Novel Therapeutic Targeting of CCL3-CCR4 Axis Mediated Apoptotic Intesitnal Injury in Necrotizing Enterocolitis

Abstract

Background: Necrotizing enterocolitis (NEC) is the leading cause of neonatal gastrointestinal-related death, while the etiology and pathogenesis are poorly understood.

Methods: The levels of CCL3 in intestinal tissue from modeling mice and patients were measured and analyzed. HE staining, TUNEL, Annexin and FCM were used to assess pathological changes and apoptosis in intestinal tissue and epithelial cells. CCL3, CCR4, cytokines, tight junction protein ZO-1, apoptosis-related genes and ERK1/2-NF-κB signaling pathway were detected by ELISA, Q-PCR, Western blotting and immunofluorescence.

Results: CCL3 levels in the intestinal tissue significantly elevated in patients with NEC and mouse models. Blockade of CCL3 significantly alleviated NEC-related intestinal tissue damage, while administration of recombinant CCL3 aggravated intestinal injury by exacerbating intestinal epithelial cell apoptosis in NEC mice. Importantly, CCR4 blockade reversed CCL3-mediated damage to intestinal tissue and intestinal epithelial cell apoptosis both in vivo and in vitro. Further mechanistic studies showed that CCL3 regulated apoptosis-related BAX/BCL-2 expression through the activation of the ERK1/2 and NF-κB pathways, which could be reversed by anti-CCR4 treatment. Furthermore, ERK1/2 inhibition reduced CCL3-mediated phosphorylation of NF-κB in IEC-6 cells, while inhibition of NF-κB had no obvious effect on ERK1/2 phosphorylation. As expected, inhibition of NF-κB regulated BAX/BCL-2 expression and alleviated CCL3-induced epithelial cell apoptosis. These results indicate that high expression of CCL3 in NEC lesions promotes intestinal epithelial apoptosis through the CCL3-CCR4-ERK1/2-NFκB-BAX/BCL2 signalling axis, thereby exacerbating NEC-related intestinal injury.

Conclusions: Our study represents an important conceptual advance that CCL3 may be one of the key culprits of intestinal tissue damage in NEC patients, and blocking either CCL3, CCR4, or NF-κB may represent a novel effective immunotherapy for NEC.

Keywords: CCL3; CCR4; NEC; apoptosis; immunotherapy.