Blocking NF‐κB activation in intestinal Lysozyme M positive cells prevents the NEC‐induced decrease in Ly6C positive cells in the neonatal intestine

Abstract

Necrotizing enterocolitis is a devastating necrotic gastro‐intestinal inflammatory disease of premature infants. We previously found that NF‐κB activation in myeloid cells but not intestinal epithelial cells mediates the intestinal injury in an experimental neonatal mouse NEC model, suggesting that myeloid cells play an important role in the pathogenesis of NEC. While myeloid cells contain various subsets, how these are affected in NEC remains unknown. Here, we examine whether NEC development is associated with alteration of monocyte phenotypes in the lamina propria (LP) of neonatal intestines, and if so, whether NF‐κB activation mediates the effect. To do so, litters containing a mixture of LysM‐Cre+/− IKKβF/F (mice lacking IKKβ, the upstream kinase responsible for NF‐κB activation in myeloid cells) and IKKβF/F mice were exposed to a NEC protocol consisting in adult commensal bacteria inoculation‐oral LPS/hypoxia/cold stress/formula feeding or allowed to be dam fed. 24 or 48 hours later, small intestinal tissues were harvested and lamina propria (LP) cells were isolated and analyzed by flow cytometry for the alive, CD11b, CD45 Ly6C and Ly6G markers. We found that, in control mice (IKKβF/F), exposure to the NEC protocol caused a significant decrease in the percentage of Ly6C+ cells (monocytes) in the intestinal LP compared to DF controls (4.2%±0.8 vs 22.3%±2.7; p<0.05), which was attenuated when IKKβ was deleted in myeloid cells (Lys‐Cre+/− IKKβF/F; 10.2%±1.2; p<0.05). To determine whether IKKβ was deleted in this preserved Ly6C+ population, we repeat the analysis with pups of Lys‐M cre IKKβF/F mice bred with mT/mG mice to identify the specific cell population undergoing cre‐recombination, as they express GFP. We found that, in dam fed pups, LysM‐cre recombination and therefore IKKβ deletion was mostly found in the Ly6C+ population (94±0.9%) and represented 17±1.5% of CD11b+ cells in Lys‐Cre+/− IKKβF/WT DF mice. However, when exposed to the NEC protocol for 24 hours, Lys‐Cre+/−IKKβF/WT pups (non‐IKKβ deleted) had only 56±3.5% of cells with cre recombination that were Ly6C+ while Lys‐Cre+/− IKKβF/F pups (IKKβ deleted) had 95% of cells with cre‐recombination/IKKβ deletion that remained LY6C+, suggesting that NF‐κB may mediate Ly6C down regulation in NEC. However, exposure to the NEC protocol did not significantly change the total percentage of cells with cre‐recombination. In conclusion, NEC exposure reduces the percentage of Ly6C+ cells, which is dependent on NF‐κB activation in the Ly6C+ population. Since loss of Ly6C is an important marker of differentiation to CX3CR1+ vessel patrolling monocytes or CX3CR1+MHCII+ intestinal macrophages, we speculate that NF‐κB dependent loss of Ly6C+ cells during NEC exposure may affect the intestinal homeostasis including the microvasculature thus predisposing to NEC.Support or Funding InformationThis study was supported by funding from NIH grant R01HD060876 (IDP).