Oral administration of surfactant protein-a reduces pathology in an experimental model of necrotizing enterocolitis

Abstract

Objectives: Necrotizing enterocolitis (NEC) frequently results in significant morbidity and mortality in premature infants. Others reported that mice deficient in pulmonary surfactant protein-A (SP-A) born and raised in a nonhygienic environment succumb to significant gastrointestinal tract pathology, and enteral administration of purified SP-A significantly reduced mortality. We hypothesized that oral administration of purified SP-A can ameliorate pathology in an experimental model of neonatal NEC.

Methods: Experimental NEC was induced in newborn Sprague-Dawley rat pups by daily formula gavage and intermittent exposure to hypoxia. Purified human SP-A (5 μg/day) was administered by oral gavage. After 4 days, surviving pups were sacrificed, and intestinal pathology was assessed by histological examination of distal terminal ileal sections. Intestinal levels of inflammatory cytokines (IL-1β, IFN-γ, and TNF-α) were assessed by enzyme-linked immunosorbent assay and levels of Toll-like receptor 4 (TLR4) by Western analysis.

Results: Sixty-one percent of the gavaged rat pups that survived to day 4 met the criteria for experimental NEC after hypoxia, whereas treatment with SP-A significantly reduced mortality and assessment of NEC. Intestinal levels of proinflammatory cytokines were significantly increased in pups exposed to hypoxia. Administration of SP-A to pups exposed to hypoxia significantly reduced IL-1β and TNF-α levels, but had little effect on elevated levels of IFN-γ. SP-A treatment of hypoxia-exposed pups significantly reduced expression of intestinal TLR4, key in NEC pathogenesis.

Conclusions: In a rat model of experimental neonatal NEC, oral administration of SP-A reduces intestinal levels of proinflammatory cytokines and TLR4 protein and ameliorates adverse outcomes associated with gastrointestinal pathologies.

Figure 1. Effect of orally administered SP-A on ileal histology and assessment of NEC in an experimental rat model of neonatal NEC

A. Representative H & E sections of ileum isolated from the various groups. DF = dam-fed, FF = formula-fed, FS = formula-fed + purified SP-A (5 μg/day), FH = formula-fed with hypoxia, FHS= formula-fed with hypoxia + SP-A (original magnification ×100). B. Assessment of NEC in sections of ileum of treated animals. A score of ≥ 2 was defined as NEC. Each point represents assessment of a single animal that survived 4 days of treatment; the graph represents animals from two independent experiments with animals treated with independent preparations of purified SP-A. P ≤ 0.05, determined by Fisher exact test was considered significant.

Figure 2. Effect of orally-administered SP-A on ileal IL-1β, TNF-α and IFN-γ cytokine levels in an experimental rat model of neonatal NEC

A. Levels of IL-1β in the ileum of rats treated for 4 days determined by ELISA; DF = dam-fed, FF = formula-fed, FS = formula-fed + purified SP-A, FH = formula-fed with hypoxia, FHS = formula-fed with hypoxia + SP-A. B. Levels of TNF-α in the ileum of rats treated for 4 days determined by ELISA. ND indicates not detectable. C. Levels of IFN-γ in the ileum of rats treated for 4 days determined by ELISA. Data represent box plots of cytokines measures in animals shown in Figure 1B. P ≤ 0.05, determined by the Kruskal-Wallis test and post hoc Mann-Whitney test, was considered significant.

Figure 3. Effect of orally-administered SP-A on ileal levels of TLR4 in an experimental rat model of neonatal NEC

A. Levels of ileal TLR4 protein in Dam-fed compared to formula-fed rat pups. Levels of ileal TLR4 protein were determined by western analysis of 2 pups. Shown is a representative immunoblot; DF = dam-fed, FF = formula-fed. B. Levels of ileal TLR4 protein determined by western analysis. Shown is a representative immunoblot. The TLR4 positive control (+ control) was purchased (Santa Cruz Biotechnology, Inc.; human erythroleukemia lysate, sc-2270). C. Densitometric quantification of TLR4 in the ileum of rats treated for 4 days; FF = formula-fed, FS = formula-fed + purified SP-A, FH = formula-fed with hypoxia, FHS = formula-fed with hypoxia + SP-A. Shown are the levels of TLR4 relative to β-actin levels (arbitrary units, means ± SEM). Data represent n = 4 – 6 animals from two independent experiments. P ≤ 0.05, determined by the Kruskal-Wallis test and post hoc Mann-Whitney test, was considered significant.

Figure 4. Expression of SP-A in the lung and ileum of adult and newly-born rats

A. Levels of SP-A in adult rat lung and ileum determined by western analysis. Shown are western blots for SP-A and β-actin. Mouse BAL represents bronchoalveolar lavage material, used as a positive control for SP-A. Arrows indicate monomer and dimerized forms of SP-A. B. Western analysis of ileal SP-A in rats treated for 4 days. Shown are western blots for SP-A and β-actin; FF = formula-fed, FS = formula-fed + purified SP-A, FH = formula-fed with hypoxia, FHS = formula-fed with hypoxia + SP-A.

Figure 5. Proposed model for the role of SP-A in ameliorating experimental NEC

Red indicates some of the factors believed to initiate NEC in the intestine. SP-A is known to dampen the activities of these factors. LPS = lipopolysaccharide, TLR4 = toll-like receptor 4.