Enterocyte Injury Research Articles

Histopathology underlying environmental enteric dysfunction in a cohort study of undernourished children in Bangladesh, Pakistan, and Zambia compared with United States children

BackgroundEnvironmental enteric dysfunction (EED) is an asymptomatic intestinal disorder associated with growth impairment, delayed neurocognitive development, and impaired oral vaccine responses. ObjectivesWe set out to develop and validate a histopathologic scoring system on duodenal biopsies from a cohort study of children with growth failure in Bangladesh, Pakistan, and Zambia (“EED”) with reference to biopsies from United States children with no clinically reported histologic pathology (referred to hereafter as “normal”) or celiac disease. MethodsFive gastrointestinal pathologists evaluated 745 hematoxylin and eosin slide images from 291 children with EED (mean age: 1.6 y) and 66 United States children (mean age: 6.8 y). Histomorphologic features (i.e., villus/crypt architecture, goblet cells, epithelial and lamina propria acute/chronic inflammation, Brunner’s glands, Paneth cells, epithelial detachment, enterocyte injury, and foveolar metaplasia) were used to score each histopathologic slide. Generalized estimating equations were used to determine differences between EED, normal, and celiac disease, and receiver operating characteristic curves were used to assess predictive value. ResultsBiopsies from the duodenal bulb showed higher intramucosal Brunner’s gland scores and lower intraepithelial lymphocyte scores than from the second or third parts of the duodenum (D2/3), so only D2/3 were included in the final analysis. Although 7 parameters differed significantly between EED and normal biopsies in regression models, only 5 (blunted villus architecture, increased intraepithelial lymphocytosis, goblet cell depletion, Paneth cell depletion, and reduced intramucosal Brunner’s glands) were required to create a total score percentage (TSP-5) that correctly identified EED against normal biopsies (AUC: 0.992; 95% CI: 0.983, 0.998). Geographic comparisons showed more severe goblet cell depletion in Bangladesh and more marked intraepithelial lymphocytosis in Pakistan. ConclusionsThis scoring system involving 5 histologic parameters demonstrates very high discrimination between EED and normal biopsies, indicating that this scoring system can be applied with confidence to studies of intestinal biopsies in EED.

Small Intestine Bacterial Overgrowth is associated with increased Campylobacter and epithelial injury in duodenal biopsies of Bangladeshi children.

Small intestine bacterial overgrowth (SIBO) has been associated with enteric inflammation, linear growth stunting, and neurodevelopmental delays in children from low-income countries. Little is known about the histologic changes or epithelial adherent microbiota associated with SIBO. We sought to describe these relationships in a cohort of impoverished Bangladeshi children. Undernourished 12-18-month-old children underwent both glucose hydrogen breath testing for SIBO and duodenoscopy with biopsy. Biopsy samples were subject to both histological scoring and 16s rRNA sequencing. 118 children were enrolled with 16s sequencing data available on 53. Of 11 histological features, we found that SIBO was associated with one, enterocyte injury in the second part of the duodenum (R = 0.21, p = 0.02). SIBO was also associated with a significant increase in Campylobacter by 16s rRNA analysis (Log 2-fold change of 4.43; adjusted p = 1.9 x 10-6). These findings support the growing body of literature showing an association between SIBO and enteric inflammation and enterocyte injury and further delineate the subgroup of children with environmental enteric dysfunction who have SIBO. Further, they show a novel association between SIBO and Campylobacter. Mechanistic work is needed to understand the relationship between SIBO, enterocyte injury, and Campylobacter.

A254 CANAGLIFLOZIN EXACERBATES CHEMICALLY INDUCED COLITIS WHEN COMBINED WITH A HIGH SUGAR DIET

Abstract Background Consumption of high-sugar diets exacerbates inflammation and hinders the healing process in animal models of acute colitis. Canagliflozin (CANA) is a sodium glucose cotransporter-2 (SGLT2) inhibitor primarily used for the management of type 2 diabetes and cardiovascular disease. CANA has demonstrated the ability to suppress human T cell and macrophage effector function and to improve inflammation in rat colitis models. Based on these findings, we hypothesized that CANA would mitigate the deleterious effects of a high sugar diet in a DSS colitis model through its effects on immune effector cells. Aims The aim of this study was to examine the effects of a high sugar diet and CANA administration on colonic inflammation in an animal model of acute colitis. Methods Adult wild-type male and female 129/SvEv mice were placed on standard chow or a high-sugar diet (HSD) containing 50% sucrose (AIN76A) ± CANA (10mg/kg) gavaged daily (n=4-6 for each group). Following three days on the respective diet, mice received 1.8-2% dextran sodium sulfate (DSS) in their drinking water for 5 days, followed by water alone for 4 days. Disease Activity Index (DAI) was computed daily based on changes in animal weight, stool consistency, and blood in stool. Colonic tissues were evaluated for histological changes, weight-to-length ratio, and cytokine levels using the MesoScale discovery platform. Results Mice on the HSD had increased severity of colitis compared with chow fed mice as evidenced by enhanced DAI, increased weight loss, and increased levels of colonic TNFα (pampersand:003C0.02). Response to CANA treatment was both sex and diet dependent. Contrary to our hypothesis, CANA treatment increased severity of colitis in male mice on the HSD as evidenced by increased DAI and delayed healing with increased enterocyte injury and increased levels of lamina propria neutrophils and lymphocytes. CANA treatment did not exacerbate disease severity in females on the HSD but did delay healing as evidenced by increased lamina propria neutrophils and lymphocytes and worse enterocyte injury. These effects were not observed in mice that were fed standard chow diets with CANA. Conclusions A high-sugar diet significantly exacerbates chemically induced colitis and delays healing. This effect is intensified when combined with CANA, especially in male mice. These findings indicate that effects of SGLT2 inhibitors on colonic inflammation can be modulated by both dietary intake and sex. Funding Agencies CCC

A272 EFFECTS OF EMPAGLIFLOZIN ON CHEMICALLY INDUCED COLITIS IN A MOUSE MODEL ARE MODULATED BY SEX AND DIETARY INTERACTIONS

Abstract Background High sugar diets have been shown to dramatically increase disease severity in mouse models of colitis. Empagliflozin (EMPA) is a highly selective sodium glucose cotransporter-2 (SGLT2) inhibitor that is used therapeutically as an antihyperglycemic agent in the management of type 2 diabetes. In human trials EMPA treatment exerts potent anti-inflammatory effects independently of glycemic control. Further, we have previously demonstrated in a genetic mouse model of colitis that EMPA treatment was highly effective in improving colonic inflammation. Based on these findings, we hypothesized that EMPA treatment may also be effective in mitigating disease severity in chemically induced colitis in mice fed a high sugar diet. Aims The aim of this study was to examine the effects of treatment with EMPA on chemically induced colitis in mice fed a high sugar diet Methods At 6-8 weeks of age, wild-type 129/SvEv mice were placed on chow (CH) or high sugar diet (HS) (50% sucrose: AIN76A) ± EMPA (10mg/kg). After two days on the diet, mice were administered dextran sodium sulfate (DSS) for 5 days in drinking water followed by water alone for 2 days (n=4-6 mice for all groups). Disease activity index (DAI) was calculated daily from animal weight change, stool consistency, and stool hemoccult. As a measurement of degree of healing, colonic tissues were evaluated at day 9 for histological changes, weight-to-length ratio, and cytokine levels by ELISA. As a measure of EMPA functionality, urinary and blood glucose levels were measured. Results Mice on the HS diet demonstrated increased susceptibility to colitis compared with chow fed mice with increased colonic levels of IL-1β, weight loss, and DAI. Significant interactions between sex and diet were seen in responses to EMPA treatment. EMPA treatment did not alter severity of colitis but did significantly delay healing in male mice on both the HS and chow diets as evidenced by increased DAI and increased enterocyte injury with increased levels of lamina propria neutrophils and lymphocytes. EMPA treatment did not increase disease severity in females on either diet. No differences were seen in colonic TNFα, IL-10, IL-12p70, or IL-22 between any of the groups. Urinary glucose levels were elevated in mice receiving EMPA and significantly higher in males compared with females (pampersand:003C0.05). EMPA treatment did not alter fasting blood glucose levels. Conclusions A high sugar diet strongly exacerbates disease severity in a sex-dependent manner in a chemically induced model of colitis. Further, contrary to our hypothesis, EMPA treatment did not improve colitis in females and worsened disease in males suggesting that the documented anti-inflammatory effects of EMPA may be condition dependent. Funding Agencies CCC

Effects of GBT1118, a voxelotor analog, on intestinal pathophysiology in sickle cell disease.

Voxelotor is an allosteric haemoglobin (Hb) modulator that binds covalently and reversibly to Hb alpha chain to facilitate improved Hb-O2 affinity and arterial oxygen. It, therefore, reduces the susceptibility of erythrocytes carrying Haemoglobin S to sickle. In this study, we have used GBT1118, an analog of voxelotor, to treat male Townes sickle cell disease (SCD) mice to investigate whether the Hb modulator could attenuate the intestinal pathophysiologic changes associated with SCD. Compared with mice fed with control chow, GBT1118-treated mice showed improvement in the intestinal pathophysiology. These mice exhibited improved small intestinal barrier functions, reduced intestinal microbial density, reduced enterocyte injury, lower serum lipopolysaccharides and smaller spleens. These improvements were observed after only 3 weeks of GBT1118 treatment. Benefits were also observed after experimentally-induced vaso-occlusive crisis (VOC). Recovery from the VOC-induced changes was faster in mice that were treated with GBT1118. The improved small intestinal barrier function was associated with higher expression of genes encoding enterocyte E-cadherin, JAM-A, ZO-1, MUC-2 and occludin while the lower intestinal microbial density associated with higher expression of genes encoding the antimicrobial peptides defensin-α 1 and defensin-α 4. Our findings provide the evidence to support the beneficial effects of GBT1118 in SCD-related intestinal pathophysiology.

5613426 LOW CHRONIC INFLAMMATION IN SCD

Introduction: Sickle Cell Disease (SCD) is a genetic disorder of hemoglobin affecting 30 million people worldwide in which the mutant HbS polymerize upon deoxygenation, causing the eritrocyte to assume a sickle morphology and reduce the fluidity of cell membrane, adhering to leucocyte immobilized to the endothelium, causing microvascular occlusion, vaso occlusive crisis and tissue ischemia (1). In additon to eritrocyte sickling, one of the hallmarks of SCD is the continuous presence of basal inflammatory process. SC patients report the following condition: higher baseline leucocyte count (2) Higher level of soluble CD62L (3) neutrofils express higher activation molecules, CD64 (4), CD11b/CD18 (5) monocytes demonstrate activated phenotypes and higer propensity to IL-1beta and TNF-alfa (6) platelets are chronically activated and express higher level of CD40L (7) patience exhibit increased intestinal permeability related to enterocyte injury (8) gut disbiosis that is associated with hospitalization and VOC (9) Ongoing inflammatory process not only render SCD patience to developing VOC, they also create vicious cicle of the pathology that represent a significant risk in the progression of the disease and in chronic damage tissue. Aims: While advance have been made to reduce the frequency of VOC in SCD, there have been very little change in the management of patience during symptom-free “steady state”. The results from various studies on treating VOC using monotherapy have so far been universally disappointing. This is not surprising in view of the complex nature of VOC. Successful treatment of VOC targeting of the downstream immunological pathways requires multipronged approaches using combination therapy, just like treatment of malignant diseases using combination chemotherapy. Methods: The reviwes of the scientific litterature is full of interesting articles in which is enlighted the interaction between the immune system and different stimuli in order to obtain a modulation of the immune reasponse toward a tolerogenic way. Results: In PubMed there are very interesting report that investigate the approach to the immune system in SCD in order to reduce the basal inflammation. There are many way to interact with the immune system to promote a tolerogenic way in SCD such as: supplement of Vitamin D to reduce resporatory illness (10) supplement of Omega-3 to reduce CRP (11) supplement of Glutamine to reduce hospitalization and pain crises (12) probiotic or enterosorbent can reduce gut disbiosis and ameliorate clinical outcome (13) moderate exercise improve cardiopulmonary function and quality life (14) (15) Conclusion: SCD is a genetic disorder caracterized by a continuous presence of low chronic inflammation and studies in human have found that in SCD patience, the more the immune system is activated, the more are likely to get involved in clinical complication such alloimmunization, hospitalization and pain crises (16). Recent evidence suggest that bioregulatory approach toward a tolerogenic way of the immune response could lead to an amelioration of clinical outcome and improvement of quality of life with cheap and feasible approaches.

Microbial gut evaluation in an angolan paediatric population with sickle cell disease.

Sickle cell disease (SCD) is one of the most common genetic conditions worldwide. It can contribute up to 90% of under-5 mortality in sub-Saharan Africa. Clinical manifestations are very heterogeneous, and the intestinal microbiome appears to be crucial in the modulation of inflammation, cell adhesion and induction of aged neutrophils, the main interveners of recurrent vaso-occlusive crisis. Enterocyte injury, increased permeability, altered microbial composition and bacterial overgrowth have all been documented as microbial and pathophysiologic changes in the gut microbiome of SCD patients in recent studies. Our aim was to sequence the bacterial 16S rRNA gene in order to characterize the gut microbiome of Angolan children with SCA and healthy siblings as a control. A total of 72 stool samples were obtained from children between 3 and 14 years old. Our data showed that the two groups exhibit some notable differences in microbiota relative abundance at different classification levels. Children with SCA have a higher number of the phylum Actinobacteria. As for the genus level, Clostridium cluster XI bacteria was more prevalent in the SCA children, whereas the siblings had a higher abundance of Blautia, Aestuariispira, Campylobacter, Helicobacter, Polaribacter and Anaerorhabdus. In this study, we have presented the first microbiota analysis in an Angolan paediatric population with SCD and provided a detailed view of the microbial differences between patients and healthy controls. There is still much to learn before fully relying on the therapeutic approaches for gut modulation, which is why more research in this field is crucial to making this a reality.

The effect of prolonged interval and continuous exercise in the heat on circulatory markers of intestinal barrier integrity.

The purpose of this study was to determine the effect of prolonged high-intensity interval (INT) and moderate-intensity continuous (CONT) treadmill exercise in the heat on markers of enterocyte injury and bacterial endotoxin translocation. Nine males completed 2 h of work-matched exercise in the heat (40°C and 15% RH) as either INT (2min at 80% VO2max and 3min at 30% VO2max) or CONT (~ 50% of VO2max). Blood samples collected pre- and post-exercise were assayed for intestinal fatty acid-binding protein (I-FABP), claudin-3 (CLDN-3), and lipopolysaccharide-binding protein (LBP). I-FABP was significantly increased from pre- to post-exercise in CONT (913.96 ± 625.13 to 1477.26 ± 760.99pg•mL-1; p = 0.014, d = 0.766) and INT (714.59 ± 470.27 to 1547.93 ± 760.99pg•mL-1; p = 0.001, d = 1.160). Pre- to post-exercise changes in I-FABP were not different between CONT and INT (p = 0.088, d = 0.414). LBP was significantly increased from pre- to post-exercise in INT (15.94 ± 2.90 to 17.35 ± 3.26μg•mL-1; p = 0.028, d = 0.459) but not CONT (18.11 ± 5.35 to 16.93 ± 5.39μg•mL-1; p = 0.070, d = 0.226), and pre- to post-exercise changes in LBP were higher in the INT compared to CONT (p < 0.001, d = 1.160). No significant changes were detected from pre- to post-exercise for CLDN-3 in CONT (14.90 ± 2.21 to 15.30 ± 3.07μg•mL-1) or INT (15.55 ± 1.63 to 16.41 ± 2.11μg•mL-1) (p > 0.05). We conclude that prolonged exercise in the heat induces enterocyte injury, but interval (or intermittent) exercise may cause greater bacterial endotoxin translocation which may increase the risk for local and systemic inflammation.

MO243: Intestinal Fatty-Acid Binding Protein: A Potential Biomarker of Enterocyte Damage in IGA Nephropathy?

Abstract BACKGROUND AND AIMS An abundance of inflammatory cells has been found in duodena of IgA nephropathy (IgAN) patients [1, 2]. The degree of intestinal inflammation was shown to correlate with the amount of proteinuria and haematuria in IgAN [3]. Serum intestinal fatty-acid-binding protein (I-FABP) has shown to correlate with the degree of enterocyte damage in untreated coeliac disease (CD) patients [4]. The median level of I-FABP in untreated CD patients was 785 pg/mL and 173 pg/mL in healthy controls [5]. We conducted this study hypothesizing that I-FABP would be elevated in IgAN as a potential biomarker for the presence of subclinical enterocyte damage and intestinal inflammation. CD was excluded with coeliac autoantibody tests. METHOD This study was carried out at the Tampere University Hospital and Tampere University, Finland. Altogether 85 IgAN patients participated (median age 55 years, 54% males). None of the patients had progressed to end-stage kidney disease, nor had they a diagnosed enteropathy. Fifteen healthy controls (median age 57 years, 47% males) were participants from our previous CD studies. Coeliac autoantibodies were determined from the serum samples by measuring IgA class endomysial antibodies (EmA) and transglutaminase antibodies (tTGAb). EmA was determined by an in-house indirect immunofluorescence method using human umbilical cord as substrate. A serum dilution of 1: ≥ 5 was considered positive. tTGAb levels were determined with EliA Celikey assay (ThermoFisher Scientific, Waltham, MA, USA, cut-off for positivity 7.0 U/mL). The serum levels of I-FABP were determined with a commercially available ELISA kit (Hycult Biotech, Uden, The Netherlands, detection limit 47 pg/mL). Current kidney function was available in 68 IgAN patients (80%), and eGFR &amp;gt;60 mL/min/1.73 m2 was regarded as preserved kidney function. RESULTS I-FABP levels among IgAN patients (median 830 pg/mL, IQR 475–1378) were higher (P &amp;lt; .001) compared with healthy controls (289 pg/mL, IQR 199–568). A total of 57% of the IgAN patients from whom the data on current kidney function were available had preserved kidney function. Also, I-FABP levels in IgAN patients with preserved kidney function (650 pg/mL, IQR 419–880) were higher compared with healthy controls (P .006). tTGAb levels in IgAN patients (1.3, 0.8–1.9 U/mL) were higher (P &amp;lt;.001) compared with healthy controls (0.6, 0.3–0.7 U/mL). One IgAN patient had a borderline elevated tTGAb of 7.1 U/mL. No one had an elevated EmA test result. I-FABP and tTGAb levels had no correlation (rS 0.182, P = .051). CONCLUSION The clear difference in the levels of I-FABP in IgAN patients with preserved kidney function compared with healthy controls was of utmost interest. Elevated I-FABP may represent enterocyte injury and intestinal inflammation present in IgAN. Even though CD was excluded serologically, also the tTGAb levels were higher among IgAN patients. These findings spur us in search for an intestinal process in the pathophysiology of IgAN.

Enhanced Bacteremia in Dextran Sulfate-Induced Colitis in Splenectomy Mice Correlates with Gut Dysbiosis and LPS Tolerance.

Because both endotoxemia and gut dysbiosis post-splenectomy might be associated with systemic infection, the susceptibility against infection was tested by dextran sulfate solution (DSS)-induced colitis and lipopolysaccharide (LPS) injection models in splenectomy mice with macrophage experiments. Here, splenectomy induced a gut barrier defect (FITC-dextran assay, endotoxemia, bacteria in mesenteric lymph nodes, and the loss of enterocyte tight junction) and gut dysbiosis (increased Proteobacteria by fecal microbiome analysis) without systemic inflammation (serum IL-6). In parallel, DSS induced more severe mucositis in splenectomy mice than sham-DSS mice, as indicated by mortality, stool consistency, gut barrier defect, serum cytokines, and blood bacterial burdens. The presence of green fluorescent-producing (GFP) E. coli in the spleen of sham-DSS mice after an oral gavage supported a crucial role of the spleen in the control of bacteria from gut translocation. Additionally, LPS administration in splenectomy mice induced lower serum cytokines (TNF-α and IL-6) than LPS-administered sham mice, perhaps due to LPS tolerance from pre-existing post-splenectomy endotoxemia. In macrophages, LPS tolerance (sequential LPS stimulation) demonstrated lower cell activities than the single LPS stimulation, as indicated by the reduction in supernatant cytokines, pro-inflammatory genes (iNOS and IL-1β), cell energy status (extracellular flux analysis), and enzymes of the glycolysis pathway (proteomic analysis). In conclusion, a gut barrier defect after splenectomy was vulnerable to enterocyte injury (such as DSS), which caused severe bacteremia due to defects in microbial control (asplenia) and endotoxemia-induced LPS tolerance. Hence, gut dysbiosis and gut bacterial translocation in patients with a splenectomy might be associated with systemic infection, and gut-barrier monitoring or intestinal tight-junction strengthening may be useful.

P101: GUT MICROBIOTA IMPACT ON ANGOLAN CHILDREN WITH SICKLE CELL DISEASE

Background: Sickle cell disease (SCD) is one of the most prevalent genetic diseases, affecting between 20 and 25 million people worldwide. In the Sub-Saharan Africa, where it is more prevalent, it contributes to 50–80% of under-5 mortality. Clinical manifestations of SCD are very heterogeneous and the intestinal microbiome appears to be crucial in the modulation of inflammation, cell adhesion and induction of aged neutrophils, which are the main interveners of recurrent vaso-occlusive crisis. Enterocyte injury, increased permeability, altered microbial composition, and bacterial overgrowth have all been documented as microbial and pathophysiologic changes in the gut microbiome of SCD patients in recent research studies. Microbiota analysis in SCD populations will be essential to demonstrate the importance of specific bacteria and their function in this disease and provide new insights for attenuating symptoms and new drug targets. Aims: Given this, our aim is to sequence by NGS bacterial 16S RNA gene in order to characterize the gut microbiome of SCD children and healthy siblings, as a control. A written informed consent was presented and explained to all the guardian participants prior to the data collection. A total of 72 stool samples were obtained from children between 3–14 years old. Results: Our preliminary results showed that the SCD and control samples exhibit some notable differences in microbiota relative abundance, at different levels of classification. Children with the disease have a higher number of the phylum Actinobacteria (p=0.013) with a mean of sequences of 5.47% (± 3.49), while the siblings have a mean of 3.25% (± 2.98). As for the genus level, only Clostridium cluster XI bacteria was more prevalent in the SCD children, whereas the siblings had higher numbers of Blautia, Aestuariispira, Campylobacter, Helicobacter, Polaribacter and Anaerorhabdus. Conclusion: There is still much to learn before fully relying on the therapeutic approaches for gut modulation, which is why more research in this field is crucial to making this a reality. This works as been supported by FCT/Aga Khan (project n°330842553) and FCT/MCTES (UIDB/05608/2020 and UIDP/05608/2020) –H&TRC.

Lactobacillus rhamnosus attenuates Thai chili extracts induced gut inflammation and dysbiosis despite capsaicin bactericidal effect against the probiotics, a possible toxicity of high dose capsaicin.

Because of a possible impact of capsaicin in the high concentrations on enterocyte injury (cytotoxicity) and bactericidal activity on probiotics, Lactobacillus rhamnosus L34 (L34) and Lactobacillus rhamnosus GG (LGG), the probiotics derived from Thai and Caucasian population, respectively, were tested in the chili-extract administered C57BL/6 mice and in vitro experiments. In comparison with placebo, 2 weeks administration of the extract from Thai chili in mice caused loose feces and induced intestinal permeability defect as indicated by FITC-dextran assay and the reduction in tight junction molecules (occludin and zona occludens-1) using fluorescent staining and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the chili extracts also induced the translocation of gut pathogen molecules; lipopolysaccharide (LPS) and (1→3)-β-d-glucan (BG) and fecal dysbiosis (microbiome analysis), including reduced Firmicutes, increased Bacteroides, and enhanced total Gram-negative bacteria in feces. Both L34 and LGG attenuated gut barrier defect (FITC-dextran, the fluorescent staining and gene expression of tight junction molecules) but not improved fecal consistency. Additionally, high concentrations of capsaicin (0.02–2 mM) damage enterocytes (Caco-2 and HT-29) as indicated by cell viability test, supernatant cytokine (IL-8), transepithelial electrical resistance (TEER) and transepithelial FITC-dextran (4.4 kDa) but were attenuated by Lactobacillus condition media (LCM) from both probiotic-strains. The 24 h incubation with 2 mM capsaicin (but not the lower concentrations) reduced the abundance of LGG (but not L34) implying a higher capsaicin tolerance of L34. However, Lactobacillus rhamnosus fecal abundance, using qRT-PCR, of L34 or LGG after 3, 7, and 20 days of the administration in the Thai healthy volunteers demonstrated the similarity between both strains. In conclusion, high dose chili extracts impaired gut permeability and induced gut dysbiosis but were attenuated by probiotics. Despite a better capsaicin tolerance of L34 compared with LGG in vitro, L34 abundance in feces was not different to LGG in the healthy volunteers. More studies on probiotics with a higher intake of chili in human are interesting.

Association between enterocyte injury and fluid balance in patients with septic shock: a post hoc exploratory analysis of a prospective observational study

BackgroundThe required fluid volume differs among patients with septic shock. Enterocyte injury caused by shock may increase the need for fluid by triggering a systematic inflammatory response or an ischemia-reperfusion injury in the presence of intestinal ischemia/necrosis. This study aimed to evaluate the association between enterocyte injury and positive fluid balance in patients with septic shock.MethodsThis study was a post hoc exploratory analysis of a prospective observational study that assessed the association between serum intestinal fatty acid-binding protein, a biomarker of enterocyte injury, and mortality in patients with septic shock. Intestinal fatty acid-binding protein levels were recorded on intensive care unit admission, and fluid balance was monitored from intensive care unit admission to Day 7. The association between intestinal fatty acid-binding protein levels at admission and the infusion balance during the early period after intensive care unit admission was evaluated. Multiple linear regression analysis, with adjustments for severity score and renal function, was performed.ResultsOverall, data of 57 patients were analyzed. Logarithmically transformed intestinal fatty acid-binding protein levels were significantly associated with cumulative fluid balance per body weight at 24 and 72 h post-intensive care unit admission both before (Pearson’s r = 0.490 [95% confidence interval: 0.263–0.666]; P < 0.001 and r = 0.479 [95% confidence interval: 0.240–0.664]; P < 0.001, respectively) and after (estimate, 14.4 [95% confidence interval: 4.1–24.7]; P = 0.007 and estimate, 26.9 [95% confidence interval: 11.0–42.7]; P = 0.001, respectively) adjusting for severity score and renal function.ConclusionsEnterocyte injury was significantly associated with cumulative fluid balance at 24 and 72 h post-intensive care unit admission. Enterocyte injury in patients with septic shock may be related to excessive fluid accumulation during the early period after intensive care unit admission.

Neutrophil extracellular traps impair intestinal barrier functions in sepsis by regulating TLR9-mediated endoplasmic reticulum stress pathway

Increased neutrophil extracellular traps (NETs) formation has been found to be associated with intestinal inflammation, and it has been reported that NETs may drive the progression of gut dysregulation in sepsis. However, the biological function and regulation of NETs in sepsis-induced intestinal barrier dysfunction are not yet fully understood. First, we found that both circulating biomarkers of NETs and local NETs infiltration in the intestine were significantly increased and had positive correlations with markers of enterocyte injury in abdominal sepsis patients. Moreover, the levels of local citrullinated histone 3 (Cit H3) expression were associated with the levels of BIP expression. To further confirm the role of NETs in sepsis-induced intestinal injury, we compared peptidylarginine deiminase 4 (PAD4)-deficient mice and wild-type (WT) mice in a lethal septic shock model. In WT mice, the Cit H3-DNA complex was markedly increased, and elevated intestinal inflammation and endoplasmic reticulum (ER) stress activation were also found. Furthermore, PAD4 deficiency alleviated intestinal barrier disruption and decreased ER stress activation. Notably, NETs treatment induced intestinal epithelial monolayer barrier disruption and ER stress activation in a dose-dependent manner in vitro, and ER stress inhibition markedly attenuated intestinal apoptosis and tight junction injury. Finally, TLR9 antagonist administration significantly abrogated NETs-induced intestinal epithelial cell death through ER stress inhibition. Our results indicated that NETs could contribute to sepsis-induced intestinal barrier dysfunction by promoting inflammation and apoptosis. Suppression of the TLR9–ER stress signaling pathway can ameliorate NETs-induced intestinal epithelial cell death.

A32 EMPAGLIFOZIN IMPROVES GASTROINTESTINAL INFLAMMATION IN A MOUSE MODEL OF COLITIS

Abstract Background Empagliflozin (EMPA) is a highly selective sodium glucose cotransporter-2 (SGLT2) inhibitor and is increasingly being utilized as an antihyperglycemic agent in the management of type 2 diabetes. Interestingly, it has been demonstrated in human trials that EMPA treatment exerts potent cardioprotective effects by reducing cardiac inflammation independently of glycemic control. Further, EMPA has also been shown to suppress LPS-induced renal and systemic inflammation in an animal model. Based on these findings, we hypothesized that EMPA treatment may also be effective in reducing gut inflammation. Aims The aim of this study was to examine the effects of treatment with EMPA on gastrointestinal inflammation in an animal model of inflammatory bowel disease and to determine mechanistic insights regarding its direct effects on gut cytokine secretion. Methods Adult male and female IL-10-/- mice with established colitis were treated with a daily gavage of EMPA (10mg/kg; n=10) or vehicle (n=10) for 14 days. Disease activity was assessed by measurement of mouse weight, colonic weight and length, histological score, cytokine levels in colonic homogenate and lipocalin-2 levels in stool. To examine for possible direct effects of EMPA, colonic explants from wild-type (n=8) and IL-10-/- (n=8) mice were incubated with increasing doses of EMPA (0.1–5 µM) ± LPS (10µg/ml) for 2 hours and tissue levels of IL-1β and TNFα protein measured by ELISA. Results After 14 days EMPA treated IL-10-/- mice had a significant improvement in colonic inflammation as evidenced by decreased colonic weight to length ratio (p=0.019), decreased fecal lipocalin-2 (p=0.03), as well as decreased enterocyte injury (p=0.01), decreased lamina propria neutrophils (p=0.01) and decreased total histological score (p=0.006). EMPA treated mice also maintained their weight over the 14 days while untreated mice continued to lose weight (p=0.04). There were no significant differences in colonic homogenate levels of TNFα, IL-1β, or IL-6 or in blood glucose levels between EMPA-treated mice and controls. In addition, EMPA did not suppress levels of basal or LPS-induced TNFα and IL-1β in colonic explants from either wild-type or IL-10-/- mice suggesting that the beneficial effects in IL-10-/- mice were not due to direct effects of EMPA on colonic TNFα or IL-1β cytokine levels. Conclusions EMPA treatment dramatically improved histologic and fecal inflammatory markers and maintained body weight in adult IL-10-/- mice with established colitis. These findings suggest further investigations into the effects of EMPA in treating gut inflammation are warranted. Funding Agencies CAG, CIHR

MtDNA-STING pathway promotes necroptosis-dependent enterocyte injury in intestinal ischemia reperfusion

Intestinal ischemia reperfusion (I/R) injury is the important pathogenesis for acute intestinal barrier disruption. The STING signaling is associated with gut homeostasis and barrier integrity. However, the biological function and regulation of STING signaling in intestinal I/R injury are not yet fully understood. As the ligand of STING signaling, the mitochondrial DNA (mtDNA) has been found to be associated with necroptosis. It still remains unknown whether mtDNA-STING signaling triggers intestinal necroptosis in intestinal I/R injury. We found that circulating RIPK3 was significantly increased and had a positive correlation with markers of enterocyte injury in critically ill patients with intestinal injury. Moreover, the levels of circulating mtDNA were also associated with the levels of circulating RIPK3. To explore the relationship between mtDNA and intestinal necroptosis, mice were treated with the intraperitoneal injection of mtDNA, and necroptosis signaling was remarkably activated and the inhibition of necroptosis alleviated mtDNA-induced intestinal injury. Furthermore, STING knockout mice showed an alleviated intestinal necroptosis. In intestinal I/R injury, mtDNA was released from IECs and necroptosis was also triggered, companied with a significant decrease of RIPK3 in the intestine. STING knockout mice markedly attenuated intestinal necroptosis and intestinal I/R injury. Finally, we found that mtDNA-mediated STING signaling triggered necroptosis through synergistic IFN and TNF-α signaling in primary IECs. Our results indicated that mtDNA-STING signaling can contribute to intestinal I/R injury by promoting IEC necroptosis. STING-mediated both IFN and TNF-α signaling can trigger intestinal nercroptosis.

MtDNA-STING Pathway Promotes Necroptosis-Dependent Enterocyte Injury in Intestinal Ischemia Reperfusion

Background: Intestinal ischemia reperfusion (I/R) injury is the important pathogenesis for acute intestinal barrier disruption. The STING signaling is associated with gut homeostasis and barrier integrity. However, the biological function and regulation of STING signaling in intestinal I/R injury are not yet fully understood. As the ligand of STING signaling, the mitochondrial DNA (mtDNA) has been found to be associated with necroptosis. It still remains unknown whether mtDNA-STING signaling triggers intestinal necroptosis in intestinal I/R injury. Methods: The levels of circulating receptor interacting protein kinase 3 (RIPK3) were first collected to explore the role of necroptosis in critically ill patients with intestinal injury. Furthermore, the relationship between mtDNA-STING signaling and necroptosis was explored through establishing a murine model of intestinal I/R injury in wild-type mice and STING knockout mice. Primary intestinal epithelial cells (IECs) were isolated to investigate downstream signaling of STING leading to intestinal necroptosis. Findings: In critically ill patients with intestinal injury, circulating RIPK3 was significantly increased and had a positive correlation with markers of enterocyte injury. Moreover, the levels of circulating mtDNA were also associated with the levels of circulating RIPK3. After the intraperitoneal injection of mtDNA in mice, necroptosis signaling was remarkably activated and the inhibition of necroptosis alleviated mtDNA-induced intestinal injury. Furthermore, STING knockout mice showed an alleviated necroptosis. In intestinal I/R injury, mtDNA released from IECs was released and necroptosis was also triggered, companied with a significant decrease of RIPK3 in the intestine. STING knockout mice markedly attenuated intestinal necroptosis and intestinal I/R injury. Finally, we found that mtDNA-mediated STING signaling triggered necroptosis through synergistic IFN and TNF-α signaling in primary IECs. Interpretation: Our results indicated that mtDNA-STING signaling can contribute to intestinal I/R injury by promoting IEC neroptosis. STING-mediated both IFN and TNF-α signaling can trigger intestinal nercroptosis. Funding Statement: This work was supported by the National Natural Science Foundation of China (81801971, 81772052), Distinguished Scholars Foundation of Jiangsu Province (JCRCB2016006). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The collection of human blood samples was approved by the Institutional Review Board Ethics Committee at Jinling Hospital. Before collecting blood samples, all patients and healthy adult volunteers provided written informed consent. The care and use of the animals were approved by Jinling Hospital Animal Care Committee.

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Association between Enterocyte Injury and Mortality in Patients on Hemodialysis Who Underwent Cardiac Surgery: An Exploratory Study

BackgroundIntestinal ischemia and enterocyte injury are significant causes of death after cardiac surgery. Hemodialysis is a well-known risk factor for intestinal ischemia. However, the relationship between enterocyte injury and mortality is unclear. This exploratory study assessed the association between intestinal fatty acid–binding protein (I-FABP), a specific marker of enterocyte injury, at intensive care unit (ICU) admission and in-hospital mortality in patients on hemodialysis who underwent cardiac surgery with cardiopulmonary bypass. Materials and methodsForty-seven consecutive patients on long-term hemodialysis who underwent elective cardiac surgery (median age, 70 y; men, 27 [57%]) were prospectively enrolled. The association between serum I-FABP levels at ICU admission and in-hospital mortality was compared with the associations between serum I-FABP levels and prognostic severity scores, vasoactive-inotropic scores, and lactate levels. ResultsOnly I-FABP levels at ICU admission were significantly related to in-hospital mortality (odds ratio, 5.54; 95% confidence interval [CI], 1.08-28.43) in the simple logistic regression analysis. Univariate and multiple linear regression analyses indicated prolonged cardiopulmonary bypass (ρ, 0.49; 95% CI, 0.15-0.83), higher mean norepinephrine dose (ρ, 0.07; 95% CI, 0.02-0.12), lower mean dopamine dose (ρ, −0.51; 95% CI, −0.94 to −0.08), and intra-aortic balloon pump use (ρ, 3.63; 95% CI, 1.68-5.59) were significant risk factors for high I-FABP levels. ConclusionsEnterocyte injury at ICU admission was associated with in-hospital mortality after cardiac surgery for patients on hemodialysis. Intraoperative hidden hypoperfusion of the intestine may impact prognoses. Enterocyte injury prevention, early diagnosis, and intervention for intestinal ischemia might be required to improve outcomes.

Immediate Post-operative Enterocyte Injury, as Determined by Increased Circulating Intestinal Fatty Acid Binding Protein, Is Associated With Subsequent Development of Necrotizing Enterocolitis After Infant Cardiothoracic Surgery.

Objectives: 1 Measure serial serum intestinal fatty acid binding protein levels in infants undergoing cardiac surgery with cardiopulmonary bypass to evaluate for evidence of early post-operative enterocyte injury. 2 Determine the association between immediate post-operative circulating intestinal fatty acid binding protein levels and subsequent development of necrotizing enterocolitis.Design: Observational cohort study. Intestinal fatty acid binding protein was measured pre-operatively, at rewarming, and at 6 and 24 h post-operatively. Percent of goal enteral kilocalories on post-operative day 5 and episodes of necrotizing enterocolitis were determined. Multivariable analysis assessed for factors independently associated with clinical feeding outcomes and suspected/definite necrotizing enterocolitis.Setting: Quaternary free-standing children's hospital pediatric cardiac intensive care unit.Patients: 103 infants <120 days of age undergoing cardiothoracic surgery with cardiopulmonary bypass.Interventions: None.Results: Median pre-operative intestinal fatty acid binding protein level was 3.93 ng/ml (range 0.24–51.32). Intestinal fatty acid binding protein levels rose significantly at rewarming (6.35 ng/ml; range 0.54–56.97; p = 0.008), continued to rise slightly by 6 h (6.57 ng/ml; range 0.75–112.04; p = 0.016), then decreased by 24 h (2.79 ng/ml; range 0.03–81.74; p < 0.0001). Sixteen subjects (15.7%) developed modified Bell criteria Stage 1 necrotizing enterocolitis and 9 subjects (8.8%) developed Stage 2 necrotizing enterocolitis. Infants who developed necrotizing enterocolitis demonstrated a significantly higher distribution of intestinal fatty acid binding protein levels at both 6 h (p = 0.005) and 24 h (p = 0.005) post-operatively. On multivariable analysis, intestinal fatty acid binding protein was not associated with percentage of goal enteral kilocalories delivered on post-operative day 5. Higher intestinal fatty acid binding protein was independently associated with subsequent development of suspected/definite necrotizing enterocolitis (4% increase in odds of developing necrotizing enterocolitis for each unit increase in intestinal fatty acid binding protein; p = 0.0015).Conclusions: Intestinal fatty acid binding protein levels rise following infant cardiopulmonary bypass, indicating early post-operative enterocyte injury. Intestinal fatty acid binding protein was not associated with percent of goal enteral nutrition achieved on post-operative day 5, likely due to protocolized feeding advancement based on clinically observable factors. Higher intestinal fatty acid binding protein at 6 h post-operatively was independently associated with subsequent development of necrotizing enterocolitis and may help identify patients at risk for this important complication.