Cellular strain amplifies LPS-induced stress signaling in immature enterocytes: potential implications for preterm infant NCPAP

Abstract

Background: Recent clinical observations of increased necrotizing enterocolitis (NEC) incidence in some nasal continuous positive airway pressure (NCPAP) patients raise concerns about whether the related abdominal distension is benign or contributes to NEC. We tested the hypothesis that mechanical strain causes an exaggerated enterocyte inflammatory response and decreased enterocyte growth and proliferation in the absence and presence of lipopolysaccharide (LPS).

Methods: First we used a confluent enterocyte (IEC-6) monolayer to investigate effects of strain on inflammatory cytokine production and Toll-like receptor 4 (TLR-4) gene expression. Then we used a low seeding density to measure cell growth and proliferation. Ten percent mechanical strain was applied.

Results: Significant increases in interleukin (IL)-8 and in IL-6 were observed after 8 and 24 h of cellular strain, respectively, and maintained throughout the study. TLR-4 expression was increased at 48 h. Mechanical strain led to slower proliferation and division whereas LPS alone had minimal effects. The responses of LPS and strain were supra-additive, suggesting synergistic cellular effects.

Conclusion: We speculate intestinal distension associated with the use of NCPAP, especially in the presence of abnormal gut colonization, may result in increased inflammatory cytokine production and be a contributing factor to neonatal intestinal morbidities.

FIGURE 1. Effects of cellular strain alone on rat neonatal enterocytes

A) Time-dependent increases in IL-6. B) Time-dependent increases in IL-8. C) Representative images of static (upper panel) vs. strained enterocyte monolayer following 48 hours. Scale bar = 100μm. D) Increased TLR-4 gene expression following 48 hours of strain vs. static conditions. All panels, n=6–12, mean±SE; *, p<0.05 when compared to time 0 static control.

FIGURE 2. Effects of LPS alone on rat neonatal enterocytes

Time-dependent increases in IL-6 (Panel A) or IL-8 (Panel B) during various LPS exposures (n=6–12, mean±SE) (●= 10μg/ml, ■ = 50μg/ml,◆ = 100μg/ml,▲ = 150μg/ml). All panels, *, p<0.05 when compared to time 0 static control.

FIGURE 3. Effects of combined treatment of LPS and cellular strain on rat neonatal enterocyte IL-6 and IL-8

Confluent monolayers were incubated with LPS for 24 (panels A and B) or 48 (panels C and D) hours in the absence (open bars) or presence (black bars) of cyclic strain. Supra-additive effects of the combined treatment were observed. (n=6–12, mean±SE). All panels, *, p<0.05 when compared to time respective static control.

FIGURE 4. Effects of cellular strain or LPS on sub-confluent cell growth and proliferation in rat neonatal enterocytes

Cells were seeded at low seeding density; numbers of cells/100x field of view (Panel A) and BrDU positive cells (Panel B) were counted (n=6–12, mean±SD) (○ =nonstrained, 0mg/ml LPS,△ =nonstrained, 10mg/ml LPS,●=strained, 0mg/ml LPS,▲ =strained, 10mg/ml LPS). All panels, *, p<0.05 when compared to respective static control.