Mucosal Organs Research Articles

Serum IL-33 as a biomarker in different diseases: useful parameter or much need for clarification?

Interleukin-33 (IL-33), a member of the IL-1 family, is critically involved in the modulation of the activity of a diverse range of immunocompetent cells. Essential roles have been implicated in cardioprotection, in both innate and adaptive immune responses in mucosal organs, and in the maintenance of adipose tissue cells. Over the past 10 years, several studies evaluated the usability of IL-33 as a biomarker in diseases of inflammatory and noninflammatory origin. Our group is currently evaluating the predictive role of serum IL-33 in acute kidney injury (AKI). The aim of the article is to discuss selected studies on IL-33 in different diseases and its potential role as a biomarker molecule.

CD4 T Cell-Mediated Immune Control of Cytomegalovirus Infection in Murine Salivary Glands.

CD4 T cells are well known for their supportive role in CD8 T cell and B cell responses during viral infection. However, during murine cytomegalovirus (MCMV) infection in the salivary glands (SGs), CD4 T cells exhibit direct antiviral effector functions to control the infection. In this mucosal organ, opposed to other infected tissues, MCMV establishes a sustained lytic replication that lasts for several weeks. While the protective function of CD4 T cells is exerted through the production of the pro-inflammatory cytokines interferon gamma (IFNγ) and tumor necrosis factor alpha (TNF), the reasons for their markedly delayed control of lytic MCMV infection remain elusive. Here, we review the current knowledge on the dynamics and mechanisms of the CD4 T cell-mediated control of MCMV-infected SGs, including their localization in the SG in relation to MCMV infected cells and other immune cells, their mode of action, and their regulation.

Beyond Immunity: Underappreciated Functions of Intestinal Macrophages.

The gastrointestinal tract hosts the largest compartment of macrophages in the body, where they serve as mediators of host defense and immunity. Seeded in the complex tissue-environment of the gut, an array of both hematopoietic and non-hematopoietic cells forms their immediate neighborhood. Emerging data demonstrate that the functional diversity of intestinal macrophages reaches beyond classical immunity and includes underappreciated non-immune functions. In this review, we discuss recent advances in research on intestinal macrophage heterogeneity, with a particular focus on how non-immune functions of macrophages impact tissue homeostasis and function. We delve into the strategic localization of distinct gut macrophage populations, describe the potential factors that regulate their identity and functional heterogeneity within these locations, and provide open questions that we hope will inspire research dedicated to elucidating a holistic view on macrophage-tissue cell interactions in the body’s largest mucosal organ.

The Role of Innate Lymphoid Cells in Chronic Respiratory Diseases.

The lung is a vital mucosal organ that is constantly exposed to the external environment, and as such, its defenses are continuously under threat. The pulmonary immune system has evolved to sense and respond to these danger signals while remaining silent to innocuous aeroantigens. The origin of the defense system is the respiratory epithelium, which responds rapidly to insults by the production of an array of mediators that initiate protection by directly killing microbes, activating tissue-resident immune cells and recruiting leukocytes from the blood. At the steady-state, the lung comprises a large collection of leukocytes, amongst which are specialized cells of lymphoid origin known as innate lymphoid cells (ILCs). ILCs are divided into three major helper-like subsets, ILC1, ILC2 and ILC3, which are considered the innate counterparts of type 1, 2 and 17 T helper cells, respectively, in addition to natural killer cells and lymphoid tissue inducer cells. Although ILCs represent a small fraction of the pulmonary immune system, they play an important role in early responses to pathogens and facilitate the acquisition of adaptive immunity. However, it is now also emerging that these cells are active participants in the development of chronic lung diseases. In this mini-review, we provide an update on our current understanding of the role of ILCs and their regulation in the lung. We summarise how these cells and their mediators initiate, sustain and potentially control pulmonary inflammation, and their contribution to the respiratory diseases chronic obstructive pulmonary disease (COPD) and asthma.

TLR3-driven IFN-β antagonizes STAT5-activating cytokines and suppresses innate type 2 response in the lung

Group 2 innate lymphoid cells (ILC2s) are involved in type 2 immune responses in mucosal organs and are associated with various allergic diseases in humans. Studies are needed to understand the molecules and pathways that control ILC2s. The aims of this study were to develop a mouse model that limits the innate type 2 immune response in the lung and to investigate the immunologic mechanisms involved in regulation of lung ILC2s. Naive BALB/c mice were administered various Toll-like receptor agonists and exposed intranasally to the fungal allergen Alternaria alternata. The mechanisms were investigated using gene knockout mice as well as cultures of lung cells and isolated lung ILC2s. Polyinosinic-polycytidylic acid, or poly (I:C), effectively inhibited innate type 2 response to A alternata. Poly (I:C) promoted production of IFNα, -β, and -γ, and its inhibitory effects were dependent on the IFN-α/β receptor pathway. IFN-β was 100 times more potent than IFN-α at inhibiting type 2 cytokine production by lung ILC2s. Signal transducer and activator of transcription 5 (STAT5)-activating cytokines, including IL-2, IL-7, and thymic stromal lymphopoietin, but not IL-33, promoted survival and proliferation of lung ILC2s invitro, while IFN-β blocked these effects. Expression of the transcription factor GATA3, which is critical for differentiation and maintenance of ILC2s, was inhibited by IFN-β. IFN-β blocks the effects of STAT5-activating cytokines on lung ILC2s and inhibits their survival and effector functions. Administration of IFN-β may provide a new strategy to treat diseases involving ILC2s.

Th cell transcription factors: Sequence characteristics and expression profiles in Epinephelus coioides after Cryptocaryon irritans infection

Mammalian T cell subset differentiation is strictly controlled by cytokines and transcription factors. T-bet, GATA-3, BCL-6 and Foxp3 are considered the key transcription factors of Th1, Th2, Tfh and Treg cells. Here, we identified the genes and predicted the protein structure of these Th cell transcription factors in the orange-spotted grouper (Epinephelus coioides). The functional domains and 3D-models of grouper T-bet, GATA-3, BCL-6 and Foxp3 all had a degree of identity with that of mammals. Tissue distribution analysis in healthy groupers demonstrated that T-bet, BCL-6 and Foxp3 were highly expressed in central lymphoid organs, whereas GATA-3 was primarily expressed in mucosal organs. After infection with the parasite Cryptocaryon irritans, in both head kidney and gills, grouper T-bet expression increased from 12 h to 1 day, and GATA-3 expression was up-regulated from day 2, suggesting that the response of grouper Th1 cells was earlier than that of Th2 cells. Furthermore, Treg cells were involved in immunotolerance from day 7 in the head kidney after parasite infection owing to the up-regulated expression of Foxp3. In brief, our study described the response characteristics of T cell transcription factors in groupers and provided a theoretical basis for subsequent immunological control.

Host-Microbiome Interaction in Lung Cancer.

Commensal microbiota has emerged as an essential biomarker and regulator of both tumorigenesis and response to cancer therapy. However, our current knowledge about microbiota in cancer has been largely limited to intestinal microbiota. As a mucosal organ harboring one of the largest surface areas in the body, the lung is exposed to a variety of microbes through inhalation and micro-aspiration, and is colonized by a diverse bacterial community in both physiological and pathological conditions. Importantly, increasing evidence has linked the lung microbiome to cancer development. Studies in lung cancer patients and mouse models have revealed tumor-associated dysregulation of the local microbiome in the lung, which in turn impacts cancer progression by shaping the tumor microenvironment and modulating the activity of tumor-infiltrating immune cells. These findings not only provide novel mechanistic insight into the biology of lung cancer but also shed light on new therapeutic targets and strategies for lung cancer prevention and treatment. The goal of this review is to discuss the key findings, remaining questions, and future directions in this new and exciting field.

Characterization of the Pelodiscus sinensis polymeric immunoglobulin receptor (P. sinensis pIgR) and its response to LPS and Aeromonas sobria

The polymeric immunoglobulin receptor (pIgR) is one of the most vital components of mucosal immunity that plays a pivotal role in mediating transcytosis of polymeric immunoglobulin (pIg) on epithelial surfaces for protection against invading pathogens. Herein, we cloned the full-length cDNA of Pelodiscus sinensis pIgR, designated as P. sinensis pIgR, made of an open reading frame (ORF) of 1848 bp, molecular weight of 68.2 kDa and estimated isoelectric point of 7.00. The deduced P. sinensis pIgR sequence had a leader peptide, extracellular region containing four immunoglobulin-like domains (Ig like domains), transmembrane and intracellular regions comparable with other vertebrates. P. sinensis pIgR contained four Ig like domains that corresponded with mammalian D1, D3, D4 and D5 similar with reptile and avian Ig like domains. It had 40 potential phosphorylation sites, four putative N-glycosylation sites and several motifs resembling mammalian pIgR motifs. Phylogenetic analysis showed a close relationship between P. sinensis pIgR with avian and reptile pIgRs. P. sinensis pIgR basal levels were higher in the esophagus, small intestine and intestinnum crissum than in other organs of health turtles. Intragastric delivery of LPS and Aeromonassobria led to significant upregulation of P. sinensis pIgR in tissues of the gastrointestinal tract. A polyclonal anti- P. sinensis pIgR antibody produced in rabbit reacted with the recombinant P. sinensis pIgR protein expressed in Escherichia coli in Western blot. These studies demonstrate the existence and immune response of P. sinensis pIgR to stimulation in mucosal organs in Chinese soft-shelled turtles.

Expansible residence decentralizes immune homeostasis.

Metazoans relegate specific tasks to dedicated organs that are established early in development, occupy discrete anatomic locations, and typically remain fixed in size. The adult immune system arises from a centralized hematopoietic niche that maintains self-renewing potential1,2, and upon maturation, becomes distributed throughout the body to monitor environmental perturbations, regulate tissue homeostasis, and mediate organism-wide defense. This study examines how immunity is integrated within adult mouse tissues while addressing issues of durability, expansibility, and contribution to organ cellularity. Focusing on antiviral T cell immunity, we observe durable maintenance of resident memory T cells (TRM) up to 450 days after infection. Once established, resident T cells did not require the T cell receptor for survival or retention of a poised effector-like state. While resident memory indefinitely dominated most mucosal organs, surgical separation of parabiotic mice unexpectedly revealed a tissue-resident provenance for bloodborne effector memory T cells, and circulating memory slowly made substantial contributions to tissue immunity in some organs. Following additional microbial experiences via serial immunizations or pet shop mice co-housing, for most tissues we find tissue pliancy allows for the accretion of tissue-resident memory, without axiomatic erosion of preexisting antiviral T cell immunity. Extending these findings, we demonstrate tissue residence and organ pliancy are generalizable aspects underlying the homeostasis of innate and adaptive immunity. The immune system-at-large grows commensurate to microbial experience reaching up to 25% of visceral organ cellularity. Regardless of location, many white blood cell populations adopted a tissue residency program within nonlymphoid organs. Thus, residence, rather than renewal or recirculation, typifies nonlymphoid immune surveillance, and organs serve as a pliant storage reservoir that can accommodate the continuous expansion of the cellular immune system throughout life. While hematopoiesis (‘to make blood’) restores certain elements of the immune system, in parallel, nonlymphoid organs sustain an accrual of durable tissue-autonomous cellular immunity, resulting in the progressive decentralization of organismal immune homeostasis.

Primary tumors from mucosal barrier organs drive unique eosinophil infiltration patterns and clinical associations

ABSTRACT Eosinophils are bone marrow-derived granulocytes that display key effector functions in allergic diseases. Nonetheless, recent data highlight important roles for eosinophils in the tumor microenvironment (TME). Eosinophils have been attributed with pleiotropic and perhaps conflicting functions, which may be attributed at least in part to variations in eosinophil quantitation in the TME. Thus, a reliable, quantitative, and robust method for the assessment of eosinophilic infiltration in the TME is required. This type of methodology could standardize the identification of these cells and promote the subsequent generation of hypothesis-driven mechanistic studies. To this end, we conducted a comprehensive analysis of multiple primary tumors from distinct anatomical sites using a standardized method. Bioinformatics analysis of 10,469 genomically profiled primary tumors revealed that eosinophil abundance within different tumors can be categorized into three groups representing tumors with high, intermediate, and low eosinophil levels. Consequently, eosinophil abundance, as well as spatial distribution, was determined in tissue tumor arrays of six tumors representing all three classifications (colon and esophagus – high; lung – intermediate; cervix, ovary, and breast – low). With the exception of breast cancer, eosinophils were mainly localized in the tumor stroma. Importantly, the tumor anatomical site was identified as the primary predictive factor of eosinophil stromal density highlighting a distinction between mucosal-barrier organs versus non-mucosal barrier organs. These findings enhance our understanding of eosinophil diversity in the TME and provide a compelling rationale for future experiments assessing the activity of these cells.

Bacillus subtilis Inhibits Viral Hemorrhagic Septicemia Virus Infection in Olive Flounder (Paralichthys olivaceus) Intestinal Epithelial Cells

Viral hemorrhagic septicemia virus (VHSV) is a highly pathogenic virus that infects a wide range of host fish species causing high economic losses in aquaculture. Epithelial cells in mucosal organs are target sites for VHSV entry into fish. To protect fish against VHSV infection, there is a need to develop antiviral compounds able to prevent establishment of infection at portals of virus entry into fish. Bacillus subtilis is a probiotic with excellent antiviral properties, of which one of its secretions, surfactin, has been shown to inhibit viral infections in mammals. Herein, we demonstrate its ability to prevent VHSV infection in olive flounder (Paralichthys olivaceus) intestinal epithelial cells (IECs) and infection in internal organs. Our findings show inhibition of VHSV infection in IECs by B. subtilis and surfactin. In addition, our findings showed inhibition of VHSV in Epithelioma Papulosum Cyprini (EPC) cells inoculated with intestinal homogenates from the fish pretreated with B. subtilis by oral exposure, while the untreated fish had cytopathic effects (CPE) caused by VHSV infection in the intestines at 48 h after the VHSV challenge. At 96 h post-challenge, samples from the untreated fish had CPE from head kidney and spleen homogenates and no CPE were observed in the intestinal homogenates, while the B. subtilis-pretreated fish had no CPE in all organs. These findings demonstrate that inhibition of VHSV infection at portals of virus entry in the intestines culminated in prevention of infection in internal organs. In summary, our results show that B. subtilis has the potential to prevent VHSV infection in fish and that its use as a probiotic in aquaculture has the potential to serve as an antiviral therapeutic agent against different viral infections.

POSTBIOTIC AND OTHER PROTECTIVE MOLECULES RECOGNIZING AND BINDING GLYCOCONJUGATES IN MUCOSAL ORGANS

New aspects of human mucosal cavity protection systems are under consideration. On the basis of own data it was suggested that mucosal cavities function as mucosal organs. New players of mucosal organ were proposed. They included system probiotic lectins and probiotic microorganisms, and system synthetic polymeric multivalence pattern glycoconjugates. Lectins and glycoconjugates (mucin-like or imitating, antigens, polysaccharides and other high molecular mass metabolites and postbiotics) were suggested to communicate to other glycoconjugates-recognizing molecules and receptors of the human higher hierarchic protection systems.
 Concept of functioning mucosal organs is supported by own proposals, strategies, approaches, methods and algorithms. The data are useful for constructing biotope ordered pro/synbiotic microbiocenoses for application in medical biotechnology.

Optimized Protocol for Characterization of Mouse Gut Innate Lymphoid Cells.

Since their discovery, innate lymphoid cells (ILCs) have gradually been gaining greater relevance in the field of immunology due to their multiple functions in the innate immune response. They can mainly be found in mucosal and barrier organs like skin, gut, and lungs, and have been classified into five main types (NKs, ILC1s, ILC2s, ILC3s, and Lti cells) according to their function and development. They all play major roles in functions such as tissue homeostasis, early pathogen defense, regulation of inflammation, or tissue remodeling. ILCs are mostly tissue-resident cells tightly bound to the tissue structure, a fact that requires long and complex protocols that do not always provide sufficient yield for analysis. This suggests the need for optimized approaches aimed at ensuring that enriched and viable ILC samples are obtained, in order to furnish quality results. Herein a detailed protocol is established for obtaining a single-cell suspension highly enriched in lymphoid cells from mouse gut in order to identify the different subsets of ILCs by means of flow cytometry. The cell marker panel and flow cytometry gating strategies for identification and quantification of all the different ILC populations are provided for simultaneous analysis. Moreover, the protocol described includes a procedure for studying the different cytokines produced by ILC3s involved in maintaining the integrity of the gut barrier and defending against extracellular pathogens. As a result, herein an efficient method is presented for studying mouse ILCs within the lamina propria of the small intestine and colon; this can constitute a useful tool for future investigations in the field.

Selective Bacterial Colonization of the Murine Larynx in a Gnotobiotic Model.

The larynx is a mucosal organ situated between the respiratory and gastrointestinal tracts. Little is known about microbial contributions to laryngeal epithelial health and pathogenesis. Developing a gnotobiotic laryngeal model will introduce new avenues for targeted explorations of microbes in laryngeal mucosal biology, allowing for enhanced understanding of host–microbe interaction in the upper airway. In this study, we first assessed the potential of using gut microbiota as a source to establish laryngeal microbiota in germ-free mice. Results demonstrated the selective nature of the upper airway and provided evidence that gut bacteria can assemble into communities that resemble the commensal resident bacteria occurring in the larynx of conventionally-raised animals phylogenetically and functionally. Then, we confirmed the reproducibility of laryngeal colonization through comparison of laryngeal microbiota in the larynx along with neighboring regions (base of tongue, esophagus, and trachea) between conventionally-raised and germ-free mice that conventionalized with cecal microbiota. Despite taxonomic differences, the established laryngeal microbiota from cecal content exhibited similarity to commensal resident microbiota in diversity within/between communities and predicted metagenomic functions. Our data also suggests little difference in bacterial distribution across the larynx and its surrounding regions and that cell motility and the ability to degrade xenobiotics is critical for bacteria colonizing upper airway. Successful colonization of laryngeal and oropharyngeal regions with gut microbiota in our study will greatly facilitate the investigation of potential localized inflammatory responses within host tissues that contribute to the disorders of essential laryngeal functions. Utilizing said gnotobiotic model to conduct future studies will allow for novel insights into direct microbial contributions to laryngeal epithelial health and pathogenesis.

Effect of booster vaccination on immunoprotection in European seabass vaccinated against vibriosis

In farmed saltwater fish, vaccination is considered the first barrier against pathogenic agents. In Egypt, the vaccine application can diminish the dilemma of summer mass kills of farmed marine fish. The efficacy of booster vaccination with a bivalent vaccine against vibriosis was studied in European seabass. Fish were vaccinated by direct immersion (DI) in water containing bivalent Vibrio alginolyticus and Vibrio parahemolyticus whole cells killed vaccine. Immersion vaccination is more applicable under semi-intensive fish culture, but less potent than injection. Accordingly, to increase antigen uptake and conferred protection, vaccinated fish randomly received booster vaccination via direct immersion (DI imm.) or intraperitoneal injection (i.p.) at 21 days post-prime vaccination (21 dpv). Vaccinated and unvaccinated fish were monitored for 3 months and challenged with V. alginolyticus and/or V. parahemolyticus at 42 dpv. Specific and non-specific immune responses were evaluated in blood samples. At 52 dpv (10 days post-challenge), the relative immunoglobulin T (IgT) expression was assessed in the gills, hindgut, head kidney and spleen. Notably, imm. + imm. and imm. + i.p. vaccination yielded significantly higher specific antibody titre at 70 dpv, whereas the former was associated with significantly higher IgT expression in the gills and hindgut (p < .05) than the latter. High IgT levels in mucosal organs (gills and hindgut) might be the first indicator of the involvement of IgT in mucosal immune responses. Relative per cent survival among booster-vaccinated fish ranged from 70% to 90%. The results support the use of this vaccination protocol as a highly effective tool to protect seabass from vibriosis and to improve fish health and environment. Booster doses are strongly recommended under farming environments.

Remote allergen exposure elicits eosinophil infiltration into allergen nonexposed mucosal organs and primes for allergic inflammation.

The natural history of allergic diseases suggests bidirectional and progressive relationships between allergic disorders of the skin, lung and gut indicative of mucosal organ cross-talk. However, impacts of local allergic inflammation on the cellular landscape of remote mucosal organs along the skin:lung:gut axis are not yet known. Eosinophils are tissue-dwelling innate immune leukocytes associated with allergic diseases. Emerging data suggest heterogeneous phenotypes of tissue-dwelling eosinophils contribute to multifaceted roles that favor homeostasis or disease. This study investigated the impact of acute local allergen exposure on the frequency and phenotype of tissue eosinophils within remote mucosal organs. Our findings demonstrate allergen challenge to skin, lung or gut elicited not only local eosinophilic inflammation, but also increased the number and frequency of eosinophils within remote, allergen non-exposed lung and intestine. Remote allergen-elicited lung eosinophils exhibited an inflammatory phenotype and their presence associated with enhanced susceptibility to airway inflammation induced upon subsequent inhalation of a different allergen. These data demonstrate, for the first time, a direct effect of acute allergic inflammation on the phenotype and frequency of tissue eosinophils within antigen non-exposed remote mucosal tissues associated with remote organ priming for allergic inflammation.

Candidiasis and Mechanisms of Antifungal Resistance.

Candidiasis can be present as a cutaneous, mucosal or deep-seated organ infection, which is caused by more than 20 types of Candida sp., with C. albicans being the most common. These are pathogenic yeast and are usually present in the normal microbiome. High-risk individuals are patients of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), organ transplant, and diabetes. During infection, pathogens can adhere to complement receptors and various extracellular matrix proteins in the oral and vaginal cavity. Oral and vaginal Candidiasis results from the overgrowth of Candida sp. in the hosts, causing penetration of the oral and vaginal tissues. Symptoms include white patches in the mouth, tongue, throat, and itchiness or burning of genitalia. Diagnosis involves visual examination, microscopic analysis, or culturing. These infections are treated with a variety of antifungals that target different biosynthetic pathways of the pathogen. For example, echinochandins target cell wall biosynthesis, while allylamines, azoles, and morpholines target ergosterol biosynthesis, and 5-Flucytosine (5FC) targets nucleic acid biosynthesis. Azoles are commonly used in therapeutics, however, because of its fungistatic nature, Candida sp. evolve azole resistance. Besides azoles, Candida sp. also acquire resistance to polyenes, echinochandins, and 5FC. This review discusses, in detail, the drug resistance mechanisms adapted by Candida sp.

Abstract A62: Directly visualizing interactions between lung microbiota, tissue-resident immune cells and tumor cells in a genetically engineered lung adenocarcinoma mouse model

Abstract Lung adenocarcinoma is closely associated with chronic inflammation, but the underlying mechanism has not been fully elucidated yet. As a mucosal organ, lung was thought to be sterile in the past due to the limitation of bacteria culture techniques, but recent 16s RNA sequencing analysis has proved that lung is colonized by a diverse bacterial community. Recent studies suggest that the dysbiosis of lung microbiota plays a critical role in lung cancer tumorigenesis. However, there is no direct evidence addressing the complex interactions between local microbiota, immune cells and tumor cells due to the limitation of traditional imaging methods. Here we developed an advanced imaging technique combining RNA fluorescent in situ hybridization, immunostaining and Clearing-enhanced 3D (Ce3D), and applied it in a genetically engineered mouse model harboring the most common mutations (activation of Kras and p53 loss of function) in patients with lung adenocarcinoma. It revealed a strong spatial-temporal correlation between lung microbiota and tissue-resident immune cells during lung adenocarcinoma development. This research was supported by the Intramural Research Programs of NIAID and NHLBI, NIH. Citation Format: Chen Zhao, Weizhe Li, Chengcheng Jin, Ronald N. Germain. Directly visualizing interactions between lung microbiota, tissue-resident immune cells and tumor cells in a genetically engineered lung adenocarcinoma mouse model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A62.

Gastrin release in a mucosal organ culture model exposed to a simulated digest of an equine dietary supplement

Horses are prone to GI diseases including colic, colitis, and gastric ulcers. Equine dietary supplements are commonly used as a means of supporting optimal equine gastrointestinal health. G’s Formula (manufactured by G’s Organic Solutions INC., BC, CAN) is an equine dietary supplement composed of dried cabbage, carrot, oat flour, and hemp meal, formulated to promote optimal GI health in horses. The objective of the current study was to investigate the impact of a simulated gastric digest of GF on gastrin (Gt) release in an in vitro mucosal organ culture model. Porcine stomachs were collected from a local abattoir (Reist and Weber, ON, CAN). A section of mucosa from the pyloric antrum was isolated and the smooth muscle removed. A 4mm punch biopsy was used to divide the mucosa, and 2 biopsies were placed into each well of a 24‐well culture dish. Each well was filled with 1.5 ml of media supplemented with glutamine. Media was changed at 24h, at which time 100 ul of either a simulated gastric digest of GF (GF), a simulated gastric digest blank (BL), or PBS (CO) was added into sample wells. Media and digests were refreshed at 48h, at which time wells were either stimulated with 10−5M carbachol (GF‐s, n=12; BL‐s, n=12; CO‐s, n=11) or sham stimulated with PBS (GF‐n, n=9; BL‐n, n=13; CO‐n, n=10). This process was repeated at 60h, and the experiment was terminated at 72h. Gt release in the media was assessed using a porcine Gt ELISA (MyBioSource INC., SD, USA) and standardized /g tissue. Data was analyzed using a repeated measures ANOVA in SAS 9.4 using PROC GLIMMIX (SAS Institute, NC, USA). There was a difference between the treatments GF and BL (16534±797 vs 14072±723 pg/ml/g, p=0.03) and a trend towards a difference between GF and CO (14581±794 pg/ml/g, p=0.09). At 60h GF‐n was higher than CO‐n (17475±1784 vs 12164±1688 pg/ml/g) and GF‐s was higher than BL‐s (16903±1543 vs 12434±1544 pg/ml/g). GF‐s was higher at 72h compared to 48h (18289±1543 vs 14051±1543, p=0.03). In conclusion, a simulated gastric digest of GF added into an in vitro organ culture of porcine pyloric antrum appears to enhance tissue Gt release.Support or Funding InformationThis project was funded by G’s Organic Solutions INC.J.L. MacNicol was funded by NSERC.

Acute cellular rejection in small-bowel transplantation impairs NCR+ innate lymphoid cell subpopulation 3/interleukin 22 axis

Acute cellular rejection (ACR) remains as one of the main causes of graft loss and death in intestinal transplant (ITx) patients. ACR promotes intestinal injury, disruption of the mucosal barrier, bacterial translocation, and organ dysfunction. As epithelial regeneration is critical in reversing these consequences, the functional axis between the innate lymphoid cell subpopulation 3 (ILC3) and interleukin 22 plays an essential role in that process. Natural-cytotoxic-receptor–positive (NCR+) ILC3 cells have been demonstrated to induce intestinal-stem-cell proliferation along with an IL-22–dependent expansion of that population in several intestinal pathologies, though thus far not after ITx. Therefore, we intended to determine the impact of chronic immunosuppression and ACR on ILC3 cells and interleukin-22 (IL-22) production in the lamina propria after that intervention. Materials and methodsWe compared biopsies from healthy volunteers with biopsies from ITx recipients without or with mild-to-moderate ACR, using flow cytometry and the quantitative-PCR. ResultsNCR+ ILC3 cells were found to be unaffected by immunosuppression at different time points posttransplant when patients did not experience ACR, but were diminished upon the occurrence of ACR independently of the post-ITx time. Moreover, IL-22–expression levels were notably reduced in ACR. ConclusionThe NCR+-ILC3/IL-22 axis is impaired during ACR contributing to a delay in or lack of a complete and efficient epithelial regeneration. Thus, our findings reveal that IL-22 analogues could potentially be used as a new complementary therapeutic approach, in conjunction with immunosuppressant drugs, in order to promote mucosal regeneration upon ACR.