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. 2022 Aug 18:2022:2291877.
doi: 10.1155/2022/2291877. eCollection 2022.

Negative Feedback of the cAMP/PKA Pathway Regulates the Effects of Endoplasmic Reticulum Stress-Induced NLRP3 Inflammasome Activation on Type II Alveolar Epithelial Cell Pyroptosis as a Novel Mechanism of BLM-Induced Pulmonary Fibrosis

Qiaohui Hong  1 , Yue Zhang  1 , Weixian Lin  1 , Wei Wang  1 , Yafei Yuan  1 , Jiajia Lin  1 , Zhanzhan Xie  1 , Xu Li  2 , Ying Meng  1
Affiliations

Negative Feedback of the cAMP/PKA Pathway Regulates the Effects of Endoplasmic Reticulum Stress-Induced NLRP3 Inflammasome Activation on Type II Alveolar Epithelial Cell Pyroptosis as a Novel Mechanism of BLM-Induced Pulmonary Fibrosis

Qiaohui Hong et al. J Immunol Res. .

Abstract

Endoplasmic reticulum stress (ER stress) contributes to the development of pulmonary fibrosis, especially in type II alveolar epithelial cells (AECs) apoptosis. ER stress also promotes NLRP3 inflammasome activation which is inhibited by upregulation of cAMP/PKA pathway. However, it is confused whether ER stress-induced NLRP3 inflammasome activation and pyroptosis in type II alveolar epithelial cells which exacerbates pulmonary fibrosis via a mechanism that is suppressed by cAMP/PKA pathway. In our research, we explored that potential links among NLRP3 inflammasome, ER stress, and cAMP/PKA pathway in type II AECs to explain the new mechanisms of pulmonary fibrosis. We found that in vivo, ER stress, NLRP3 inflammasome, and PKA upregulated in the alveolar epithelial area in animal models of pulmonary fibrosis. In addition, immunofluorescence staining further confirmed that ER stress, NLRP3 inflammasome, and cAMP/PKA had potential links on type II AECs in BLM group. In vitro, ER stress stimulated NLRP3 inflammasome activation, promoted pyroptosis, and also upregulated cAMP/PKA pathway. Upregulation of cAMP/PKA pathway inhibited ER stress-induced pyroptosis of A549 cells and vice versa. These results initially supported conclusion that ER stress may stimulate NLRP3 inflammasome activation and pyroptosis in type II AECs, which exacerbated pulmonary fibrosis, and cAMP/PKA pathway may act as a feedback regulator.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
(a) H&E and Masson's trichrome staining (magnification 200×). (b) Relative Ashcroft score. ∗P < 0.05 vs. control group.
Figure 2
Figure 2
ER stress and NLRP3 inflammasome activation increased in the alveolar region in the BLM group. (a–c) Immunochemical staining of ER stress markers Grp78, Grp94, and CHOP and (d–g) NLRP3 inflammasome-related proteins NLRP3, IL1β, ASC, and N-GSMDM.
Figure 3
Figure 3
Coexpression of ER stress marker and NLRP3 inflammasome-related proteins in type II alveolar epithelial cells in BLM-induced pulmonary fibrosis. Immunofluorescence staining showing (a, b) colocalization of SPC with the ER stress marker CHOP and the NLRP3 inflammasome-related protein NLRP3 and (c, d) colocalization of the ER stress marker CHOP with the NLRP3 inflammasome-related protein NLRP3 and IL-1β.
Figure 4
Figure 4
Endoplasmic reticulum stress promoted NLRP3 inflammasome activation and pyroptosis in type II alveolar epithelial cells. A549 cell lines were pretreated 4PBA (5 mM) for 1 h or NLRP3 expression was knocked down by siRNA transfection before exposure to tunicamycin (1 μg/mL) for 24 h. (a–e) Protein levels of the ER stress markers Grp94 and CHOP and the NLRP3 inflammasome-related proteins NLRP3, ASC, cleaved caspase-1, cleaved IL1β, and N-GSDMD were detected by Western blot analysis. (f, g) Colocalization of Grp94/NLRP3 and CHOP/NLRP3 was detected by immunofluorescence staining. (h) LDH release assay of cell viability. (i) Cell viability was assessed by CCK8 assay. ∗P < 0.05 vs. control group, ∗∗P < 0.05 vs. tunicamycin group, (j) LDH release assay of cell viability, (k) cell viability was assessed by CCK8 assay. n.s.: not significant vs. control group, ∗P < 0.05 vs. tunicamycin group.
Figure 5
Figure 5
Upregulation of the cAMP/PKA pathway in BLM-induced pulmonary fibrosis. (a) Immunohistochemical staining of PKA protein. Immunofluorescence staining showing colocalization of (b, c) SPC/PKA and SPC/cAMP and (d) cAMP/NLRP3.
Figure 6
Figure 6
ER stress induced upregulation of the cAMP/PKA pathway. (a) A549 cells were pretreated with 4PBA (5 mM) for 1 h before exposure to tunicamycin (1 μg/mL) for 24 h. cAMP was detected by ELISA. (b) Western blot showed PKA protein level. (c, d) Immunofluorescence staining showed colocalization of cAMP/Grp94 and cAMP/NLRP3. ∗P < 0.05 vs. control group, ∗∗P < 0.05 vs. tunicamycin group.
Figure 7
Figure 7
Inhibition of the cAMP/PKA pathway exacerbated type II alveolar epithelial cell death induced by ER stress. A549 cells were pretreated KH7 (10 μM) or H89 (8 μM) or forskolin (100 μM) for 1 h before exposure to tunicamycin for 24 h. (a) LDH release assay of cell viability. (b) Cell viability was assessed by CCK8 assay. (c) LDH release assay of cell viability. (d) Cell viability was assessed by CCK8 assay. (e) LDH release assay of cell viability. (f) Cell viability was assessed by CCK8 assay. ∗(g) Western blot showing NLRP3 and ASC protein levels; ∗P < 0.05 vs. control group, ∗∗P < 0.05 vs. tunicamycin group.
Figure 8
Figure 8
Negative feedback of the cAMP/PKA pathway regulates the effects of ER stress-induced NLRP3 inflammasome activation on type II alveolar epithelial cell pyroptosis, which alleviates pulmonary fibrosis.
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