Suppression of NLRP3 inflammasome by a small molecule targeting CK1α–β-catenin–NF-κB and CK1α–NRF2–mitochondrial OXPHOS pathways during mycobacterial infection

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  Figure 1. PP suppresses NLRP3–ASC–Casp-1 inflammasome activation in M.tb H37Rv-infected THP-1 cells. (A) RT-qPCR assay of the mRNA expression of NLRP3 in THP-1 cells. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with different concentrations of PP for another 24 h. (B) WB assay of the NLRP3 and pro-caspase-1 protein expression in THP-1 cells. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with different concentration of PP for another 24 h. (C) WB assay of the NLRP3 and pro-caspase-1 protein expression in THP-1 cells. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with PP, Str and Rif for another 24 h. Str, streptomycin; Rif, Rifampicin. (D) THP-1 cells infected with H37Rv (MOI=10) for 4 h and then treated with PP for another 24 h were subjected to IP with anti-NLRP3 antibody and IB with antibodies against NLRP3, ASC, pro-caspase-1 and caspase-1 p10. (E) Relative cell viabilities of THP-1 after treatment with different concentrations of PP. (F) CFUs of M.tb H37Rv in THP-1 cells after PP, Str or Rif treatment. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with PP, Str or RIF for 24 h. Str,…

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  Figure 2. PP suppresses M.tb H37Rv-induced GSDMD cleavage and IL-1β production in THP-1 cells. (A-C) RT-qPCR assay of GSDMD (A), IL-1β (B) and IL-18 (C) mRNA expression in THP-1 cells. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with different concentrations of PP for another 24 h. (D) WB assay of pro-IL-1β, IL-1β, IL-18, GSDMD and N-GSDMD protein expression in THP-1 cells. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with different concentrations of PP for another 24 h. (E) WB assay of pro-IL-1β, IL-1β, IL-18, GSDMD and N-GSDMD protein expression in THP-1 cells. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with PP, Str or Rif for another 24 h. Str, streptomycin; Rif, Rifampicin. Data represent mean ± SD for three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test was used to assess the statistical difference for (A<C). **P < 0.01; ****P < 0.0001.

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  Figure 3. PP suppresses M.tb H37Rv-induced NLRP3 activation via repressing the β-catenin–NF-κB signal pathway. (A) Volcano plot of RNA-seq results. (B) KEGG enrichment of differentially expressed genes identified in transcriptome sequencing of H37Rv-infected THP-1 cells that were treated with PP. (C) WB assay of p-p65 and β-catenin protein expression in THP-1 cells. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with different concentrations of PP for another 24 h. (D) WB assay of p-p65 and β-catenin protein expression in THP-1 cells. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with PP, Str or Rif for another 24 h. PP, pyrvinium pamoate; Str, streptomycin; Rif, Rifampicin.

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  Figure 4. PP suppresses mitochondrial OXPHOS-related mRNA expression in H37Rv-infected THP-1 cells. (A) Heatmap of differentially expressed mitochondrial OXPHOS-related genes identified in transcriptome sequencing of THP-1 cells. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with PP at 3 µg/ml for another 24 h. (B-M) RT-qPCR verification of mitochondrial OXPHOS-related mRNA expression. Data represent mean ± SD for three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test was used to assess the statistical difference for B-M. **P < 0.01.

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  Figure 5. PP inhibits NRF2–mitochondrial OXPHOS pathway during H37Rv infection in macrophages. (A) RT-qPCR assay of NRF2 mRNA expression in THP-1 cells that infected with H37Rv(MOI=10) for 4 h and then treated with different concentrations of PP for another 24 h. (B) WB assay of NRF2 and mitochondrial OXPHOS-related protein expression in THP-1 cells. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with different concentrations of PP for another 24 h. (C) WB assay of NRF2 and mitochondrial OXPHOS-related protein expression in THP-1 cells. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with PP, Str or Rif for another 24 h. Str, streptomycin; Rif, Rifampicin. (D) WB assay of p-p65 and OXPHOS-related protein expression in THP-1 cells. THP-1 cells were infected with H37Rv (MOI=10) for 4 h and then treated with NRF2-IN-1 (NRF2 inhibitor) at 30 µM for another 24 h. (E) Schematic of NRF2 regulating OXPHOS-related gene expression and thus upregulating ROS. Data represent mean ± SD for three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test was used to assess the statistical difference for A. ****P < 0.0001.

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  Figure 6. CK1α siRNA reverses the inhibitory effects of PP on M.tb H37Rv-induced inflammatory responses in macrophages. (A) Docking model of CK1α and pyrvinium. Protein CK1α is shown as cartoon, the ligand pyrvinium and the key ligand-binding residues are shown as sticks. (B) WB assay of CK1α protein expression in THP-1 cells that were transfected with each of the three CK1α siRNAs, respectively. (C) WB assay of CK1α, NLRP3, GSDMD, N-GSDMD, pro-IL-1β and IL-1β protein expression in H37Rv-infected THP-1 cells that were treated with/without PP and/or CK1α siRNA-2. Cells were transiently transfected for 24 h with CK1α siRNA-2 and then replaced with fresh culture medium and cultured for another 12 h. Then H37Rv was respectively used to infect THP-1 cell for 4 h, the culture was abandoned and cells were washed with PBS for 3 times, and then PP was added and cultured for another 24 h. (D) WB assay of CK1α, p-p65, β-catenin, NRF2 and mitochondrial OXPHOS-related protein expression in H37Rv-infected THP-1 cells that were treated with/without PP and/or CK1α siRNA-2.

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  Figure 7. The mechanism of PP suppressing NLRP3 inflammasome by targeting CK1α–β-catenin–NF-κB–NRF2–mitochondrial OXPHOS pathways during bacterial infection. M.tb could induce NLRP3–ASC–Casp-1 inflammasome activation and GSDMD–IL-1β inflammatory pyroptosis through suppressing casein kinase 1A1 (CK1α)–β-catenin/NF-κB signaling pathway and CK1α–NRF2–mitochondrial oxidative phosphorylation (OXPHOS) pathway. PP could suppress this effect by targeting CK1α.