Effects of Different Probiotic Strains B. Lactis, L. Rhamnosus and L. Reuteri on Brain-Intestinal Axis Immunomodulation in an Endotoxin-Induced Inflammation

Aim: The study evaluated the effects of supplementation with three different probiotic strains B. lactis (LACT GBTM), L. rhamnosus (RHAM GBTM) and L. reuteri (REUT GBTM) on brain-intestinal immunomodulation in an animal model of LPS-induced inflammation. Methods: 50 mice Balb/C were distributed into five groups: Control; lipopolysaccharide (LPS); LPS + B. lactis (LACT GBTM); LPS + L. rhamnosus (RHAM GBTM); LPS + L. reuteri (REUT GBTM). The animals were supplemented with their respective probiotic microorganisms daily, for 30 days, at a concentration of 1x109 CFU/animal/day. After 30 days of supplementation, animals received the inflammatory insult by LPS (15mg/kg). Behavioral tests, oxidative stress and inflammation were performed, as well as gut and brain histology. Results: In the behavioral test, LPS+ B. lactis group was less anxious than the other groups. Serum interleukin IL-1β and IL-6 levels increased in all groups that received the LPS insult and there was a reduction in inflammation in the supplemented groups when compared to the LPS group in brain and gut. A reduction in myeloperoxidase activity and oxidative stress in groups supplemented with probiotics. Intestine histological analysis, damage to tissue integrity in the LPS group and preservation of integrity in the supplemented animals. In the brain, infiltrates of perivascular inflammatory cells can be seen in the LPS group. Conclusion: The three probiotic studies showed efficient immunomodulating activity and ensured integrity of the intestinal barrier function, even after the severe insult by LPS. These results show the important role of probiotics in the gut-brain axis.

Cells and Mediators of Inflammation as Effectors of Epithelial Repair in the Inflamed Intestine

Mucosal and histological healing have become the gold standards for assessing the efficacy of therapy in patients living with inflammatory bowel diseases (IBD). Despite these being the accepted goals in therapy, the mechanisms that underlie the healing of the mucosa after an inflammatory insult are not well understood, and many patients fail to meet this therapeutic endpoint. Here we review the emerging evidence that mediators (e.g. prostaglandins, cytokines, proteases, reactive oxygen and nitrogen species) and innate immune cells (e.g. neutrophils and monocytes/macrophages), that are involved in the initiation of the inflammatory response, are also key players in the mechanisms underlying mucosal healing to resolve chronic inflammation in the colon. The dual function mediators comprise an inflammation/repair program that returns damaged tissue to homeostasis. Understanding details of the dual mechanisms of these mediators and cells may provide the basis for the development of drugs that can help to stimulate epithelial repair in patients affected by IBD.

Effects of Different Probiotic Strains B. Lactis, L. Rhamnosus and L. Reuteri on Brain-Intestinal Axis Immunomodulation in an Endotoxin-Induced Inflammation

Aim: The study evaluated the effects of supplementation with three different probiotic strains B. lactis (LACT GBTM), L. rhamnosus (RHAM GBTM) and L. reuteri (REUT GBTM) on brain-intestinal immunomodulation in an animal model of LPS-induced inflammation. Methods: 50 mice Balb/C were distributed into five groups: Control; lipopolysaccharide (LPS); LPS + B. lactis (LACT GBTM); LPS + L. rhamnosus (RHAM GBTM); LPS + L. reuteri (REUT GBTM). The animals were supplemented with their respective probiotic microorganisms daily, for 30 days, at a concentration of 1x109 CFU/animal/day. After 30 days of supplementation, animals received the inflammatory insult by LPS (15mg/kg). Behavioral tests, oxidative stress and inflammation were performed, as well as gut and brain histology. Results: In the behavioral test, LPS+ B. lactis group was less anxious than the other groups. Serum interleukin IL-1β and IL-6 levels increased in all groups that received the LPS insult and there was a reduction in inflammation in the supplemented groups when compared to the LPS group in brain and gut. A reduction in myeloperoxidase activity and oxidative stress in groups supplemented with probiotics. Intestine histological analysis, damage to tissue integrity in the LPS group and preservation of integrity in the supplemented animals. In the brain, infiltrates of perivascular inflammatory cells can be seen in the LPS group. Conclusion: The three probiotic studies showed efficient immunomodulating activity and ensured integrity of the intestinal barrier function, even after the severe insult by LPS. These results show the important role of probiotics in the gut-brain axis.

Abstract P333: The Vascular Barrier: A Common Anti-arrhythmic Target In Atrial Fibrillation And Myocardial Infarction

Vascular leak is a major sequela of inflammation, which is associated with arrhythmic pathologies such as atrial fibrillation (AF) and myocardial infarction (MI). We recently demonstrated that the vascular leak-inducing cytokine vascular endothelial growth factor (VEGF; 90-580 pg/ml - levels found in AF patients) induces acute remodeling (30-60 minutes) of sodium channel (Na V 1.5) -rich intercalated disk (ID) nanodomains, disrupting their ultrastructure and prompting translocation of Na V 1.5 from these sites. This in turn disrupted impulse propagation and promoted arrhythmias in murine atria. Here, we tested the hypotheses that i) similar acute pro-arrhythmic remodeling occurs in the ventricles of MI patients, and ii) protecting the vascular barrier may prevent arrhythmias following an acute inflammatory insult. First, we examined myocardial samples from five human MI patients. VEGF was overexpressed in both cardiomyocytes and vascular endothelium in the border zone surrounding <6 month-old infarcts. Notably, co-localization analysis showed significantly reduced Na V 1.5 near both connexin43 and N-cadherin within the border zone in 1-, 3-, and 9-day-old infarcts, paralleling our observations in mouse atria. Next, we returned to our murine model of AF induced by acute inflammatory insult (100 pg/ml VEGF for 60 minutes) to test the antiarrhythmic efficacy of protecting the vascular endothelial barrier. Overall, median in vivo arrhythmia burden was higher in VEGF-treated mice relative to vehicle controls (7.5±11 vs. 0±6 s/hr). We tested two strategies shown to prevent vascular barrier breakdown: Blocking connexin43 hemichannels (αCT11 peptide) decreased in vivo arrhythmia burden to 0 ± 6.07 s/hr. Panx1-IL2 (a peptide inhibitor of Panx1 channels) treatment decreased also in vivo arrhythmia burden (0 ± 15.57 s/hr with 1.6 μM Panx1-IL2). Similar antiarrhythmic efficacy was also achieved with small molecule inhibitors of Cx43 and Panx1. These results highlight VEGF-induced vascular leak as a novel mechanism for acute arrhythmias both in the early stage AF and following MI. Indeed, this mechanism may contribute to post-MI AF. Importantly, vascular-barrier protection may be a viable strategy to prevent these arrhythmias.

Hypoxia Inducible Factor-1α Is a Regulator of Autophagy in Osteoarthritic Chondrocytes

Objective To investigate the relationship between hypoxia inducible factor-1α (HIF-1α) and the autophagic response in osteoarthritic chondrocytes (OA), under inflammatory insult as represented by in vitro OA model. Methods Human chondrocyte cell line C28/I2 was cultured in both normoxic and hypoxic conditions and treated with interleukin-1β (IL1β) to emulate OA inflammatory insult in vitro. Cellular HIF-1α expression was silenced using siRNA transfection and cellular autophagic (P62/LC3II) response and OA chondrocyte damage (COL2A1/MMP13) related proteins were examined using western blotting. Cellular mitophagic (BNIP3/PINK1/Parkin) and apoptotic (Caspase/Cleaved Caspase 3) were also evaluated to assess mitophagy-mediated cell death due to HIF-1α silencing. Results Chondrocyte basal autophagy levels were higher in a HIF-1α elevated environment and was more resistant to IL1β-induced inflammatory insult. Increase in autophagic proteins showed better chondrocyte repair, which resulted a lower level of reactive oxygen species production, and lesser damage to chondrocyte integrity. Silencing HIF-1α activates cellular PINK1/Parkin and BNIP3 mitophagic proteins, which leads to the activation of Caspase/Cleaved Caspase 3 apoptotic cascade. Conclusion Our results show that chondrocyte autophagy is dependent on HIF-1α expression, showing the importance of HIF-1α in hypoxic chondrocyte function in OA. Dysregulation of HIF-1α expression results in the activation of mitophagy-mediated apoptosis.

Indoleamine 2,3-dioxygenase 1 limits hepatic inflammatory cells recruitment and promotes bile duct ligation-induced liver fibrosis

Liver fibrosis is a course of chronic liver dysfunction, can develop into cirrhosis and hepatocellular carcinoma. Inflammatory insult owing to pathogenic factors plays a crucial role in the pathogenesis of liver fibrosis. Indoleamine 2,3-dioxygenase 1 (IDO1) can affect the infiltration of immune cells in many pathology processes of diseases, but its role in liver fibrosis has not been elucidated completely. Here, the markedly elevated protein IDO1 in livers was identified, and dendritic cells (DCs) immune-phenotypes were significantly altered after BDL challenge. A distinct hepatic population of CD11c+DCs was decreased and presented an immature immune-phenotype, reflected by lower expression levels of co-stimulatory molecules (CD40, MHCII). Frequencies of CD11c+CD80+, CD11c+CD86+, CD11c+MHCII+, and CD11c+CD40+ cells in splenic leukocytes were reduced significantly. Notably, IDO1 overexpression inhibited hepatic, splenic CD11c+DCs maturation, mature DCs-mediated T-cell proliferation and worsened liver fibrosis, whereas above pathological phenomena were reversed in IDO1−/− mice. Our data demonstrate that IDO1 affects the process of immune cells recruitment via inhibiting DCs maturation and subsequent T cells proliferation, resulting in the promotion of hepatic fibrosis. Thus, amelioration of immune responses in hepatic and splenic microenvironment by targeting IDO1 might be essential for the therapeutic effects on liver fibrosis.

Characterisation of EFV12 a bio-active small peptide produced by the human intestinal isolate Lactobacillus gasseri SF1109

EFV12 is a small bioactive peptide produced by Lactobacillus gasseri SF1109, a human intestinal isolate with probiotic features. In this study, EFV12 antimicrobial and anti-inflammatory properties are characterised. In particular, we propose a possible mechanism of action for EFV12 involving bacterial membranes targeting. Moreover, we show that this small peptide is able to bind lipopolysaccharides (LPS) and to counteract its inflammatory insult preventing LPS action on Toll-like receptor 4, thus interfering with extracellular signal-regulated kinase, p38 and Jun N-terminal kinase, mitogen-activated protein kinases signalling pathways. Altogether these observations suggest that the bioactive peptide EFV12 is a good candidate to promote L. gasseri induced gut homeostasis and counteracting intestinal pathogens.

Maternal Obesity Does Not Exacerbate the Effects of LPS Injection on Pregnancy Outcomes in Mice

Obesity increases the risk of a number of pregnancy complications, potentially due to chronic inflammation. We predicted that an obesogenic high-fat diet (HFD) in mice would create an inflammatory environment that would exacerbate the effects of lipopolysaccharide (LPS), an inflammatory insult, administered during pregnancy. Females were placed on a HFD or a low-fat diet (LFD) prior to mating, injected with 2 µg LPS or control on gestational day 7 and collected on day 14. Treatment with LPS increased the odds that a female thought to be pregnant at injection had no conceptuses at day 14 (p = 0.024), suggesting that injection with LPS was more likely to induce complete abortion. However, there was no effect of diet on the odds of having no conceptuses at day 14 and no interaction between diet and LPS injection. Diet and LPS injection had no effect on the number of viable fetuses in females still pregnant at day 14. For fetal weight, there was a significant interaction between diet and treatment (p = 0.017), whereby LPS reduced fetal weight in HFD females but not in LFD females. However, LPS treatment of HFD females reduced fetal weight to that observed in control-injected LFD females. Although LPS increased the odds of abortion, there was little evidence that a HFD exacerbated the effects of LPS.