Expression In Intestinal Tissue Research Articles

A ratiometric fluorescent probe with dual-targeting capability for heat shock imaging

HSO3− is an important reactive sulfur species that maintains the normal physiological activities of living organisms and participates in a variety of redox homeostatic processes. It has been found that changes in HSO3− levels is closely related to the heat stroke phenomenon of the organism. Heat stroke causes damage to normal cells, which in turn causes damage to the body and even death. It is crucial to accurately monitor and track the physiological behavior of HSO3− during heat stroke. Herein, a ratiometric multifunctional fluorescent probe DRM-SO2 with dual-targeting ability to rapidly and precisely recognize HSO3− being constructed based on the FRET mechanism. DRM-SO2 has extra Large Stokes shift (216 nm), very high sensitivity (DL = 12.2 nM), fast response time and good specificity. When DRM-SO2 undergoes Michael addition with HSO3−, the fluorescence emission peak was blue-shifted from 616 nm to 472 nm, and a clear ratiometric signal appeared. The interaction between lysosomes and mitochondria in maintaining cellular homeostasis was investigated by the dual-targeting ability of the probe using HSO3− as a mediator. DRM-SO2 achieved successful targeting and real-time monitoring of exogenous and endogenous HSO3− in the cells. More importantly, imaging experiments in heat stroke mice revealed high HSO3− expression in intestinal tissues. This provides new ideas and research tools for early prevention of heat stroke-induced diseases such as intestinal injuries. In addition, the semi-quantitative monitoring experiments for paper-based visualization of HSO3− make the probe promising for the design of portable detectors.

The intestinal microbial metabolite acetyl l-carnitine improves gut inflammation and immune homeostasis via CADM2

Intestinal symbiotic bacteria play a key role in the regulation of immune tolerance in inflammatory bowel disease (IBD) hosts. However, the bacterial strains directly involved in this regulation and their related metabolites are largely unknown. We sought to investigate the effects of intestinal microbial metabolites on intestinal epithelium and to elucidate their therapeutic potential in regulating intestinal mucosal inflammation and immune homeostasis. Here, we used metagenomic data from Crohn's disease (CD) patients to analyze the composition of intestinal flora and identify metabolite profiles associated with disease behavior, and used the mouse model of dextran sodium sulfate (DSS)-induced colitis to characterize the therapeutic effects of the flora metabolite acetyl l-carnitine (ALC) on DSS-induced colitis. We found that intraperitoneal injection of ALC treatment could significantly alleviate the symptoms of DSS-induced colitis in mice, including prevention of weight loss, reduction in disease activity index (DAI) scores, increasing of colonic length, reduction in histological scores, and improvement in intestinal barrier function. Further, transcriptome sequencing analysis and gene silencing experiments revealed that the absence of CADM2 abolished the inhibitory effect of ALC on the TLR-MyD88 pathway in colonic epithelial cells, thereby reducing the release of inflammatory factors in colon epithelial cells. And we confirmed a significant downregulation of CADM2 expression in intestinal tissues of CD patients compared to healthy people in a population cohort. In addition, we also found that ALC increased the ratio of Treg cells in colon, and decreased the ratio of Th17 cells and macrophages, thereby improving the immune tolerance of the organism. The proposed study could be a potential approach for the treatment of CD.

DOP22 Predictive biomarkers of therapeutic response in Inflammatory Bowel Disease: a step towards personalized medicine

Abstract Background Inflammatory bowel diseases are complex conditions characterized by heterogeneity at clinical, immunological, molecular, genetic, and microbial levels. The most effective approaches to induce clinical remission and control inflammation in these diseases are biologic and JAK-inhibitor therapies. However, a significant proportion of patients do not respond to treatment. Consequently, the identification of predictive biomarkers of clinical response to the different available therapies could improve the selection of patients who may benefit from the most appropriate treatment. Methods A multiomic study (transcriptomic, proteomic, metabolomic and metagenomic) was performed in 53 patients with active Crohn´s disease (CD) and 50 patients with active ulcerative colitis (UC) before and after 14 weeks of treatment (anti-TNFα, ustekinumab, vedolizumab or tofacitinib). Responders and non-responders patients were categorized based on endoscopic findings. Samples (serum, urine, stool and intestinal biopsies) were used for RNA-seq analysis, liquid chromatography-mass spectrometry, nuclear magnetic resonance, and 16S rRNA gene sequencing. Results Our findings revealed multiple mRNAs, proteins and metabolites associated with the response to different treatments (Table 1). Differential gene expression analyses in intestinal tissue showed that the main differences were detected between responders versus non-responders UC patients to anti-TNF, vedolizumab and tofacitinib. Proteomic analysis found numerous differentially expressed proteins between the study groups. Additionally, ROC curves revealed two proteins with good predictive value to discriminate between CD and UC patients responders versus non-responders to anti-TNF treatment (AUC=0.81 and AUC=0.96, respectively). Metabolomics showed up-regulation of 21 lipoproteins in serum from CD patients responders to ustekinumab compared to non-responders. Metabolic analysis pathways identified enrichment in ketone and butyrate metabolism, mitochondrial electron transport chain, carnitine synthesis, and oxidation of chain fatty acids. Regarding metagenomic results, only differences were found in microbial composition in responders versus non-responders UC patients treated with anti-TNF (Figure 1). Conclusion This study provides early insights into shifts in gene and protein expression in intestinal tissue, alterations in serum and urine metabolites, and changes in gut microbiota as potential predictors of response to biologics and JAK-inhibitor treatment. Further research is needed to validate these results and asses their clinical significance in identifying patients most likely to benefit from each therapeutic approach.

Argininyl-Fructosyl-Galactose: A Novel Amino Acid Derivative improves Cisplatin-induced Intestinal Toxicity via Reactive Oxygen Species- mediated Apoptosis Pathway.

Based on the Maillard reaction principle of red ginseng, this study innovatively synthesized a new amino acid derivative by combining arginine with lactose through simulated synthesis and was separated and purified through repeated silica gel and polyacrylamide gel (Bio-gel P-II) column chromatography. The work was aimed at elucidating the synthesis of a novel amino acid derivative and investigating the intestinal protective activity of the novel amino acid derivative and possible molecular mechanism by establishing the intestinal injury model induced by cisplatin in mice. The purity and molecular weight of the amino acid derivatives were determined to be by electrospray ionization mass spectrometry (ESI-MS). Subsequently, by establishing cisplatin (20 mg/kg)-induced intestinal injury in vivo for 10 days and IEC-6 cell model. The biochemical indexes and histopathological analysis were used to evaluate the oxidative stress and inflammatory and pathological changes of intestinal tissue in mice. The protein expression levels of p-Nuclear transcription factor-κB (p-NF-κB), cleaved caspase 3/caspase 3, cleaved caspase 9/caspase-9, Bcl-2, Bax, cytochrome C, phosphatidylinositol 3-kinase (PI3K), Protein Kinase B (Akt), p-PI3K, p-Akt were quantified through immunofluorescence staining and western blot analysis. The new amino acid derivatives of chemical structure were identified to be 1- (arginine-Nαgroup)-1-deoxy-4-O-(β-D-galactopyranosyl)-D-fructose, named Argininylfructosyl- galactose (AFGA, C18H34N4O12). The results showed that pretreatment with a single AFGA dose remarkably alleviated cisplatin-evoked intestinal oxidative stress injury, and the levels of reactive oxygen species (ROS) were lessening in IEC-6 cells (p<0.05, p<0.01) and could effectively reduce the secretion of TNF-α and IL-1β in serum and the expression level of NF-κB protein in intestinal tissues (p<0.01). Meantime, AFGA also significantly suppressed the caspase 3, caspase 9, cytochrome C and Bax protein expression in intestinal tissue in mice (p<0.01), and regulated the PI3K/Akt pathway (p<0.05, p<0.01). Importantly, the molecular docking results of AFGA also suggested a better binding ability with the above-mentioned related target proteins. The results clearly revealed AFGA as a potential multifunctional therapeutic agent with a clear protective effect against cisplatin-induced intestinal injury may be related to the PI3K/Akt signaling pathway.

EFFECT OF MIR-21-3P ON INTESTINAL INJURY IN RATS WITH TRAUMATIC HEMORRHAGIC SHOCK RESUSCITATED WITH THE SODIUM BICARBONATE RINGER'S SOLUTION.

Background : This study aims to determine the impact and mechanism of miR-21-3p on intestinal injury and intestinal glycocalyx during fluid resuscitation in traumatic hemorrhagic shock (THS), and the different impacts of sodium lactate Ringer's solution (LRS) and sodium bicarbonate Ringer's solution (BRS) for resuscitation on intestinal damage. Methods : A rat model of THS was induced by hemorrhage from the left femur fracture. The pathological changes of intestinal tissues and glycocalyx structure were observed by hematoxylin-eosin staining and transmission electron microscope. MiR-21-3p expression in intestinal tissues was detected by real-time quantitative polymerase chain reaction. The expression of glycocalyx-, cell junction-, and PI3K/Akt/NF-κB signaling pathway-related proteins was analyzed by western blot. Results : MiR-21-3p expression was increased in THS rats, which was suppressed by resuscitation with BRS. BRS or LRS aggravated the intestinal injury and damaged intestinal glycocalyx in THS rats. The expression of SDC-1, HPA, β-catenin, MMP2, and MMP9 was upregulated, the expression of E-cad was downregulated, and the PI3K/Akt/NF-κB signaling pathway was activated in THS rats, which were further aggravated by BRS or LRS. The adverse effect of LRS was more serious than BRS. MiR-21-3p overexpression deteriorated the injury of intestinal tissues and intestinal glycocalyx; increased the expression of SDC-1, HPA, β-catenin, MMP2, and MMP9 while decreasing E-cad expression; and activated the PI3K/Akt/NF-κB signaling pathway in BRS-resuscitated THS rats. Conclusion : MiR-21-3p aggravated intestinal tissue injury and intestinal glycocalyx damage through activating PI3K/Akt/NF-κB signaling pathway in rats with THS resuscitated with BRS.

Probiotics mitigated sepsis-related mortality through antioxidant and inflammatory-mediated mechanisms

The current therapies used to treat sepsis are ineffective and sepsis-related mortality is high. Therefore, it is important to find new treatment options that reduce mortality from that disease. In the present study, the effects of probiotic pretreatment in septic rats were studied on morality, hematological parameters, oxidative stress, antioxidant capacity, and inflammation. After preparation of rats and administration of probiotic VSL # 3, cecal ligation and puncture (CLP) surgery was used to induce sepsis. Then, the animals were divided into four groups: sham, sham + VSL # 3, septic, and septic + VSL # 3. Sepsis-related mortality, hematological parameters, and biochemical factors were assessed. Also, the expression of inflammatory cytokines IL-6, TNF-α and IL-10 in serum were studied using enzyme-linked immunosorbent assay (ELISA). Then, the NF-κB gene and protein expression in intestinal tissue was studied by RT-qPCR and western blot techniques, respectively. Daily pretreatment with VSL # 3 in septic rats resulted in a 20 % reduction in mortality over 10 days and a significant modulation in hematological parameters. Evaluation of oxidant and antioxidant indices showed a decrease in oxidative stress and an increase in antioxidant capacity in septic rats receiving VSL # 3. Also, decreased expressions of inflammatory cytokines IL-6, TNF-α, IL-10, and NF-κB were observed in septic rats receiving VSL # 3. In general, it can be concluded that the administration of probiotic VSL # 3, which resulted in the improved survival of septic rats and better antioxidant and anti-inflammatory conditions, can be considered as a new adjutant therapeutic approach in the treatment of sepsis.

Fatty acid translocase (FAT/CD36) in silver pomfret (Pampus argenteus): Molecular cloning and functional characterization

Understanding the mechanisms of lipid transport and metabolism in fish is crucial to enhance dietary lipid utilization. Here, fatty acid translocase (CD36) gene was characterized in silver pomfret (Pampus argenteus). The open reading frame of silver pomfret cd36 gene was 1395 bp, encoding 464 amino acids. The silver pomfret CD36 protein contained typical transmembrane regions and N-glycosylation modification sites, and was localized to the cytomembrane. The cd36 gene was ubiquitously expressed in all tested tissues, with the highest expression observed in brain tissue. In vivo, both fasting and short-term high-fat feeding could increase cd36 expression in intestinal tissue. In vitro, cd36 expression was induced by palmitic acid, oleic acid, linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid treatment in intestinal tissue. Furthermore, dual-luciferase reporter assay results indicated that peroxisome proliferator-activated receptor gamma (PPARγ) could enhance cd36 promoter activity, and the co-expression of cd36 and pparγ was observed in EPA-incubated intestine, suggesting that EPA may regulate the expression of cd36 via PPARγ to maintain the homeostasis of intestinal lipid metabolism in silver pomfret. These results highlighted the crucial role of CD36 in silver pomfret, and suggested that the cd36 expression may be regulated by PPARγ. This study could contribute to a greater understanding of lipid metabolism and the development of effective strategies for nutrient requirements in fish.

BRAFV600E-mutated serrated colorectal neoplasia drives transcriptional activation of cholesterol metabolism

BRAF mutations occur early in serrated colorectal cancers, but their long-term influence on tissue homeostasis is poorly characterized. We investigated the impact of short-term (3 days) and long-term (6 months) expression of BrafV600E in the intestinal tissue of an inducible mouse model. We show that BrafV600E perturbs the homeostasis of intestinal epithelial cells, with impaired differentiation of enterocytes emerging after prolonged expression of the oncogene. Moreover, BrafV600E leads to a persistent transcriptional reprogramming with enrichment of numerous gene signatures indicative of proliferation and tumorigenesis, and signatures suggestive of metabolic rewiring. We focused on the top-ranking cholesterol biosynthesis signature and confirmed its increased expression in human serrated lesions. Functionally, the cholesterol lowering drug atorvastatin prevents the establishment of intestinal crypt hyperplasia in BrafV600E-mutant mice. Overall, our work unveils the long-term impact of BrafV600E expression in intestinal tissue and suggests that colorectal cancers with mutations in BRAF might be prevented by statins.

Inhibiting sorting nexin 10 promotes mucosal healing through SREBP2-mediated stemness restoration of intestinal stem cells.

Intestinal stem cell (ISC) is a promising therapeutic target for inflammatory bowel disease. Cholesterol availability is critical for ISC stemness. Low plasma cholesterol is a typical feature of Crohn's disease (CD); however, its impact on mucosal healing remains unclear. Here, we identified an essential role of sorting nexin 10 (SNX10) in maintaining the stemness of ISCs. SNX10 expression in intestinal tissues positively correlates with the severity of human CD and mouse colitis. Conditional SNX10 knockout in intestinal epithelial cells or ISCs promotes intestinal mucosal repair by maintaining the ISC population associated with increased intracellular cholesterol synthesis. Disassociation of ERLIN2 with SCAP by SNX10 deletion enhances the activation of SREBP2, resulting in increased cholesterol biosynthesis. DC-SX029, a small-molecule inhibitor of SNX10, was used to verify the druggable potential of SNX10 for the treatment of patients with CD. Our study provides a strategy for mucosal healing through SREBP2-mediated stemness restoration of ISCs.

Transcriptome and the gut microbiome analysis of the impacts of Brucella abortus oral infection in BALB/c mice

Brucellosis is a zoonotic infectious disease caused by Brucella spp, which could cause serious economic losses to animal husbandry and threaten human public health. Ingestion of contaminated animal products is a common way to acquire Brucella infection in humans, while research on effect of oral Brucella infection on host gut microbiota and the gene expression in intestinal tissues is limited. In the present study, 16S rRNA sequencing and RNA sequencing were conducted to explore gut microbiota and expression profiles of mRNAs in the colon of BALB/c mice, which were infected by Brucella abortus 2308. The fecal samples were collected at 7 and 28 days post infection to observe changes in the gut microbiota during Brucella infection. In the alpha diversity analysis, significantly increased Chao 1 index was observed at 28 days after Brucella infection. The Bray–Curtis distancebased principal coordinate analysis indicated that the WT group showed a separation from the Brucella infection groups. In addition, analysis of composition of microbes revealed that Prevotellaceae_NK3B31_group were more abundant in 1 week and 4 week infection groups, while Turicibacter was only more abundant in 4 week infection group. Based on the RNA-seq assay, a total of 45 differentially expressed genes were detected between Brucella abortus infection group and control group. Furthermore, KEGG pathway enrichment analysis showed that protein processing in endoplasmic reticulum, Legionellosis, Spliceosome, Hippo signaling pathway and Influenza A were significantly enriched in response to Brucella abortus infection. Our finding will help to improve the knowledge of the mechanisms underlying Brucella infection and may provide novel targets for future treatment of this pathogen infection.

Murine Models of Salmonella Infection.

The pathogen Salmonella enterica encompasses a range of bacterial serovars that cause intestinal inflammation and systemic infections in humans. Mice are a widely used infection model due to their relative simplicity and versatility. Here, we provide standardized protocols for culturing the prolific zoonotic pathogen S. enterica serovar Typhimurium for intragastric inoculation of mice to model colitis or systemic dissemination, along with techniques for direct extraintestinal infection. Furthermore, we present procedures for quantifying pathogen burden and for characterizing the immune response by analyzing tissue pathology, inflammatory markers, and immune cells from intestinal tissues. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Murine colitis model utilizing oral streptomycin pretreatment and oral S. Typhimurium administration Basic Protocol 2: Intraperitoneal injection of S. Typhimurium for modeling extraintestinal infection Support Protocol 1: Preparation of S. Typhimurium inoculum Support Protocol 2: Preparation of mixed S. Typhimurium inoculum for competitive infection Basic Protocol 3: Assessment of S. Typhimurium burden Support Protocol 3: Preservation and pathological assessment of S. Typhimurium-infected tissues Support Protocol 4: Measurement of inflammatory marker expression in intestinal tissues by qPCR Support Protocol 5: Preparation of intestinal content for inflammatory marker quantification by ELISA Support Protocol 6: Immune cell isolation from Salmonella-infected intestinal tissues.

Lafutidine Ameliorates Indomethacin-Induced Small Intestinal Damage in Rats by Modifying the Intestinal Mucosal Barrier, Inflammation, and Microbiota

Introduction: Nonsteroidal anti-inflammatory drug (NSAID)-induced small intestinal damage is a serious and escalating clinical problem without effective treatment. Lafutidine (LAF) is a novel histamine H2 receptor antagonist with a mucosal protective action. This study aimed to investigate the protective effect of LAF on indomethacin (IND)-induced enteropathy in rats. Methods: Rats were treated with LAF for 10 days with concomitant IND treatment on the final 5 days. Changes in metabolism and hematological and biochemical parameters were measured, and intestinal damage was blindly scored. Intestinal mucosal tissue and luminal contents were collected for transcriptome and microbiota sequencing. Intestinal inflammation and barrier function were also evaluated. Results: LAF treatment prevented anorexia and weight loss in rats and ameliorated reductions in hemoglobin, hematocrit, total protein, and albumin levels. LAF reduced the severity of IND-induced intestinal damage including macroscopic and histopathological damage score. Transcriptome sequencing results indicated that LAF might have positive effects on intestinal inflammation and the intestinal mucosal barrier. Further research revealed that LAF decreased neutrophil infiltration, and IL-1β and TNF-α expression in intestinal tissue. Besides, the treatment increased mucus secretion, MUC2, Occludin, and ZO-1 expression, and decreased serum D-lactate levels. LAF treatment also ameliorates microbial dysbiosis in small intestine induced by IND and increased the abundance of Lactobacillus acidophilus. Conclusion: LAF may protect against NSAID enteropathy via enhancing the intestinal mucosal barrier, inhibiting inflammation, and regulating microbiota.

P247 Oncostatin M and its receptor are markers of endoscopic and histological disease activity in Inflammatory Bowel Disease

Abstract Background Oncostatin M (OSM) and its receptor (OSMR) have been associated with active disease in patients with inflammatory bowel disease (IBD). This study compared the expression of OSM and OSMR in intestinal tissue according to clinical, endoscopic, and histological disease activity. Methods A multi-centre IBD database containing RNA-sequencing datasets, was used to analyse OSM and OSMR expression in ileal and/or colonic tissue, expressed as transcripts per million (TPM). Clinical scoring systems included the Harvey Bradshaw Index for Crohn’s disease (CD) and partial Mayo score for ulcerative colitis (UC). Endoscopic scoring systems included the simple endoscopic score for CD (SES-CD) and UC endoscopic index of severity (UCEIS). Histological scoring included the modified Naini and Cortina, and Riley scores. Graphs were presented in log transformed TPM+1 values. Results 155 patients (108 CD, 47 UC) and 38 controls were included. For IBD patients, 49% were men, median age was 35 years and intestinal tissue was collected at endoscopy (73%) and surgery (27%). Compared to controls, patients with CD and UC had significantly elevated median expression of OSM (0.08, 0.29 and 0.34 TPM respectively) and OSMR (1.21, 1.69 and 3.53 TPM respectively). OSMR expression was higher in UC patients than CD (p&amp;lt;0.001). There was no difference in OSM or OSMR between Harvey Bradshaw Index categories (0-4 vs 5-7 vs 8-16) or partial Mayo scores (0 vs 1-3 vs 4-7). Endoscopically, OSM was higher for SES-CD scores of 16-51 (1.11 TPM), compared to SES-CD scores of 0-3 (0.14 TPM, p=0.003) and controls (0.08 TPM, p&amp;lt;0.0001). Median OSMR increased non-significantly with increasing SES-CD scores (score 0-3: 1.59 TPM; score 4-15: 1.90 TPM; score 16-51: 2.04 TPM). There was no difference in OSM and OSMR expression between UCEIS scores, but the most severe disease (UCEIS score 5-8) had higher expression than controls for both OSM (3.92 vs 0.08 TPM, p&amp;lt;0.001) and OSMR (6.10 vs 1.21 TPM, p&amp;lt;0.0001). Increasing expression of OSM and OSMR was found with increasing histological scores, see Figure. Using the modified Naini and Cortina score for CD, OSM and OSMR were most expressed in the highest scores compared to the lowest scores (OSM: 1.48 vs 0.09 TPM, p&amp;lt;0.001; OSMR: 5.50 vs 1.27 TPM, p=0.003). For the modified Riley score for UC, both OSM and OSMR were also able to differentiate between the highest and lowest score groups (OSM: 3.14 vs 0.12 TPM, p=0.0001; OSMR: 6.61 vs 1.58 TPM, p&amp;lt;0.0001). Conclusion This study showed that OSM and OSMR expression in intestinal tissue is associated with inflammation both histologically and endoscopically. Further investigations are needed to confirm our observations and explore the OSM pathway’s relevance in intestinal inflammation.

P917 Intestinal complement interacts with commensal microbiota and provides pattern recognition in Inflammatory Bowel Disease

Abstract Background The complement system is a central humoral part of innate immunity and provides essential pattern recognition capabilities as well as effector functions such as opsonisation and target cell lysis. Its prominent role in infection and immunity is undisputed but its involvement in the pathogenesis of inflammatory bowel diseases remains less clear. Published data point to a role of complement within the intestinal lumen supporting clearance of pathogens1. Methods Expression of complement proteins within the intestinal epithelium was quantified by real-time qPCR on lesional and non-lesional biopsy samples from patients with IBD and healthy controls. The concentration of complement proteins was measured in fecal supernatants using ELISA. Flow cytometry of fecal bacterial single cell suspensions was used to assess complement (C3) and antibody (IgA) opsonisation status. Finally, cytometrically distinct populations were isolated by fluorescence-assisted cell sorting (FACS) and microbial community composition was investigated using 16S rDNA sequencing. Prediction of functional profiles from amplicon data was performed using Tax4Fun2. Results Complement expression in intestinal tissue was increased during active inflammation but certain targets were significantly less expressed in lesional mucosal sites (e.g. C6, C7). Free complement proteins in fecal supernatants were mostly undetectable by ELISA. In contrast, complement opsonisation was verified on fecal bacteria using flow cytometry. The degree of complement deposited on bacterial surfaces was significantly correlated with fecal calprotectin (p &amp;lt; 0.01). The percentage of complement-opsonized bacteria ranged from 0 to 5.3 % and did not correlate with intestinal inflammation while percentages of IgA opsonisation were generally higher (0.4 - 90.1%) and increased with fecal calprotectin (p &amp;lt; 0.01). FACS followed by 16S amplicon sequencing detected differences in bacterial taxa abundance between complement-opsonized, IgA-opsonized and non-opsonized fractions. Functional in-silico profiling revealed associations of complement-opsonization with bacterial virulence factors. Conclusion Luminal and mucosal complement expression and activity is evident in patients with IBD. Complement opsonisation profiles are distinct from IgA opsonisation and show features of specificity regarding recognition of microbial features.

P064 High intestinal Secretory Leukocyte Protease Inhibitor (SLPI) expression correlates with abundant neutrophilic inflammation and identifies Inflammatory Bowel Disease patients with a strong IL-17 response.

Abstract Background Inflammatory bowel disease, comprising Crohn’s disease (CD) and ulcerative colitis (UC), is driven by aberrant host-microbial immune interactions. Disease heterogeneity and severity hamper effective treatment, thus new therapeutic strategies tailored to the patient’s underlying immune defect are required. In mice, upon epithelial barrier breach and microbiota driven inflammation, colonic intestinal epithelial cells increase Secretory Leukocyte Protease Inhibitor (SLPI) expression. SLPI can exert anti-microbial activity and inhibit NF-kB activation. Therefore, we hypothesized that SLPI expression is increased in the intestine of IBD patients and may differentiate a subgroup of patients with a distinctive underlying immune disease. Methods Intestinal biopsies and plasma from therapy-naive pediatric patients (age 6-18) with CD (n=41), UC (n=22) or IBD-negative controls (n=15) were collected. Intestinal SLPI protein was detected by immunohistochemistry (IHC), scored semi-quantitatively, and patients were dichotomized into ‘SLPI-high’ and ‘SLPI-low’ groups. RNA sequencing and quantitative PCR (qPCR) were performed. Plasma protein concentrations were assessed with Olink technology®. Results We observed increased SLPI mRNA and protein expression in intestinal tissue of CD and UC patients, compared to IBD-negative controls. Expression was the strongest in macroscopically inflamed tissue and more dominant in colon than in small intestinal ileum. Colonic SLPI expression was associated with endoscopic severity of inflammation, with histologic disease activity, and with clinical disease activity in CD and UC patients. Furthermore, high epithelial SLPI expression associated with abundant neutrophil infiltration and calprotectin expression. RNA-seq revealed that biopsies of SLPI-high patients had a signature of inflammatory neutrophilic inflammation including the genes OSM, IL-1B, and CXCL8, which have been associated with severe disease progression and resistance to anti-TNF treatment. Most notably, biopsies of SLPI-high patients had a signature of increased IL-17 signaling, which was supported by IHC detection of high frequencies of IL-17 producing cells in the paired biopsies and higher plasma concentrations of CCL20, MMP10 and IL-17A. Conclusion Together, these results demonstrate that intestinal SLPI expression identifies severe IBD patients with high intestinal SLPI expression and immune features of therapy resistance, who have increased IL-17A responses and subsequent neutrophilic inflammation compared to SLPI-low patients. As such, epithelial SLPI expression is a histological parameter that could support tailored treatment strategies in pediatric IBD patients at the time of diagnosis.

Expression of tolerogenic dendritic cells in the small intestinal tissue of patients with celiac disease

Tolerogenic dendritic cells (tolCDs) play an important role in the regulation of inflammation in autoimmune diseases such as celiac disease (CeD). Dendritic cells express CD207, CD11c, and CD103 on their surface. In addition to the receptors mentioned above, tolCDs can express the immune-regulating enzyme indoleamine 2,3-dioxygenase (IDO). This study aimed to determine the mRNA and protein expression of CD11c, CD103 and CD207 markers, and also IDO gene expression in intestinal tissues of CeD patients in comparison to the healthy individuals. Duodenal biopsies were collected from 60 CeD patients and 60 controls. Total RNA was extracted and gene expression analysis was performed using Real-time PCR SYBR® Green method. Additionally, biopsy specimens were paraffinized and protein expression was evaluated using immunohistochemistry (IHC) for expression of CD11c+, CD207+and CD103+. Gene expression levels of CD11c (P = 0.045), CD103 (P < 0.001), CD207 (P < 0.001) and IDO (P = 0.01) were significantly increased in CeD patients compared to the control group. However, only CD103 protein expression was found to be significantly higher in CeD patients in comparison to the control group (P < 0.001). The result of this study showed that the expresion levels of CD11c, CD103, CD207 and IDO markers were higher in CeD patients compared to the controls, indicating the effort of dendritic cells to counterbalance the gliadin-triggered abnormal immune responses in CeD patients.

Morphine mediated neutrophil infiltration in intestinal tissue play essential role in histological damage and microbial dysbiosis

ABSTRACT The gut microbial ecosystem exhibits a complex bidirectional communication with the host and is one of the key contributing factors in determining mucosal immune homeostasis or an inflammatory state. Opioid use has been established to induce gut microbial dysbiosis consistent with increased intestinal tissue inflammation. In this study, we investigated the role of infiltrated immune cells in morphine-induced intestinal tissue damage and gut microbial dysbiosis in mice. Results reveal a significant increase in chemokine expression in intestinal tissues followed by increased neutrophil infiltration post morphine treatment which is direct consequence of a dysbiotic microbiome since the effect is attenuated in antibiotics treated animals and in germ-free mice. Neutrophil neutralization using anti-Ly6G monoclonal antibody showed a significant decrease in tissue damage and an increase in tight junction protein organization. 16S rRNA sequencing on intestinal samples highlighted the role of infiltrated neutrophils in modulating microbial community structure by providing a growth benefit for pathogenic bacteria, such as Enterococcus, and simultaneously causing a significant depletion of commensal bacteria, such as Lactobacillus. Taken together, we provide the first direct evidence that neutrophil infiltration contributes to morphine-induced intestinal tissue damage and gut microbial dysbiosis. Our findings implicate that inhibition of neutrophil infiltration may provide therapeutic benefits against gastrointestinal dysfunctions associated with opioid use.

Cecum microbiome and metabolism characteristics of Silky Fowl and White Leghorn chicken in late laying stages.

Cecal microflora plays a key role in the production performance and immune function of chickens. White Leghorn (WL) is a well-known commercial layer line chicken with high egg production rate. In contrast, Silky Fowl (SF), a Chinese native chicken variety, has a low egg production rate, but good immune performance. This study analyzed the composition of cecal microbiota, metabolism, and gene expression in intestinal tissue of these varieties and the correlations among them. Significant differences were observed in the cecal microbes: Bacteroides was significantly enriched in WL, whereas Veillonellaceae and Parabacteroides were significantly enriched in SF. Carbohydrate biosynthesis and metabolism pathways were significantly upregulated in WL cecum, which might provide more energy to the host, leading to persistently high levels of egg production. The higher Parabacteroides abundance in SF increased volicitin content, enhanced α-linolenic acid metabolism, and significantly negatively correlated with metabolites of propanoate metabolism and carbohydrate metabolism. Genes related to lipid metabolism, immunity, and melanogenesis were significantly upregulated in the SF cecum, regulating lipid metabolism, and participating in the immune response, while genes related to glucose metabolism and bile acid metabolism were expressed at higher levels in WL, benefiting energy support. This study provided a mechanism for intestinal microorganisms and metabolic pathways to regulate chicken egg-laying performance and immunity.

I-FABP as a Potential Marker for Intestinal Barrier Loss in Porcine Polytrauma.

Polytrauma and concomitant hemorrhagic shock can lead to intestinal damage and subsequent multiple organ dysfunction syndrome. The intestinal fatty acid-binding protein (I-FABP) is expressed in the intestine and appears quickly in the circulation after intestinal epithelial cell damage. This porcine animal study investigates the I-FABP dynamics in plasma and urine after polytrauma. Furthermore, it evaluates to what extent I-FABP can also act as a marker of intestinal damage in a porcine polytrauma model. Eight pigs (Sus scrofa) were subjected to polytrauma which consisted of lung contusion, tibial fracture, liver laceration, and hemorrhagic shock followed by blood and fluid resuscitation and fracture fixation with an external fixator. Eight sham animals were identically instrumented but not injured. Afterwards, intensive care treatment including mechanical ventilation for 72 h followed. I-FABP levels in blood and urine were determined by ELISA. In addition, immunohistological staining for I-FABP, active caspase-3 and myeloperoxidase were performed after 72 h. Plasma and urine I-FABP levels were significantly increased shortly after trauma. I-FABP expression in intestinal tissue showed significantly lower expression in polytraumatized animals vs. sham. Caspase-3 and myeloperoxidase expression in the immunohistological examination were significantly higher in the jejunum and ileum of polytraumatized animals compared to sham animals. This study confirms a loss of intestinal barrier after polytrauma which is indicated by increased I-FABP levels in plasma and urine as well as decreased I-FABP levels in immunohistological staining of the intestine.

MEFV and NLRP3 Inflammasome Expression Is Attributed to Immature Macrophages and Correlates with Serum Inflammatory Proteins in Crohn´s Disease Patients

Inflammasomes are intracellular protein complexes whose activation results in proinflammatory cytokines. Inflammasomes are implicated in Crohn´s disease (CD) pathogenesis, yet the contribution of inflammasomes in intestinal epithelial cells (IECs) versus lamina propria (LP) macrophages is poorly understood. Whether inflammasome expression in intestinal tissue reflects the serum inflammatory protein profile of patients is also not known. We aimed to determine the intestinal cell types where inflammasome expression is increased in CD and if they correlate with the serum protein profile. RT-PCR and NanoString nCounter technology were used to characterize inflammasome gene expression in CD patients and controls. The mucosa, LP and IEC cell fractions and FACS-sorted cells were analyzed. Proximity extension assay with a 92-protein panel was used to determine the serum inflammatory protein profile. Compositional analysis was used to correlate ileum inflammasome gene expression with intestinal mononuclear phagocyte populations. We show that NLRP3 and MEFV inflammasome sensors and downstream effector expression including IL-1β are increased in inflamed mucosa of IBD patients and correlate with disease activity. Inflammasome gene expression increased with the abundance of immature intestinal macrophages, and increased IL-1β released by CD LP cells correlated with immature macrophage frequency. Inflammasome gene expression was also increased in circulating monocytes, the precursors of immature intestinal macrophages. Finally, the serum inflammatory profile of CD patients correlates with ileal expression of genes related to NLRP3 and MEFV inflammasomes. Overall, we show that MEFV and NLRP3 inflammasome expression in CD intestine is attributed to the accumulation of immature macrophages and correlates with serum inflammatory proteins.