Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: The MT3-Zn 2+ axis suppresses TRIF signaling resulting in decreased IRF3 phosphorylation. When MT3 is absent, TRIF-IRF3-STAT1 signaling and non-canonical inflammasome activation are exaggerated. A lack of MT3 augments immunity to gram-negative bacteria, an effect, that is further enhanced by the combined absence of MT3 and caspase-11 in vivo . Thus, while MT3 curtails caspase-11 activation, the two molecules act together in compromising antibacterial immunity.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Phospho-proteomics, Activation Assay, Bacteria, In Vivo

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Table S1 and Files S1 , S2 |Protein interaction network of Mus musculus MT3 to determine functionally enriched GO BP categories using the STRING database.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Transduction, Cell Differentiation

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S1 MT3 suppresses caspase-11 inflammasome activation in BMDMϕ. qRT-PCR analysis of Mt3 expression in WT BMDMϕ stimulated with (A) iLPS (2 μg/ml) or vehicle control, 3-5 independent experiments and (B) exLPS (10 μg/ml) for 48h, 3 independent experiments, two-tailed t-test. (C) Western Blots of pro- and active-caspase-11, pro-caspase-1, pro-IL1β and β-actin in cell lysates and active-caspase-1 and active-IL-1β in supernatants of WT and Mt3 -/- BMDMϕ stimulated with iLPS (10 μg/ml) or vehicle for 48h. Bar graphs are densitometric analysis of targets normalized to β-actin, 3-4 independent experiments, one-way ANOVA, data are mean ± SEM. (D) Western Blots of pro- and active-caspase-11 and β-actin in lysate + supernatant samples from WT and Mt3 -/- BMDMϕ stimulated with iLPS (2 μg/ml) or vehicle for 48h. Bar graphs are densitometric analysis of targets normalized to β-actin. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Control, Two Tailed Test, Western Blot

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S2 . MT3 curtails CASPASE-4 and caspase-11 signaling and antibacterial immunity in hMϕ and in vivo . (A) MT3 and MT2A expression analyzed by qRT-PCR in hMϕ transfected with scramble siRNA or MT3 siRNA for 24h, 3 independent experiments, two-tailed t-test. (B) Scramble siRNA or MT3 siRNA treated hMϕ stimulated with iLPS (10 μg/ml) or vehicle for 48h. Immunoblots of pro-CASPASE-4 and active-CASPASE-4 in cell extracts, 3 independent experiments, one-way ANOVA. (C) Active-IL-1β measured by ELISA in supernatants of hMϕ treated as above, 3 independent experiments, one-way ANOVA. (D) E . coli growth inhibition in hMϕ transfected with MT3 siRNA and infected with 25 E . coli (K12): 1 hMϕ for 24h compared to scramble siRNA treated hMϕ, 3 independent experiments, two-tailed t-test. (E) E . coli growth inhibition in WT and Mt3 -/- BMDMϕ infected with 25 E . coli (K12):1 hMϕ for 24h, 4 independent experiments, two-tailed t-test. (F) WT and Mt3 -/- mice infected i.p. with 1X10 9 E . coli for 6h, log CFUs of E . coli in blood, kidney and peritoneal lavage samples, n = 12-15 per group, two-tailed t-test. (G) Western blots of inflammasome mediators in kidney homogenates of WT and Mt3 -/- mice infected as above, n = 6 per group, two-tailed t-test. (H) WT and Mt3 -/- mice infected i.p. with 1 X10 9 E . coli for 1h and IL-1β measured in peritoneal lavage and serum by ELISA. n = 3 per group, two-tailed t-test. (I) WT and Mt3 -/- mice primed i.p. with poly(I:C) (10 mg/kg) for 6h and challenged with LPS (2 mg/kg) i.p. After 18h, IL-1β was measured in peritoneal lavage and serum by ELISA, n = 3/group, two-tailed t-test. (J) Bacterial growth in spleen, lung and kidney of WT and Mt3 -/- mice infected i.n. with K. pneumoniae (4 X10 4 CFUs/mouse) for 48h, n = 8-12 per group, two-tailed t-test, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Transfection, Two Tailed Test, Western Blot, Enzyme-linked Immunosorbent Assay, Inhibition, Infection

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S3 Caspase-11 synergizes with MT3 in impairing bacterial clearance. WT, C asp-11 -/- , Mt3 -/- and Casp-11 -/- Mt3 -/- mice were infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h. (A) Bacterial CFUs measured in kidney, blood and peritoneal lavage, n = 3-6 per group, one-way ANOVA. (B) Western blots of pro-GSDMD, active-GSDMD (p31), pro-caspase-1, active-caspase-1, pro-IL1β and active-IL-1β in kidney homogenates, n = 3-6 per group, one-way ANOVA, data are mean ± SEM. (C) WT and Mt3 -/- mice treated i.p. with MCC950 (1 mg/mouse) or PBS and infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h. IL1β was measured by ELISA in peritoneal lavage, n = 6 per group, one-way ANOVA, data are mean ± SEM. Bacterial CFUs in whole blood and kidney, n = 4 per group, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S4 Myeloid-MT3 suppresses non-canonical inflammasome activation and blunts gram-negative bacterial clearance in vivo . (A) Generation of Mt3 fl/fl mice by inserting loxp sites flanking exon 3 of the Mt3 gene using the CRISPR-Cas9 gene targeting approach. Mt3 fl/fl mice crossed with Lys2Cre mice to obtain Lys2Cre Mt3 fl/fl mice. (B) Efficacy of myeloid Mt3 deletion assessed by genotyping peritoneal Mϕ (PMϕ) and BMDMϕ from Lys2Cre , Mt3 fl/fl and Lys2Cre Mt3 fl/fl mice. Gel electrophoresis analysis demonstrating efficient deletion of the Mt3 gene from BMDMϕ and PMϕ of Lys2Cre Mt3 fl/fl mice. (C) Western blots of pro-caspase-11, active-caspase-11, pro-GSDMD, active-GSDMD (p31), pro-caspase-1, active-caspase-1, pro-IL1β and active-IL-1β in whole kidney homogenates of mice infected as above, n = 3-5 per group, two-tailed t-test. (D) Bacterial CFUs in kidney and whole blood of Lys2Cre and Lys2Cre Mt3 fl/fl mice infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h, n = 3-5 per group, two-tailed t-test, data are mean ± SEM. **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, In Vivo, CRISPR, Nucleic Acid Electrophoresis, Western Blot, Infection, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S5 MT3 thwarts TRIF-IRF3-STAT1 signaling to suppress non-canonical inflammasome activation. (A) Functional enrichment analysis of differentially expressed genes using RNA-seq data from resting WT and Mt3 -/- BMDMϕ (NCBI SRA: PRJNA533616) FDR, false detection rates. (B, C) Heat map (left) and table (right) show differentially expressed IFN-related genes in resting Mt3 -/- BMDMϕ compared to resting WT BMDMϕ obtained from RNA-seq analysis. (D) Western blots of pIRF3, pSTAT1, STAT1, GBP2 and GBP5 in vehicle or iLPS (10 μg/ml)-treated WT and Mt3 -/- BMDMϕ lysates, 3-4 independent experiments, one-way ANOVA. (E) Western blots of TRIF in lysates from WT and Mt3 -/- BMDMϕ stimulated as above, 3 independent experiments, one-way ANOVA. (F) Scramble and Ticam1 siRNA treated WT and Mt3 -/- BMDMϕ treated with iLPS (10 μg/ml) or vehicle for 48h. Immunoblots of TRIF (2 independent experiments), pro-caspase-11, and active-caspase-11 in lysates and active-IL-1β in supernatants, 3 independent experiments, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, Functional Assay, RNA Sequencing, Western Blot

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figures S6 , 7 MT3-Zn 2+ axis drives negative regulation of the non-canonical inflammasome. (A) SEC-ICP-MS of WT and Mt3 -/- BMDMϕ exposed to vehicle or iLPS (10 ug/ml) for the indicated time points, chromatograms depict Zn 2+ distribution in cell lysates across various molecular masses, arrow indicates Zn 2+ associated with the MT-peak (18-21 min.) on the chromatogram, Y axis is off-set to allow easy comparison under the same scale. (B) Bar graphs of total Zn 2+ and MT-Zn 2+ in WT and Mt3 -/- BMDMϕ post iLPS (10 μg/ml) or vehicle exposure. Two-way t-test against respective BMDMϕ controls at each time point, 3 independent experiments, data are mean ± SD. (C) WT BMDMϕ treated with iLPS (10 μg/ml) or vehicle for 24h in Zn 2+ sufficient or Zn 2+ deficient Opti-MEM media, immunoblots of pIRF3, pro-caspase-11, active-caspase-11 and pro-IL-1β in lysates and active-IL-1β in media supernatants, one-way ANOVA, data are mean ± SEM. (D, E) Mt3 -/- BMDMϕ transfected with Pro-Ject™ or Pro-Ject™ complexed with apo-MT3, 4Zn 2+ MT3 or 6Zn 2+ MT3 and treated with iLPS (10 μg/ml) or vehicle for 24h in Zn 2+ deficient Opti-MEM media. (D) Chromatograms depict Zn 2+ distribution in cell lysates across various molecular masses, arrow indicates Zn 2+ signal associated with the MT-peak (18-21 min.) on the chromatogram, Y axis is off-set to allow easy comparison under the same scale. (E) Western blots of pIRF3, pro-caspase-11, active-caspase-11 and pro-IL1β in lysates and active-IL-1β in supernatants, 3 independent experiments, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Comparison, Western Blot, Transfection

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: Reagents and resources.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Expressing, Plasmid Preparation, Control, Extraction, Blocking Assay, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Filtration, Transfection, Reverse Transcription, Cytotoxicity Assay, Isolation, Software, Imaging, Real-time Polymerase Chain Reaction