Chronic Intestinal Inflammation Research Articles

SLC44A4 Is the Predominant Transport System for Uptake of the Microbiota-Generated Phosphorylated Form of Vitamin B1 (Thiamin Pyrophosphate; TPP) in Colonocytes: Role in Colonocyte's Physiology

Background: Humans/mammals obtain vitamin B1 from diet and the gut-microbiota. Considerable amount of the microbiota-generated vitamin B1 exists in the form of TPP. We have previously shown that colonocytes are capable of absorbing intact TPP via a specific and effcient carrier-mediated process ( JBC 289:4405-16, 2014); we have also identified the SLC44A4 as a colonic TPP transporter (cTPPT) in both human and mouse and have characterized different aspects of its regulation and cell biology ( Plos One 14: e0224234, 2019). Little, however, is known about the relative contribution of the cTPPT toward total colonic carrier-mediated TPP uptake. Aims: To determine the relative contribution of cTPPT toward total colonic carrier-mediated TPP uptake, and to investigate the effect of its knockout/down on colonocyte's physiology. Methods: An Slc44a4 knockout (KO) mouse model was generated by crossing homozygous Slc44a4 LoxP (+/+) animals with CMV promoter-driven Cre-transgenic mice. Knocking down the SLC44A4 in human colonic epithelial CCD841 cells and in human primary differentiated colonocyte monolayers (from organ donors) was achieved using gene-specific siRNAs. [3H]-TPP was used in uptake studies. Results: Body weight and colonic length of the Slc44a4 KO mice were significantly lower than those of WT-littermates. Near complete (and specific) inhibition in colonic carrier-mediated [3H]-TPP uptake was observed in Slc44a4 KO mice compared to WT-littermates. Similarly, knocking down SLC44A4 of human CCD841 cells and that of human primary colonocyte monolayers led to significant reduction in [3H]-TPP uptake. Since colonocytes have limited ability to synthesize TPP endogenously [they express a very low level of the enzyme thiamin pyrophosphokinase; BBA 1517:320-2, 2001] and that recent genomic studies have suggested that the SLC44A4 gene could be a potential “ulcerative colitis susceptibility” gene ( Genes Immun 17:105-9, 2016), we also examined the effect of Slc44a4 KO on colonocytes physiology. Results of our RNA-seq and Ingenuity Pathway Analysis showed an increase in several genes related to chronic colitis and intestinal inflammation, and changes in genes involved in energy metabolism. Also, the Slc44a4 KO mice were found to be more susceptible to developing colitis when exposed to a low dose of dextran sodium sulfate compared to their WT-littermates. Conclusion: The SLC44A4 is the predominant/only transport system for uptake of the microbiota-generated TPP and that its loss compromise normal colonocyte's physiology. Supported by NIH grants DK-56061 & AA-018071, and VA grants 101BX001142 & IK6BX006189. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mesenteric adipose-derived exosomal TINAGL1 enhances intestinal fibrosis in Crohn's Disease via SMAD4

IntroductionCrohn's Disease (CD) is a chronic inflammatory condition characterized by intestinal fibrosis, severely impacting patient quality of life. The molecular mechanisms driving this fibrosis remain inadequately understood. Recent evidence implicates mesenteric adipose tissue (MAT) in CD pathogenesis, particularly through its exosome secretion, which may influence fibrogenic pathways. Understanding the role of MAT-derived exosomes is crucial for unraveling these molecular processes. ObjectivesThis study aims to elucidate the role of MAT-derived exosomes in CD-related intestinal fibrosis. We focus on investigating their molecular composition and the potential impact on fibrosis progression, with an emphasis on identifying novel therapeutic targets. MethodsWe induced chronic intestinal inflammation in mice using dinitrobenzene sulfonic acid (DNBS), simulating CD-like fibrosis. Exosomes were isolated from DNBS-treated mice (MG) and normal controls (NG) for characterization using electron microscopy and proteomic analysis. Additionally, human colonic fibroblasts were exposed to exosomes from CD patients and healthy individuals, with subsequent assessment of fibrogenesis through proteomic and RNA sequencing analyses. ResultsProteomic analyses revealed a significant activation of the TGF-β signaling pathway in MG-treated mice compared to controls, correlating with enhanced intestinal fibrosis. In vitro experiments demonstrated that colonic fibroblasts exposed to CD patient-derived exosomes exhibited increased fibrogenic activity. Protein docking and co-immunoprecipitation studies suggested a critical interaction between TINAGL1 and SMAD4, enhancing fibrosis. Importantly, in vivo experiments corroborated that recombinant TINAGL1 protein exacerbated DNBS-induced intestinal fibrosis. ConclusionOur findings highlight the pivotal role of MAT-derived exosomes, particularly those carrying TINAGL1, in the progression of intestinal fibrosis in CD. The involvement of the TGF-β signaling pathway, especially the SMAD4 protein, offers new insights into the molecular mechanisms of CD-related fibrosis and presents potential targets for therapeutic intervention.

Quiescent ulcerative colitis and Crohn’s disease have potential effect on cognitive function

BackgroundUlcerative colitis (UC) and Crohn’s disease (CD) imply chronic intestinal inflammation with both local and systemic manifestations. Cognition is a lifelong process of learning and memory processing, which has been identified to be affected by chronic systemic illnesses.AimTo evaluate the cognitive functions in inflammatory bowel disease (IBD) patients in remissionPatients and methodsInclusion criteria are as follows: 70 IBD patients in remission and a group of 50 healthy control. Mini-mental state examination (MMSE) was used for assessment of global cognitive function and Trail Making Test (TMT) for assessment of executive functions. TMT consists of part A which measures attention and performance speed and part B which measures mental flexibility. Both tests have been previously validated on Arabic-speaking populations.ResultsThe study included 70 patients (50 UC and 20 CD), who have been in remission for 21 ± 9 months. Of the included cases, about 51 were already on steroids ± azathioprine, and 19 patients were on biologics. Males represented 60% (n = 42) while females 40% (n = 28), and their mean age was 34 ± 8 years. Both cases and controls were matched for age and sex. The mean score of MMSE among IBD cases was significantly worse than controls (28.5 ± 3 versus 30, P < 0.001). Also, the duration of TMT parts A and B was significantly longer in cases than controls. The cases group scored 32 ± 19.5 s in TMT part A, versus 23 s by the controls. In TMT B, the recorded scores were 255 ± 48, versus 234 s in cases and control groups respectively (P < 0.001). In the cases group, 6 patients (8%) had below normal MMSE score of less than 24, with mild (3 patients, mean score was 21) to moderate cognitive impairment (3 patients, mean score was 15). The presence of extraintestinal manifestation was the only disease-related factor that was associated with cognitive impairment. Of the patient-related factor: older age, being divorced, and living in rural areas were associated with poorer cognitive functions.ConclusionIBD patients potentially suffer from cognitive impairment. In our study, factors as extraintestinal complications, older age, marital status, and residence in rural areas could be contributing factors to this impairment.

Exploring the molecular mechanisms and shared gene signatures between celiac disease and ulcerative colitis based on bulk RNA and single-cell sequencing: Experimental verification

Many immune-mediated diseases have the common genetic basis, as an autoimmune disorder, celiac disease (CeD) primarily affects the small intestine, and is caused by the ingestion of gluten in genetically susceptible individuals. As for ulcerative colitis (UC), which most likely involves a complex interplay between some components of the commensal microbiota and other environmental factors in its origin. These two autoimmune diseases share a specific target organ, the bowel. The etiology and immunopathogenesis of both conditions characterized by chronic intestinal inflammation, ulcerative colitis and celiac disease, are not completely understood. Both are complex diseases with genetics and the environmental factors contributing to dysregulation of innate and adaptive immune responses, leading to chronic inflammation and disease. This study is designed to further clarify the relationship between UC and CeD. The GEO database was used to download gene expression profiles for CeD (GSE112102) and UC (GSE75214). The GSEA KEGG pathway analysis revealed that immune-related pathways were significantly associated with both diseases. Further, we screened 187 shared differentially expressed genes (DEGs) of the two diseases. Gene Ontology (GO) and WikiPathways were carried out to perform the biological process and pathway enrichment analysis. Subsequently, based on the DEGs, the least absolute shrinkage and selection operator (LASSO) analysis was performed to screen for the diagnostic biomarkers of the diseases. Moreover, single-cell RNA-sequencing (RNA-seq) data from five colonic propria with UC showed that REG4 expression was present in Goblet cell, Enteroendocrine cell, and Epithelial. Finally, our work identified REG4 is the shared gene of UC and CeD via external data validation, cellular experiments, and immunohistochemistry. In conclusion, our study elucidated that abnormal immune response could be the common pathogenesis of UC and CeD, and REG4 might be a key potential biomarker and therapeutic target for the comorbidity of these two diseases.

Factors associated with stunting in under-five children with environmental enteropathy in slum areas of Jimma town, Ethiopia.

Stunting is a major public health issue with a significant influence on the health and development of children in low-income countries, where it affects up to 32% of children. Nutritional intake is impacted by alterations in intestinal permeability and underlying chronic inflammation, which hinder children's normal linear growth. Environmental enteropathy is a poorly understood condition with chronic intestinal inflammation. The purpose of this study was to identify the magnitude of stunting, change in growth, and factors associated with stunting and change in height for the age Z-score of children with an elevated lactulose-to-mannitol ratio. An observational follow-up study was conducted to follow children with an elevated lactulose-to-mannitol ratio for changes in their linear growth. A lactulose-mannitol test was performed to identify children with elevated lactulose-to-mannitol ratios, indicative of environmental enteropathy. After a 1-year follow-up, anthropometry was repeated to assess their linear growth. A multivariable logistic regression analysis was performed to identify the independent predictors for stunting in children with elevated lactulose-to-mannitol ratios. All tests were two-sided, and a p-value of <0.05 was considered significant. The prevalence of stunting in children with an elevated L:M at baseline and end line was found to be 72.4% (95% CI: 60.3, 84.5) and 78.4% (95% CI: 66.7, 90.2), respectively. In a multivariate analysis, a low dietary diversity score (<4 food groups), presence of flies and insects in the toilet area, poor handwashing practices during a critical time, and MUAC z < -2 were significantly associated with stunting. Flies and insects in the toilet area and unsafe disposal of feces were significantly associated with changes in HAZ in children with elevated lactulose-to-mannitol ratios, an indicator of environmental enteropathy. Most of the children with an elevated lactulose-to-mannitol ratio in the study population were stunted, and no significant change in their linear growth was observed after 1-year follow-up. Therefore, further investigation and urgent intervention are needed to prevent environmental enteropathy and stunting among under-five children in this community who are exposed to very poor sanitary conditions and other risk factors for malnutrition.

Recombinant probiotic Lactococcus lactis delivering P62 mitigates moderate colitis in mice.

p62 is a human multifunctional adaptor protein involved in key cellular processes such as tissue homeostasis, inflammation, and cancer. It acts as a negative regulator of inflammasome complexes. It may thus be considered a good candidate for therapeutic use in inflammatory bowel diseases (IBD), such as colitis. Probiotics, including recombinant probiotic strains producing or delivering therapeutic biomolecules to the host mucosal surfaces, could help prevent and mitigate chronic intestinal inflammation. The objective of the present study was to combine the intrinsic immunomodulatory properties of the probiotic Lactococcus lactis NCDO2118 with its ability to deliver health-promoting molecules to enhance its protective and preventive effects in the context of ulcerative colitis (UC). This study was realized in vivo in which mice were supplemented with the recombinant strain. The intestinal barrier function was analyzed by monitoring permeability, secretory IgA total levels, mucin expression, and tight junction genes. Its integrity was evaluated by histological analyses. Regarding inflammation, colonic cytokine levels, myeloperoxidase (MPO), and expression of key genes were monitored. The intestinal microbiota composition was investigated using 16S rRNA Gene Sequencing. No protective effect of L. lactis NCDO2118 pExu:p62 was observed regarding mice clinical parameters compared to the L. lactis NCDO2118 pExu: empty. However, the recombinant strain, expressing p62, increased the goblet cell counts, upregulated Muc2 gene expression in the colon, and downregulated pro-inflammatory cytokines Tnf and Ifng when compared to L. lactis NCDO2118 pExu: empty and inflamed groups. This recombinant strain also decreased colonic MPO activity. No difference in the intestinal microbiota was observed between all treatments. Altogether, our results show that recombinant L. lactis NCDO2118 delivering p62 protein protected the intestinal mucosa and mitigated inflammatory damages caused by dextran sodium sulfate (DSS). We thus suggest that p62 may constitute part of a therapeutic approach targeting inflammation.

Ferulic Acid Derivatives Ameliorate Intestine Barrier Destruction by Alleviating Inflammatory Responses in Dextran Sulfate Sodium-Induced Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), a chronic disorder affecting the colon and rectum, involves the overproduction of pro-inflammatory cytokines causing damage to tight junctions (TJ) in the intestinal epithelial cells and chronic inflammation. The current mainstay of treatment, sulfasalazine, often causes adverse effects, thereby necessitating the exploration of alternative herbal medicines with fewer side effects. Portulaca oleracea L. (P. oleracea), a traditional medicinal herb, contains feruloyl amide compounds. We synthesized new compounds by conjugating ferulic acid (FA) with (±)-octopamine. Our study focused on novel FA derivatives that demonstrate protective effects against the intestinal epithelial barrier and inflammatory responses. In lipopolysaccharide-induced cells, C1 and C1a inhibited the production of inflammatory mediators. In Caco-2 cells, these compounds maintained the TJ protein expression, thereby demonstrating their protective effects on the epithelial barrier. In a mouse model of dextran sulfate sodium-induced IBD, a treatment with these compounds ameliorated features including a body weight reduction, colon shortening, an increased disease activity index, and histopathological changes. Furthermore, C1a demonstrated greater efficacy than C1 at the same concentration. These findings suggest that the novel FA derivative (C1a) effectively alleviates clinical signs and inflammatory mediators in IBD, making these compounds potential candidates as natural medicines for the treatment of IBD.

New insights into the interactions between the gut microbiota and the inflammatory response to ulcerative colitisin a mouse model of dextran sodium sulfate and possible mechanisms of action for treatment with PE&AFWE.

Inflammatory bowel disease (IBD), comprising Crohn's disease (CD) and ulcerative colitis (UC), is a heterogeneous state of chronic intestinal inflammation. Intestinal innate immunity, including innate immune cells, defends against pathogens and excessive entry of gut microbiota, while preserving immune tolerance to resident intestinal microbiota, and may be characterized by its capacity to produce a rapid and nonspecific reaction. The association between microbiota dysbiosis and the pathogenesis of IBD is complex and dynamic. When the intestinal ecosystem is in dysbiosis, the reduced abundance and diversity of intestinal gut microbiota make the host more vulnerable to the attack of exogenous and endogenous pathogenic gut microbiota. The aim of our study was to comprehensively assess the relationship between microbial populations within UC, the signaling pathways of pathogenic gut microbe therein and the inflammatory response, as well as to understand the effects of using PE&AFWE (poppy extract [Papaver nudicaule L.] and Artemisia frigida Willd. extract) on UC modulation. A UC mouse model was established by inducing SPF-grade C57BL/6 mice using dextrose sodium sulfate (DSS). Based on metagenomic sequencing to characterize the gut microbiome, the relationship between gut microbiota dysbiosis and gut microbiota was further studied using random forest and Bayesian network analysis methods, as well as histopathological analysis. (1) We found that the 5 gut microbiota with the highest relative abundance of inflammatory bowel disease UC model gut microbiota were consistent with the top 5 ranked natural bacteria. There were three types of abundance changes in the model groups: increases (Chlamydiae/Proteobacteria and Deferribacteres), decreases (Firmicutes), and no significant changes (Bacteroidetes). The UC model group was significantly different from the control group, with 1308 differentially expressed species with abundance changes greater than or equal to 2-fold. (2) The proportion of the fecal flora in the UC group decreased by 37.5% in the Firmicutes and increased by 14.29% in the proportion of Proteobacteria compared to the control group before treatment. (3) The significantly enriched and increased signaling pathways screened were the 'arachidonic acid metabolic pathway' and the 'phagosomal pathway', which both showed a decreasing trend after drug administration. (4) Based on the causal relationship between different OTUs and the UC model/PE&AFWE administration, screening for directly relevant OTU networks, the UC group was found to directly affect OTU69, followed by a cascade of effects on OTU12, OTU121, OTU93, and OTU7, which may be the pathway of action that initiated the pathological changes in normal mice. (5) We identified a causal relationship between common differentially expressed OTUs and PE&AFWE and UC in the pre- and post-PE&AFWE-treated groups. Thereby, we learned that PE&AFWE can directly affect OTU90, after which it inhibits UC, inhibiting the activity of arachidonic acid metabolic pathway by affecting OTU118, which in turn inhibits the colonization of gut microbiota by OTU93 and OTU7. (6) Histopathological observation and scoring (HS) of the colon showed that there was a significant difference between the model group and the control group (p < 0.001), and that there was a significant recovery in both the sulfasalazine (SASP)and the PE&AFWE groups after the administration of the drug (p < 0.0001). We demonstrated causal effects and inflammatory metabolic pathways in gut microbiota dysbiosis and IBD, with five opportunistic pathogens directly contributing to IBD. PE&AFWE reduced the abundance of proteobacteria in the gut microbiota, and histopathology showed significant improvement.

Abstract 1440: Retinoblastoma binding protein 9 regulates intestinal inflammation and inflammation-associated carcinogenesis

Abstract The intestinal epithelium constitutes the mucosal barrier, and its dysfunction promotes microbial invasion from the gut lumen, which further induces the intestinal inflammation. Chronic intestinal inflammation, as observed in poorly controlled inflammatory bowel diseases (IBD), leads to the development of inflammation-associated cancer. Thus, a deeper understanding of the regulatory system that controls intestinal homeostasis is essential. Retinoblastoma-binding protein 9 (RBBP9) is expressed in a range of human cancer cells. RBBP9 was initially reported to have the ability to regulate the cell cycle pathway. More recent studies have identified RBBP9 as a serine hydrolase (SH) that promotes cancer progression by subverting the anti-proliferative function of TGFβ in a pancreatic cancer model. However, little is known about the roles RBBP9 plays in the development of intestinal cancer. In this study, we investigated the role of RBBP9 in inflammation and inflammation-associated cancer in the intestine. Analyses of surgically resected human samples and publicly available datasets demonstrated that RBBP9 expression was reduced in patient samples of ulcerative colitis (UC) and colitis-associated cancer (CAC). Genetically knockout mice for Rbbp9 (Rbbp9−/−) were susceptible to experimental colitis induced by dextran sulfate sodium (DSS). Furthermore, Rbbp9−/− mice exhibited enhanced azoxymethane/DSS-induced tumorigenesis, a model for human CAC. Mechanistically, RNA sequencing of these tumors showed increased enrichment of inflammation-associated signatures, including interferon signaling pathways. Ex vivo organoid experiments revealed the suppressive function of RBBP9 in interferon signaling-mediated apoptosis of the intestinal epithelium. Simultaneous inhibition of RBBP9 and STAT1, a downstream transcription factor of interferon signaling, reverted the apoptotic phenotype of Rbbp9−/− organoids. Collectively, our data provide insight into the understanding of the crucial role of RBBP9 in protecting against intestinal inflammation and inflammation-related tumorigenesis via regulating the interferon signaling-mediated apoptotic pathway. Citation Format: Yuki Nakanishi, Kensuke Hamada, Mayuki Omatsu, Kosuke Iwane, Hiroki Kitamoto, Maokoto Okabe, Yu Muta, Shuji Yamamoto, Akihisa Fukuda, Hiroaki Kasashima, Hiroshi Seno. Retinoblastoma binding protein 9 regulates intestinal inflammation and inflammation-associated carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1440.

Immunoregulatory Effects of Elemental Diet and Its Ingredient, Tryptophan, via Activation of the Aryl Hydrocarbon Receptor in Mice.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation and its treatment varies widely; however, when inflammation is high, a complete nutrient containing pre-digested elemental diet (ED) is used to preserve the intestinal tract. In this study, we investigated the mechanisms underlying the effectiveness of EDs for IBD using mice. C57BL/6 mice were orally treated with the ED (5 mL/day) and its ingredient L-tryptophan (Trp) (1-100 mg/kg), respectively. Flow cytometry analysis revealed that treatment with the ED and Trp (10 and 100 mg/kg) significantly increased the percentage of splenic CD4+-/CD25+-/Foxp3+ regulatory T cells (Tregs). In the 2% DSS-induced colitis-mouse model, Trp administration (100 mg/kg) led to a significant decrease in TNF-α and increase in IL-10 in the serum as well as a significant decrease in the inflammation score. Furthermore, the aryl hydrocarbon receptor (AhR) agonistic activity, which is a key function of Treg induction, of Trp and 15 Trp metabolites was characterized using a highly sensitive DR-EcoScreen cell assay. Five Trp metabolites, including L-kynurenine, acted as AhR agonists, while Trp did not. Taken together, these results suggest that the ED treatment has a Trp-dependent immunoregulatory effect, and several Trp metabolites that activate the AhR might contribute to induction of remission in patients with IBD.

Miniaturized implantable temperature sensors for the long-term monitoring of chronic intestinal inflammation.

Diagnosing and monitoring inflammatory bowel diseases, such as Crohn's disease, involves the use of endoscopic imaging, biopsies and serology. These infrequent tests cannot, however, identify sudden onsets and severe flare-ups to facilitate early intervention. Hence, about 70% of patients with Crohn's disease require surgical intestinal resections in their lifetime. Here we report wireless, miniaturized and implantable temperature sensors for the real-time chronic monitoring of disease progression, which we tested for nearly 4 months in a mouse model of Crohn's-disease-like ileitis. Local measurements of intestinal temperature via intraperitoneally implanted sensors held in place against abdominal muscular tissue via two sutures showed the development of ultradian rhythms at approximately 5 weeks before the visual emergence of inflammatory skip lesions. The ultradian rhythms showed correlations with variations in the concentrations of stress hormones and inflammatory cytokines in blood. Decreasing average temperatures over the span of approximately 23 weeks were accompanied by an increasing percentage of inflammatory species in ileal lesions. These miniaturized temperature sensors may aid the early treatment of inflammatory bowel diseases upon the detection of episodic flare-ups.

Characterization of intestinal O-glycome in reactive oxygen species deficiency.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation resulting from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host. Reactive oxygen species (ROS) generated by NADPH oxidases (NOX) provide antimicrobial defense, redox signaling and gut barrier maintenance. NADPH oxidase mutations have been identified in IBD patients, and mucus layer disruption, a critical aspect in IBD pathogenesis, was connected to NOX inactivation. To gain insight into ROS-dependent modification of epithelial glycosylation the colonic and ileal mucin O-glycome of mice with genetic NOX inactivation (Cyba mutant) was analyzed. O-glycans were released from purified murine mucins and analyzed by hydrophilic interaction ultra-performance liquid chromatography in combination with exoglycosidase digestion and mass spectrometry. We identified five novel glycans in ileum and found minor changes in O-glycans in the colon and ileum of Cyba mutant mice. Changes included an increase in glycans with terminal HexNAc and in core 2 glycans with Fuc-Gal- on C3 branch, and a decrease in core 3 glycans in the colon, while the ileum showed increased sialylation and a decrease in sulfated glycans. Our data suggest that NADPH oxidase activity alters the intestinal mucin O-glycans that may contribute to intestinal dysbiosis and chronic inflammation.

Strategies for targeting cytokines in inflammatory bowel disease.

Cytokines produced by immune cells contribute to the development and perpetuation of inflammatory bowel disease (IBD), namely Crohn's disease and ulcerative colitis, by regulating various aspects of the inflammatory response. Pro-inflammatory cytokines trigger chronic intestinal inflammation, tissue damage, carcinogenesis and perpetuation of disease and suppress the resolution of inflammation in IBD. The clinical success of antibodies that neutralize tumour necrosis factor (TNF) and thecytokineIL-12p40 in individuals with IBD has underscored this concept. Moreover, genetic and preclinical studies have emphasized the crucial role of IL-23 in IBD, leading to clinical approval of antibodies targeting this cytokine. Multiple studies have also investigated the administration of cytokines with assumed anti-inflammatory effects, but this approach has yet to show any real clinical benefit in individuals with IBD. Recent studies have targeted the cytokine network through the use of multi-cytokine blockers (for example, Janus kinase (JAK) inhibitors), IL-2-induced regulatory T cells or advanced combination therapies that use multiple cytokine blockers simultaneously (for example, anti-TNF along with anti-IL-23 antibodies). This Review will focus on our current understanding of how cytokines produced by innate and adaptive immune cells contribute to IBD pathogenesis and discuss how their modulation may inform future treatments for IBD.

Peripheral and intestinal T lymphocyte subsets in dogs with chronic inflammatory enteropathy.

Dysregulated T lymphocyte response is thought to play a key role in chronic intestinal inflammation (CIE). To evaluate the presence of changes in peripheral and intestinal T lymphocyte subsets and to describe potential immune and inflammatory biomarkers in dogs with CIE. Sixteen healthy dogs and 26 dogs were diagnosed with CIE. Prospective case-control study evaluating peripheral and intestinal T lymphocytes using flow cytometry and inflammatory markers obtained from complete blood cell counts. Dogs with CIE had higher peripheral activated T helper (Th) lymphocytes (87/μL [18-273] CIE, 44/μL [16-162] healthy control (HC, P = .013) and regulatory T cells (Treg; 108/μL [2-257] CIE, 34/μL [1-114] HC, P = .004). In the intestinal epithelium, CIE dogs presented lower percentages of Th (4.55% [1.75-18.67] CIE, 8.77% [3.79-25.03] HC, P = .002), activated Th cells (0.16% [0.02-0.83] CIE, 0.33% [0.05-0.57] HC, P = .03) and CD4/CD8 ratio (0.08 [0.02-0.39] CIE, 0.21 [0.07-0.85] HC, P = .003). Conversely, higher percentage of activated T cytotoxic cells (20.24% [3.12-77.12] CIE, 12.32% [1.21-39.22] HC, P = .04) and interferon-gamma (IFN-γ) producing T lymphocytes (7.36% [0.63-55.83] CIE, 1.44% [0.00-10.56] HC, P = .01) within the epithelium was observed. In the lamina propria the percentage of Treg lymphocytes was higher (6.02% [1.00-21.48] CIE, 3.52% [0.18-10.52] HC, P = .02). Systemic and intestinal immune alterations occur in dogs with CIE suggesting that blood IFN-γ producing T lymphocytes and the systemic immune-inflamation index (SII)could potentially serve as biomarkers for the disease.

Chronic Liver Disease and Promising Therapeutic Strategy: A Concise Review

Abstract Background: Chronic liver dysfunction (CLD) is a significant global health issue; alcohol use, obesity, drugs, and antibiotic and viral infections contribute to its development. However, changes in gut microbiota composition, primarily as people grow older, can also play a role in initiating and progressing liver dysfunction. Purpose: This review summarizes the altered gut microbiota–mediated chronic liver dysfunction and promising therapeutic strategies for NAFLD, ALD, NASH, and liver cirrhosis. Discussion on the chronic liver disease and its therapeutic aspects: The gut microbiota influences physiological processes, including immune system regulation, by promoting the production of pro-inflammatory markers and elevating the expression of toll-like receptors (TLRs) on hepatocytes, metabolisms of bile acids and choline, alteration in saturated aliphatic organic acid (SCFA) production, and food absorption. These alterations increase intestinal permeability, chronic inflammation, and liver dysfunction. Dysbiosis triggers inflammatory pathways, oxidative stress, and the production of toxic compounds that damage liver cells and promote chronic liver diseases such as alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and cirrhosis. Age-related changes in gut motility, secretory function, and immunological function can further decline beneficial bacterial diversity and abundance and shifts in pathogenic bacterial species’ relative abundance. Conclusion: Dysbiosis resulting from these age-related changes may contribute to the development of CLD. Numerous therapeutic approaches are available to restore the altered gut microbiota and reduce the risk of chronic liver dysfunction.

Dietary inclusion of a Saccharomyces cerevisiae metabolite improved reproductive performance but did not affect intestinal permeability in two chicken meat breeder lines

Gastrointestinal dysbiosis is a disturbance in mucosal homeostasis, producing low-grade chronic intestinal inflammation and impaired intestinal barrier function. It is induced by several factors, including nutrition and stress, which are both significant factors when considering current broiler breeder practices. A great grandparent (GGP) chicken meat line was identified displaying clinical signs characteristic of potential dysbiosis, including wet droppings and litter, in addition to reduced reproductive performance when compared to a consistently high performing line. This study aimed to determine whether the reduced reproductive performance observed in these hens was a result of dysbiosis and whether dietary supplementation with a Saccharomyces cerevisiae (SC) fermentation product would alleviate clinical signs. Dietary inclusion of SC did not influence intestinal permeability, WBC differentials, or corticosterone concentration in either the wet litter (WL) or high-performing (HP) breeder lines. Compared to hens from the HP line, WL line hens had a significant increase in intestinal permeability at 26 wk (onset of lay). WL hen heterophil counts were increased markedly at week 26 before declining. At weeks 26, 32, and 37 there were also significant increases in monocytes. Higher plasma corticosterone was also observed in WL hens at 37 wk. No significant differences in heterophil to lymphocyte (H:L) ratios or feather corticosterone were observed between lines. Dietary inclusion of SC supplementation to breeder diets had some benefit in regards to reducing hen mortality, improving egg production and hatchability but only in the WL line. Results from this study did not indicate that hens from the wet litter line were experiencing gut dysbiosis. Chronic intestinal inflammation may be a possible reason for the increase in intestinal permeability. These results do indicate that both breeder lines may be exhibiting physiological stress. Future investigation into the physiology and behavior around point of lay is required to find novel strategies to alleviate this stress and in turn, potentially improve welfare and production outcomes.

A12 ROLE OF INTESTINAL EPITHELIAL HNF4A IN PROTECTING AGAINST ACUTE INFLAMMATORY STIMULI

Abstract Background Inflammatory bowel disease is a group of chronic resulting from complex interactions between host susceptibility genes, the host microbiome, the immune system, and environmental triggers. The gastrointestinal epithelium acts as a barrier against harmful stimuli, and defects in its integrity are a precursor for the development of IBD. Our laboratory has shown that the conditional deletion of Hnf4a in the mouse intestinal epithelium leads to chronic intestinal inflammation. However, the functional role of HNF4A on epithelial barrier integrity is still controversial. Aims To evaluate the impact of Hnf4a deletion on barrier integrity during acute inflammation. Methods We used a tamoxifen-inducible CreER-loxP system to delete Hnf4a in the adult intestinal epithelium (Hnf4aΔIEC-ind). Intestinal permeability was assessed in Hnf4aΔIEC-ind and control mice in the collected serum after oral gavage of FITC-dextran. Mice were infected with an invasion-attenuated strain of Salmonella Typhimurium (SB103). S. Typhimurium loads were scored in the feces, liver, and spleen tissue. Histological analysis was carried out by various staining. Also, we used a model of T-cell-induced enteropathy triggered by a T-cell activating monoclonal anti-CD3ε Ab (145-2C11). Mice were assessed for clinical signs of enteropathy and sacrificed 1-, 3-, and 5 days post-injection. Gene expression of selected targets was studied by RT-qPCR. Results FITC-dextran assay showed a significant increase of the intestinal permeability in uninfected mutant mice compared to controls. In contrast, an oral infection with an invasion-deficient S. Typhimurium strain did not differentially impact in tissue bacterial loads or epithelial damage between groups. Likewise, the loss of epithelial HNF4A led to increased Muc2 transcripts expression in the ileum of infected mutant mice but not in the colon. Histological examinations at four days post-infection showed an elevated number and size of goblet cells in the ileal crypts of mutant mice. Bacterial localization using the general bacterial probe EUB388-cy3 (red) staining showed that infected mutant mice tended to enhance barrier mucus layer thickness compared to the controls. On the contrary, acute T cell activation with anti-CD3 mAb induced a significant increase of mRNA expression of Cxcl1 in the small intestine of mutant mice. H&amp;E staining showed elongated ileal crypts in mutant mice compared to controls after anti-CD3 injection. Conclusions Altogether, these results support that HNF4A could play a crucial adaptive role in modulating the small intestine epithelial barrier function and controlling the immune system during acute inflammatory stress. Funding Agencies CCC, CIHR

A196 CSF2 AUTOANTIBODIES AS A SEROLOGICAL MARKER FOR CROHN'S DISEASE

Abstract Background Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract that severely affects quality of life. Despite technological advances, CD diagnoses remain difficult and invasive, with delayed diagnoses correlated with worse prognosis and complications. CD is multifactorial, involving complex interactions between genetic and environmental risk factors, which leads to difficulty in identifying a cause and cure. However, research alludes to an underlying dysregulation of immune activity that leads to chronic intestinal inflammation. Mononuclear phagocytes (MNP) are essential immune cells that promote intestinal homeostasis through supporting immune tolerance, antimicrobial activity, and barrier integrity. A crucial cytokine that promotes the survival and function of intestinal MNP is Colony Stimulating Factor 2 (CSF2). Intriguingly, previous work in our lab demonstrated that CSF2 autoantibodies (CSF2-Ab) can be detected in the serum of every third CD patient and antedate the onset of CD by up to 6 years. In contrast, these titers are not seen in healthy donors or ulcerative colitis. Moreover, CSF2-Ab were predictive of disease location and severity, and are able to neutralize CSF2 by binding to glycosylations, impacting downstream signaling in MNP. These data suggest a role for CSF2-Ab in promoting intestinal immune dysregulation in CD. Aims Our work aims to characterize CSF2-Ab and their role in CD. Methods We developed a bead-based flow cytometric assay to screen CD patient serum for CSF2-Ab in several cohorts that contain samples at time points prior to, at, and after diagnosis. Results Our preliminary data demonstrate that our assay is rapid and specific for detecting CSF2-Ab of various isotypes in human serum and can be validated using ELISA. Furthermore, we show that this assay can be used to determine the epitope specificity of CSF2-Ab in CD patients in just one single sample. Conclusions We have developed a rapid and accessible assay to predict CD development years before diagnosis using minimal serum samples. Moreover, this screen will enable subclassification of patients based on their autoantibody reactivity to glycovariants of CSF2. Glycovariants that escape recognition by CD-specific CSF2-Ab could potentially be used as a therapeutic to ameliorate disease. Beyond treatment, understanding how CSF2-Ab epitope specificity and isotypes may change over the course of disease development will serve as a roadmap for elucidating the role of CSF2-Ab in CD. Funding Agencies CAG, CIHR

STERILE NEUTROPHILIA INDUCED VIA CXCR4 ANTAGONISM MITIGATES COLONIC INFLAMMATION BY ENHANCING IMMUNOSUPPRESSIVE REGULATORY T CELLS IN MICE

Abstract Sterile neutrophilia (SN) is commonly associated with chronic diseases including inflammatory bowel diseases (IBD), but its defined role is largely unknown. Plerixafor (AMD3100) is a CXCR4 antagonist that promotes neutrophil egress from bone marrow resulting in SN. We first confirmed that C57BL6/J mice administered Plerixafor (10 mg/kg/bw, i.p.,) increased their circulating neutrophils by 50% within 2.5 h. Further, to study the role of SN in IBD, we induced chronic colitis by neutralizing IL-10 signaling via αIL-10R mAb (1.0 mg/mouse, i.p., 4 weekly injections) with or without weekly twice Plerixafor. Control mice were given isotype control mAb. As expected, αIL-10R+Plerixafor-treated mice had increased peripheral and splenic neutrophils. Further, these mice displayed remarkably reduced epithelial damage, hyperplasia, immune cell infiltration, and disease activity index when compared to αIL-10R+vehicle treated mice, which had typical colitis symptoms. To delineate the mechanism for such protection by Plerixafor, we profiled immune cells in circulation, spleen and lamina propria. In circulation αIL-10R+Plerixafor-treated mice had elevated CD3+CD4+ helper T cells (Th) but reduced CD3+CD8+ cytotoxic T (Tc) cells and CD3+CD4+FoxP3+ cells (Tregs) and CD3+CD4+RORγt+ cells (Th17) remained unchanged. While in the spleens of αIL-10R+Plerixafor-treated mice displayed significant increased Tregs, but decreased Th and Tc cells. Interestingly, markedly elevated Tregs, RORγt+ Tregs and tolerogenic IL-10+ Tregs was observed in lamina propria when compared to αIL-10R+ vehicle given mice. Interestingly, the proinflammatory IL-17+ Th cells were decreased in αIL-10R+Plerixafor-treated mice which showed mild colitis. Moreover, to further elucidate the role of Plerixafor in intestinal barrier function in colitic mice, we noticed treatment with Plerixafor increased the expression of occludin and claudins (1, 2), which are the principal transmembrane tight junctions (TJ) proteins; however Plerixafor treatment reduced the expression of zonula occludens (ZO), a peripheral membrane TJ protein. Collectively, our results demonstrate that SN could be an adaptive and beneficial immune response during chronic intestinal inflammation.

Interleukin 21 Drives a Hypermetabolic State and CD4+ T-Cell–Associated Pathogenicity in Chronic Intestinal Inflammation

Incapacitated regulatory T cells (Tregs) contribute to immune-mediated diseases. Inflammatory Tregs are evident during human inflammatory bowel disease; however, mechanisms driving the development of these cells and their function are not well understood. Therefore, we investigated the role of cellular metabolism in Tregs relevant to gut homeostasis. Using human Tregs, we performed mitochondrial ultrastructural studies via electron microscopy and confocal imaging, biochemical and protein analyses using proximity ligation assay, immunoblotting, mass cytometry and fluorescence-activated cell sorting, metabolomics, gene expression analysis, and real-time metabolic profiling utilizing the Seahorse XF analyzer. We used a Crohn's disease single-cell RNA sequencing dataset to infer the therapeutic relevance of targeting metabolic pathways in inflammatory Tregs. We examined the superior functionality of genetically modified Tregs in CD4+ T-cell-induced murine colitis models. Mitochondria-endoplasmic reticulum appositions, known to mediate pyruvate entry into mitochondria via voltage-dependent anion channel 1 (VDAC1), are abundant in Tregs. VDAC1 inhibition perturbed pyruvate metabolism, eliciting sensitization to other inflammatory signals reversible by membrane-permeable methyl pyruvate supplementation. Notably, interleukin (IL) 21 diminished mitochondria-endoplasmic reticulum appositions, resulting in enhanced enzymatic function of glycogen synthase kinase 3 β, a putative negative regulator of VDAC1, and a hypermetabolic state that amplified Treg inflammatory response. Methyl pyruvate and glycogen synthase kinase 3 β pharmacologic inhibitor (LY2090314) reversed IL21-induced metabolic rewiring and inflammatory state. Moreover, IL21-induced metabolic genes in Tregs invitro were enriched in human Crohn's disease intestinal Tregs. Adoptively transferred Il21r-/- Tregs efficiently rescued murine colitis in contrast to wild-type Tregs. IL21 triggers metabolic dysfunction associated with Treg inflammatory response. Inhibiting IL21-induced metabolism in Tregs may mitigate CD4+ T-cell-driven chronic intestinal inflammation.