Suppression of NLRP3 Inflammasome by Erythropoietin via the EPOR/JAK2/STAT3 Pathway Contributes to Attenuation of Acute Lung Injury in Mice

Abstract

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common and devastating clinical disorders with high mortality and no specific therapy. An excessive inflammatory response results in the progression of ALI/ARDS, and the NLRP3 inflammasome is a key participant in inflammation. Erythropoietin (EPO), which is clinically used for anemia, reportedly exerts pleiotropic effects in ALI. However, whether EPO could protect against lipopolysaccharide (LPS)-induced ALI by regulating the NLRP3 inflammasome and its underlying mechanisms remain poorly elucidated. This study aimed to explore whether the therapeutic effects of EPO rely on the suppression of the NLRP3 inflammasome and the specific mechanisms in an LPS-induced ALI mouse model. ALI was induced in C57BL/6 mice by intraperitoneal (i.p.) injection of LPS (15 mg/kg). EPO was administered intraperitoneally at 5 U/g after LPS challenge. The mice were sacrificed 8 h later. Our findings indicated that application of EPO markedly diminished LPS-induced lung injury by restoring histopathological changes, lessened lung wet/dry (W/D) ratio, protein concentrations in bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) levels. Meanwhile, EPO evidently decreased interleukin-1β (IL-1β) and interleukin-18 (IL-18) secretion, the expression of NLRP3 inflammasome components including pro-IL-1β, NLRP3, and cleaved caspase-1 as well as phosphorylation of nuclear factor-κB (NF-κB) p65, which may be associated with activation of EPO receptor (EPOR), phosphorylation of Janus-tyrosine kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). However, all the beneficial effects of EPO on ALI and modulation NLRP3 inflammasome were remarkably abrogated by the inhibition of EPOR/JAK2/STAT3 pathway and knockout (KO) of NLRP3 gene. Taken together, this study indicates that EPO can effectively attenuate LPS-induced lung injury in mice by suppressing the NLRP3 inflammasome, which is dependent upon activation of EPOR/JAK2/STAT3 signaling and inhibition of the NF-κB pathway.

Keywords: EPOR; JAK2/STAT3; NF-κB; NLRP3 inflammasome; acute lung injury; erythropoietin.

FIGURE 1

EPO mitigated LPS-induced acute lung injury in mice. EPO (5 U/g) was injected into the peritoneal cavity of mice after LPS (15 mg/kg) stimulation. The mice were sacrificed 8 h later, and the effects of EPO were assessed in (A,B) hematoxylin and eosin-stained sections (original magnification × 100, × 400). Lung injury scores (C) were recorded from 0 (no damage) to 16 (maximum damage) according to the criteria described in Section “Materials and Methods.” The lung wet/dry ratio (D) was tested to evaluate pulmonary edema. The total protein concentrations in the BALF (E) were measured to reflect the integrity of the pulmonary alveolar-capillary barrier. Myeloperoxidase (MPO) concentrations in lung tissues (F) were measured by ELISA to quantitatively determine the resolution of infiltrated cells. The lung injury score data are presented as medians and ranges (5th –95th percentile), and the differences among groups were assessed by Kruskal–Wallis test and the post hoc test (Dunn’s method) was applied to investigate the differences one by one. n = 4 per group. The other data are presented as the mean ± SD. The differences of D–F were assessed by one-way ANOVA and the post hoc test (Turkey method) was applied to investigate the differences one by one. n = 8 per group.

FIGURE 2

EPO downregulated the expression and concentration of IL-1β and IL-18 in LPS-induced ALI. EPO (5 U/g) was injected into the peritoneal cavity of mice after LPS (15 mg/kg) stimulation. The mice were sacrificed 8 h later, and the serum was obtained to measure the concentrations of IL-1β and IL-18 (A,D) by ELISA. The lung tissues were harvested to measure the concentrations of IL-1β and IL-18 (B,E) by ELISA, and the mRNA expression levels of pro-IL-1β and pro-IL-18 (C,F) were measured by QPCR. The data are presented as the mean ± SD. The differences among groups were assessed by one-way ANOVA and the post hoc test (Turkey method) was applied to investigate the differences one by one. n = 8 per group.

FIGURE 3

EPO suppressed the NLRP3 inflammasome in LPS-induced ALI. EPO (5 U/g) was injected into the peritoneal cavity of mice after LPS (15 mg/kg) stimulation. The mice were sacrificed 8 h later, and the lung tissues were obtained to measure the mRNA expression levels of caspase-1, ASC, and NLRP3 (A–C) by QPCR. In addition, the protein expression levels of pro-caspase-1, pro-IL-1β, ASC, NLRP3, and cleaved caspase-1 (D–I) were detected by Western blotting. The data are presented as the mean ± SD. The differences among groups were assessed by one-way ANOVA and the post hoc test (Turkey method) was applied to investigate the differences one by one. n = 8 per group.

FIGURE 4

EPO activated the EPOR/JAK2/STAT3 pathway and inhibited NF-κB activation in LPS-induced ALI. EPO (5 U/g) was injected into the peritoneal cavity of mice after LPS (15 mg/kg) stimulation, and the mice were sacrificed 8 h later. The lung tissues were harvested to measure the protein expression levels of p-JAK2, t-JAK2, p-STAT3, t-STAT3, NF-κB p-p65, and NF-κB t-p65 (A–D) by Western blotting. The data are presented as the mean ± SD. The differences among groups were assessed by one-way ANOVA and the post hoc test (Turkey method) was applied to investigate the differences one by one. n = 8 per group.

FIGURE 5

The protective effects of EPO on acute lung injury were EPOR/JAK2/STAT3/NF-κB signaling axis dependent. EMP9 (an EPOR antagonist, 1 mg/mouse), Fedratinib (a JAK2 inhibitor, 100 mg/kg), NSC 74859 (a STAT3 inhibitor, 5 mg/kg), BAY 11-7082 (an NF-κB inhibitor, 20 mg/kg), and DMSO (solvent of inhibitors) were injected into the peritoneal cavity 30 min before EPO treatment. Then, EPO (5 U/g) was injected into the peritoneal cavity of mice after LPS (15 mg/kg) stimulation, and the mice were sacrificed 8 h later. The lung tissues were harvested to measure the protein expression of p-JAK2, t-JAK2, p-STAT3, t-STAT3, NF-κB p-p65, and NF-κB t-p65 (A–D) by Western blotting. The data are presented as the mean ± SD. The differences among groups were assessed by one-way ANOVA and the post hoc test (Turkey method) was applied to investigate the differences one by one. n = 8 per group.

FIGURE 6

Inhibitors of the EPOR/JAK2/STAT3 pathway reversed the suppressing effect of EPO on the NLRP3 inflammasome. EMP9 (an EPOR antagonist, 1 mg/mouse), Fedratinib (a JAK2 inhibitor, 100 mg/kg), NSC 74859 (a STAT3 inhibitor, 5 mg/kg), BAY 11-7082 (an NF-κB inhibitor, 20 mg/kg), and DMSO (solvent of the inhibitors) were injected into the peritoneal cavity 30 min before EPO treatment. Then, EPO (5 U/g) was injected into the peritoneal cavity of mice after LPS (15 mg/kg) stimulation. The mice were sacrificed 8 h later, and the lung tissues were harvested to measure the protein expression levels of pro-caspase-1, pro-IL-1β, NLRP3, cleaved caspase-1 (A–E) by Western blotting. The data are presented as the mean ± SD. The differences among groups were assessed by one-way ANOVA and the post hoc test (Turkey method) was applied to investigate the differences one by one. n = 8 per group.

FIGURE 7

EPO protected against lung injury through the EPOR/JAK2/STAT3 and NF-κB signaling pathways. EMP9 (an EPOR antagonist, 1 mg/mouse), Fedratinib (a JAK2 inhibitor, 100 mg/kg), NSC 74859 (a STAT3 inhibitor, 5 mg/kg), BAY 11-7082 (an NF-κB inhibitor, 20 mg/kg), and DMSO (solvent of the inhibitors) were injected into the peritoneal cavity 30 min before EPO treatment. Then, EPO (5 U/g) was injected into the peritoneal cavity of mice after LPS (15 mg/kg) stimulation. The mice were sacrificed 8 h later, and the sera were obtained to measure the concentrations of IL-1β and IL-18 (A,B) by ELISA. The BALF was obtained to measure the total protein concentrations (F). In addition, the lung tissues were harvested to measure the lung wet/dry ratio (E), and the concentrations of IL-1β, IL-18, and MPO (C,D,G) were measured by ELISA. The data are presented as the mean ± SD. The differences among groups were assessed by one-way ANOVA and the post hoc test (Turkey method) was applied to investigate the differences one by one. n = 8 per group.

FIGURE 8

The knockout of NLRP3 mimicked the effects of EPO on the NLRP3 inflammasome in ALI. EPO (5 U/g) was injected into the peritoneal cavity of mice after LPS (15 mg/kg) stimulation of NLRP3 knockout (KO) mice and wild-type (WT) mice, and the mice were sacrificed 8 h later. The lung tissues were harvested to measure the protein expression levels of pro-caspase-1, pro-IL-1β, cleaved caspase-1, and NLRP3 (A–E) by Western blotting. The data are presented as the mean ± SD. The differences among groups were assessed by two-way ANOVA and the post hoc test (Turkey method) was applied to investigate the differences one by one. n = 7 per group.

FIGURE 9

EPO attenuated lung injury and inhibited IL-1β and IL-18 secretion, which was dependent on NLRP3 in LPS-induced ALI. EPO (5 U/g) was injected into the peritoneal cavity of mice after LPS (15 mg/kg) stimulation in NLRP3 knockout (KO) mice and wild-type (WT) mice, and the mice were sacrificed 8 h later. The lung tissues were harvested to measure the lung wet/dry ratio (A) and the concentrations of IL-1β and IL-18 (D,E) by ELISA. The sera were obtained to measure the concentrations of IL-1β and IL-18 (B,C) by ELISA. The data are presented as the mean ± SD. The differences among groups were assessed by two-way ANOVA and the post hoc test (Turkey method) was applied to investigate the differences one by one. n = 7 per group.

FIGURE 10

EPO attenuated LPS-induced acute lung injury by suppressing the NLRP3 inflammasome via the EPOR/JAK2/STAT3/NF-κB pathway in mice. EPO binds to EPOR, further phosphorylates JAK2 to p-JAK2, and controls the kinase activity of JAK2 to phosphorylate STAT3 to p-STAT3. Subsequently, p-STAT3 inhibits the phosphorylation of NF-κB p65 and the transcriptional activity of NF-κB p-p65 to attenuate the transcription and expression of NLRP3 and pro-IL-1β, which consequently leads to a decrease in mature IL-1β levels and the mitigation of lung injury.