Activation of NLRP3 inflammasome in alveolar macrophages contributes to mechanical stretch-induced lung inflammation and injury

Abstract

Mechanical ventilation of lungs is capable of activating the innate immune system and inducing sterile inflammatory response. The proinflammatory cytokine IL-1β is among the definitive markers for accurately identifying ventilator-induced lung inflammation. However, mechanisms of IL-1β release during mechanical ventilation are unknown. In this study, we show that cyclic stretch activates the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasomes and induces the release of IL-1β in mouse alveolar macrophages via caspase-1- and TLR4-dependent mechanisms. We also observed that NADPH oxidase subunit gp91(phox) was dispensable for stretch-induced cytokine production, whereas mitochondrial generation of reactive oxygen species was required for stretch-induced NLRP3 inflammasome activation and IL-1β release. Further, mechanical ventilation activated the NLRP3 inflammasomes in mouse alveolar macrophages and increased the production of IL-1β in vivo. IL-1β neutralization significantly reduced mechanical ventilation-induced inflammatory lung injury. These findings suggest that the alveolar macrophage NLRP3 inflammasome may sense lung alveolar stretch to induce the release of IL-1β and hence may contribute to the mechanism of lung inflammatory injury during mechanical ventilation.

FIGURE 1. Cyclic stretch induces the release of active forms of IL-1β and IL-18 from mouse AMs

A, Mouse AMs were cyclically stretched at the levels of 0, 8, 15 and 20% for 4 h. Following cyclic stretch (CS), the release of mature IL-1β and IL-18 was measured in the culture supernatants (SN) of AMs by Western blot analysis. The levels of Pro-IL-1β and Pro-IL-18 were determined in AMs cell lysates (Lys) after cyclic stretch. B, Mouse AMs were exposed to 20% cyclic stretch for the indicated times. Following cyclic stretch (CS), the release of mature IL-1β and IL-18 was measured in the culture supernatants (SN) of AMs by Western blot analysis. The levels of Pro-IL-1β and Pro-IL-18 were determined in AMs cell lysates (Lys) after cyclic stretch. C, The levels of IL-1β in the culture medium following different magnitudes of cyclic stretch were detected by ELISA. D, The levels of IL-1β in the culture medium following 20% cyclic stretch for different time intervals were detected by ELISA. E. Mouse AMs were cyclically stretched at the levels of 0, 8, 15 and 20% for 4 h. The concentrations of LDH were measured in supernatants to evaluate cell cytoxicity. Data are means from three independent experiments. *p < 0.05 vs. control group (static).

FIGURE 2. Cyclic stretch (CS)-induced IL-1β release is caspase-1-dependent

A, Mouse AMs were cyclically stretched at the levels of 0, 8, 15 and 20% for 4 h. The levels of caspase-1 (Casp-1) were detected by Western blotting. B, Mouse AMs were exposed to 20% cyclic stretch for the indicated times. After stretch, the levels of caspase-1 were detected by Western blotting. C, Effects of Casp-1 specific inhibitor ac-YVAD-FMK (YVAD) on IL-1β release, and expressions of Pro-IL-1β, Pro-Casp-1, and mature Casp-1. D. The levels of IL-1β were detected by ELISA. E. Effect of Casp-1 siRNA on IL-1β release, Pro-IL-1β, Pro-Casp-1, mature Casp-1. A scrambled siRNA (si Sc) was used as a negative control. F. The levels of IL-1β were detected by ELISA. *p <0.05, vs. control group (static). Data are means from three independent experiments. † p <0.05 vs CS control group (stretched).

FIGURE 3. Cyclic stretch stimulates IL-1β production via NLRP3-dependent signaling pathway

A. Cyclic stretch (CS) induced NLRP3 inflammasome activation in AMs. AMs were exposed to 20% cyclic stretch for the indicated time. The assembly of NLRP3 inflammasome was detected using immunoprecipitation with anti-ASC Ab followed by immunoblotting for NLRP3, ASC, and caspase-1 cleavage product p10 fragments (Casp-1). The NLRP3 activation was confirmed by using immunoprecipitation with anti-NLRP3 Ab followed by immunoblotting for ASC, NLRP3, and Casp-1. B. Effects of depletion of NLRP3, IPAF, or AIM2 with respective siRNAs on IL-1β, IL-18 and mature Casp-1 production following 20% cyclic stretch for 4 h. IL-1β and IL-18 release in the culture supernatants (SN) of AMs and levels of Pro-IL-1β, Pro-IL-18, Pro-Casp-1 and Casp-1 in cell lysate (Lys) were determined by Western blot analysis. A scrambled siRNA (si Sc) was used as a negative control. C. The levels of IL-1β in cell-culture media were measured by ELISA. The graph shows the mean and SEM from 3 independent experiments. *p < 0.05 vs. the control (static) group. † p <0.05 vs. si Sc+CS group.

FIGURE 4. Mitochondrial ROS are required for NLRP3 inflammasome activation following cyclic stretch

A. Representative histograms of flow cytometry experiments demonstrating the effects of cyclic stretch and SS-31 on mitochondrial ROS generation. AMs were pretreated with SS-31 peptide (10 μM) for 24 h prior to stretch. AMs were stained with MitoSOX for 30 min and analyzed by flow cytometry. B. Quantitative data showing changes in mean fluorescent intensity (MFI) of MitoSOX following stretch and SS-31 treatment (n=3). C. SS-31 dose-dependently inhibited IL-1β release and caspase-1 activation following 20% cyclic stretch for 4 h. IL-1β release in the culture supernatants (SN) of AMs and levels of Pro-IL-1β, Pro-Casp-1 and Casp-1 in cell lysate (Lys) were determined by Western blot analysis. D. IL-1β in cell-culture media was measured by ELISA (n=3). E. Effects of rotenone on IL-1β release and caspase-1 activation following 20% cyclic stretch for 4 h. F. IL-1β in cell-culture media was measured by ELISA (n=3). *p < 0.05 vs. the control (static) group. † p <0.05 vs. CS alone group.

FIGURE 5. NADPH oxidase-derived ROS do not contribute to cyclic stretch-induced NLRP3 inflammasome activation

A. AMs isolated from gp91^phox−/−mice show no change in IL-1β release following stretch. Wild type and gp91^phox−/−AMs were subjected to 20% cyclic stretch for 4 h. IL-1β release in the culture supernatants (SN) of AMs and levels of Pro-IL-1β, Pro-Casp-1 and Casp-1 in cell lysate (Lys) were determined by Western blot analysis. B. The levels of IL-1β in cell-culture media were measured by ELISA (n=3). C. Representative histograms of flow cytometry experiments demonstrating the effects of cyclic stretch on mitochondrial ROS generation in gp91^phox−/− and wild type AMs. D. Quantitative data showing changes in mean fluorescent intensity (MFI) of MitoSOX following stretch (n=3). *p < 0.05 vs. the control (static) group.

FIGURE 6. Uric acid production by cyclic stretch contributes to NLRP3 inflammasome activation partially through mitochondrial ROS

A. Effects of cyclic stretch and allopurinol on uric acid production. AMs were exposed to 20% cyclic stretch for 4 h in the absence and presence of allopurinol pretreatment for 4 h. Uric acid concentrations were measured in the media of cultured AMs post stretch as described in Materials and Methods. B. Effects of allopurinol on IL-1β release and caspase-1 activation. AMs were subjected to 20% cyclic stretch for 4 h. IL-1β release in the culture supernatants (SN) of AMs and levels of Pro-IL-1β, Pro-Casp-1 and Casp-1 in cell lysate (Lys) were determined by Western blot analysis. C. Representative histograms of flow cytometry experiments demonstrating the effects of MSU on mitochondrial ROS generation. D. Quantitative data showing the effects of MSU on mean fluorescent intensity (MFI) of MitoSOX (n=3). E. Effects of MSU on IL-1β release measured by ELISA (n=3). F. Representative histograms of flow cytometry experiments demonstrating the effects of allopurinol on cyclic stretch-induced mitochondrial ROS generation. *p < 0.05 vs. the control (static, A) or non-MSU treated (D, E) group. † p <0.05 vs. CS alone group.

FIGURE 7. IL-1β production following cyclic stretch occurs via TLR4 signaling. A

AMs isolated from tlr4^−/− mice show less IL-1β release following stretch. Wild type and tlr4^−/− AMs were subjected to 20% cyclic stretch for 4 h. IL-1β release in the culture supernatants (SN) of AMs and levels of Pro-IL-1β, Pro-Casp-1 and Casp-1 in cell lysate (Lys) were determined by Western blot analysis. B. The levels of IL-1β in cell-culture media were measured by ELISA (n=3). C. Representative histograms of flow cytometry experiments demonstrating the effects of cyclic stretch on mitochondrial ROS generation in tlr4^−/− and wild type AMs. D. Quantitative data showing changes in mean fluorescent intensity (MFI) of MitoSOX following stretch (n=3). *p < 0.05 vs. the control (static) group. † p <0.05 vs. WT+CS group.

FIGURE 8. Mechanical ventilation with a high tidal volume activates NLRP3 inflammasome in mouse lungs

Wild type and gp91^phox−/− mice were ventilated with a normal (L) or high (H) tidal volume for the indicated times. At the end of experiments, lung tissue and BAL fluid were recovered. A. Effects of mechanical ventilation and SS-31 on NLRP3 inflammasome activation and subsequent release of IL-1β and IL-18. Top: The assembly of NLRP3 inflammasome was detected using immunoprecipitation with anti-ASC Ab followed by immunoblotting for NLRP3, ASC, and caspase-1 cleavage product p10 fragments (Casp-1). Bottom: The levels of mature IL-1β, mature IL-18, Pro-IL-1β, Pro-IL-18, Pro-Casp-1 and Casp-1 in lung homogenates were determined by Western blot analysis. B. The levels of IL-1β in BAL fluid were measured by ELISA. The graph shows the mean and SEM from six mice. C. Effects of depletion of AMs with liposome on mechanical ventilation-induced release of IL-1β and IL-18, and caspase-1 activation. The levels of mature IL-1β, mature IL-18, Pro-IL-1β, Pro-IL-18, Pro-Casp-1 and Casp-1 in lung homogenates were determined by Western blot analysis. D. The levels of IL-1β in BAL fluid were measured by ELISA (n=6). *p < 0.05 vs. the control group (L). † p <0.05 vs. WT+H group (B, 2h alone) or PBS+H group (D).

FIGURE 9. Model of inflammasome activation by mechanical stretch

ASC, apoptosis-associated speck-like protein containing a CARD domain; Casp-1, caspase-1; NLRP3, NOD-like receptor containing pyrin domain 3; ROS, reactive oxygen species.