Pediatric Resident University of California Davis Children's Hospital Sacramento, California, United States
Background: Necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality in premature infants. Antimicrobial peptides (AMP) are a vital component of intestinal defense through direct antimicrobial activity and regulation of epithelial homeostasis. The AMP LL-37 decreases experimental neonatal sepsis mortality; however, the mechanism is unclear, and studies in NEC are lacking. Objective: We hypothesize that LL-37 decreases NEC mortality and enhances epithelial wound closure. Design/Methods: NEC-like injury was induced using the well-described Paneth cell disruption model. 14-day-old mice were injected with 75 µg/kg dithizone followed by a gastric gavage of 10^8 CFU/g K. pneumoniae and compared to controls for mortality. Mice in the NEC-induction group were exposed to 100 µg/kg body weight LL-37 twice daily three days prior to NEC induction (pre-treatment) or once one hour after K. pneumoniae administration (post-treatment). Epithelial wounding was achieved by applying a rotating circular silicone disk to IEC-18 cell cultures. After wounding, cells were treated with LL-37 or epithelial growth factor (EGF) and compared to sham controls. Cell closure was quantified with Image J at 0h, 6h, 12h, and 24h and analyzed with GraphPad Prism. Results: There were no significant differences in survival between LL-37-treated and non-treated groups. However, while NEC and pre-treatment group survival were similar (85% and 82%), there was a clinically relevant increase in survival in the post-treatment group (95%, n>19). LL-37 treatment (1ug/ml) significantly closed epithelial wounds faster than sham controls (79% vs. 51%, p< 0.001) and was similar to EGF treatment (55.7 %). However, higher doses of LL-37 (12.5 and 25 ug/ml) significantly decreased rates of wound healing (43.1% and 10.9%, p< 0.0001, n>18).
Conclusion(s): LL-37 treatment following but not before NEC induction improves survival in experimental NEC. While this improvement is not statistically significant, a three-fold decrease in mortality compared to non-treatment is clinically relevant. A possible mechanistic rationale for this is its impact on epithelial wound healing. Our cell culture data shows LL-37 significantly aids wound closure, similar to the impact of the growth hormone EGF. However, this is dose-dependent as administration of higher concentrations of LL-37 significantly worsened wound closure, likely secondary to LL-37 disruption of the cell wall of the epithelial cells. In conclusion, our data suggest that LL-37 may help reduce NEC mortality and improve gut healing, but dosing is critical to prevent further injury to the infant.
