Fig 1.
Identification of SLC30A1 by a genome-wide CRISPR-Cas9-mediated screen.
(A) Microscopy of pTRE3G-NLRP3 Tet-on THP-1 cells before and after Dox treatment (100 ng/mL, 8 h). Scale bar, 20 μm. (B) Immunoblotting analysis of the indicated proteins in pTRE3G-NLRP3 Tet-on THP-1 cells treated with Dox (100 ng/mL) for 8 h. (C) Schematic illustration of the genome-wide CRISPR-Cas9-mediated screen. pTRE3G-NLRP3 Tet-on THP-1 cells stably expressing a genome-wide sgRNA library and Cas9 (Round 0) were subjected to three rounds (Round 1-Round 3) of selection. Enriched genes in the surviving cells (Round 3) were identified by next-generation sequencing (NGS) analysis. (D) Genes identified by the screen were ranked and plotted. The x axis shows the fold change of genes (Round 3/Round 0). The y axis shows the p-value. Genes ranking in the front that involved in NLRP3 inflammasome pathway were plotted with green dots. SLC30A1, was plotted with a red dot.
Fig 2.
SLC30A1 deficiency suppresses inflammasome activation.
(A and B) Immunoblotting analysis of the indicated proteins in WT and SLC30A1−/− THP-1 cells pretreated with LPS (1 μg/mL) for 3 h, followed by Nigericin (2.5 μM) (A) for 1 h or ATP (2.5 mM) (B) for 3 h. L + N, LPS + Nig; L + A, LPS + ATP. (C) Immunoblotting analysis of the indicated proteins in WT, SLC30A1−/− and SLC30A1 re-expression THP-1 cells pretreated with LPS (1 μg/mL) for 3 h, followed by Nigericin (2.5 μM) for 1 h or ATP (2.5 mM) for 3 h. L + N, LPS + Nig; L + A, LPS + ATP. (D) Immunoblotting analysis of the indicated proteins in WT, SLC30A1−/− and SLC30A1 re-expression THP-1 cells pretreated with LPS (1 μg/mL) for 3 h, followed by Salmonella Typhimurium (MOI = 5) for 1 h. L + S, LPS + S. Typhimurium. (E) Immunoblotting analysis of the indicated proteins in WT and SLC30A1−/− THP-1 cells treated with Pam2CSK4 (10 ng/mL) for the indicated times. (F) Fluorescence microscopy of WT and SLC30A1−/− THP-1 cells stably expressing BFP-ASC pretreated with LPS (1 μg/mL) for 3 h, followed by Nigericin (2.5 μM) for 1 h or Salmonella Typhimurium (MOI = 5) for 1 h. ASC specks were pointed by white arrows. Scale bar, 20 μm. (G) Quantification of percentage of WT and SLC30A1−/− THP-1 cells stably expressing BFP-ASC containing ASC speck after the indicated treatment. Data are the mean ± SD. Student’s t-test was used to analyze data. N.S., not significant.
Fig 3.
SLC30A1 deficiency increases intracellular zinc content.
(A) Fluorescence microscopy of WT, SLC30A1−/− and SLC30A1 re-expression THP-1 cells treated with TSQ (5 μg/mL) for 30 min. The intracellular zinc content is characterized by blue fluorescence. Scale bar, 20 μm. (B) Flow cytometry analysis of WT, SLC30A1−/− and SLC30A1 re-expression THP-1 cells treated with TSQ (5 μg/mL) for 30 min. P5 represents positive cell population.
Fig 4.
Zinc inhibits inflammasome activation by suppressing caspase-1.
(A and D) Immunoblotting analysis of the indicated proteins in WT THP-1 cells pretreated with LPS (1 μg/mL) for 3 h and the indicated concentration of ZnCl2 for 1 h in turn, followed by Nigericin (5 μM) for 1 h. (B) Immunoblotting analysis of the indicated proteins in WT THP-1 cells pretreated with LPS (1 μg/mL) for 3 h and the indicated concentration of ZnCl2 for 1 h in turn, followed by Salmonella Typhimurium (MOI = 10) for 1 h. (C) Immunoblotting analysis of the indicated proteins in WT THP-1 cells and Chelex-100-treated WT THP-1 cells pretreated with LPS (1 μg/mL) for 3 h, followed by Nigericin (5 μM) for 1 h or Salmonella Typhimurium (MOI = 10) for 1 h. L + N, LPS + Nig; L + S, LPS + S. Typhimurium. (E) Immunoblotting analysis of the indicated protein from in vitro cleavage assay with 250 ng caspase-1, 1 μg GSDMD or pro-IL-1β and the indicated concentration of ZnCl2, CuCl2, FeCl2 or MnCl2 at 37°C for 2 h.
Fig 5.
Zinc binds inflammatory caspases at a conserved His-Cys-Cys triad.
(A) Zn2+-binding site of caspase-1. The protein structure data was downloaded from PDB (1ICE) and opened by ChimeraX. (B) Sequence alignment of caspase-1 from different species. Protein sequences were obtained from UniProt. Casp1_HUMAN: P29466; Casp1_MOUSE: P29452; Casp1_RAT: P43527; Casp1_HORSE: Q9TV13; Casp1_PIG: Q9N2I1; Casp1_DOG: Q9MZV7. (C) Zn2+/Cu2+/Mn2+-binding stoichiometry of caspase-1 and variants identified by ICP-MS. ANOVA test was used to analyze data. ***p < 0.001. (D) Immunoblotting analysis of the indicated proteins in CASP1−/− THP-1 cells pretreated with ZnCl2 (50 μM) or CuCl2 (50 μM) for 1 h, followed by LPS (1 μg/106 cells) electroporation. (E) Cell viability analysis of CASP1−/− THP-1 cells pretreated with ZnCl2 (50 μM) or CuCl2 (50 μM) for 1 h, followed by LPS (1 μg/106 cells) electroporation. Data are the mean ± SD. ANOVA test was used to analyze data. N.S., not significant, p > 0.05; ****p < 0.0001.
Fig 6.
Zinc inhibits inflammatory response in vivo.
(A) Immunoblotting analysis of the indicated proteins in WT and Slc30a1−/− iBMDM cells pretreated with LPS (1 μg/mL) for 3 h, followed by ATP (2.5 mM) for 3 h or Salmonella Typhimurium (MOI = 5) for 1 h. L + A, LPS + ATP; L + S, LPS + S. Typhimurium. (B and C) Immunoblotting analysis of the indicated proteins in WT iBMDM cells pretreated with LPS (1 μg/mL) for 3 h and the indicated concentration of ZnCl2 for 1 h in turn, followed by ATP (5 mM) (B) for 3 h or Salmonella Typhimurium (MOI = 10) (C) for 1 h. (D) Survival of the mice (n = 8) pretreated (i.n.) with saline or ZnCl2 (2 mg Zn/kg) for 24 h, followed by cecal ligation and puncture operation. Survival curves were compared using Mantel-Cox test. (E) IL-1β, IL-18, IL-6 and TNF-α concentration of sera from the mice pretreated (i.n.) with saline or ZnCl2 (2 mg Zn/kg) for 24 h, followed by cecal ligation and puncture operation. Blood samples were collected at 6 h after the operation. Data are the mean ± SD (n = 4). Student’s t-test was used to analyze data. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01. (F) Survival of the mice (n = 6) pretreated (i.p.) with vehicle (5% DMSO + 95% saline), VX-765 (25 mg/kg), z-VAD-FMK (25 mg/kg) or ZnCl2 (2 mg Zn/kg) for 24 h, followed by LPS (30 mg/kg) injection (i.p.). Survival curves were compared using Mantel-Cox test. (G) IL-1β and IL-18 concentration of sera from the mice pretreated (i.p.) with vehicle (5% DMSO + 95% saline), VX-765 (25 mg/kg), z-VAD-FMK (25 mg/kg) or ZnCl2 (2 mg Zn/kg) for 24 h, followed by LPS (30 mg/kg) injection (i.p.). Blood samples were collected at 12 h after the injection. Data are the mean ± SD (n = 6). ANOVA test was used to analyze data. *p < 0.05; **p < 0.01; ****p < 0.0001.
Fig 7.
Zinc homeostasis regulates autoimmune diseases.
(A) Photographs of the control (Con) and Zn-insufficient (-Zn) psoriasis mice with scaling skin. Scale bar, 5 mm. (B) PASI score of the control (Con) and Zn-insufficient (-Zn) psoriasis mice. Higher score indicates severer symptoms. Data are the mean ± SD (n = 4). Student’s t-test was used to analyze data. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01. (C) Photographs of the control (Con) and Zn-rich (+Zn) psoriasis mice with scaling skin. Scale bar, 5 mm. (D) PASI score of the control (Con) and Zn-rich (+Zn) psoriasis mice. Higher score indicates severer symptoms. Data are the mean ± SD (n = 4). Student’s t-test was used to analyze data. N.S., not significant, p > 0.05; *p < 0.05. (E) Immunoblotting analysis of the indicated proteins in the brain from the control (Con) and Zn-rich (+Zn) APP/PS1 mice (n = 4). (F) Glycine silver staining for the brain from the control (Con) and Zn-rich (+Zn) APP/PS1 mice. The axons were pointed by black arrows. Scale bar, 100 μm. (G) Axonal density of brains from the control (Con) and Zn-rich (+Zn) APP/PS1 mice. Data are the mean ± SD (n = 3). Student’s t-test was used to analyze data. **p < 0.01. (H and I) Quantification of latency to platform during the training phase (H) and percentage of time spent in the target quadrant during the probe test (I) by Morris water maze test of the control (Con) and Zn-rich (+Zn) APP/PS1 mice. Data are the mean ± SD (n = 6). Student’s t-test was used to analyze data. *p < 0.05.