Gut Microbiota Restricts NETosis in Acute Mesenteric Ischemia-Reperfusion Injury

Abstract

Objective: Recruitment of neutrophils and formation of neutrophil extracellular traps (NETs) contribute to lethality in acute mesenteric infarction. To study the impact of the gut microbiota in acute mesenteric infarction, we used gnotobiotic mouse models to investigate whether gut commensals prime the reactivity of neutrophils towards formation of neutrophil extracellular traps (NETosis). Approach and Results: We applied a mesenteric ischemia-reperfusion (I/R) injury model to germ-free (GF) and colonized C57BL/6J mice. By intravital imaging, we quantified leukocyte adherence and NET formation in I/R-injured mesenteric venules. Colonization with gut microbiota or monocolonization with Escherichia coli augmented the adhesion of leukocytes, which was dependent on the TLR4 (Toll-like receptor-4)/TRIF (TIR-domain–containing adapter-inducing interferon-β) pathway. Although neutrophil accumulation was decreased in I/R-injured venules of GF mice, NETosis following I/R injury was significantly enhanced compared with conventionally raised mice or mice colonized with the minimal microbial consortium altered Schaedler flora. Also ex vivo, neutrophils from GF and antibiotic-treated mice showed increased LPS (lipopolysaccharide)-induced NETosis. Enhanced TLR4 signaling in GF neutrophils was due to elevated TLR4 expression and augmented IRF3 (interferon regulatory factor-3) phosphorylation. Likewise, neutrophils from antibiotic-treated conventionally raised mice had increased NET formation before and after ischemia. Increased NETosis in I/R injury was abolished in conventionally raised mice deficient in the TLR adaptor TRIF. In support of the desensitizing influence of enteric LPS, treatment of GF mice with LPS via drinking water diminished LPS-induced NETosis in vitro and in the mesenteric I/R injury model. Conclusions: Collectively, our results identified that the gut microbiota suppresses NETing neutrophil hyperreactivity in mesenteric I/R injury, while ensuring immunovigilance by enhancing neutrophil recruitment.

The Commensal Microbiota Enhances ADP-Triggered Integrin αIIbβ3 Activation and von Willebrand Factor-Mediated Platelet Deposition to Type I Collagen

“…Remarkably, MAMPs derived from the gut microbiota can signal to distant vascular beds, influencing both endothelial cell function and platelet responses [ 18 , 19 , 20 ]. In the vasculature, remote signaling of microbial patterns is mediated by TLRs and nucleotide-binding oligomerization domain (NOD)-like receptors [ 15 , 21 ], impacting on hematopoiesis but also affecting the development of vascular disease states such as atherosclerosis, ischemia-reperfusion injury, and arterial thrombosis [ 22 , 23 , 24 ].…”

Colonization with Altered Schaedler Flora Impacts Leukocyte Adhesion in Mesenteric Ischemia-Reperfusion Injury

“…Since our gnotobiotic approach recently demonstrated a pro-adhesive role of a complex microbiota and of Escherichia coli JP313 and Bacillus subtilis PY79 in leukocyte adhesion in the mesenteric venules [ 44 ], we wondered whether the presence of the defined minimal microbial consortium ASF would impact on leukocyte adherence. Using high-speed fluorescence intravital microscopy, we observed a significantly reduced adhesion of leukocytes to the wall of mesenteric venules pre- and post-ischemia in ASF-colonized mice as compared to CONV-R mice that were colonized from birth with a complex gut microbiota ( Figure 2 A).…”

“…Acute mesenteric infarction is characterized by a disrupted gut barrier and dysregulation of the host immune response [ 40 , 41 ]. It requires immediate treatment to prevent life-threatening complications, such as peritonitis and multiple organ failure [ 42 , 43 ]. In recent work with gnotobiotic mouse models, we revealed that the presence of gut microbiota enhances leukocyte adhesion to ischemia-reperfusion injured mesenteric venules, but efficiently suppresses the formation of neutrophil extracellular traps (NETs) [ 44 ].…”

“…It requires immediate treatment to prevent life-threatening complications, such as peritonitis and multiple organ failure [ 42 , 43 ]. In recent work with gnotobiotic mouse models, we revealed that the presence of gut microbiota enhances leukocyte adhesion to ischemia-reperfusion injured mesenteric venules, but efficiently suppresses the formation of neutrophil extracellular traps (NETs) [ 44 ]. This was regulated through toll-like receptor-4 (TLR4) signaling.…”

“…Furthermore, the probiotic formulation VSL#3, mediated via myeloid differentiation primary response 88 (MyD88)-mediated signaling, delayed thrombus formation in cremaster muscle arterioles at conditions of acute intestinal inflammation [ 46 ]. While the pro-adhesive role of a complex gut microbiota for leukocyte tethering in ischemia-reperfusion injury is well-recognized [ 44 , 47 ], the protective function of defined gut bacterial strains remains unresolved.…”