aims Intestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice. Methods We generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces. Results Mcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development. Conclusion The antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases., Graphical abstract

The bronchial epithelium is continuously exposed to a multitude of noxious challenges in inhaled air. Cellular contact with most damaging agents is reduced by the action of the mucociliary apparatus and by formation of a physical barrier that controls passage of ions and macromolecules. In conjunction with these defensive barrier functions, immunomodulatory cross-talk between the bronchial epithelium and tissue-resident immune cells controls the tissue microenvironment and barrier homeostasis. This is achieved by expression of an array of sensors that detect a wide variety of viral, bacterial, and nonmicrobial (toxins and irritants) agents, resulting in production of many different soluble and cell-surface molecules that signal to cells of the immune system. The ability of the bronchial epithelium to control the balance of inhibitory and activating signals is essential for orchestrating appropriate inflammatory and immune responses and for temporally modulating these responses to limit tissue injury and control the resolution of inflammation during tissue repair. In asthmatic patients abnormalities in many aspects of epithelial barrier function have been identified. We postulate that such abnormalities play a causal role in immune dysregulation in the airways by translating gene-environment interactions that underpin disease pathogenesis and exacerbation.

Background & Aims: Gut dysbiosis is closely involved in the pathogenesis of inflammatory bowel disease (IBD). However, it remains unclear whether IBD-associated gut dysbiosis contributes to disease pathogenesis or is merely secondary to intestinal inflammation. We established a humanized gnotobiotic (hGB) mouse system to assess the functional role of gut dysbiosis associated with 2 types of IBD: Crohn's disease (CD) and ulcerative colitis (UC). Methods: Germ-free mice were colonized by the gut microbiota isolated from patients with CD and UC, and healthy controls. Microbiome analysis, bacterial functional gene analysis, luminal metabolome analysis, and host gene expression analysis were performed in hGB mice. Moreover, the colitogenic capacity of IBD-associated microbiota was evaluated by colonizing germ-free colitis-prone interleukin 10âdeficient mice with dysbiotic patients' microbiota. Results: Although the microbial composition seen in donor patients' microbiota was not completely reproduced in hGB mice, some dysbiotic features of the CD and UC microbiota (eg, decreased diversity, alteration of bacterial metabolic functions) were recapitulated in hGB mice, suggesting that microbial community alterations, characteristic for IBD, can be reproduced in hGB mice. In addition, colonization by the IBD-associated microbiota induced a proinflammatory gene expression profile in the gut that resembles the immunologic signatures found in CD patients. Furthermore, CD microbiota triggered more severe colitis than healthy control microbiota when colonized in germ-free interleukin 10âdeficient mice. Conclusions: Dysbiosis potentially contributes to the pathogenesis of IBD by augmenting host proinflammatory immune responses. Transcript profiling: GSE73882. Keywords: Dysbiosis, Microbiota, Crohn's Disease, Ulcerative Colitis

Background & Aims Unregulated activity of interleukin (IL) 22 promotes intestinal tumorigenesis in mice. IL22 binds the antagonist IL22 subunit alpha 2 (IL22RA2, also called IL22BP). We studied whether alterations in IL22BP contribute to colorectal carcinogenesis in humans and mice. Methods We obtained tumor and nontumor tissues from patients with colorectal cancer (CRC) and measured levels of cytokines by quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. We measured levels of Il22bp messenger RNA in colon tissues from wild-type, Tnf–/–, Lta–/–, and Ltb–/– mice. Mice were given azoxymethane and dextran sodium sulfate to induce colitis and associated cancer or intracecal injections of MC38 tumor cells. Some mice were given inhibitors of lymphotoxin beta receptor (LTBR). Intestine tissues were analyzed by single-cell sequencing to identify cell sources of lymphotoxin. We performed immunohistochemistry analysis of colon tissue microarrays from patients with CRC (1475 tissue cores, contained tumor and nontumor tissues) and correlated levels of IL22BP with patient survival times. Results Levels of IL22BP were decreased in human colorectal tumors, compared with nontumor tissues, and correlated with levels of lymphotoxin. LTBR signaling was required for expression of IL22BP in colon tissues of mice. Wild-type mice given LTBR inhibitors had an increased tumor burden in both models, but LTBR inhibitors did not increase tumor growth in Il22bp–/– mice. Lymphotoxin directly induced expression of IL22BP in cultured human monocyte–derived dendritic cells via activation of nuclear factor κB. Reduced levels of IL22BP in colorectal tumor tissues were associated with shorter survival times of patients with CRC. Conclusions Lymphotoxin signaling regulates expression of IL22BP in colon; levels of IL22BP are reduced in human colorectal tumors, associated with shorter survival times. LTBR signaling regulates expression of IL22BP in colon tumors in mice and cultured human dendritic cells. Patients with colorectal tumors that express low levels of IL22BP might benefit from treatment with an IL22 antagonist.

Background Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a receptor for prostaglandin D2 (PGD2), is expressed in human ILC2. However, the function of CRTH2 in these cells is unclear. Objectives We sought to determine the role of PGD2 and CRTH2 in human ILC2 and compare it with that of established ILC2 activators, IL-25 and IL-33. Methods The effects of PGD2, IL-25, IL-33 on the cell migration, cytokine production, gene regulation and receptor expression of ILC2 were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR and QuantiGene assays. The effects of PGD2 under physiological conditions were evaluated by using the supernatant from activated mast cells. Results PGD2 binding to CRTH2 induced ILC2 migration, production of type 2 cytokines and many other cytokines. ILC2 activation via CRTH2 also up-regulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD2 on ILC2 could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. Conclusions PGD2 is an important and potent activator of ILC2 via CRTH2 mediating strong pro-allergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges innate and adaptive pathways in human ILC2.

Background Group 2 innate lymphoid cells (ILC2s) are involved in the initial phase of type 2 inflammation and can amplify allergic immune responses by orchestrating other type 2 immune cells. Prostaglandin (PG) E2 is a bioactive lipid that plays protective roles in the lung, particularly during allergic inflammation. Objective We set out to investigate how PGE2 regulates human ILC2 function. Methods The effects of PGE2 on human ILC2 proliferation and intracellular cytokine and transcription factor expression were assessed by means of flow cytometry. Cytokine production was measured by using ELISA, and real-time quantitative PCR was performed to detect PGE2 receptor expression. Results PGE2 inhibited GATA-3 expression, as well as production of the type 2 cytokines IL-5 and IL-13, from human tonsillar and blood ILC2s in response to stimulation with a combination of IL-25, IL-33, thymic stromal lymphopoietin, and IL-2. Furthermore, PGE2 downregulated the expression of IL-2 receptor α (CD25). In line with this observation, PGE2 decreased ILC2 proliferation. These effects were mediated by the combined action of E-type prostanoid receptor (EP) 2 and EP4 receptors, which were specifically expressed on ILC2s. Conclusion Our findings reveal that PGE2 limits ILC2 activation and propose that selective EP2 and EP4 receptor agonists might serve as a promising therapeutic approach in treating allergic diseases by suppressing ILC2 function., Graphical abstract

Highlights • Type 2 immunity is associated with both helminth infection and responses to injury. • Pathways involved in tissue repair and helminth immunity overlap. • The IL-4Rα is central to accelerating both repair and helminth control. • Adaptive immunity contributes to more rapid wound repair., Metazoan parasites typically induce a type 2 immune response, characterized by T helper 2 (Th2) cells that produce the cytokines IL-4, IL-5 and IL-13 among others. The type 2 response is host protective, reducing the number of parasites either through direct killing in the tissues, or expulsion from the intestine. Type 2 immunity also protects the host against damage mediated by these large extracellular parasites as they migrate through the body. At the center of both the innate and adaptive type 2 immune response, is the IL-4Rα that mediates many of the key effector functions. Here we highlight the striking overlap between the molecules, cells and pathways that mediate both parasite control and tissue repair. We have proposed that adaptive Th2 immunity evolved out of our innate repair pathways to mediate both accelerated repair and parasite control in the face of continual assault from multicellular pathogens. Type 2 cytokines are involved in many aspects of mammalian physiology independent of helminth infection. Therefore understanding the evolutionary relationship between helminth killing and tissue repair should provide new insight into immune mechanisms of tissue protection in the face of physical injury.

Background Respiratory syncytial virus (RSV) is a major health care burden with a particularly high worldwide morbidity and mortality rate among infants. Data suggest that severe RSV-associated illness is in part caused by immunopathology associated with a robust type 2 response. Objective We sought to determine the capacity of RSV infection to stimulate group 2 innate lymphoid cells (ILC2s) and the associated mechanism in a murine model. Methods Wild-type (WT) BALB/c, thymic stromal lymphopoietin receptor (TSLPR) knockout (KO), or WT mice receiving an anti-TSLP neutralizing antibody were infected with the RSV strain 01/2-20. During the first 4 to 6 days of infection, lungs were collected for evaluation of viral load, protein concentration, airway mucus, airway reactivity, or ILC2 numbers. Results were confirmed with 2 additional RSV clinical isolates, 12/11-19 and 12/12-6, with known human pathogenic potential. Results RSV induced a 3-fold increase in the number of IL-13–producing ILC2s at day 4 after infection, with a concurrent increase in total lung IL-13 levels. Both thymic stromal lymphopoietin (TSLP) and IL-33 levels were increased 12 hours after infection. TSLPR KO mice did not mount an IL-13–producing ILC2 response to RSV infection. Additionally, neutralization of TSLP significantly attenuated the RSV-induced IL-13–producing ILC2 response. TSLPR KO mice displayed reduced lung IL-13 protein levels, decreased airway mucus and reactivity, attenuated weight loss, and similar viral loads as WT mice. Both 12/11-19 and 12/12-6 similarly induced IL-13–producing ILC2s through a TSLP-dependent mechanism. Conclusion These data demonstrate that multiple pathogenic strains of RSV induce IL-13–producing ILC2 proliferation and activation through a TSLP-dependent mechanism in a murine model and suggest the potential therapeutic targeting of TSLP during severe RSV infection., Graphical abstract

Background & Aims: Interleukin (IL)33 is a recently described alarmin that is highly expressed in the gastric mucosa and potently activates Th2 immunity. It may play a pivotal role during Helicobacter pylori infection. Here, we delineate the role of IL33 in the normal gastric mucosa and in response to gastropathy. Methods: IL33 expression was evaluated in mice and human biopsy specimens infected with H pylori and in mice after dosing with aspirin. IL33 expression was localized in the gastric mucosa using immunofluorescence. Mice were given 1 or 7 daily doses of recombinant IL33 (1 μg/dose), and the stomach and the spleen responses were quantified morphologically, by flow cytometry and using quantitative reverse-transcription polymerase chain reaction and immunoblotting. Results: In mice, the IL33 protein was localized to the nucleus of a subpopulation of surface mucus cells, and co-localized with the surface mucus cell markers Ulex Europaeus 1 (UEA1), and Mucin 5AC (Muc5AC). A small proportion of IL33-positive epithelial cells also were Ki-67 positive. IL33 and its receptor Interleukin 1 receptor-like 1 (ST2) were increased 4-fold after acute (1-day) H pylori infection, however, this increase was not apparent after 7 days and IL33 expression was reduced 2-fold after 2 months. Similarly, human biopsy specimens positive for H pylori had a reduced IL33 expression. Chronic IL33 treatment in mice caused systemic activation of innate lymphoid cell 2 and polarization of macrophages to the M2 phenotype. In the stomach, IL33-treated mice developed transmural inflammation and mucous metaplasia that was mediated by Th2/signal transducer and activator of transcription 3 signaling. Rag-1-/- mice, lacking mature lymphocytes, were protected from IL33-induced gastric pathology. Conclusions: IL33 is highly expressed in the gastric mucosa and promotes the activation of T helper 2âcytokineâexpressing cells. The loss of IL33 expression after prolonged H pylori infection may be permissive for the T helper 1âbiased immune response observed during H pylori infection and subsequent precancerous progression. Keywords: IL33, Helicobacter pylori, Inflammatory Response, Gastric Cancer

Background: The IL-1 family cytokine IL-33 is involved in the induction of airway inflammation in allergic patients and after viral infection. Several cell types, including CD4 1 TH2 cells and the recently described type 2 innate lymphoid cells (ILCs), are targets for IL-33, yet the mechanisms by which this cytokine modulates their activation are not clear. Objectives: Our goal was to investigate a role for mammalian target of rapamycin (mTOR) signaling in the activation of TH2 and ILC responses and the induction of airway inflammation by IL-33. Methods: We biochemically determined the effect of IL-33 on mTOR activation in TH2 cells and ILCs and examined the effect of this signaling pathway in vivo using a murine model of IL-33‐induced lung inflammation. Results: We found that IL-33 induces mTOR activation through p110d phosphoinositide 3-kinase and that blockade of the mTOR pathway inhibited IL-33‐induced IL-5 and IL-13 production by TH2 cells and ILCs. Furthermore, use of a ribosomal protein S6 kinase 1 inhibitor implicated a role for ribosomal protein S6 kinase 1 in IL-33‐induced mTOR-dependent cytokine production. Intranasal administration of IL-33 to wild-type mice induced airway inflammation, whereas adoptive transfer of wildtype ILCsto IL-33receptor‐deficient(St2 2/2 )mice recapitulated this response. Importantly, coadministration of the mTOR inhibitor rapamycin reduced IL-33‐dependent ILC, macrophage, and eosinophil accumulation; cytokine secretion; and mucus deposition in the airways. Conclusion: These data reveal a hitherto unrecognized role of mTOR signaling in IL-33‐driven, ILC-dependent inflammation in vivo and suggest that manipulation of this pathway might represent a target for therapeutic intervention for airway inflammation. (J Allergy Clin Immunol 2012;130:1159-66.)

Background & Aims Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation. Methods ILCs were isolated from colons of Tbx21-/- × Rag2-/- mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota. Results IL17A- and IL22-producing, natural cytotoxicity receptor-negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor-positive cells, in a dose-dependent manner. Conclusions IL6 contributes to activation of colonic natural cytotoxicity receptor-negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor-positive cells.