GSK2656157, a PERK Inhibitor, Alleviates Pyroptosis of Macrophages Induced by Mycobacterium Bacillus Calmette-Guerin Infection

Abstract

Tuberculosis (TB) is the leading cause of human death worldwide due to Mycobacterium tuberculosis (Mtb) infection. Mtb infection can cause macrophage pyroptosis. PERK, as a signaling pathway protein on the endoplasmic reticulum, plays an important role in infectious diseases. It is not clear whether PERK is involved in the regulation of pyroptosis of macrophages during Mtb infection. In this study, Bacillus Calmette-Guerin (BCG) infection resulted in high expression of pro-caspase-1, caspase-1 p20, GSDMD-N, and p-PERK in the THP-1 macrophage, being downregulated with the pre-treatment of GSK2656157, a PERK inhibitor. In addition, GSK2656157 inhibited the secretion of IL-1β and IL-18, cell content release, and cell membrane rupture, as well as the decline in cell viability induced by BCG infection. Similarly, GSK2656157 treatment downregulated the expressions of pro-caspase-1, caspase-1 p20, caspase-11, IL-1β p17, IL-18 p22, GSDMD, GSDMD-N, and p-PERK, as well as reducing fibrous tissue hyperplasia, inflammatory infiltration, and the bacterial load in the lung tissue of C57BL/6J mice infected with BCG. In conclusion, the inhibition of PERK alleviated pyroptosis induced by BCG infection, which has an effect of resisting infection.

Keywords: Bacillus Calmette–Guerin; PERK; macrophages; pyroptosis.

Figure 1

BCG infection induced THP-1 macrophage pyroptosis. The THP-1 macrophages were infected with BCG at MOI of 10 for 0, 2, 6, 12, 24, and 48 h. (A) qRT-PCR results of GSDMD mRNA expression. (B) Western blot results of GSDMD-N protein expression. (C) GSDMD-N protein expression analysis histogram. (D,E) ELISA results of IL-1β (D) and IL-18 (E) secretion levels. (F) LDH kits detection results. (G) CCK-8 assay results of cell viability. The gray value of a Western blot was determined using ImageJ. ^ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA analysis).

Figure 2

BCG infection activated PERK of the THP-1 macrophage. The THP-1 macrophages were infected with BCG at MOI of 10 for 0, 2, 6, 12, 24, and 48 h. (A) qRT-PCR results of PERK mRNA expression. (B) Western blot assay results of p-PERK and PERK protein expression. (C) p-PERK protein expression analysis histogram. ^ns p > 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA analysis).

Figure 3

GSK2656157 suppressed BCG-infection-induced pyroptosis of THP-1 macrophages. The THP-1 macrophages were pretreated with GSK2656157 for 2 h and then infected with BCG at MOI of 10 for 24 hpi. (A,B) qRT-PCR results of GSDMD (A) and pro-caspase-1 (B) mRNA expression in control, BCG, GSK, and BCG + GSK groups, respectively. (C) Western blot analysis of p-PERK, PERK, GSDMD-N, pro-caspase-1, and caspase-1 p20 protein expression levels in control, BCG, GSK, and BCG + GSK groups, respectively. (D–G) Expression histogram of p-PERK (D), GSDMD-N (E), pro-caspase-1 (F), and caspase-1 p20 (G) protein. (H,I) ELISA results of IL-1β (H) and IL-18 (I) secretion levels in control, BCG, GSK, and BCG + GSK groups, respectively. (J) LDH kit detection results in control, BCG, GSK, and BCG + GSK groups. (K) CCK-8 assay results of cell viability in control, BCG, GSK, and BCG + GSK groups. (L) Immunofluorescence staining results of GSDMD protein expression in control, BCG, and BCG + GSK groups; GSDMD is green, DAPI is blue, and the scale was 20 μm. (M) GSDMD protein analysis histogram. (N) The results of cell morphology photographed by TEM in control, BCG, and BCG + GSK groups. Scale bar: 2 μm. Nucleus (N), mitochondria (Mi), intact and continuous cell membranes (green arrow), broken cell membranes (red arrow), cell contents released (yellow arrow). * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA analysis).

Figure 4

GSK2656157 inhibited pyroptosis in the lung tissue of BCG-infected C57BL/6J mice. The C57BL/6J mice were infected with BCG and then treated with GSK2656157. (A–C) qRT-PCR results GSDMD (A), pro-caspase-1 (B), and caspase-11 (C) mRNA expression in control, BCG, and BCG + GSK groups, respectively. (D) Western blot analysis of p-PERK, PERK, GSDMD-N, pro-caspase-1, caspase-1 p20, and caspase-11 protein expression in control, BCG, and BCG + GSK groups, respectively. (E–I) Expression histogram of p-PERK (E) GSDMD-N (F), pro-caspase-1 (G), caspase-1 p20 (H), and caspase-11 (I) protein. (J–M) IHC staining results of lung tissues with p-PERK (J) and GSDMD (L) protein expression in control, BCG, and BCG + GSK groups, respectively; the scale is 40 μm, ^ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA analysis).

Figure 5

GSK2656157 reduced the expression of IL-1β p17 and IL-18 p22 in the lung tissue of BCG-infected C57BL/6J mice. The C57BL/6J mice were infected with BCG and then treated with GSK2656157. (A,B). qRT-PCR results of pro-IL-1β (A) and pro-IL-18 (B) mRNA expression in control, BCG, and BCG + GSK groups, respectively; (C) Western blot analysis of IL-1β p17 and IL-18 p22 in control, BCG, and BCG + GSK groups, respectively. (D,E) IL-1β p17 (D) and IL-18 p22 (E) protein expression analysis histogram. (F,G) IHC results of IL-1β protein expression in control, BCG, and BCG + GSK groups; the scale is 40 μm. * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA analysis).

Figure 6

GSK2656157 inhibited fibrous tissue hyperplasia, inflammatory infiltration, and bacterial load in the lung tissue of BCG-infected C57BL/6J mice. The C57BL/6J mice were infected with BCG and then treated with GSK2656157. (A) HE staining results of fibrous tissue hyperplasia and inflammatory infiltration in control, BCG, and BCG + GSK groups. (B) Statistical results of lung tissue injury scores in C57BL/6J mice; the scale is 40 μm. (C) Analysis results of bacterial load in the lung tissue of C57BL/6J mice; ** p < 0.01, *** p < 0.001 (one-way ANOVA analysis).