Regulation of the NLRP3 inflammasome and macrophage pyroptosis by the p38 MAPK signaling pathway in a mouse model of acute lung injury

Abstract

Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) is characterized by uncontrolled progressive lung inflammation. Macrophages serve a key role in the pathogenesis of ALI/ARDS. Macrophage pyroptosis is a process of cell death releasing the proinflammatory cytokines interleukin (IL)‑1β and IL‑18. It was hypothesized that macrophage pyroptosis may partially account for the uncontrolled lung inflammation of ALI/ARDS. In the present study, greater macrophage pyroptosis in lipopolysaccharide (LPS)‑treated macrophages and the ALI/ARDS mouse model was observed. The expression of nucleotide‑binding domain, leucine‑rich‑containing family, pyrin domain‑containing (NLRP)3 and IL‑1β and cleavage of caspase‑1 were significantly elevated following LPS treatment accompanied by greater activation of p38 mitogen‑activated protein kinase (MAPK) signaling in vitro and in vivo. However, blocking p38 MAPK signaling through the inhibitor SB203580 significantly suppressed the acute lung injury and excessive lung inflammation in vivo, consistent with the reduced expression of the NLRP3 inflammasome and IL‑1β and cleavage of caspase‑1. Pretreatment of the rat NR8383 macrophage cell line with SB203580 significantly decreased the population of caspase‑1+PI+ pyroptotic cells and expression of NLRP3/IL‑1β. However, a larger population of Annexin V+PI‑ apoptotic cells was observed following blocking of the p38 MAPK signaling pathway. The results indicated that blockage of p38 MAPK signaling pathway skewed macrophage cell death from proinflammatory pyroptosis towards non‑inflammatory apoptosis. These effects may contribute to attenuated acute lung injury and excessive inflammation in the SB203580‑treated mice. The results may provide a novel therapeutic strategy for the treatment of uncontrolled lung inflammation in patients with ALI/ARDS.

Figure 1.

p38 MAPK inhibitor SB203580 suppresses acute lung injury and inflammation in mice after i.t. injection of LPS. All parameters were measured at 24 h following the i.t. LPS treatment. (A) Representative images of the histology of lung tissues by H&E staining. Black arrows: Thickened alveolar septal; blue arrows: Alveolar hemorrhage and red arrows: infiltrated polymorphonuclear leukocytes (magnification, ×200). (B) Lung pathological score in terms of lung injury and inflammation. (C) Total protein content and (D) total cell counts in BAL; (E) weight loss and (F) ratio of lung Wet/Dry were analyzed. Data are presented as the mean ± standard error of the mean. **P<0.01, ***P<0.001 vs. PBS group; ^#P<0.05, ^##P<0.01 vs. LPS group, n=5 per group. BAL, bronchoalveolar lavage fluid; i.t., intratracheal; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase.

Figure 2.

p38 MAPK inhibitor SB203580 suppresses IL-1β, TNF-α and IL-6 expression in mice following the i.t. injection of LPS. Twenty-four hours following SB203580 i.p. and LPS i.t. treatment, the lung and BAL fluids were collected. IL-1β (A) mRNA and (B) protein expression levels; TNF-α (C) mRNA and (D) protein expression levels and IL-6 (E) mRNA and (F) protein expression levels were analyzed by RT-qPCR analysis and by ELISA analysis, respectively. Data are presented as the mean ± standard error of the mean. **P<0.01, ***P<0.001 vs. PBS group; ^#P<0.05, ^##P<0.01 vs. LPS group, n=5 mice per group. BAL, bronchoalveolar lavage fluid; i.p., intraperitoneal; i.t., intratracheal; IL, interleukin; LPS, lipopolysaccharide; TNF, tumor necrosis factor.

Figure 3.

p38 MAPK inhibitor SB203580 suppresses caspase-1, NLRP3 and TLR2 expression in mice following i.t. injection of LPS. The pro-caspase-1, cleaved caspase-1, NLRP3 (A), NF-κB and TLR2 and TLR4 (B) were analyzed by western blot analysis following LPS treatment. NLRP3 (C) and TLR2 (D) mRNA expression levels were analyzed by RT-qPCR analysis. **P<0.01, ***P<0.001 vs. PBS group; ^#P<0.05 vs. LPS group, n=5 per group. IL, interleukin; i.t., intratracheal; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κ-light-chain-enhancer of activated B cells; NLRP, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing; TLR, Toll-like receptor.

Figure 4.

LPS treatment increases NLRP3 expression in macrophages in vitro in a time-dependent manner. RAW264.7 were treated with 1 µg/ml LPS for 4, 8, 12, and 24 h. The untreated cells were used as controls. The expression of NLRP3 protein was analyzed by immunofluorescence staining. (A) Representative image of NLRP3 expression levels in cells treated at different time points and (B) quantification of them following LPS treatment. Data are presented as a ratio of NLRP3+ cells to the number of nuclei in 7 random selected fields n=5 per group. *P<0.05, **P<0.01 vs. untreated cells. (C) LPS treatment increases the formation of NLRP3 inflammasomes, and NLRP3 protein was colocalized with the cleaved caspase-1. Red: NLRP3; Green: Cleaved caspase-1; Blue: DAPI-stained nuclei. White arrow indicates NLRP3 inflammasome. LPS, lipopolysaccharide; NLRP, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing.

Figure 5.

p38 MAPK inhibitor SB203580 suppresses caspase-1 expression but increased Annexin V+ cell population in vitro. The rat macrophage cell line NR8383 was treated with 1 µg/ml LPS for 4 h with/without pretreatment with 10 µM SB203580. The caspase-1 and Annexin V+ cells were measured by flow cytometry. (A) LPS increased caspase-1 expression which was reversed by pretreatment with SB203580. (B) LPS increased Annexin V+PI- early apoptotic cells and pretreatment with p38 MAPK inhibitor SB203580 enhanced this. (C) Quantitative analysis of caspase-1+PI+ pyroptotic cells and of (D) Annexin V+PI- apoptotic cells. Data are presented as the mean ± standard error of the mean. **P<0.01 vs. the untreated control group; ^#P<0.05, ^##P<0.01 vs. the LPS-treated group, n=3 per group. LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase.

Figure 6.

p38 MAPK inhibitor SB203580 suppresses the expression of NLRP3 and cleavage of caspase-1 in vitro. The rat macrophage cell line NR8383 was treated with 1 µg/ml LPS for 4 h with/without pretreatment with 10 µM SB203580. Protein expression levels of (A) p-p38, (B) NLRP3 and TLR2/TLR4 and (C) caspase-3 and cleaved-caspase-3, were analyzed by western blot analysis. Quantitative analysis for (D) p-p38, (E) TLR2, (F) cleaved-caspase-3 and (G) NLRP3 was performed, and data are presented as the mean ± standard error of the mean. *P<0.05, **P<0.01 vs. the untreated control. ^#P<0.05 vs. the LPS treated group. n=3 per sample. LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NLRP, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing; p, phosphorylated; TLR, Toll-like receptor.

Figure 7.

p38 MAPK inhibitor SB203580 suppresses the expression of IL-1β and IL-6 in the LPS-treated macrophages. The rat macrophage cell line NR8383 was treated with 1 µg/ml LPS for 4 h with/without pretreatment with 10 µM SB203580. The mRNA expression levels of (A) IL-1β and (B) IL-6 in the treated cells were measured by RT-qPCR. The protein expression levels of (C) IL-1β and (D) IL-6 in the cell supernatants were measured by ELISA. Data are presented as the mean ± standard error of the mean, n=3 per group. ***P<0.001 vs. the untreated control group; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. the LPS-treated group, n=3 per group. IL, interleukin; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase.

Figure 8.

Schematic diagram of the p38 MAPK signaling pathway in the regulation of the NLRP3 inflammasome and macrophage pyroptosis. LPS activates p38 MAPK signaling pathway in macrophages through the TLR2/NF-κB signaling pathway, subsequently upregulating protein expression and promoting the formation of the NLRP3/caspase-1/ASC protein complex (NLRP3 inflammasome). The activated NLRP3 inflammasome cleaves pro-IL-1β and pro-IL-18 to become active IL-1β and IL-18, which ultimately induces macrophage pyroptosis. The signaling pathway is suppressed by p38 MAPK inhibitor SB203580. In addition, SB203580 upregulates caspase-3 and enhances macrophage apoptosis. ASC, apoptosis-associated speck-like protein; IL, interleukin; LPS, lipopolysaccharide; MAPK, Mitogen-Activated Protein Kinase; NF-κB, nuclear factor-κ-light-chain-enhancer of activated B cells; NLRP leucine-rich-containing family, pyrin domain-containing; TLR, Toll-like receptor.