Reduced IL-10 production in fetal type II epithelial cells exposed to mechanical stretch is mediated via activation of IL-6-SOCS3 signaling pathway.

An imbalance between pro-inflammatory and anti-inflammatory cytokines is a key factor in the lung injury of premature infants exposed to mechanical ventilation. Previous studies have shown that lung cells exposed to stretch produces reduced amounts of the anti-inflammatory cytokine IL-10. The objective of these studies was to analyze the signaling mechanisms responsible for the decreased IL-10 production in fetal type II cells exposed to mechanical stretch. Fetal mouse type II epithelial cells isolated at embryonic day 18 were exposed to 20% stretch to simulate lung injury. We show that IL-10 receptor gene expression increased with gestational age. Mechanical stretch decreased not only IL-10 receptor gene expression but also IL-10 secretion. In contrast, mechanical stretch increased release of IL-6. We then investigated IL-10 signaling pathway-associated proteins and found that in wild-type cells, mechanical stretch decreased activation of JAK1 and TYK2 and increased STAT3 and SOCS3 activation. However, opposite effects were found in cells isolated from IL-10 knockout mice. Reduction in IL-6 secretion by stretch was observed in cells isolated from IL-10 null mice. To support the idea that stretch-induced SOCS3 expression via IL-6 leads to reduced IL-10 expression, siRNA-mediated inhibition of SOCS3 restored IL-10 secretion in cells exposed to stretch and decreased IL-6 secretion. Taken together, these studies suggest that the inhibitory effect of mechanical stretch on IL-10 secretion is mediated via activation of IL-6-STAT3-SOCS3 signaling pathway. SOCS3 could be a therapeutic target to increase IL-10 production in lung cells exposed to mechanical injury

A role for caveolin-1 in mechanotransduction of fetal type II epithelial cells

Mechanical forces are critical for fetal lung development. Using surfactant protein C (SP-C) as a marker, we previously showed that stretch-induced fetal type II cell differentiation is mediated via the ERK pathway. Caveolin-1, a major component of the plasma membrane microdomains, is important as a signaling protein in blood vessels exposed to shear stress. Its potential role in mechanotransduction during fetal lung development is unknown. Caveolin-1 is a marker of type I epithelial cell phenotype. In this study, using immunocytochemistry, Western blotting, and immunogold electron microscopy, we first demonstrated the presence of caveolin-1 in embryonic day 19 (E19) rat fetal type II epithelial cells. By detergent-free purification of lipid raft-rich membrane fractions and fluorescence immunocytochemistry, we found that mechanical stretch translocates caveolin-1 from the plasma membrane to the cytoplasm. Disruption of the lipid rafts with cholesterol-chelating agents further increased stretch-induced ERK activation and SP-C gene expression compared with stretch samples without disruptors. Similar results were obtained when caveolin-1 gene was knocked down by small interference RNA. In contrast, adenovirus overexpression of the wild-type caveolin-1 or delivery of caveolin-1 scaffolding domain peptide inside the cells decreased stretch-induced ERK phosphorylation and SP-C mRNA expression. In conclusion, our data suggest that caveolin-1 is present in E19 fetal type II epithelial cells. Caveolin-1 is translocated from the plasma membrane to the cytoplasm by mechanical stretch and functions as an inhibitory protein in stretch-induced type II cell differentiation via the ERK pathway

Mechanical stretch decreases IL-10 receptor and IL-10 gene expression and increases SOCS3.

<p>E18 type II epithelial cells were exposed to 20% cyclic stretch for 24 hours. (<b>A</b>) Samples were analyzed by real-time PCR for IL-10 receptor mRNA expression (n = 5, *P<0.0001, **P<0.0001 vs their respective controls). (<b>B</b>) By Western blot for IL-10 R1 protein abundance (n = 4, *P<0.05 vs control). (<b>C</b>) By real-time PCR for IL-10 gene expression (n = 4, *P<0.05). (<b>D</b>) By real-time PCR for SOCS3 gene expression (n = 3, *P<0.05).</p

IL-10 receptor and SOCS3 expression levels increase with gestational age.

<p>Fetal type II cells were isolated at different gestational ages (E17–E19). Samples were processed and analyzed by real-time PCR for IL-10 receptor mRNA expression (<b>A</b>), by Western blot for IL-10 receptor protein abundance (<b>B</b>) and by real-time PCR for SOCS3 gene expression (<b>C</b>). <i>Upper panels</i> in <b>B</b> are representative blots. (*P<0.05 and **P<0.05 versus E17).</p

SOCS3 decreases IL-10 secretion and increases IL-6 release in fetal type II cells exposed to mechanical stretch.

<p><b>A)</b> E18 type II epithelial cells were transfected with nothing (negative), no target or siRNA for SOCS3 [1 µM] and exposed to 20% mechanical stretch for 24 h. Supernatant was collected and IL10 concentration was determined by ELISA (n = 5, *P<0.05 vs their respective controls). The <i>upper panel</i> shows a representative Western blot demonstrating efficient inhibition of SOCS3 protein in samples treated with siRNA SOCS3 at 1 µM. <b>B)</b> E18 type II cells were processed as described in <b>A</b> and exposed to mechanical stretch for 24 h to measure release of IL-6 by ELISA (n = 4, *P<0.05 vs control; **P<0.05 vs stretch no target). <b>C)</b> E18 type II cells were infected with adenovirus expressing SOCS3 at multiplicity of infection (MOI) of 15 or negative control green fluorescent protein (GFP) at the same MOI. Two days later, monolayers were exposed to 20% stretch for 24 h. Supernatants were collected and IL-6 concentration was quantified by ELISA (n = 4, *P<0.05 vs control; **P<0.05 vs stretch GFP). The <i>upper panel</i> shows a representative Western blot showing increased expression of SOCS3 protein in samples infected with adenovirus encoding SOCS3.</p

Effect of mechanical stretch on IL-10 and IL-6 release into the supernatant of type II epithelial cells.

<p><b>A)</b> E18 type II cells were exposed to 20% cyclic stretch for 24 hours. Supernatant was collected and measured for concentrations of IL-10 by ELISA, as described in methods. Results were normalized to the cell lysate concentration in each sample (n = 5, *P<0.02 vs control). <b>B)</b> Samples were processed as above, except that IL-6 release was also measured in type II cells isolated from IL-10 knockout mice (n = 3, *P<0.05 vs control wild-type; n = 3, **P<0.05 vs control IL-10 KO).</p

Effect of mechanical stretch on IL-10 signaling proteins in wild-type cells.

<p>E18 type II epithelial cells were exposed to 20% cyclic stretch for 24 h. Samples were analyzed by Western blot using phospho-JAK1 (<b>A</b>), phospho-TYK2 (<b>B</b>), phospho-STAT3 (<b>C</b>) and total SOCS3 (<b>D</b>) antibodies. Blots were reprobed with total antibodies and GAPDH (for SOCS3) to control for protein loading. <i>Top panels</i> are representative Western blots. Results are from 4 independent experiments. (*P<0.05 vs controls).</p