Surfactant protein A reduces TLR4 and inflammatory cytokine mRNA levels in neonatal mouse ileum

Lidan Liu,Joseph L Alcorn,Nicole Y Fatheree,J Marc Rhoads,Yuying Liu,Cullen M Grable,Adrian Robles,Xiangli Liu,Chaim Z Aron

doi:10.1038/s41598-021-82219-y

Abstract

Levels of intestinal toll-like receptor 4 (TLR4) impact inflammation in the neonatal gastrointestinal tract. While surfactant protein A (SP-A) is known to regulate TLR4 in the lung, it also reduces intestinal damage, TLR4 and inflammation in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. We hypothesized that SP-A-deficient (SP-A−/−) mice have increased ileal TLR4 and inflammatory cytokine levels compared to wild type mice, impacting intestinal physiology. We found that ileal TLR4 and proinflammatory cytokine levels were significantly higher in infant SP-A−/− mice compared to wild type mice. Gavage of neonatal SP-A−/− mice with purified SP-A reduced ileal TLR4 protein levels. SP-A reduced expression of TLR4 and proinflammatory cytokines in normal human intestinal epithelial cells (FHs74int), suggesting a direct effect. However, incubation of gastrointestinal cell lines with proteasome inhibitors did not abrogate the effect of SP-A on TLR4 protein levels, suggesting that proteasomal degradation is not involved. In a mouse model of experimental NEC, SP-A−/− mice were more susceptible to intestinal stress resembling NEC, while gavage with SP-A significantly decreased ileal damage, TLR4 and proinflammatory cytokine mRNA levels. Our data suggests that SP-A has an extrapulmonary role in the intestinal health of neonatal mice by modulating TLR4 and proinflammatory cytokines mRNA expression in intestinal epithelium.

Highlights

Despite vast improvements in short-term survival with modern neonatal intensive care, necrotizing enterocolitis (NEC) continues to be one of the most dangerous complications for premature infants, occurring in 10% of extremely low birth weight infants with a death rate over 20%1
We previously reported that orogastric gavage of purified human surfactant protein A (SP-A) to neonatal rat pups significantly reduced intestinal damage, levels of intestinal pro-inflammatory cytokines and levels of intestinal toll-like receptor 4 (TLR4) levels in a rat pup model of neonatal NEC35
Because wild type rat pups express endogenous SP-A, we wished to determine if ileal TLR4 and IL-1β levels were altered in the absence of SP-A by assessing levels using SP-A−/− mice compared to wild type mice

Summary

Introduction

Despite vast improvements in short-term survival with modern neonatal intensive care, necrotizing enterocolitis (NEC) continues to be one of the most dangerous complications for premature infants, occurring in 10% of extremely low birth weight infants with a death rate over 20%1. Intestinal epithelium and bacterial pathogens interact through toll-like receptor 4 (TLR4), which recognizes pathogen-associated molecules such as lipopolysaccharides (LPS)[8,9] These receptors are a crucial part of the innate immune system’s rapid response to pathogens to ward off infection[10]. Injury to the premature intestinal allows inappropriate translocation of bacteria from the lumen into the mucosa and the binding of bacterial LPS to mucosal TLR4, which initiates an innate immune signaling cascade, resulting in increased injury, defective repair, excessive inflammation, apoptosis of enterocytes and intestinal stem cells and inhibition of enterocyte migration and proliferation[16,17,18,19]. SP-A has the unique ability to reduce short-term inflammation by dampening LPS-induced TLR4 activation as well as reducing long-term excessive inflammation by enhancing of negative regulators of TLR4 activity to signal expression of pro-inflammatory cytokines

Methods

Results

Conclusion