Restoration of the Mucosal IgA Response by Improving CD4+ T Pyroptosis Fails to Attenuate Gut Bacterial Translocation and Organ Damage After LPS Attack.

BACKGROUND: Currently, the mechanisms of impaired gut mucosal immunity in sepsis remain unclear. Gut immunoglobulin A (IgA) is an important defense mechanism against invasive pathogens, and CD4+ T cells regulate the IgA response. AIM: We aimed to verify the hypothesis indicating that CD4+ T pyroptosis induced by lipopolysaccharide (LPS) leads to an impaired gut IgA response and subsequent bacterial translocation and organ damage. METHODS: Cultured CD4+ T cells and mice were manipulated with LPS, and pyroptosis was improved by A438079 or adoptive CD4+ T cell transfer. The changes demonstrated in pyroptosis-related molecules, cytotoxicity and CD4+ T cells were examined to determine CD4+ T pyroptosis. The changes demonstrated in IgA+ B cells, AID (key enzyme for immunoglobulins) and IgA production and function were examined to evaluate the IgA response. Serum biomarkers, bacterial colonies and survival analysis were detected for bacterial translocation and organ damage. RESULTS: LPS attack induced CD4+ T pyroptosis, as evidenced by increased expression of P2X7, Caspase-11 and cleaved GSDMD, which elevated cytotoxicity and decreased CD4+ T cells. Decreased CD4+ T subsets (Foxp3+ T and Tfh cells) influenced the IgA response, as evidenced by lower AID expression, which decreased IgA+ B cells and IgA production and function. A438079 or cell transfer improved the IgA response but failed to reduce the translocation of gut pathogens, damage to the liver and kidney, and mortality of mice. CONCLUSION: LPS attack results in CD4+ T pyroptosis. Improvement of pyroptosis restores the mucosal IgA response but fails to ameliorate bacterial translocation and organ damage.

Dexmedetomidine Alleviates Gut-Vascular Barrier Damage and Distant Hepatic Injury Following Intestinal Ischemia/Reperfusion Injury in Mice.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) challenge often results in gut barrier dysfunction and induces distant organ injury. Dexmedetomidine has been shown to protect intestinal epithelial barrier against I/R attack. The present study aims to investigate the degree to which intestinal I/R attack will contribute to gut-vascular barrier (GVB) damage, and to examine the ability of dexmedetomidine to minimize GVB and liver injuries in mice. METHODS: In vivo, intestinal ischemic challenge was induced in mice by clamping the superior mesenteric artery for 45 minutes. After clamping, the mice were subjected to reperfusion for either 2, 4, 6, or 12 hours. Intraperitoneal injection of dexmedetomidine 15, 20, or 25 µg·kg-1 was performed intermittently at the phase of reperfusion. For the in vitro experiments, the challenge of oxygen-glucose deprivation/reoxygenation (OGD/R) was established in cultured vascular endothelial cells, and dexmedetomidine (1 nM) was used to treat the cells for 24 hours. Moreover, in vivo and in vitro, SKL2001 (a specific agonist of ß-catenin) or XAV939 (a specific inhibitor of ß-catenin) was applied to determine the role of ß-catenin in the impacts provided by dexmedetomidine. RESULTS: The attack of intestinal I/R induced GVB damage. The greatest level of damage was observed at 4 hours after intestinal reperfusion. There was a significant increase in plasmalemma vesicle-associated protein-1 (PV1, a specific biomarker for endothelial permeability) expression (5.477 ± 0.718 vs 1.000 ± 0.149; P < .001), and increased translocation of intestinal macromolecules and bacteria to blood and liver tissues was detected (all P < .001). Liver damages were observed. There were significant increases in histopathological scores, serum parameters, and inflammatory factors (all P < .001). Dexmedetomidine 20 µg·kg-1 reduced PV1 expression (0.466 ± 0.072 vs 1.000 ± 0.098; P < .001) and subsequent liver damages (all P < .01). In vitro, dexmedetomidine significantly improved vascular endothelial cell survival (79.387 ± 6.447% vs 50.535 ± 1.766%; P < .001) and increased the productions of tight junction protein and adherent junction protein (all P < .01) following OGD/R. Importantly, in cultured cells and in mice, ß-catenin expression significantly decreased (both P < .001) following challenge. Dexmedetomidine or SKL2001 upregulated ß-catenin expression and produced protective effects (all P < .01). However, XAV939 completely eliminated the protective effects of dexmedetomidine on GVB (all P < .001). CONCLUSIONS: The disruption of GVB occurred following intestinal I/R. Dexmedetomidine alleviated I/R-induced GVB impairment and subsequent liver damage.

Activation of PD-1 Protects Intestinal Immune Defense Through IL-10/miR-155 Pathway After Intestinal Ischemia Reperfusion.

BACKGROUND: To date, mechanisms of intestinal immunoglobulin (Ig) dysfunction following intestinal ischemia/reperfusion (I/R) remain unclear. Programmed death 1 (PD-1) is associated with immune responses of lymphocytes. AIM: We aimed to verify the hypothesis that activation of PD-1 may improve intestinal immune dysfunction by regulating IL-10/miR-155 production after intestinal IR injury. METHODS: Intestinal I/R injury was induced in mice by clamping the superior mesenteric artery for 1 h followed by 2-h reperfusion. PD-L1 fusion Ig, anti-interleukin (IL)-10 monoclonal antibody (mAb), and microRNA (miR)-155 agomir were administered. PD-1 expression, IL-10 mRNA, and protein expression in Peyer's patches (PP) CD4+ cells were measured. MiR-155 levels, tumor necrosis factor (TNF)-α and IL-1ß concentration, and activation-induced cytidine deaminase (AID), a key enzyme for intestinal immune antibodies, in PP tissues were measured, respectively. Importantly, the production and cecal bacteria-binding capacity of IgA and IgM were detected. RESULTS: Intestinal I/R led to decreased PD-1 expression, imbalanced production, and impaired bacteria-binding capacity of IgA and IgM. Activating PD-1 by PD-L1 Ig facilitated IL-10 synthesis, then decreased miR-155 levels, and subsequently promoted AID expression and reduced TNF-α, IL-1ß concentration. Upregulation of AID improved the disruptions of intestinal immune barrier caused by IgA and IgM dysfunction. Anti-IL-10 mAb and miR-155 agomir abolished the protective effects of PD-L1 Ig on the intestinal immune defense. CONCLUSION: Activation of PD-1 with PD-L1 Ig relieves intestinal immune defensive injury through IL-10/miR-155 pathway following intestinal I/R attack. PD-1, IL-10, and miR-155 may be potential targets for the damages of intestinal barrier and immunity.

Propofol Does Not Reduce Pyroptosis of Enterocytes and Intestinal Epithelial Injury After Lipopolysaccharide Challenge.

BACKGROUND: To date, mechanisms of sepsis-induced intestinal epithelial injury are not well known. P2X7 receptor (P2X7R) regulates pyroptosis of lymphocytes, and propofol is usually used for sedation in septic patients. AIMS: We aimed to determine the occurrence of enterocyte pyroptosis mediated by P2X7R and to explore the effects of propofol on pyroptosis and intestinal epithelial injury after lipopolysaccharide (LPS) challenge. METHODS: A novel regimen of LPS challenge was applied in vitro and in vivo. Inhibitors of P2X7R (A438079) and NLRP3 inflammasome (MCC950), and different doses of propofol were administered. The caspase-1 expression, caspase-3 expression, caspase-11 expression, P2X7R expression and NLRP3 expression, extracellular ATP concentration and YO-PRO-1 uptake, and cytotoxicity and HMGB1 concentration were detected to evaluate enterocyte pyroptosis in cultured cells and intestinal epithelial tissues. Chiu's score, diamine oxidase and villus length were used to evaluate intestinal epithelial injury. Moreover, survival analysis was performed. RESULTS: LPS challenge activated caspase-11 expression and P2X7R expression, enhanced ATP concentration and YO-PRO-1 uptake, and led to increased cytotoxicity and HMGB1 concentration. Subsequently, LPS resulted in intestinal epithelial damage, as evidenced by increased levels of Chiu's score and diamine oxidase, and shorter villus length and high mortality of animals. A438079, but not MCC950, significantly relieved LPS-induced enterocyte pyroptosis and intestinal epithelial injury. Importantly, propofol did not confer the protective effects on enterocyte pyroptosis and intestinal epithelia although it markedly decreased P2X7R expression. CONCLUSION: LPS attack leads to activation of caspase-11/P2X7R and pyroptosis of enterocytes. Propofol does not reduce LPS-induced pyroptosis and intestinal epithelial injury, although it inhibits P2X7R upregulation.

TGF-ß1 improves mucosal IgA dysfunction and dysbiosis following intestinal ischaemia-reperfusion in mice.

Intestinal ischaemia/reperfusion (I/R) severely disrupts gut barriers and leads to high mortality in the critical care setting. Transforming growth factor (TGF)-ß1 plays a pivotal role in intestinal cellular and immune regulation. However, the effects of TGF-ß1 on intestinal I/R injury remain unclear. Thus, we aimed to investigate the effects of TGF-ß1 on gut barriers after intestinal I/R and the molecular mechanisms. Intestinal I/R model was produced in mice by clamping the superior mesenteric artery for 1 hr followed by reperfusion. Recombinant TGF-ß1 was intravenously infused at 15 min. before ischaemia. The results showed that within 2 hrs after reperfusion, intestinal I/R disturbed intestinal immunoglobulin A class switch recombination (IgA CSR), the key process of mucosal IgA synthesis, and resulted in IgA dysfunction, as evidenced by decreased production and bacteria-binding capacity of IgA. Meanwhile, the disruptions of intestinal microflora and mucosal structure were exhibited. Transforming growth factor-ß1 activated IgA CSR as evidenced by the increased activation molecules and IgA precursors. Strikingly, TGF-ß1 improved intestinal mucosal IgA dysfunction, dysbiosis and epithelial damage at the early stage after reperfusion. In addition, SB-431542, a specific inhibitor of activating mothers against decapentaplegic homologue (SMAD) 2/3, totally blocked the inductive effect of TGF-ß1 on IgA CSR and almost abrogated the above protective effects on intestinal barriers. Taken together, our study demonstrates that TGF-ß1 protects intestinal mucosal IgA immunity, microbiota and epithelial integrity against I/R injury mainly through TGF-ß receptor 1/SMAD 2/3 pathway. Induction of IgA CSR may be involved in the protection conferred by TGF-ß1.

Decreased PD-1/PD-L1 Expression Is Associated with the Reduction in Mucosal Immunoglobulin A in Mice with Intestinal Ischemia Reperfusion.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) disrupts intestinal mucosal integrity and immunoglobulin A (IgA) generation. It has recently been shown that the programmed cell death-1 receptor (PD-1) plays a crucial role in regulating intestinal secreted IgA (sIgA). AIMS: To evaluate changes in PD-1 and PD-ligand 1 (PD-L1) expression on Peyer's patches (PP) CD4(+) T cells and to investigate the correlation between PD-1/PD-L1 and intestinal IgA production/mucosal integrity in mice following intestinal I/R. METHODS: I/R injury was induced by clamping the superior mesenteric artery for 1 h followed by 2-h reperfusion. PD-1/PD-L1 expression on PP CD4(+) T cells was measured in I/R and sham-operated mice. Additionally, transforming growth factor-ß1 (TGF-ß1) and interleukin-21 (IL-21) mRNA in CD4(+) T cells and IgA(+) and IgM(+) in PP B cells, as well as intestinal mucosal injury and sIgA levels, were assessed. RESULTS: PD-1/PD-L1, TGF-ß1, and IL-21 expression was down-regulated after intestinal I/R. Furthermore, IgA(+) B cells decreased and IgM(+) B cells increased in mice with intestinal I/R. Importantly, decreased PD-1/PD-L1 expression was correlated with increased mucosal injury and decreased IgA levels, as well as with decreased TGF-ß1 and IL-21 expression. CONCLUSIONS: Intestinal I/R inhibits PD-1/PD-L1 expression on PP CD4(+) T cells, which was associated with an impaired intestinal immune system and mechanical barriers. Our study indicates that PD-1/PD-L1 expression on CD4(+) T cells may be involved in the pathogenesis of intestinal I/R injury.