MT3 attenuates cell death in neuronal and glial cells, but the precise underlying mechanisms are not fully understood (21). As non-canonical inflammasome activation leads to pyroptotic cell death, we investigated if MT3 effector function is related to caspase-11 activation in Mϕ. We explored whether MT3 is involved in inflammatory and cell-death processes using functional enrichment analysis. We assessed protein-protein interaction networks of MT3 in Mus musculus and Homo sapiens using the STRING database (22). The MT3 interaction partners significantly enriched 15 mouse and 43 human gene ontology categories for biological processes (GO BP) related to programmed cell death (PCD), LPS responses, signaling via TRIF, IL-1 and type-I IFN, cytokine responses and several immune processes. A complete network of MT3 interactions and GO BP categories is in Table 1 , Supplementary Table S1 , Supplementary Files S1 and S2 . PCD and LPS responses are linked to inflammasome activation and more specifically, TRIF and type-I IFN signaling are tied to the non-canonical inflammasome pathway. A lack of TRIF signaling ablates non-canonical inflammasome activation in response to iLPS without impacting Mϕ response to canonical NLRP3 triggers such as ATP and nigericin (20). Thus, our bioinformatics analysis together with our previously reported role for MT3 in suppressing proinflammatory responses in Mϕ led us to investigate whether MT3 negatively regulates the non-canonical inflammasome pathway.

In Mϕ, exposure to iLPS triggers caspase-11 activation and cell death by pyroptosis (2). We directly assessed if MT3 regulates non-canonical inflammasome activation using WT and Mt3^-/- mice. BMDMϕ were exposed to iLPS or vehicle control and time-dependent changes in the gene expression of Mt1, Mt2 and Mt3 were examined. Mt3 expression increased gradually from 1 hour (h) and peaked at 48h in WT BMDMϕ challenged with iLPS ( Figure 1A ). The expression of Mt1 and Mt2 peaked at 6h, but receded over time in both WT and Mt3^-/- BMDMϕ ( Supplementary Figures S1A, B ). To determine whether exogenous LPS had an effect on MT3, we stimulated WT BMDMϕ with extracellular LPS (exLPS) for 48h. While iLPS increased Mt3 expression by 10-fold, the fold increase observed with exLPS challenge was much lower ( Figure 1B ).

Next, we examined if MT3 regulated non-canonical inflammasome activation by assessing pro- and active forms of caspase-11, caspase-1 and IL-1β in cell lysates and supernatants of WT and Mt3^-/- BMDMϕ challenged with iLPS. A lack of MT3 exacerbated activation of caspase-11 in cell lysates, and caspase-1 and IL-1β in culture supernatants. Pro-caspase-11, pro-caspase-1 and pro-IL-1β proteins in cell lysates were similar between iLPS treated WT and Mt3^-/- BMDMϕ ( Figure 1C ). We further examined combined lysate and supernatant samples and found increased caspase-11 activation in Mt3^-/- BMDMϕ challenged with iLPS ( Figure 1D ). Thus, the differences observed were not merely a result of reduced release of the active forms from cells, but an increase in non-canonical inflammasome activation in Mt3^-/- BMDMϕ. Intracellular transfection of LPS at two different concentrations (2 and 10 μg/ml) yielded similar results ( Figures 1C, D ). We then examined the levels of IL-1α in supernatants of WT and Mt3^-/- BMDMϕ challenged with iLPS. In contrast to IL-1β, IL-1α was moderately reduced in the supernatants of Mt3^-/- BMDMϕ ( Supplementary Figures S1C, D ). We note that although IL-1α is activated by caspase-11, it is characteristically different from IL-1β, as it exists in both membrane-bound and secreted forms that are differentially regulated, and both pro- and cleaved IL-1α are bioactive (23–25).

Gram-negative bacteria activate the non-canonical inflammasome via CASPASE-4 in hMϕ (26). We investigated whether human MT3, similar to mouse MT3, suppressed non-canonical inflammasome activation and antibacterial immunity. Human monocyte-derived Mϕ obtained from PBMCs were transfected with scramble siRNA or MT3 siRNA followed by transfection with iLPS. To assess siRNA specificity, we analyzed the expression of MT3 and MT2A genes. MT3, but not MT2A expression was silenced in MT3 siRNA transfected hMϕ ( Figure 2A ). MT3 deficiency resulted in elevated activation of CASPASE-4 and heightened release of IL-1β from hMϕ ( Figures 2B, C ). We previously showed that a lack of MT3 increased resistance of mouse BMDMϕ to Escherichia coli (19). We therefore queried whether silencing MT3 in hMϕ impaired bacterial clearance in vitro. hMϕ treated with scramble siRNA or MT3 siRNA were infected with E. coli K12 for 24h. MT3-deficient hMϕ exerted a sharp decline in intracellular bacterial survival compared to control hMϕ ( Figure 2D ).

We examined whether MT3 regulated antibacterial immunity and non-canonical inflammasome activation in vitro and in vivo. WT and Mt3^-/- BMDMϕ were infected with E. coli. After 24h, bacterial survival was reduced in Mt3^-/- BMDMϕ ( Figure 2E ). Next, we infected WT and Mt3^-/- mice in vivo intraperitoneally (i.p.) with E. coli for 6h. Compared to WT mice, MT3 deficiency bolstered bacterial elimination from the blood and moderately improved bacterial clearance in the kidney and peritoneal lavage ( Figure 2F ). Caspase-11, GSDMD (N-terminal) and caspase-1 activation were heightened in kidney homogenates of infected Mt3^-/- mice compared to infected WT mice ( Figure 2G ). The decrease in pro-GSDMD of Mt3^-/- mice may be explained by increased conversion of pro- to active-GSDMD form ( Figure 2G ). IL-1β in the peritoneal lavage was significantly elevated (p<0.01) and serum IL-1β exhibited a trend towards increase in infected Mt3^-/- mice compared to WT controls ( Figure 2H ). To determine if this response is consistent upon LPS challenge in vivo, we primed mice i.p. with poly(I:C) for 6h and challenged them i.p. with ultrapure LPS. After 18h, IL-1β was elevated in the peritoneal lavage and serum of Mt3^-/- mice compared to WT mice ( Figure 2I ). We further queried the impact of MT3 on LPS-induced sepsis. WT and Mt3^-/- mice were challenged with ultrapure LPS (20 mg/kg) and assayed for weight loss, murine sepsis scores (MSS) as reported previously (27) and survival. MT3 deficiency resulted in greater weight loss and increased sepsis scores, but both genotypes similarly succumbed to septic shock ( Supplementary Figures S2A-C ).

We then investigated whether MT3 increased susceptibility to other gram-negative bacteria. WT and Mt3^-/- mice were infected intranasally (i.n.) with a virulent, heavily encapsulated strain of Klebsiella pneumoniae (KP2 2-70). MT3 deficiency significantly (p<0.05) improved K. pneumoniae clearance in the spleen, but no changes were observed in the lung and kidney ( Figure 2J ). Gram-positive bacteria activate caspase-11 via the NLRP6 inflammasome (28). We determined whether the increased non-canonical inflammasome activation and antibacterial resistance observed in Mt3^-/- mice extended to gram-positive bacterial infection. WT and Mt3^-/- mice were challenged subcutaneously (s.q.) with a clinical isolate of Group-A-Streptococcus GAS5448 (29). After 72h, Mt3^-/- mice manifested significantly (p<0.05) reduced GAS burden in the kidney and spleen compared to WT mice. Bacterial CFUs in the blood exhibited a similar trend ( Supplementary Figure S2D ). Importantly, although Mt3^-/- mice exhibited higher activation of caspase-1 and IL-1β, the levels of active caspase-11 and pro-caspase-11 were diminished in GAS-infected Mt3^-/- mice compared to WT mice ( Supplementary Figure S2E ). Thus, although MT3 compromises resistance to both gram-negative and gram-positive bacteria, it specifically suppresses non-canonical inflammasome signaling in response to gram-negative microbial triggers.

Caspase-11 is crucial in antibacterial defenses particularly against gram-negative bacteria, although some studies have suggested a detrimental role for caspase-11 in bacterial elimination (30–35). MT3 suppressed antibacterial immunity as well as non-canonical inflammasome activation. Thus, we investigated whether the heightened immunity to E. coli in Mt3^-/- mice was due to increased non-canonical inflammasome activation. We infected WT, Casp-11^-/- , Mt3^-/- , and Mt3^-/-Casp-11^-/- mice ( Supplementary Figure S3A ) i.p. with E. coli and assessed bacterial burden 6h post-infection. Compared to WT mice, bacterial elimination was enhanced in Mt3^-/- and Casp-11^-/- mice but this response was further exacerbated in Mt3^-/- mice lacking caspase-11 ( Figure 3A ). These data indicate that the combined absence of MT3 and caspase-11 improves resistance to gram-negative bacterial infection. We then analyzed caspase-11 inflammasome mediators in kidney homogenates harvested 6h post-infection. Mice lacking MT3, or caspase-11, exhibited elevated activation of GSDMD (N-terminal), caspase-1 and IL-1β compared to WT mice ( Figure 3B ). These changes were also observed in the Mt3^-/-Casp-11^-/- mice. Since GSDMD is a target of caspase-11 as well as caspase-1 (36), an elevation in active GSDMD may result from higher caspase-1 activation observed in mice lacking MT3, caspase-11 or both. Caspase-8, a pro-apoptotic caspase, collaborates with caspase-11 to mediate systemic inflammation and septic shock (37, 38). Moreover, caspase-8, in addition to caspase-1 can directly cleave IL-1β. We therefore analyzed caspase-8 in kidney homogenates from E. coli infected WT, Mt3^-/- , Casp-11^-/- and Mt3^-/-Casp-11^-/- mice. Caspase-11 negatively influenced the activation of caspase-8 ( Supplementary Figure S3B ).

The above data demonstrate that even when caspase-11 is absent, Mt3^-/- mice exert heightened levels of active caspase-1, active IL-1β and antibacterial immunity. We therefore queried whether improved bacterial elimination in the absence of MT3 was facilitated by the canonical NLRP3 inflammasome. WT and Mt3^-/- mice were treated i.p. with MCC950 (NLRP3 inhibitor) followed by infection i.p. with E. coli. Treatment with MCC950 reduced IL-1β levels and sharply blunted antibacterial resistance in the blood and kidney of Mt3^-/- mice compared to vehicle-treated controls. Bacterial burdens were also elevated in WT mice by NLRP3 inhibition ( Figure 3C ). Collectively, these data demonstrate that MT3 negatively controls activation of the non-canonical inflammasome and that both MT3 and caspase-11 cripple resistance to bacterial infection.

To affirm that the effects on caspase-11 inflammasome activation observed in the Mt3^-/- mice were dependent on myeloid-MT3, we generated mice specifically lacking MT3 in myeloid cells (Lys2Cre Mt3^fl/fl ) ( Figure 4A and Supplementary Figures S4A, B ). Genotyping analysis of BMDMϕ and peritoneal Mϕ (PMϕ) from Lys2Cre, Mt3^fl/fl and Lys2Cre Mt3^fl/fl mice demonstrated efficient removal of the Mt3 gene from BMDMϕ and PMϕ only in Lys2Cre Mt3^fl/fl mice ( Figure 4B ). To determine if myeloid MT3 deficiency augmented non-canonical inflammasome activation in vivo, we infected Lys2Cre and Lys2Cre Mt3^fl/fl mice i.p. with E. coli. After 6h, caspase-11 inflammasome targets and bacterial burden were examined in kidney and blood. Lys2Cre Mt3^fl/fl mice exerted increased activation of caspase-11, GSDMD (N-terminal), caspase-1, and IL-1β in kidney homogenates and improved bacterial elimination compared to Lys2Cre control mice ( Figures 4C, D ). Thus, myeloid MT3 facilitates subversion of non-canonical inflammasome activation and contributes to antibacterial immunity in vivo.

Signaling via the TRIF pathway is crucial for caspase-11 activation and synergistic engagement of the NLRP3 inflammasome leading to activation of caspase-1 and IL-1β (20). Downstream of TRIF, IRF3 and IRF7 induce IFNβ production that activates STAT1 signaling and promotes transcription of inflammasome components including caspase-11 and guanylate binding proteins (GBPs). GBP2 and GBP5 facilitate LPS release into the cytosol from intracellular vacuoles containing bacteria (39, 40). Our functional enrichment data based on protein-protein interaction network analyses revealed a potential involvement of MT3 in LPS, TRIF, type-I IFN and IL-1 signaling ( Table 1 , Supplementary Table S1 and Supplementary Files S1 , S2 ). We further examined our published RNA-seq data (NCBI SRA: PRJNA533616) to determine differentially expressed genes by comparing resting WT and Mt3^-/- BMDMϕ (19). The derived list of differentially expressed genes significantly enriched 12 GO BP categories directly related to cytokine and chemokine signaling and regulation of inflammatory responses based on DAVID functional enrichment analysis ( Figure 5A ) ( 41 ). These analyses suggested that MT3 deficiency perturbed the expression of immune-related genes even at the resting state. We reported that a lack of MT3 augments IFNγ responsiveness (19). Herein, from our RNA-seq analysis, we identified 20 genes related to IFN-signaling that were upregulated in resting Mt3^-/- BMDMϕ compared to resting WT BMDMϕ (p adj <0.05) ( Figures 5B, C ). Among these, Isg15, Mx1 and Ifit (Ifit1bl1, Ifit3, Ifit3b, Ifit2, Ifit1, Ifit1bl2) family of genes are known targets of type-I IFNs (42–45). These observations led us to posit that MT3 regulated the cellular response to LPS challenge by modulating the TRIF-IRF3-STAT1 axis upstream of non-canonical inflammasome activation. LPS engages the TRIF-IRF3-STAT1 axis via toll-like receptor 4 (TLR4) signaling in Mϕ. To address this hypothesis, we challenged WT and Mt3^-/- BMDMϕ with iLPS or vehicle and examined activation of the TRIF-IRF3-STAT1 pathway. Mϕ lacking MT3 exerted increased activation of phospho-IRF3 (pIRF3), pSTAT1, GBP2 and GBP5 ( Figure 5D ). TRIF protein levels were unaltered by MT3 deficiency ( Figure 5E ). Type-I IFN signaling is required for activation of the caspase-11 inflammasome cascade by gram-negative bacteria (20). As MT3 deficiency augmented the expression of genes involved in IFN signaling ( Figures 5B, C ), we blocked the interferon-α/β receptor (IFNAR)1 using a monoclonal antibody prior to iLPS challenge in WT and Mt3^-/- Mϕ. IFNAR1 blockade resulted in decreased pro-caspase-11 (p43 subunit). Total STAT1 and pro-caspase-1 (p38 subunit) were not greatly affected ( Supplementary Figure S5 ). We found robust attenuation of pSTAT1, active-caspase-11 and active-caspase-1, but secretion of active-IL-1β in both WT and Mt3^-/- Mϕ was increased upon blockade of IFNAR1 signaling. This finding corresponded with high pro-IL-1β levels in Mϕ treated with the IFNAR1 antibody ( Supplementary Figure S5 ). These data indicate that although IFNAR1 signaling is required for fueling the non-canonical inflammasome cascade and activation of caspase-1, pro-IL-1β and its activation are suppressed by IFNAR1.

We queried if MT3 exerted a brake on TRIF signaling to downmodulate non-canonical inflammasome activation. WT and Mt3^-/- BMDMϕ were treated with scramble or Ticam1 (gene encoding TRIF) siRNA, and challenged with iLPS ( Figure 5F ). Ticam1 silencing reversed the effects of MT3 deficiency resulting in a sharp reduction in caspase-11 and IL-1β activation in Mϕ ( Figure 5F ). These data reveal a central role for MT3 in attenuating the crosstalk between TRIF signaling and the caspase-11 activation cascade.

MTs are master regulators of intracellular Zn²⁺ availability and distribution (46, 47). We determined if negative control of the non-canonical inflammasome by MT3 was Zn²⁺-dependent. First, we systematically assessed Zn²⁺ changes in WT and Mt3^-/- BMDMϕ upon challenge with iLPS over time using SEC-ICP-MS. Activation of the non-canonical inflammasome in WT Mϕ was associated with profound changes in the intracellular Zn²⁺ pool. iLPS exposure led to a gradual increase in total Zn²⁺ largely associated with the chromatogram peak(s) between 18-21 min. that we previously identified as MTs ( Figures 6A, B ) (9, 11). The time-dependent elevation in Zn²⁺ corresponded with kinetics of Mt3 induction ( Figures 1A and 6A, B ). Mϕ lacking MT3 failed to elevate total Zn²⁺ and MT-associated Zn²⁺ in response to iLPS ( Figure 6B ). In contrast to the increase in Zn²⁺ pool observed in WT Mϕ challenged with iLPS, resting Mt3^-/- Mϕ harbored higher Zn²⁺ content that reduced over time post iLPS challenge ( Figure 6B ). These data indicate that MT3 drives an elevation in intracellular Zn²⁺ in Mϕ during non-canonical inflammasome activation.

Zn²⁺ chelation in human monocytes increases IRF3 activation (48). We reasoned that if the effects of MT3 were Zn²⁺ dependent, altering the intracellular Zn²⁺ concentration will at least in part reverse the heightened non-canonical inflammasome signaling observed in Mt3^-/- cells. To test this postulate, we exposed WT and Mt3^-/- BMDMϕ to increasing amounts of ZnSO₄ and challenged them with iLPS in vitro. Exogenous ZnSO₄ supplementation remarkably reduced the ability of Mt3^-/- Mϕ to respond to iLPS. pIRF3, pSTAT1 and activation of caspase-11 were reduced in ZnSO₄-supplemented Mt3^-/- Mϕ ( Supplementary Figure S6A ). A similar effect of Zn²⁺ was also observed in WT Mϕ ( Supplementary Figure S6A ). We investigated if exposing WT Mϕ to a Zn²⁺-deficient environment would mimic the effects MT3 deficiency on the non-canonical inflammasome. WT BMDMϕ were cultured in Zn²⁺-sufficient or Zn²⁺-deficient Opti-MEM media prior to iLPS exposure. WT Mϕ exposed to a Zn²⁺-deficient milieu manifested higher pIRF3 and caspase-11 activation accompanied by increased activation and release of IL-1β ( Figure 6C ). The amount of TRIF, pro-caspase-11 and pro-IL-1β were not affected by Zn²⁺ deficiency ( Figures 6C and Supplementary Figure S6B ).

Next, we directly addressed whether Zn²⁺ is required for the suppressive function of MT3 on the caspase-11 inflammasome. We first overexpressed the MT3 gene in Mt1^-/-Mt2^-/- Mϕ and isolated the protein. Mt1^-/-Mt2^-/- BMDMϕ were transfected with the Mt3 overexpressing vector (pCMV6-Ac-MT3-GFP) or an empty vector (pCMV6-Ac-GFP) control. Mt1^-/-Mt2^-/- Mϕ were used so as to exclude any contribution of these MTs in the MT3 purification process. The MT-associated peak from MT3-overexpressed Mϕ was identified by SEC-ICP-MS ( Supplementary Figure S7 ) and collected. We complexed MT3 with the ⁶⁶Zn²⁺ isotope to acquire an MT3-Zn²⁺ saturation of 4 Zn²⁺ ions per MT3 (MT3-4Zn²⁺) and 6 Zn²⁺ ions per MT3 (MT3-6Zn²⁺). The ⁶⁶Zn²⁺ isotope was used to monitor changes in the ratio of ⁶⁶Zn²⁺/⁶⁴Zn²⁺ post-transfection of the MT3-⁶⁶Zn²⁺ complexes in Mϕ. We transfected apo-MT3, MT3-4Zn²⁺ or MT3-6Zn²⁺ into Mt3^-/- BMDMϕ in Zn²⁺-deficient media. The use of Mt3^-/- BMDMϕ and Zn²⁺-deficient media enabled exclusion of any possible contribution from endogenous MT3 and exogenous Zn²⁺ in our analysis. Post-transfection of apo-MT3 or MT3-Zn²⁺ complexes, Mϕ were challenged with iLPS to activate the non-canonical inflammasome. To confirm that intracellular Zn²⁺ changes occurred upon transfection of the MT3- ⁶⁶Zn²⁺ complexes, we analyzed BMDMϕ lysates by SEC-ICP-MS. Mt3^-/- cells transfected with MT3-4Zn²⁺ and MT3-6Zn²⁺ but not apo-MT3 exhibited an increase in the ⁶⁶Zn²⁺/⁶⁴Zn²⁺ ratio in the MT-peak region at 18-21 mins. in the chromatogram indicating an elevation in the intracellular ⁶⁶Zn²⁺ isotope ( Figure 6D ). These data confirm that transfection of the MT3-Zn²⁺ complexes resulted in an increase in intracellular ⁶⁶Zn²⁺ in Mϕ. In parallel, we isolated cell lysates and supernatants proteins from these Mϕ to determine whether apo-MT3 or the MT3-Zn²⁺ complexes modulated the non-canonical inflammasome pathway. Transfection of MT3-4Zn²⁺ and MT3-6Zn²⁺ but not apo-MT3, dampened pIRF3, active caspase-11 and active IL-1β in response to iLPS ( Figure 6E ). TRIF levels were unaffected by MT3 transfection ( Figure 6E ). Pro-caspase-11 and pro-IL-1β were modestly diminished by MT3-4Zn²⁺ and MT3-6Zn²⁺ exposure, but these changes were not significant ( Figure 6E ). The effect of MT3-6Zn²⁺ was more profound than that of MT3-4Zn²⁺ indicating that a higher Zn²⁺ saturation on MT3 corresponded with a stronger suppressive effect on the non-canonical inflammasome ( Figure 6E ).

Taken together, these findings reveal a previously undescribed interplay between the non-canonical inflammasome and its negative regulator, whereby the MT3-Zn²⁺ axis suppresses caspase-11 inflammasome, but the two molecules concur in compromising immunological fitness of the host during bacterial pathogenesis.

Reagents and resources can be found in Table 2.

E. coli (K12) and K. pneumoniae were kindly provided by Dr. Jason Gardner at the University of Cincinnati. Group A Streptococcus (GAS 5448) was kindly provided by Dr. Suba Nookala at the University of North Dakota.

All mice used in this study were on the C57BL/6 background. WT and Casp4^tm1Yuan /J (Casp-11^-/- ) mice were acquired from the Jackson Laboratory. Lys2Cre mice were kindly provided by Dr. George S. Deepe. Jr. (University of Cincinnati). Mt3^-/- (exon 3 deleted) and Mt3^fl/fl mice were generated using clustered regularly interspaced short palindromic repeats (CRISPR) by the Transgenic Animal and Genome Editing Core facility at the Cincinnati Children’s Hospital Medical Center (CCHMC). In mice, the Mt gene cluster is located on chromosome 8. The Mt3 gene consists of 3 exons and is preceded by Mt1 and Mt2 genes. We targeted exon 3 of Mt3 by flanking it with loxp sites (Mt3^fl/fl ) using the CRISPR-Cas9 gene targeting approach. Lys2Cre Mt3^fl/fl mice that exhibit myeloid Mt3 deficiency were generated by crossing Lys2Cre mice to Mt3^fl/fl mice. Deletion of the Mt3 gene was confirmed in BMDMϕ and PMϕ of Lys2Cre Mt3^fl/fl mice by genotyping as detailed in the CRISPR/Cas9 generation of Mt3^fl/fl mice section. To confirm 3’ loxp site insertion or deletion, genomic DNA amplified using forward (5' TAG GCT TCC CAC CTG TTT GG 3') and reverse (5' GCC AAG ATA AAG TCC GGG GT 3') primers. To confirm 5’ loxp site insertion or deletion, genomic DNA amplified using forward (5' TCG AAC TAC CTC CAA ACA GAG AAC 3') and reverse (5' TCA GTT TGG TCC AAA CGG GAT G 3') primers. To confirm Lys2Cre gene insertion, genomic DNA amplified using mutant (5' CCC AGA AAT GCC AGA TTA CG 3'), common (5' CTT GGG CTG CCA GAA TTT CTC 3') and WT (5' TTA CAG TCG GCC AGG CTG AC 3') primers. Casp-11^-/-Mt3^-/- mice were generated by crossing Casp-11^-/- mice to Mt3^-/- mice. Casp-11 was amplified using tail genomic DNA by mutant reverse (5' CGC TTC CTC GTG CTT TAC GGT AT 3'), common forward (5' ACA ATT GCC ACT GTC CAG GT 3') and WT reverse (5' CAT TGC TGA CCT TAT TTC TGT ATG G 3') primers. Mt3 was amplified using tail genomic DNA by forward (5' TTG GGG TGA GGT GTA GAG GT 3') and reverse (5 GCC AAG ATA AAG TCC GGG GT ' 3') primers. Mice used in this study had ad-libitum access to food and water. All mice were housed in the Department of Laboratory Animal Medicine, University of Cincinnati, accredited by American Association for Accreditation of Laboratory Animal Care (Frederick, MD) and experiments were conducted in accordance with Animal Welfare Act guidelines of the National Institutes of Health.

The methods for the design of sgRNAs, donor oligos and the production of Mt3^fl/fl (loxp sites surrounding exon 3 of the murine Mt3 gene) animals were as described previously (82). The sgRNAs were selected according to the on- and off-target scores from the CRISPR design web tool (http://genome-engineering.org) as well as CRISPOR (http://crispor.tefor.net) (83). The selected sgRNA target sequences were cloned, according to the published method (84), into the pX458 vector (addgene #48138) that was modified by us to contain an optimized sgRNA scaffold (85) and a Cas9-2A-GFP. Their editing activity were validated by the T7E1 assay in mouse mK4 cells (86), compared side-by-side with Tet2 sgRNA that was known to work in mouse embryos efficiently (87). Validated sgRNA was transcribed in vitro using the MEGAshorscript T7 kit (ThermoFisher) and purified by the MEGAclear Kit (ThermoFisher), and stored at -80˚C. To prepare the injection mix, we incubated sgRNA and Cas9 protein (ThermoFisher) at 37˚C for 5 mins. to form the ribonucleoprotein complex and then added the donor oligos to it. The initial attempt was to insert both loxP sites simultaneously via piezo-driven cytoplasmic injection (88) of 100 ng/ul Cas9 protein, 50 ng/ul 5’ sgRNA, 50 ng/ul 3’ sgRNA, 50 ng/ul 5’ donor, and 50 ng/ul 3’ donor into fertilized eggs. Injected eggs were transferred into the oviductal ampulla of pseudo-pregnant CD-1 females on the same day. Pups were born and genotyped by PCR and Sanger sequencing. However, only 3’ loxP-containing mice were obtained from this attempt. After breeding them to homozygosity for the 3’ loxP, a new set of 5’ sgRNA and the donor oligo was designed and injected into the zygotes with the mix containing 150 ng/ul Cas9 protein, 75 ng/ul 5’ sgRNA, and 100 ng/ul ssDNA donor oligo. Injected eggs were transferred into the oviductal ampulla of pseudopregnant CD-1 females on the same day. Pups were born and genotyped by PCR and Sanger sequencing. Founder mice carrying both 5’ and 3’ loxP sites in cis were finally obtained. Animals were housed in a controlled environment with a 12-h light/12-h dark cycle, with free access to water and a standard chow diet. All animal procedures were carried out in accordance with the Institutional Animal Care and Use Committee-approved protocol of Cincinnati Children’s Hospital and Medical Center.

hMϕ were prepared from peripheral blood mononuclear cells (PBMCs). Briefly, human blood obtained from the Hoxworth Blood Center, University of Cincinnati was diluted (1:2) with calcium- and magnesium-free 1X Dulbecco’s phosphate-buffered saline (DPBS) and inverted gently to mix. Ficoll-Paque (10 ml for the total volume of 40 ml diluted blood) was layered at the bottom of the tube. The tubes were centrifuged at 400 X g for 30 mins. without break at 20˚C. The PBMC interface was transferred to sterile tubes and washed three times using 40 ml of DPBS containing 2mM EDTA and centrifuged at 120 X g for 10 mins. without break at 4˚C. A final wash was performed with DPBS (without EDTA). Isolated PBMCs were resuspended in complete RPMI 1640 medium. PBMCs (5 X 10⁶) were plated in 24 well plates containing complete RPMI medium. After 24h, adherent monocytes were washed three times with DPBS and plated in complete RPMI 1640 medium (Corning^®) containing 10 ng/ml macrophage-colony stimulating factor (M-CSF), 10%FBS, 10 μg/ml gentamycin sulfate (Alkali Scientific Inc.) and 2-mercaptoethanol. Cells were differentiated by exposure to human recombinant M-CSF on days 0, 2 and 4. After 6 days, hMϕ were washed with DPBS prior to use for the experiment.

Mouse BMDMϕ were prepared by differentiating bone marrow cells in complete RPMI 1640 medium containing 10 ng/ml mouse M-CSF, 10% fetal bovine serum (FBS) (HyClone Laboratories, Utah), gentamycin sulfate (10 μg/ml) and 2-mercaptoethanol. BMDMϕ were fed on days 0, 2 with complete RPMI 1640 medium containing 10 ng/ml M-CSF and were supplemented on day 4 with M-CSF. After 6 days, adherent Mϕ were harvested by washing with DPBS followed by trypsinization and centrifuged at 1600 rpm for 5 mins. at 4˚C. Mϕ were washed again with DPBS at 1600 rpm for 5 mins. at 4˚C and counted under the microscope. Dead cells were excluded from enumeration using Trypan Blue stain. BMDMϕ (0.5 X 10⁶ in 24 well or 1 X 10⁶ in 12 well plate) were seeded.

The STRING database was used to review the protein-protein interaction networks of MT3 in Mus musculus and Homo sapiens (22). A full network analysis was conducted based on text mining, experiments, databases, co-expression, neighborhood, gene fusion and co-occurrence data with a minimum required interaction score of 0.4 and a maximum of 50 interactors in the first shell and 50 interactors in the second shell. Statistical significance of the enriched biological processes (GO BP categories) in the MT3 network was set with a false discovery rate (FDR) <0.05.

Identification of differentially expressed genes in resting WT compared to resting Mt3^-/- Mϕ was based on our previously published RNA-seq data (NCBI SRA: PRJNA533616) (19). The number of biological replicates used in the analysis was 3 per group. Genes differentially expressed with a fold change FC>2 and adjusted p value q<0.05 were considered significant. The Benjamini-Hochberg correction was used to adjust p values for multiple hypothesis testing. Differentially expressed genes in Mt3^-/- BMDMϕ compared to WT BMDMϕ with q<0.05 were queried using the functional annotation clustering tool DAVID to identify statistically enriched GO categories (using the GO terms, BP direct, CC direct and MF direct) in Mt3^-/- BMDMϕ compared to WT BMDMϕ (41). Significance of enrichment was set to FDR <0.05.

For gene silencing, Mϕ were transfected with the transfection complex (50 μl) of siRNA and TransIT-TKO^® (0.5%) transfection reagent (Mirus Bio™) in 500 μl of complete RPMI 1640 medium without antibiotics as per the manufacturer’s instructions. Concentration of siRNAs used for gene silencing were 100 nM each of the non-targeting pool (ON-TARGETplus™ scramble siRNA), human MT3 (MT3 Silencer^® Pre-designed siRNA) and mouse Ticam1 (ON-TARGETplus SMARTpool). All siRNAs were purchased from Dharmacon (GE Healthcare). Both BMDMϕ and hMϕ were incubated with the siRNA containing transfection complexes for 24h in RPMI medium and washed prior to transfection with LPS in Opti-MEM medium. TRIF silencing was assessed by protein expression using Western blots. Human MT3 silencing was assessed by gene expression using qRT-PCR.

IFNAR1 on WT and Mt3^-/- BMDMϕ was neutralized using 10 μg/ml monoclonal anti-IFNAR1 antibody (BioLegend; Clone: MAR1-5A3) 1h prior and 24h after iLPS (10 μg/ml) stimulation. Negative control groups were treated with the same dose of isotype control IgG antibody (BioLegend; Clone: MOPC-21). After a total 48h, cell lysates and supernatants were harvested for the molecular analysis.

To activate the non-canonical inflammasome, 1 X 10⁶ Mϕ were transfected with a transfection complex (50 μl) of 0.3% TransIT™-LT1 (Mirus Bio™) transfection reagent (vehicle) and 2 μg/ml or 10 μg/ml ultrapure LPS-B5 (In vivo Gen) prepared from E. coli 055:K59(B5) in 500 μl Opti-MEM medium (Thermo Fisher Scientific-US) as per manufacturer’s instructions for 24h.

Molecular biology grade chelex-100 resin (BioRad) was washed three times with metal free ddiH₂O prior to use. To prepare Zn²⁺-deficient Opti-MEM medium, washed Chelex-100 resin (3 g per 100 ml) was added to Opti-MEM media and vigorously shaken for 1h on an orbital shaker at room temperature. After this time, media was filtered using a 0.22 μm filter and mixed with fresh washed chelex-100 resin and the same procedure was repeated for a total of 3 times to eliminate metals from Opti-MEM media. Chelex was removed from the media by a final filtration step. During each of these stages, an aliquot of the media was saved to monitor the efficiency of Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺, Cu²⁺, Ni²⁺ and Fe²⁺ elimination by ICP-MS. The amount of Zn²⁺ in Opti-MEM media was decreased by 95% by the above chelation method. To prepare Zn²⁺-sufficient media, chelexed Opti-MEM was reconstituted with Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺ and Zn²⁺ at the original concentrations as measured by ICP-MS. To prepare Zn²⁺-deficient media, all measured elements except Zn²⁺ were added to the chelexed Opti-MEM media at the original measured concentrations. Finally, the pH of Zn²⁺-sufficient and Zn²⁺-deficient Opti-MEM media was adjusted to 7.4 and filtered prior to use.

The pCMV6-Ac-GFP vector containing the mouse Mt3 gene (pCMV6-Ac-MT3-GFP) and empty pCMV6-Ac-GFP vectors were acquired from Origene and dissolved in nuclease-free sterile H2O. Plasmid DNA (5 ng) was added to 50 μl of thawed Novablue competent E. coli cells (EMD Millipore) and transformation was performed as per manufacturer’s instructions. E. coli cells were serially diluted in S. O. C media (ThermoFisher Scientific) and plated onto Luria-Bertani (LB) plates with 50 μg/ml carbenicillin and grown for 24h at 37˚C. A single colony was isolated and inoculated in LB media containing carbenicillin and grown at 37˚C for 5h in a shaker. The culture was further amplified by passaging for another 24h. E. coli cells were then harvested by centrifugation at 2000 rpm for 10 mins. and plasmid was extracted using the EndoFree plasmid MAXI kit (Qiagen) as per the manufacturer’s instructions. The plasmid was reconstituted in endotoxin-free TE buffer and OD readings obtained were in the range of 1.8-1.9. The resulting endotoxin-free plasmid DNA was set to a concentration of 1 mg/ml in filter-sterilized EndoFree TE buffer and frozen into aliquots until further use.

Mt1^-/-Mt2^-/- BMDMϕ were transfected with pCMV6-Ac-GFP control vector or pCMV6-Ac-MT3-GFP vector using the LT1 transfection reagent (Mirus Bio) in RPMI media containing 10% serum without antibiotics as per the manufacturer’s instructions. After 48h, BMDMϕ cultures were lysed with 250 μl of 0.1% SDS prepared in double-deionized (ddi) H₂O for 20 min. on ice with intermittent mixing. Cell lysates were transferred to 0.22 μm filter tubes and centrifuged at 13000 rpm for 5 mins. Filtered cell lysates were subjected to SEC-ICP-MS to isolate the MT3 protein as described below.

Cell lysates from above were analyzed by SEC-ICP-MS to detect the MT3-associated peak, followed by collection of the fraction of interest (18-21 mins.). The collected fraction was concentrated by freeze drying in Millrock lyophilizer (Millrock, NY). The concentrated fraction was treated with 1 g of Chelex X-100 resin to remove the divalent metals associated to the protein. The total MT concentration was calculated by the total sulfur concentration in the sample with a 1:20 stoichiometry. The sample was divided into 3 fractions, and each fraction was incubated for 2 h in 50 mM Tris-HCl with the appropriate concentration of ⁶⁶Zn²⁺ nitrate to obtain an MT3-Zn²⁺ saturation of 0, 4 or 6 Zn²⁺ ions per MT3 molecule. After incubation, samples were filtered using a 3 kDa MWCO filter to remove the unbound Zn²⁺ and reconstituted in 1X PBS.

In a 24 well plate, 5 X 10⁵ Mt3^-/- BMDMϕ were transfected with the transfection complex (10 μl) containing 500 ng of apo-MT3, 4Zn²⁺-MT3 or 6Zn²⁺-MT3 and Pro-Ject™ (1.75 μl) protein transfection reagent (ThermoScientific-US) in 250 μl of 2% FBS containing antibiotic free RPMI 1640 medium. Control Mt3^-/- BMDMϕ were treated with Pro-Ject™ alone. Cells were incubated with the transfection complexes for 3.5h and washed two times using HBSS prior to challenge with iLPS in Zn²⁺ free Opti-MEM medium. At the experiment end point, cell lysates were either prepared for SEC-ICP-MS analysis or both cell lysates and supernatants were collected for the analysis of non-canonical inflammasome activation.

SEC-ICP-MS-MS analysis of WT and Mt3^−/− BMDMϕ was performed as described previously (11). Mϕ were plated in Opti-MEM media, and either left untreated or transfected with 10 μg/ml LPS for 1, 24 and 48h. After this time, Mϕ were washed twice in HBSS and cells were lysed with 0.1% SDS on ice for 20 mins. Cell lysates were then centrifuged in 0.22 mm filter tubes at 13000 rpm for 10 mins. Filtered cell lysates were frozen at -80°C until further analysis by SEC-ICP-MS. 50-80 µl of cell lysates were injected to the HPLC-ICP-MS system according to protein concentration. To normalize the response of ICP-MS-MS signal from SEC separations on different days, 50 μl of 0.5 mg/ml carbonic anhydrase was injected into the liquid chromatography system, and area of Zn²⁺ signals from samples was normalized to area of the carbonic anhydrase peak from each day. The absorbance of carbonic anhydrase at 280 nm was followed to ensure protein integrity.

The instrumentation consisted of an Agilent 1100 HPLC equipped with a degasser, a binary pump, a thermostated auto sampler, a column oven compartment and a diode array detector. For the Mϕ lysates, a TSK gel 3000SW gel filtration column (TSK Tokyo Japan) 7.8 × 300 mm, 10 mm particle size was used. The mobile phase was ammonium acetate pH 7.4, 0.05% MeOH at 0.5 ml/minute. The HPLC system was coupled to the ICP-MS-MS nebulizer via a short polyether ether ketone capillary of 0.17 mm internal diameter. An Agilent 7500ce ICP-MS system equipped with a micromist quartz nebulizer, a chilled double pass Scott spray chamber and a standard 2 mm insert quartz torch with shield torch was used for all experiments. The ICP-MS was operated by the Agilent Mass Hunter integrated chromatographic software in the helium collision mode as reported previously (11). The isotope dilution experiments were processed by exporting the chromatographic data to Origin (Origin labs, CA) and the signal, in the form of counts per second, was used to calculate the ratio of ⁶⁶Zn^2+/64Zn²⁺ at every point in the chromatograms. This was used to generate a new chromatogram that reflected the input of ⁶⁶Zn²⁺ from the MT3-Zn²⁺ complex at every molecular mass region in the chromatogram. The total area under the chromatograms was used to calculate the concentration of total Zn²⁺ (⁶⁴Zn²⁺) and ⁶⁶Zn²⁺ from the MT3-⁶⁶Zn²⁺ complexes (⁶⁶Zn^2+/64Zn²⁺) against a calibration curve of Zn²⁺ based on carbonic anhydrase.

All metal analysis experiments were performed using trace metal grade reagents with acid washed plastic vials. Reagent blanks were used to correct the background signal. The analysis was performed through a metal free encased auto sampler. The concentration of Zn²⁺ in the blanks was always below 100 parts per trillion (ppt), the blank estimate concentration on the calibration curves was always below 50 ppt, while the detection limits were below 30 ppt.

For chromatographic analysis, the mobile phase was cleaned using a Chelex 100 resin, using the batch method. In brief, 3 g of Chelex-100 was added to a liter of mobile phase, stirred for 30 mins. and passed through a 0.45 mm membrane. This decreased the Zn²⁺ concentration below 200 ppt (measured as total). By this method, the base line ICP-MS-MS ⁶⁶Zn²⁺ signal was below 1000 counts per second, which represents sub-ppb levels. The SEC column was cleaned using 10 volumes of 0.2 M NaCl and equilibrated with the mobile phase, followed by injection of 50 μl of 2% HNO₃ three times to remove any accumulated Zn²⁺ in the column. With this procedure, Zn²⁺ distribution in the samples never deviated more than 10% compared to the theoretical natural Zn²⁺ isotope distribution in the control Mϕ samples. Four blanks and four carbonic anhydrase standards were injected after the cleaning procedure for monitoring Zn²⁺ signal by ICP-MS-MS to ensure optimal column performance. Typically, the column was cleaned every 30-40 samples.

Thirteen-week-old WT and Mt3^-/- mice were primed with i.p. injection of 10 mg/kg poly(I:C) for 6h followed by 2 mg/kg ultrapure LPS-B5 (In vivoGen) prepared from E. coli 055:K59(B5) (i.p. injection) for 18h. At the experiment end point, blood was collected by cardiac puncture, allowed to clot, and centrifuged at 2000 rpm for 30 mins. at 4˚C to isolate serum. Serum was used to measure cytokines by enzyme-linked immunosorbent assay (ELISA).

For in vitro infection, E. coli (K12) was grown in LB broth at 37˚C overnight in an orbital shaker. The culture was pelleted, washed and resuspended with ice-cold DPBS. Optical Density (OD) of the culture was measured at 600nm using a spectrophotometer. To analyze E. coli burden in in vitro, hMϕ were transfected with scramble siRNA or MT3 siRNA as described above. hMϕ, WT and Mt3^-/- BMDMϕ were infected with a multiplicity of infection (MOI) of 25 E. coli: 1 hMϕ for 3.5h in Opti-MEM media. Mϕ were washed 3 times with 10 μg/ml gentamycin sulfate containing DPBS to kill extracellular bacteria and incubated in Opti-MEM media with antibiotic for 24h. Mϕ were again washed 3 times with antibiotic-free DPBS, diH₂O was added and cells were incubated for 30 min. to induce osmotic lysis. Cells were scraped and lysates were diluted in DPBS followed by plating on LB agar plates and incubated at 37˚C for 24h. Colonies were enumerated as above. Intracellular bacterial burden was represented as percent inhibition of bacterial growth in MT3-silenced hMϕ compared to scramble siRNA treated hMϕ and in Mt3^-/- BMDMϕ compared to WT BMDMϕ.

To analyze antibacterial immunity in vivo, 10 to12 week-old mice were used. Mice were infected with E. coli 1 X 10⁹ CFUs via i.p. injection (300 µl/mouse) for 1h or 6h. To investigate the role of NLRP3 inflammasome in antibacterial immunity, mice were treated with 1 mg/mouse MCC950 (an inhibitor of NLRP3 inflammasome) via i.p. injection (100 µl/mouse) for 1h followed by E. coli 1 X 10⁹ CFUs via i.p. injection (300 µl/mouse) for 6h. K. pneumoniae KP2 2-70, a virulent, heavily encapsulated gram-negative bacterial strain (89, 90), was grown overnight in brain heart infusion (BHI) broth. The following morning bacteria were washed with DPBS, and administered at 4 x 10⁴ CFUs in 50 µl per mouse by the i.n. to isoflurane-anesthetized mice for 48h. At the infection end point, blood was collected by cardiac puncture. A portion of the blood sample was acquired in anticoagulant (3% Na-citrate or EDTA) containing tubes to determine bacterial CFUs in blood. The remaining blood was allowed to clot, and centrifuged at 2000 rpm for 30 mins. at 4˚C to isolate serum. Peritoneal lavage was collected using ice-cold 10 ml DPBS. Kidney, lung, and spleen were collected after perfusion with 3 ml of DPBS, indicated organs and skin was rinsed in DPBS and ground with 5 ml DPBS using a glass grinder. Bacterial growth was measured in blood, peritoneal lavage, kidney, lung, skin and spleen samples. Serum and peritoneal lavage were used to measure cytokines by enzyme-linked immunosorbent assay (ELISA).

Septic shock was induced in mice via i.p. injection with ultrapure LPS-B5 (20 mg/kg) (In vivoGen) prepared from E. coli 055:K59(B5). Mice were weighed and sepsis scores were determined at various intervals. The MSS scoring method assesses sepsis severity with scores ranging from 1-4 based on 7 parameters (appearance, consciousness, activity, stimulus (sounds/touch), eyes aspect, respiration rate and respiration quality) (27). Survival analysis was conducted using the Kaplan-Meier analysis method and log-rank (Mantel-Cox test) was used to determine statistical differences in survival.

A representative M1T1 clonal Group-A-Streptococcus GAS5448 was used for subcutaneous infections (91). GAS was grown at 37°C under static conditions in Todd-Hewitt broth (BD, MD, USA) supplemented with 1.5% yeast extract (BD Biosciences, MD, USA) and in vivo infections were performed as described previously (29, 92). WT and Mt3^-/- mice (n=8/group) were used in this study as a model for subcutaneous GAS infections. One day prior to infection, the hair on the back of the mice was depilated (using Nair cream) and mice were infected subcutaneously with 0.1 ml of GAS suspension prepared in sterile DPBS (Ca²⁺/Mg²⁺ free, low endotoxin, Mediatech, VA, USA, DPBS) (OD₆₀₀ adjusted to yield ~1-5x10 (8) CFUs). Actual inoculum was determined by plating on trypticase soy agar containing 5% sheep blood (BD Biosciences, MD, USA). Mice were monitored twice daily for body weight, lesions, and mortality. To determine GAS dissemination and load, mice were humanely euthanized 72h post-infection. Blood was drawn through cardiac puncture; necrotic skin, kidney, and spleen were recovered aseptically and weighed. One ml of DPBS was added per 100 mg tissue and homogenized (Omni International, Marietta, GA) followed by plating of ten-fold dilutions on blood agar plates. GAS burden was calculated as colony-forming units (CFUs) per ml (blood) or per mg of tissue. The remaining homogenates were centrifuged for 15 mins. at 12,000 x g at 4°C, and supernatants were stored at -80°C for western blot analysis.

RNA was isolated from Mϕ after elimination of genomic DNA using RNeasy Plus Mini kit (Qiagen) or QUICK-RNA™ MINIPREP KIT (Thomas Scientific). cDNA was prepared using Reverse Transcription Systems Kit (Promega, WI) or rAmp First Strand cDNA Synthesis Flex Kit (Thomas Scientific). Taqman primer/probe sets (Applied Biosystems, CA) were used for real-time gene expression analysis using ABI Prism 7500. For time course analysis of expression of murine Mt genes, Mϕ were left unstimulated or stimulated with 2 μg/ml iLPS for 0h, 1h, 6h, 24h and 48h. Data are presented as fold change in gene expression normalized to unstimulated Mϕ at the 0h time point. To examine the effects of extracellular ultrapure LPS (exLPS) on murine Mt3 gene expression, BMDMϕ were left unstimulated or stimulated with 10 μg/ml exLPS. Data are presented as fold change in gene expression normalized to unstimulated Mϕ at the 48h time point. For MT2A gene expression analysis in MT3 silenced hMϕ, cells were treated with either MT3 siRNA or scramble siRNA as mentioned above. Data are presented as fold change in gene expression normalized to control siRNA treated hMϕ. Hypoxanthine guanine phosphoribosyl transferase (Hprt) was used as an internal control to compare target gene expression.

TRIF (Proteintech), pIRF3 (BIOSS), STAT1, pSTAT1 (Abcam), GBP2, GBP5 (Proteintech), caspase-11 (Abcam and eBioscience™), CASPASE-4 (MBL), Gasdermin D (Proteintech and Cell Signaling Technologies), caspase-1 (AdipoGen Life Sciences), IL-1β (R&D Systems) and caspase-8 (Enzo Life Sciences) were assessed in kidney homogenates of E. coli infected mice. Cell lysates were prepared using Denaturing Cell Extraction Buffer (Invitrogen) containing protease & phosphatase inhibitor cocktail (ThermoScientific). Culture supernatants were frozen at -80˚C until use and processed using methanol-chloroform protein extraction method. Briefly, supernatants were mixed with equal volume of 100% ice-cold methanol and 0.25 times of the total volume of chloroform followed by gentle vortexing and centrifugation at 20,000 X g at 4˚C for 10 min. Upper-phase was discarded without disturbing inter-phase proteins. Ice-cold methanol (500 μl) was added to the tube, gently vortexed and centrifuged at 20,000 X g at 4˚C for 10 mins. Supernatants were discarded and pellet was dried at 37˚C for 3-5 mins. Urea (8 M, pH-8.0) was used to dissolve the pellet and extracted proteins were stored at -80˚C. Total cell lysates, supernatants proteins, cell lysates + supernatants and kidney homogenates were boiled in SDS-PAGE 1X sample buffer at 95˚C for 5 mins. Kidney homogenates were centrifuged at 20,000 X g at 4˚C for 15 mins. Supernatants were collected for protein analysis. Reduced proteins were run on 8%,10% or 12% SDS-PAGE gels and transferred on to 0.22 μm nitrocellulose membranes (GE Healthcare Life Sciences). Membranes were blocked using 5% skim milk in 1X Tris-buffered saline and 0.1% Tween 20 (1XTBST) and probed overnight with primary antibodies at 4˚C. Membranes were washed 3 times for 10 mins. each with 1XTBST and probed with corresponding HRP conjugated or IRDyes (LI-COR) secondary antibodies, washed and developed using BrightStar™ Femto HRP Chemiluminescent 2-Component Substrate Kit (Alkali Scientific Inc. β-actin was used as an internal loading control. Western blots were analyzed using ImageJ software and densitometry data were normalized to β-actin.

Human and mouse IL-1β (BioLegend) concentration in media supernatants, serum and in peritoneal lavage and mouse IL-1α (BioLegend) in media supernatants were determined using commercial ELISA kits according to the manufacturer’s instructions.

Data were analyzed using Sigma plot or GraphPad Prism by one-way ANOVA for multiple comparisons using the indicated ad-hoc methods with at least 3 or more independent biological replicates. Where two groups were compared, two-tailed t-test was used. For in vivo infection, bacterial CFUs were log-transformed for statistical analysis. p values were calculated, *p < 0.05, **p < 0.01, ***p< 0.001; NS, not significant, ND, not detected.