Background: Acute mesenteric ischemia (AMI) is a life-threatening clinical situation, potentially leading to bowel wall necrosis and multiple organ failure. Intestinal ischemia, caused by the occlusion of the superior mesenteric artery, breaks the intestinal barrier and results in the translocation of bacterial products. The impact of the gut microbiota as a trigger of Toll-like receptor (TLR) signaling for the recruitment of leukocytes to ischemia/reperfusion (I/R) damaged mesenteric venules and its influence on the formation of neutrophil extracellular traps (NETs) is unexplored.

Aims: We studied gnotobiotic mouse models to resolve the role of gut commensals on NETosis in AMI.

Methods: We applied a mesenteric I/R injury model to germ-free (GF) and C57BL/6J mice colonized with a gut microbiota, Escherichia coli, or Bacillus subtilis. By intravital imaging, we quantified leukocyte adherence and NET formation in I/R-injured mesenteric venules. Comparing GF with conventionally raised (CONV-R) mice, lipopolysaccharide (LPS)-triggered NETosis was analyzed in vivo and with bone marrow-derived neutrophils ex vivo.

Results: Colonization with a complex gut microbiota as well as monocolonization with Escherichia coli JP313 and Bacillus subtilis augmented the adhesion of leukocytes to I/R-injured mesenteric venules, which was dependent on the Toll-like receptor-4 (TLR4) / TIR-domain-containing adapter-inducing interferon-β (TRIF) pathway. Conversely, antibiotic depletion of gut commensals reduced leukocyte adhesion. In contrast, NETosis was significantly increased in I/R-injured mesenteric venules of GF mice and in an ex vivo assay. Likewise, neutrophils from CONV-R mice treated with broad-spectrum antibiotics showed increased NETosis. LPS administration to GF mice suppressed I/R-induced NETosis. In line with augmented TLR4 / TRIF signaling, isolated bone marrow-derived neutrophils from GF mice showed enhanced LPS-induced NETosis. Furthermore, NETosis in I/R injury was reduced in Trif^-/- and Myd88^-/-xTrif^-/- mice, but not in Myd88^-/- mice.

Conclusions: Collectively, our results identified that the gut microbiota restricts NETing neutrophils in mesenteric I/R injury, while ensuring immunovigilance by enhancing neutrophil recruitment.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/the-gut-microbiota-restricts-netosis-in-acute-mesenteric-ischemia-reperfusion-injury/