ADAR1 protects pulmonary macrophages from sepsis-induced pyroptosis and lung injury through miR-21/A20 signaling

Abstract

Acute lung injury is a serious complication of sepsis with high morbidity and mortality. Pyroptosis is a proinflammatory form of programmed cell death that leads to immune dysregulation and organ dysfunction during sepsis. We previously found that adenosine deaminase acting on double-stranded RNA 1 (ADAR1) plays regulatory roles in the pathology of sepsis, but the mechanism of ADAR1 in sepsis-induced pyroptosis and lung injury remains unclear. Here, we mainly investigated the regulatory effects and underlying mechanism of ADAR1 in sepsis-induced lung injury and pyroptosis of pulmonary macrophages through RNA sequencing of clinical samples, caecal ligation and puncture (CLP)-induced septic mouse models, and in vitro cellular experiments using RAW264.7 cells with lipopolysaccharide (LPS) stimulation. The results showed that pyroptosis was activated in peripheral blood mononuclear cells (PBMCs) from patients with sepsis. In the CLP-induced septic mouse model, pyroptosis was mainly activated in pulmonary macrophages. LPS-stimulated RAW264.7 cells showed significantly increased activation of the NLRP3 inflammasome. ADAR1 was downregulated in PMBCs of patients with sepsis, and overexpression of ADAR1 alleviated CLP-induced lung injury and NLRP3 inflammasome activation. Mechanistically, the regulatory effects of ADAR1 on macrophage pyroptosis were mediated by the miR-21/A20/NLRP3 signalling cascade. ADAR1 attenuated sepsis-induced lung injury and hindered the activation of pyroptosis in pulmonary macrophages in sepsis through the miR-21/A20/NLRP3 axis. Our study highlights the role of ADAR1 in protecting pulmonary macrophages against pyroptosis and suggests targeting ADAR1/miR-21 signalling as a therapeutic opportunity in sepsis-related lung injury.

Keywords: A20; ADAR1; lung injury; macrophage; pyroptosis; sepsis.

Figure 1

PBMCs from patients with sepsis showed a pyroptosis-like phenotype. (A) Representative SEM images of PBMCs from healthy volunteers and patients with sepsis. Scale bar indicates 5 μm and 2.5 μm. The red arrow indicates pore formation. N=3. (B) Flow cytometry showing the percentage of cleaved GSDMD-positive cells in PBMCs from healthy subjects and patients with sepsis. N=5. (C) Luminex assay showing the serum concentration of several cytokines in healthy subjects (N=6) and patients with sepsis (N=18). Data are shown as the mean ± SD. *P< 0.05, **P< 0.01 versus Nor. Nor: healthy volunteer.

Figure 2

Sequencing analysis of PBMCs from healthy subjects and patients with sepsis. (A) PCA plot of PBMC samples from healthy volunteers and patients with sepsis (N=4). (B) KEGG analysis of the enriched pathways related to the cell cycle and death from transcriptome sequencing of PBMCs in healthy subjects and patients with sepsis (N=4). (C) GSEA of cell cycle and death-related pathways in the two groups according to KEGG analysis of DEGs from transcriptome sequencing of PBMCs in healthy subjects and patients with sepsis (N=4). (D) Western blotting of human PBMC samples to show the relative expression levels of NLRP3, caspase-1, cleaved-GSDMD, and IL-1β between the two groups. N=6. Data are shown as the mean ± SD. *P< 0.05, **P< 0.01 versus Nor. Nor: healthy volunteer.

Figure 3

Activation of the NLRP3 inflammasome and pyroptosis in a mouse septic model. (A) Representative HE images of mouse lung tissues after CLP treatment over time. Scale bar means 200 μm. The disease score and lung injury score of lung tissues in each group were evaluated. N=6. (B) Double staining of F4/80 (red, macrophages), CK7 (red, epithelial cells), CD3 (red, T lymphocytes), VEad (red, endotheliocytes), and cleaved GSDMD (green) in the lung tissues of the sham and CLP-treated mice (24 h). DAPI is blue. N=6. Scale bar means 20 μm. (C) Relative protein expression levels of NLRP3, caspase-1, GSDMD, and cleaved GSDMD in the mouse lung tissues of the two groups as determined by Western blotting. N=3. (D) Relative mRNA expression levels of Nlrp3, Gsdmd, A20, Il1β, Il6, Tnf, and Il10 in the mouse lung tissues of the two groups, as determined by qRT‒PCR. N=6. Data are shown as the mean ± SD. *P< 0.05, **P< 0.01, ***P< 0.001 versus the sham group.

Figure 4

Bone marrow-derived macrophages (BMDMs) stimulated with LPS demonstrated an elevated level of cell death. (A-B) Flow cytometry analysis was used to analyse the polarization of BMDMs after LPS stimulation. N=6 in every group. BMDMs were first gated on FSC and SSC to remove debris and conjugates and then defined as CD11b⁺F4/80⁺ subpopulations (upper right), with the purity displayed as the percentage of the parent population gated on FSC/SSC. Tissue-infiltrated macrophages are defined as CD11b⁺F4/80⁺ cells; M1 macrophages are CD11b⁺F4/80⁺CD11c⁺CD206^- cells. (C) Annexin V/PI staining was used to identify and measure cell death in BMDMs following exposure to LPS. N=3. (D) Annexin V/PI staining was used to identify and measure cell death in RAW264.7 cells following exposure to LPS. N=3.

Figure 5

LPS-stimulated BMDMs and RAW264.7 cells exhibited increased pyroptosis. (A) The alterations in gene expression levels of Adar1, Nlrp3, Gsdmd, caspase1, and Il1β were assessed in BMDMs following 24 hours of LPS stimulation using qRT‒PCR. (B) Flow cytometry showing the percentage of cleaved GSDMD-positive cells in the control and LPS-treated (1 μg/ml) RAW264.7 cells. N=3. (C) qRT‒PCR analysis of Nlrp3, Gsdmd, caspase1, A20, Il1β, Il6, Tnf, and Il10 levels in different cell groups. N=3. Data are shown as the mean ± SD. *P< 0.05, **P< 0.01, ***P< 0.001, ns=no statistical significance.

Figure 6

ADAR1 expression was decreased in septic CLP-treated mice. (A) ADAR1 mRNA expression levels in human PBMCs from healthy subjects (N=10) and patients with sepsis (N=10). ***P< 0.001 versus Nor. (B) The distribution and expression of ADAR1 in various cells in normal lung tissues. (C) ADAR1 expression in macrophages decreased 24 h after CLP. (D) Double staining of F4/80 (green) and ADAR1 (red) in the mouse lung tissues of the sham and CLP-treated mice. DAPI is blue. N=6. Scale bar represents 50 μm.

Figure 7

ADAR1 alleviates bacterial burden in septic mice, increases the CD4⁺/CD8⁺ ratio in peripheral blood, and promotes immune restoration. N = 6 in each group. (A) A comprehensive analysis was conducted on peripheral blood samples from mice in the sham, CLP, and CLP+OE-A groups to compare and evaluate the levels of leukocytes, lymphocytes, and neutrophils. (B) Flow cytometry was used to analyse the effect of ADAR1 on the CD4⁺/CD8⁺ ratio in mouse peripheral blood. (C) The levels of bacteria in blood and lung tissues after sham, CLP, and CLP+OE-A interventions using bacterial culture. The injection of the ADAR1 virus reduced the bacterial burden in the blood and lung compared to that in the CLP group. Data are shown as the mean ± SD. *P< 0.05, **P< 0.01, ***P< 0.001 versus CLP.

Figure 8

Overexpression of ADAR1 alleviated CLP-induced pyroptosis in mice. (A) HE staining of mouse lung tissues in the sham, CLP, and CLP with OE-A groups was performed. The disease score and lung injury score were evaluated. N=6. Scale bar represents 50 μm. (B) Immunofluorescence staining of ADAR1 (red) and cleaved GSDMD (red) in the mouse lung tissues of the sham, CLP, and CLP+OE-A groups. DAPI is blue. N=3. Scale bar represents 50 μm. (C) qRT‒PCR analysis of Adar1, Nlrp3, Gsdmd, caspase1, A20, and Il1β levels in different groups. N=3. Data are shown as the mean ± SD. *P< 0.05, **P< 0.01, ***P< 0.001, ns=no statistical significance.

Figure 9

ADAR1 regulated pyroptosis in RAW264.7 cells after LPS stimulation through NLRP3 signalling. (A) The alteration of relative mRNA expression of Adar1 in RAW264.7 cells with LPS stimulation over time. N=4. (B) RAW264.7 cells were transfected with ADAR1 siRNA (si-A), and the relative mRNA expression of Adar1, Nlrp3, and A20 was measured by qRT‒PCR. N=6. ***P< 0.001 versus Ctrl. (C) Relative protein expression of ADAR1, NKRP3, and A20 was also assessed through Western blotting. N=3. (D) RAW264.7 cells were transfected with ADAR1 siRNA (si-A) and ADAR1 overexpression plasmid (OE-A) under LPS administration. SEM images showing the RAW264.7 cell morphology of the control, LPS, LPS+si-A, and LPS+OE-A groups. Scale bar indicates 10 μm and 2.5 μm. The red arrow indicates pore formation. N=6. (E) Immunofluorescence staining of cleaved GSDMD (red) and caspase-1 (red) in the four groups. DAPI is blue. N=6. Scale bar indicates 75 μm. (F) Western blotting images and quantification of NLRP3, A20, cleaved GSDMD, caspase-1, and IL-1β in the four groups. N=3. (G) Relative mRNA expression levels of Nlrp3, A20, and Il1β in the four groups determined by qRT‒PCR. N=3. Data are shown as the mean ± SD. *P< 0.05, **P< 0.01, ***P< 0.001.

Figure 10

ADAR1 regulated NLRP3 inflammasome activity through the miR-21/A20 signalling pathway in sepsis. (A) The alteration of relative mRNA expression of miR-21 in RAW264.7 cells with LPS stimulation over time. N=4. (B) Relative expression of miR-21 in RAW264.7 cells in the control, LPS, LPS+si-A, and LPS+OE-A groups. N=3. (C) RIP assay showing the binding effects of ADAR1 with pre-miR-21 in RAW264.7 cells through isolation of RNA‒protein binding complexes via anti-ADAR1 and further qRT‒PCR measuring the pre-miR-21 levels. N=3. ***P< 0.001 versus anti-IgG. (D) RAW264.7 cells were transfected with miR-21 mimic or inhibitors, and the relative protein levels of A20, NLRP3, GSDMD, caspase-1, and IL-1β were assessed using Western blotting. N=3. (E) Above: relative mRNA levels of miR-21, A20, and NLRP3 in PBMCs from healthy subjects (N=4) and patients with sepsis (N=8). **P< 0.01, ***P< 0.001 versus Nor. Below: relative mRNA levels of miR-21, A20, and Nlrp3 in mouse lung tissues of the sham and CLP groups. N=3. **P< 0.01 versus sham. (F) Relative mRNA expression levels of several potential miR-21 target genes in RAW264.7 cells transfected with miR-21 mimic or inhibitors are shown. The genes related to pyroptosis and inflammation were screened from TargetScan. N=3. (G) Dual luciferase activity assay showing the direct binding effects of miR-21-5p with its target gene A20 in RAW264.7 cells. N=3. **P< 0.01. (H) Relative mRNA expression levels of A20, Nlrp3, caspase1, Gsdmd, Il1β, and Il6 in Raw264.7 cells transfected with A20 siRNA (si-A20) determined by qRT‒PCR. N=4. (I) Western blotting of A20, caspase-1, cleaved GSDMD, and IL-1β in Raw264.7 cells transfected with si-A20. N=3. Data are shown as the mean ± SD. *P< 0.05, **P< 0.01, ***P< 0.001 versus Ctrl. ns=no statistical significance.