Neutrophil KLF2 regulates inflammasome-dependent neonatal mortality from endotoxemia

Preterm neonates die at a significantly higher rate from sepsis than full-term neonates, attributable to their dysregulated immune response. In addition to tissue destruction caused directly by bacterial invasion, an overwhelming cytokine response by the immune cells to bacterial antigens also results in collateral damage. Sepsis leads to decreased gene expression of a critical transcription factor, Kruppel-like factor-2 (KLF2), a tonic repressor of myeloid cell activation. KLF2 gene expression is also lower in murine pups at an earlier postnatal age. Using a murine model of myeloid-KLF2 deletion, we show that loss of KLF2 leads to decreased survival after endotoxemia in a developmentally dependent manner, with increased mortality at postnatal day 4 (P4) compared to P12 pups. This survival is significantly increased by neutrophil depletion before endotoxemia. P4 knockout pups have increased pro-inflammatory cytokine levels after endotoxemia compared to P4 controls or P12 pups, with significantly increased levels of IL-1b, an end product of the activation of the NLRP3 inflammasome complex. Both loss of myeloid KLF2 and a younger postnatal age lead to increased NLRP3 protein expression and release of IL-1b in bone marrow neutrophils. Inhibition of NLRP3 inflammasome activation by MCC950 significantly increased survival after endotoxemia in P4 pups. Transcriptomic analysis of bone marrow neutrophils showed that loss of myeloid-KLF2 is associated with gene enrichment of pro-inflammatory pathways in a developmentally dependent manner. These data suggest that targeting KLF2 could be a novel strategy to decrease the pro-inflammatory cytokine storm in neonatal sepsis and improve survival in neonates with sepsis. Bone marrow neutrophil total RNA from postnatal day 4 andn 12 pups from two genotypes, Klf2fl/fl Lyz2Cre and Lyz2Cre. Bone marrows were isolated from bilateral femurs and tibias, crushed in RPMI-1640 media, and neutrophils were isolated using immunomagnetic negative selection (StemCell Mouse Neutrophil Enrichment Kit, Vancouver, BC, Canada). Total RNA was extracted using Qiagen RNeasy Micro Kit.