Background: While the role of neutrophil extracellular traps (NETs) in sepsis is well-defined, the impact of the commensal gut flora and related activation of innate immune signaling for NETosis in the mesenteric microcirculation during mesenteric ischemia-reperfusion (MIR) is entirely unresolved. Under this condition, gut microbes leak into the portal circulation and need to be sequestered by activated neutrophils to prevent systemic dissemination. To investigate the impact of gut microbiota on this issue in a controlled experimental environment, Altered Schaedler Flora (ASF) mice, which have a defined minimal microbial consortium of eight gut bacteria, were studied.

Aims: To explore how the defined ASF affects NET-formation and leukocyte recruitment in acute MIR injury.

Methods: In the MIR model, laparatomy was applied to 6-8 weeks old mice under anesthesia, inducing ischemia in the small intestine by ligating the A. mesenterica cranialis with a microclamp. After 1 hour, reperfusion was allowed by removing the clamp. Adherent leukocytes (stained with Acridine Orange) and NETs (stained with SYTOX Orange) were counted in vivo in a 0.06 mm² area of interest with a high-speed epifluorescence microscope both before the ligation and after reperfusion. Germ-free (GF) C57BL/6J mice, ASF mice and ASF mice treated with Neomycin and Ampicillin (ASF+Abx) were studied. Statistical analyses were performed with the independent samples Student’s t-test (age-matched groups).

Results: Post ischemia, GF mice showed significantly increased numbers of adherent leukocytes in mesenteric venules, whereas ASF mice showed a decrease. NET formation was significantly increased in GF, ASF and ASF+Abx mice after reperfusion. ASF mice showed less NETosis compared to GF controls. As a confirm, depletion of the Altered Schaedler Flora by antibiotics restored a trend of elevated NET formation.

Conclusions: The observed reduction in NETing neutrophils in ASF mice relative to GF controls demonstrates a role of the gut microbiota as an environmental pre-conditioning factor in MIR-induced NETosis.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/colonization-with-the-minimal-microbial-consortium-altered-schaedler-flora-hampers-the-formation-of-neutrophil-extracellular-traps-in-acute-mesenteric-ischemia-reperfusion-injury/