CD40L+ neutrophils: a brain inflammatory link from gut ischemia/reperfusion injury

Gut ischemia/reperfusion (I/R) causes distant organ dysfunction, including brain injury, through gut-brain crosstalk. Here, we discovered a novel subset of neutrophils (CD40L⁺ PMNs) that accumulates in both the gut and brain following intestinal I/R. We hypothesized that these CD40L⁺ PMNs are primed in the gut by B cell-expressed OX40L and subsequently migrate to the brain, where they activate microglia and promote brain dysfunction. Intestinal I/R was induced in C57BL/6 mice by superior mesenteric artery occlusion for 30 min followed by reperfusion. Brain infiltration of CD40L⁺ PMNs and microglial TNFα production were assessed. In vitro co-cultures of brain neutrophils with BV2 microglia (± anti-CD40L antibody) and bone marrow-derived neutrophils with B cells (± anti-OX40L antibody) elucidated the mechanisms underlying CD40L⁺ PMN function and generation. Mesenteric lymph duct ligation (MLDL) was used to examine CD40L⁺ PMNs trafficking, and adoptive transfer of CD40L-blocked neutrophils followed by open-field and Y-maze testing evaluated neurological outcomes. Intestinal I/R markedly increased CD40L⁺ PMN infiltration into the brain and enhanced microglial TNFα production, both of which were attenuated by neutrophil depletion. CD40L-dependent microglial activation was confirmed in vitro. Brain CD40L⁺ PMNs originated from Peyer’s patches, as MLDL reduced their brain accumulation, and their generation required B cell OX40L. Importantly, blockade of CD40L on neutrophils reduced anxiety-like behavior and preserved working memory after intestinal I/R. These findings identify B cell OX40L-induced CD40L⁺ PMNs as a key mediator of gut-brain inflammation and a promising therapeutic target for I/R-induced brain dysfunction.