Neutrophil Extracellular Traps Caused by Gut Leakage Trigger the Autoimmune Response in Nonobese Diabetic Mice

Increased formation of neutrophil extracellular traps (NETs) is associated with gut leakage in type 1 diabetes (T1D). To explore the mechanism of how enteropathy exacerbated by NETs triggers pancreatic autoimmunity in T1D, we carried out a correlation analysis for NET formation with gut barrier functions and autoimmunity in nonobese diabetic (NOD) mice. Inducing chronic colitis or knocking out of peptidyl arginine deiminase type 4 (PAD4) in NOD mice were used to further study the effect of NET formation on the progression of T1D. Microbial alterations in Deferribacteres and Proteobacteria, along with the loss of gut barrier function, were found to be associated with increased endotoxin and abnormal formation of NETs in NOD mice. Both DSS-induced colitis and knockout of PAD4 in NOD mice indicated that PAD4-dependent NET formation was involved in the aggravation of gut barrier dysfunction, the production of autoantibodies, and the activation of enteric autoimmune T cells, which then migrated to pancreatic lymph nodes (PLNs) and caused self-damage. The current study thus provides evidence that PAD4-dependent NET formation is engaged in leaky gut triggering pancreatic autoimmunity and suggests that either degradation of NETs or inhibition of NET formation may be helpful for innovative therapeutic interventions in T1D.

Impact of Dietary Sodium Butyrate and Salinomycin on Performance and Intestinal Microbiota in a Broiler Gut Leakage Model

Unfavorable alterations of the commensal gut microbiota and dysbacteriosis is a major health problem in the poultry industry. Understanding how dietary intervention alters the microbial ecology of broiler chickens is important for prevention strategies. A trial was conducted with 672 Ross 308 day-old male broilers fed a basic diet (no additives, control) or the basic diet supplemented with 500 mg/kg encapsulated butyrate or 68 mg/kg salinomycin. Enteric challenge was induced by inclusion of 50 g/kg rye in a grower diet and oral gavage of a 10 times overdose of a vaccine against coccidiosis. Compared to control and butyrate-supplemented birds, salinomycin supplementation alleviated growth depression. Compared to butyrate and non-supplemented control, salinomycin increased potentially beneficial Ruminococcaceae and reduced potentially pathogenic Enterobacteriaceae and counts of Lactobacillus salivarius and Clostridium perfringens. Further, salinomycin supplementation was accompanied by a pH decrease and succinic acid increase in ceca, while coated butyrate (0.5 g/kg) showed no or limited effects. Salinomycin alleviated growth depression and maintained intestinal homeostasis in the challenged broilers, while butyrate in the tested concentration showed limited effects. Thus, further investigations are required to identify optimal dietary inclusion rates for butyrate used as alternative to ionophore coccidiostats in broiler production.

NEIL3-deficiency increases gut permeability and contributes to a pro-atherogenic metabolic phenotype

Atherosclerosis and its consequences cause considerable morbidity and mortality world-wide. We have previously shown that expression of the DNA glycosylase NEIL3 is regulated in human atherosclerotic plaques, and that NEIL3-deficiency enhances atherogenesis in Apoe−/− mice. Herein, we identified a time point prior to quantifiable differences in atherosclerosis between Apoe−/−Neil3−/− mice and Apoe−/− mice. Mice at this age were selected to explore the metabolic and pathophysiological processes preceding extensive atherogenesis in NEIL3-deficient mice. Untargeted metabolomic analysis of young Apoe−/−Neil3−/− mice revealed significant metabolic disturbances as compared to mice expressing NEIL3, particularly in metabolites dependent on the gut microbiota. 16S rRNA gene sequencing of fecal bacterial DNA indeed confirmed that the NEIL3-deficient mice had altered gut microbiota, as well as increased circulating levels of the bacterially derived molecule LPS. The mice were challenged with a FITC-conjugated dextran to explore gut permeability, which was significantly increased in the NEIL3-deficient mice. Further, immunohistochemistry showed increased levels of the proliferation marker Ki67 in the colonic epithelium of NEIL3-deficient mice, suggesting increased proliferation of intestinal cells and gut leakage. We suggest that these metabolic alterations serve as drivers of atherosclerosis in NEIL3-deficient mice.

Gut leakage markers in response to strenuous exercise in patients with suspected coronary artery disease

Introduction Although regular physical activity is associated with reduced risk of cardiovascular disease (CVD), acute vigorous exercise seems to transiently increase the risk of acute coronary events in patients with underlying CVD. Some studies have reported regular physical activity to associate with microbial diversity, whereas elevated levels of gut leakage markers have been shown after strenuous exercise in healthy individuals. Any predictive value of a temporary increase in gut leakage markers on the risk of coronary events in susceptible individuals is unknown. Purpose We aimed to explore gut leakage markers in response to a bout of strenuous exercise in patients with symptoms of chronic coronary syndrome (CCS). We hypothesized that gut leakage markers would increase after acute strenuous exercise, and that the increase would be higher in patients with angiographically verified CAD. Methods Patients referred to exercise stress testing or coronary angiography due to symptoms suggestive of CCS were included (n=327). A maximal exercise ECG stress test was performed using a bicycle ergometer. Venous blood samples were drawn at rest prior to the test and within 5 min after the test ended, for analysis of soluble cluster of differentiation 14 (sCD14), lipopolysaccharide-binding protein (LBP) and intestinal fatty-acid binding protein (I-FABP) by ELISAs. Quantification of lipopolysaccharide (LPS) and relative quantification of gene expression of the toll-like receptor 4 (TLR4) in circulating leukocytes was performed in a subset of patients (n=101). Patients then underwent coronary angiography, and were grouped according to the degree of CAD. Results Of the 287 patients who completed the exercise stress test and coronary angiography, 69 (24%) had no CAD, 88 (31%) had non-significant CAD and 130 (45%) had significant CAD. Mean exercise duration was 10:05±4:46 min and the duration did not differ between the groups. There were no significant differences in resting levels of gut leakage markers between the groups. In the total population, sCD14, LBP and LPS increased significantly after exercise (p<0.0001, all), whereas I-FABP did not. The gene expression of TLR4 decreased significantly after exercise (p<0.0001). There were no differences in exercise-induced changes in any of the measured markers between groups with no CAD, non-significant CAD and significant CAD. Conclusion In patients with symptoms suggestive of CCS, LPS, LBP and sCD14 increased significantly after strenuous exercise, suggesting that even short bouts of vigorous exercise are associated with gut leakage. The decrease in gene expression of TLR4 may be discussed to be compensatory to the increase in LPS or possibly reflecting an increase in TLR4 translation in response to LPS. The presence of CAD or not did not seem to impact exercise-induced increase in gut leakage markers. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Stein Erik Hagen Foundation for Clinical Heart Research, Olso, Norway

Gut Leakage Markers in Response to Strenuous Exercise in Patients with Suspected Coronary Artery Disease

Elevated levels of gut leakage markers have been shown after strenuous exercise in healthy individuals. Any association between a temporary increase in these markers and the presence of coronary artery disease (CAD) is unknown. We therefore aimed to explore circulating gut leakage markers in response to a bout of strenuous exercise in patients with symptoms of CAD. Patients referred to exercise stress testing due to symptoms of CAD were included (n = 287). A maximal exercise ECG stress test was performed and venous blood samples were drawn at rest and within five minutes after, for analysis of soluble cluster of differentiation 14 (sCD14), lipopolysaccharide-binding protein (LBP), intestinal fatty-acid binding protein (I-FABP), lipopolysaccharide (LPS) and gene expression of toll-like receptor 4 (TLR4) in circulating leukocytes. Patients then underwent coronary angiography. LPS, LBP and sCD14 increased significantly after strenuous exercise in patients with symptoms of CAD, suggesting that even short bouts of vigorous exercise are associated with gut leakage. The gene expression of TLR4 decreased significantly after exercise, possibly as a negative feedback to the increase in LPS. There were no differences in exercise-induced changes between the groups of CAD, suggesting gut leakage to be independent of the presence of CAD.

Increased susceptibility to dextran sulfate-induced mucositis of iron-overload β-thalassemia mice, another endogenous cause of septicemia in thalassemia

Enterocyte damage and gut dysbiosis are caused by iron-overload in thalassemia (Thl), possibly making the gut vulnerable to additional injury. Hence, iron-overload in the heterozygous β-globin deficient (Hbbth3/+) mice were tested with 3% dextran sulfate solution (DSS). With 4 months of iron-gavage, iron accumulation, gut-leakage (fluorescein isothiocyanate dextran (FITC-dextran), endotoxemia, and tight junction injury) in Thl mice were more prominent than WT mice. Additionally, DSS-induced mucositis in iron-overloaded mice from Thl group was also more severe than the WT group as indicated by mortality, liver enzyme, colon injury (histology and tissue cytokines), serum cytokines, and gut-leakage (FITC-dextran, endotoxemia, bacteremia, and the detection of Green-Fluorescent Producing Escherichia coli in the internal organs after an oral administration). However, Lactobacillus rhamnosus GG attenuated the disease severity of DSS in iron-overloaded Thl mice as indicated by mortality, cytokines (colon tissue and serum), gut-leakage (FITC-dextran, endotoxemia, and bacteremia) and fecal dysbiosis (microbiome analysis). Likewise, Lactobacillus conditioned media (LCM) decreased inflammation (supernatant IL-8 and cell expression of TLR-4, nuclear factor κB (NFκB), and cyclooxygenase-2 (COX-2)) and increased transepithelial electrical resistance (TEER) in enterocytes (Caco-2 cells) stimulated by lipopolysaccharide (LPS) and LPS plus ferric ion. In conclusion, in the case of iron-overloaded Thl, there was a pre-existing intestinal injury that wask more vulnerable to DSS-induced bacteremia (gut translocation). Hence, the prevention of gut-derived bacteremia and the monitoring on gut-leakage might be beneficial in patients with thalassemia.

Gut Leakage of Fungal‐Related Products: Turning Up the Heat for HIV Infection

The intestinal epithelial layer serves as a physical and functional barrier between the microbiota in the lumen and immunologically active submucosa. Th17 T-cell function protects the gut epithelium from aggression from microbes and their by-products. Loss of barrier function has been associated with enhanced translocation of microbial products which act as endotoxins, leading to local and systemic immune activation. Whereas the inflammatory role of LPS produced by Gram-negative bacteria has been extensively studied, the role of fungal products such as β-D-glucan remains only partially understood. As HIV infection is characterized by impaired gut Th17 function and increased gut permeability, we critically review mechanisms of immune activation related to fungal translocation in this viral infection. Additionally, we discuss markers of fungal translocation for diagnosis and monitoring of experimental treatment responses. Targeting gut barrier dysfunction and reducing fungal translocation are emerging strategies for the prevention and treatment of HIV-associated inflammation and may prove useful in other inflammatory chronic diseases.

Gut related inflammation and cardiorespiratory fitness in patients with CAD and type 2 diabetes: a sub-study of a randomized controlled trial on exercise training

Aim Gut leakage has been shown to associate with low-grade inflammation and lower cardiorespiratory fitness in diabetic subjects. We aimed to investigate whether gut leakage markers related to cardiorespiratory fitness in patients with both coronary artery disease and type 2 diabetes, and whether these were affected by long-term exercise training. Methods Patients with angiographically verified coronary artery disease and type 2 diabetes mellitus (n = 137) were randomized to either 12 months exercise intervention or conventional follow-up. A cardiopulmonary exercise test and fasting blood samples were obtained before and after intervention to assess VO2peak and the biomarkers soluble CD14, lipopolysaccharide-binding protein and intestinal fatty-acid binding protein as markers of gut leakage. Results 114 patients completed the intervention satisfactory. VO2peak correlated inversely to sCD14 (r = − 0.248, p = 0.004) at baseline. Dividing sCD14 into quartiles (Q), VO2peak was significantly higher in Q1 vs. Q2–4 (p = 0.001), and patients in Q2-4 (sCD14 > 1300 ng/mL) had an OR of 2.9 (95% CI 1.2–7.0) of having VO2peak below median (< 23.8 ml/kg/min) at baseline. There were no statistically significant differences in changes in gut leakage markers between the two randomized groups (all p > 0.05) after 12 months. Conclusions Cardiorespiratory fitness related inversely to sCD14, suggesting physical capacity to be associated with gut leakage in patients with CAD and T2DM. Long-term exercise training did not affect circulating gut leakage markers in our population. Trial registration NCT01232608, Registered 02 November 2010—Retrospectively registered at https://clinicaltrials.gov/ct2/show/NCT01232608?term=NCT01232608&draw=2&rank=1

Prominent Indomethacin-Induced Enteropathy in Fcgriib Defi-cient lupus Mice: An Impact of Macrophage Responses and Immune Deposition in Gut

A high dose of NSAIDs, a common analgesic, might induce lupus activity through several NSAIDs adverse effects including gastrointestinal permeability defect (gut leakage) and endotoxemia. Indomethacin (25 mg/day) was orally administered for 7 days in 24-wk-old Fc gamma receptor IIb deficient (FcgRIIb-/-) mice, an asymptomatic lupus model (increased anti-dsDNA without lupus nephritis), and age-matched wild-type (WT) mice. Severity of indomethacin-induced enteropathy in FcgRIIb-/- mice was higher than WT mice as demonstrated by survival analysis, intestinal injury (histology, immune-deposition, and intestinal cytokines), gut leakage (FITC-dextran assay and endotoxemia), serum cytokines, and lupus characteristics (anti-dsDNA, renal injury, and proteinuria). Prominent responses of FcgRIIb-/- macrophages toward lipopolysaccharide (LPS) compared to WT cells due to the expression of only activating-FcgRs without inhibitory-FcgRIIb were demonstrated. Extracellular flux analysis indicated the greater mitochondria activity (increased respiratory capacity and respiratory reserve) in FcgRIIb-/- macrophages with a concordant decrease in glycolysis activity when compared to WT cells. In conclusion, gut leakage-induced endotoxemia is more severe in indomethacin-administered FcgRIIb-/- mice than WT, possibly due to the enhanced indomethacin toxicity from lupus-induced intestinal immune-deposition. Due to a lack of inhibitory-FcgRIIb expression, mitochondrial function, and cytokine production of FcgRIIb-/- macrophages were more prominent than WT cells. Hence, lupus disease-activation from NSAIDs-enteropathy-induced gut leakage is possible.