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

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.

Impact of Surfactant Protein-A on Immunomodulatory Properties of Murine and Human Breast Milk

“…• These results suggest that SP-A in milk reduces intestinal inflammation either directly or indirectly (by altering immunomodulatory components of milk). (23). We have also shown using a rat model of experimental NEC that human SP-A gavage feeds reduced inflammatory cytokine and TLR4 expression, histological damage, and mortality (27).…”

“…cDNA was synthesized from 1 μL mRNA via iScript cDNA Synthesis Kit (#170-8891; Bio-Rad Laboratories, Hercules, CA). RT-PCR analysis was conducted for TLR4, IL-1β, TNF-α, IL-6, and CXCL-1 using primers described previously (23). RT-PCR was performed using LightCycler 96 System (Roche Diagnostics, Indianapolis, IN).…”

“…SP-A deficient (SPAKO) mice raised in non-hygenic environments exhibit higher mortality than wild type (WT) mice raised under same conditions; surprisingly mortality was attributable to intestinal distension and necrosis, suggesting a NEC-like illness; however, when SPAKO pups are reared with WT dams, mortality was reduced (22). Our lab reported SPAKO mice have increased TLR4 and inflammatory cytokine expression compared to WT mice, which is reduced by gavaged human SP-A (23). We have also shown using a rat model of experimental NEC that human SP-A gavage feeds reduced inflammatory cytokine and TLR4 expression, histological damage, and mortality (27).…”

Surfactant Protein A Attenuates Generalized and Localized Neuroinflammation In Neonatal Mice

“…Assessment of SP-A and cytokine expression. Expression of SP-A was determined via standard reverse transcription PCR analysis, while expression of cytokines IL-1β, IL-6, CXCL1, TNF-α, and IL-10 was determined via real-time quantitative reverse transcription PCR (qRT-PCR) as described previously [17]. Brains were harvested, ash frozen in liquid nitrogen, then transferred to -80°C freezer until ready for RNA isolation.…”

“…SP-A and SP-D were found to have pro-in ammatory effects in the colon [15] and vascular system [16]. On the other hand, SP-A and SP-D have been found to attenuate in ammation in the intestine [17][18][19], kidney [20], pancreas [21], fetal tissue [22,23], and vascular system [16]. While the anti-in ammatory effects of SP-A in the CNS have not been directly investigated, several previous studies have explored SP-A expression in the context of multiple CNS disease processes [16, [24][25][26].…”