Anti-eCIRP Strategy Protects Against Intestinal Dysfunction and Mortality in Necrotizing Enterocolitis

Author(s):
Colleen Nofi; Monowar Aziz; Jose Prince; Ping Wang

Background:

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease characterized by bowel ischemia and high mortality risk. Extracellular cold-inducible RNA-binding protein (eCIRP) is an alarmin that exaggerates tissue injury in inflammatory states and is elevated in neonatal sepsis.

Hypothesis:

We hypothesized that targeting eCIRP with the novel eCIRP scavenger, MFG-E8-derived oligopeptide (MOP3), prevents intestinal dysfunction and mortality in a preclinical model of NEC.

Methods:

NEC was induced in 5-7-day-old wild-type (WT) and CIRP^-/- mouse pups utilizing a combination of hypercaloric formula gavage (supplemented with LPS 4 μg/g/day), and hypoxia. WT pups were treated with MOP3 (20 μg/g i.p.) or vehicle daily. To test intestinal permeability, mice were administered FITC-dextran by orogastric gavage 3 hours prior to sera collection. TUNEL staining was quantified on representative sections to evaluate intestinal cell death. Intestinal sections were immunostained for tight junctional protein, zona occludens-1 (ZO-1). Differences in overall mortality were assessed in a 5-day survival study.

Results:

CIRP^-/- NEC pups were protected against intestinal cell death vs WT (apoptotic cells reduction of 56% vs. WT, p<0.05). Sera fluorescence was significantly reduced in CIRP^-/- pups compared to WT in NEC, indicating reduction in intestinal permeability (Fig. 1A). ZO-1 immunostaining was restored in CIRP^-/- NEC pups compared to the reduction observed in WT NEC (Fig. 1B). CIRP^-/- pups had significantly improved 5-day NEC survival at 100% (compared to 65% for WT) (Fig. 1C). MOP3 recapitulated the benefits afforded by CIRP-knockdown in NEC, evidenced by protection against intestinal cell death (apoptotic cell reduction of 59% vs. vehicle, p<0.05), reduction of sera fluorescence, and restoration of ZO-1 immunostaining (Fig. 1D, E). Finally, MOP3 treatment improved 5-day NEC survival to 80% compared to vehicle at 50% (Fig. 1F).

Conclusions:

eCIRP deficiency protects against intestinal injury, barrier dysfunction, and mortality in NEC. Targeting of eCIRP by scavenger peptide, MOP3, further improves outcomes in experimental NEC, highlighting an exciting new therapeutic strategy to promote survival in NEC.