miR-146a regulates mechanotransduction and pressure-induced inflammation in small airway epithelium

Abstract

Mechanical ventilation generates biophysical forces, including high transmural pressures, which exacerbate lung inflammation. This study sought to determine whether microRNAs (miRNAs) respond to this mechanical force and play a role in regulating mechanically induced inflammation. Primary human small airway epithelial cells (HSAEpCs) were exposed to 12 h of oscillatory pressure and/or the proinflammatory cytokine TNF-α. Experiments were also conducted after manipulating miRNA expression and silencing the transcription factor NF-κB or toll-like receptor proteins IRAK1 and TRAF6. NF-κB activation, IL-6/IL-8/IL-1β cytokine secretion, miRNA expression, and IRAK1/TRAF6 protein levels were monitored. A total of 12 h of oscillatory pressure and TNF-α resulted in a 5- to 7-fold increase in IL-6/IL-8 cytokine secretion, and oscillatory pressure also resulted in a time-dependent increase in IL-6/IL-8/IL-1β cytokine secretion. Pressure and TNF-α also resulted in distinct patterns of miRNA expression, with miR-146a being the most deregulated miRNA. Manipulating miR-146a expression altered pressure-induced cytokine secretion. Silencing of IRAK1 or TRAF6, confirmed targets of miR-146a, resulted in a 3-fold decrease in pressure-induced cytokine secretion. Cotransfection experiments demonstrate that miR-146a's regulation of pressure-induced cytokine secretion depends on its targeting of both IRAK1 and TRAF6. MiR-146a is a mechanosensitive miRNA that is rapidly up-regulated by oscillatory pressure and plays an important role in regulating mechanically induced inflammation in lung epithelia.

Figure 1.

Oscillatory transmural pressure activates the NF-κB pathway and results in increased proinflammatory cytokine expression and secretion in HSAEpCs. A, B) Four hours of oscillatory pressure induced increased NF-κB DNA binding activity (A) and inhibition of IκB-α (B). IκB-α expression level was assessed by Western blot and densitometry. C–F) Four hours of oscillatory pressure also increased IL-6, IL-8, and TNF-α mRNA expression (C), while 12 h of pressure induced significant secretion of IL-6 (D) and IL-8 (E). Twelve hours of 30 ng/ml TNF-α also induced significant IL-6 (D) and IL-8 (E) secretion in HSAEpCs, but 12 h of pressure resulted in minimal TNF-α secretion (F). Combined TNF-α and pressure stimulation induced higher cytokine release than either stimulus alone (D, E). qRT-PCR data were normalized to GAPDH. HSAEpCs were exposed to 0.2-Hz, 0- to 20-cmH₂O oscillatory pressure. Results are presented as means ± sd; n = 2 (A, C), 3 (B–F). *P < 0.05 vs. 0 h or static control; ^⋀P < 0.05 vs. 12 h pressure and 12 h TNF-α.

Figure 2.

Oscillatory transmural pressure results in a time-dependent increase in proinflammatory cytokine secretion. IL-6 and IL-1β cytokine production continually increases during exposure to 1, 4, 8, and 12 h of oscillatory pressure. IL-8 cytokine production also increases but saturates after 8 h of oscillatory pressure. All means are statistically different (P<0.01) except as indicated (n=4–6).

Figure 3.

Effect of TNF-α, oscillatory pressure, and both TNF-α and pressure on miRNA expression. A, B) Heatmaps of miRNAs with statistically significant differences in expression levels (P<0.05) during TNF-α treatment (A) and pressure treatment (B). Data also shown for combined TNF-α and pressure treatment (B). Data generated in triplicate using Nanostring nCounter System with 12 h exposure time. C) Fold change of miRNA expression in HSAEpCs exposed to 12 h of 30 ng/ml TNF-α, 0.2-Hz oscillatory pressure, or both TNF-α and pressure compared to untreated cells; n = 2–3. Data were generated using RT-PCR. *P < 0.05 vs. untreated cells.

Figure 4.

Effect of silencing NF-κB on miR-146a expression and cytokine release. A) NF-κB expression was silenced by transfection with 50 nM or 100 nM NF-κB p65 siRNA. B, C) Silencing NF-κB results in a small decrease in miR-146a expression during oscillatory pressure (B) and significantly reduces pressure-induced increases in IL-6 and IL-8 secretion (C). HSAEpCs were transfected with scramble siRNA (control) or NF-κB p65 siRNA and subjected to 12 h of oscillatory pressure 48 h after transfection. n = 4 (B); 6 (C).*P < 0.05 vs. control; ^⋀P < 0.05 vs. no pressure; ^$P = 0.064 vs. control.

Figure 5.

Effect of miR-146a knockdown and overexpression on NF-κB activation (A, D), IL-6 secretion (B, E), and IL-8 secretion (C, F). NF-κB activation was assessed after 4 h of oscillatory pressure, while cytokine secretion was assessed after 12 h of pressure. A–C) Knocking down miR-146a with 100 nM anti-miR-146a resulted in minimal changes in pressure induced NF-κB activation (A) and a statistically significant increase in IL-6 (B) and IL-8 (C) secretion during oscillatory pressure. D–F). Overexpression of miR-146a with 0.5 nM pre-miR-146a resulted in a statistically significant decrease in NF-κB activation (D) and dramatic reductions in pressure-induced IL-6 (E) and IL-8 (F) secretion. Experiments were conducted 2 or 3 d after transfection; n = 2 (A, D), 6 (B, C, E, F). *P < 0.05 vs. anti-miR or pre-miR control.

Figure 6.

Effect of silencing miR-146a targets IRAK1 and TRAF6 on pressure-induced cytokine secretion. A) IRAK1 and TRAF6 protein expression was efficiently silenced using either 50 or 100 nM IRAK1 and TRAF6 siRNA. B) Silencing IRAK1 significantly reduced both baseline cytokine levels and pressure-induced IL-6 and IL-8 cytokine secretion. C) Silencing TRAF6 also significantly reduced baseline and pressure-induced IL-6 and IL-8 cytokine secretion. n = 2–5. *P < 0.05 vs. siRNA control; ^$P = 0.06 vs. siRNA control.

Figure 7.

Effect of simultaneously knocking down IRAK1 or TRAF6 and miR-146a on pressure-induced cytokine secretion. Oscillatory pressure induced more cytokine production in cells treated with anti-miR-146a + scrambled siRNA (open bars) than in control cells treated with negative anti-miR + scrambled siRNA (solid bars). Cells treated with siIRAK1 or siTRAF6 exhibited significant reductions in IL-6 and IL-8 secretion compared to the negative anti-miR + scrambled siRNA control. However, no statistically significant difference was found in cytokine production between the anti-miR146a + siIRAK1 and the negative anti-miR + siIRAK1 samples (light shaded bars) or between the anti-miR146a + siTRAF6 and the negative anti-miR + siTRAF6 samples (dark shadedbars). These data indicate that regulation of pressure-induced cytokine secretion by miR-146a depends on its targeting of IRAK1 and TRAF6. n = 4–6. *P < 0.01 vs. scrambled siRNA and negative anti-miR controls (solid bars).

Figure 8.

Schematic diagram of pathways involved in pressure-induced cytokine production in HSAEpCs and mechanisms of miR146a regulation. Dashed line indicates minor levels of activation.