Chemokine-receptor-dependent inflammatory responses contribute to two-hit-induced experimental necrotizing enterocolitis and represent a potential therapeutic target.

Necrotizing enterocolitis (NEC) is a severe multifactorial inflammatory disorder of the preterm intestine for which effective preventive and therapeutic strategies remain limited. Using formula feeding (FF)-associated dysbiosis combined with subsequent viral inflammation, we previously demonstrated that this clinically relevant two-hit strategy induces NEC through a complex mechanism involving the Klebsiella oxytoca-derived metabolite tilivalline (TV) and NK1.1 + cells. Here, we investigated whether chemokine-receptor-dependent inflammatory responses associated with inflammatory cell recruitment contribute to NEC pathogenesis and whether pharmacologic blockade of chemokine-receptor signaling attenuates disease progression. NEC was induced in C57BL/6 mouse pups using two novel and clinically relevant two-hit models: FF combined with R837-triggered viral inflammation, or TV gavage in dam-fed pups followed by R837 treatment. In some experiments, pups were pretreated subcutaneously with TAK-779, a pharmacologic inhibitor of CCR2/CCR5/CXCR3 signaling. NEC-like intestinal injury was assessed by intestinal permeability and histological analysis. RT-qPCR and flow cytometry were used to evaluate chemokine-receptor expression and inflammatory cell infiltration, respectively. A human NEC RNA-seq dataset (E-MTAB-15683) was used to evaluate the expression of chemokine-receptor genes. Following the R837-induced second hit, mouse pups previously subjected to the FF-associated first hit exhibited robust induction of chemokines and chemokine receptors, accompanied by recruitment of inflammatory cells and the development of NEC. TAK-779 markedly reduced the expression of chemokines and their receptors, attenuated inflammatory cell recruitment, and protected against NEC induced by the sequential FF- and viral inflammation-associated two-hit process. Similarly, subjecting BF pups to TV (first hit) followed by R837 treatment (second hit) resulted in heightened expression of chemokines and their receptors, accompanied by NEC development; both effects were prevented by TAK-779 pretreatment. Notably, analysis of human NEC transcriptomic data revealed increased expression of multiple chemokines and chemokine receptors in diseased intestinal tissues. Our findings demonstrate that chemokine-receptor-dependent inflammatory responses contribute to the propagation of NEC by promoting the accumulation of NK1.1+ cells and other inflammatory cells. Pharmacologic inhibition of chemokine-receptor signaling attenuates NEC in clinically relevant experimental two-hit models that recapitulate key features of disease pathogenesis, highlighting these pathways as potential therapeutic targets warranting further investigation.