Non-canonical inflammasome activation by mouse caspase-11 (or human CASPASE-4/5) is crucial for the clearance of certain gram-negative bacterial infections, but can lead to severe inflammatory damage. Factors that promote non-canonical inflammasome activation are well recognized, but less is known about the mechanisms underlying its negative regulation. Herein, we identify that the caspase-11 inflammasome in mouse and human macrophages (Mϕ) is negatively controlled by the zinc (Zn2+) regulating protein, metallothionein 3 (MT3). Upon challenge with intracellular lipopolysaccharide (iLPS), Mϕ increased MT3 expression that curtailed the activation of caspase-11 and its downstream targets caspase-1 and interleukin (IL)-1β. Mechanistically, MT3 increased intramacrophage Zn2+ to downmodulate the TRIF-IRF3-STAT1 axis that is prerequisite for caspase-11 effector function. In vivo, MT3 suppressed activation of the caspase-11 inflammasome, while caspase-11 and MT3 synergized in impairing antibacterial immunity. The present study identifies an important yin-yang relationship between the non-canonical inflammasome and MT3 in controlling inflammation and immunity to gram-negative bacteria.

Non-canonical inflammasome activation by mouse caspase-11 (or human CASPASE-4/5) is crucial for the clearance of certain gram-negative bacterial infections, but can lead to severe inflammatory damage. Factors that promote non-canonical inflammasome activation are well recognized, but less is known about the mechanisms underlying its negative regulation. Herein, we identify that the caspase-11 inflammasome in mouse and human macrophages (Mϕ) is negatively controlled by the zinc (Zn2+) regulating protein, metallothionein 3 (MT3). Upon challenge with intracellular lipopolysaccharide (iLPS), Mϕ increased MT3 expression that curtailed the activation of caspase-11 and its downstream targets caspase-1 and interleukin (IL)-1β. Mechanistically, MT3 increased intramacrophage Zn2+ to downmodulate the TRIF-IRF3-STAT1 axis that is prerequisite for caspase-11 effector function. MT3 suppressed activation of the caspase-11 inflammasome, while caspase-11 and MT3 synergized in impairing antibacterial immunity. The present study identifies an important yin-yang relationship between the non-canonical inflammasome and MT3 in controlling inflammation and immunity to gram-negative bacteria.

Group A Streptococcus (GAS, Streptococcus pyogenes) is an important human pathogen that can cause a broad spectrum of diseases ranging from self-limiting pharyngitis and impetigo to deeper, life-threatening invasive infections such as streptococcal toxic shock syndrome and necrotizing fasciitis (NF). An overarching pro-inflammatory cytokine storm and a rapidly progressive tissue destruction lead to significant morbidity and lethal outcomes of GAS NF. Transcriptome profiling of uninfected and GAS infected skin from HLA-II transgenic mouse models of GAS NF revealed strong activation of inflammatory signaling and inhibition of peroxisome proliferator-activated receptor (PPAR) signaling pathways. Specifically, PPARgamma (PPARγ), a lipid ligand-activated transcription factor driving adipogenic and anti-inflammatory effects, was a significantly inhibited upstream regulator and this inhibition was reflected in downregulation of PPARγ-dependent genes. So far, there are no reports on the role of PPARγ in GAS infections. Here, we explored the potential use of the PPARγ selective synthetic agonist, pioglitazone (PZ), in combination with clindamycin (CLN), as a combined therapeutic approach in vivo in an HLA-II mouse model of GAS NF. Although the mechanism of synergy is not understood, our data demonstrate that combined CLN PZ intervention offers significant advantages over treatment with CLN alone, and uncover a new role for PPARγ in attenuating skin lesions, GAS burden, IL-6 and IFN-γ levels, and weight loss during GAS NF. To our knowledge, this is the first report to demonstrate the benefits of a combined host- and pathogen-directed treatment approach to ameliorate severity and inflammation during GAS NF.

Despite prompt intervention with antibiotics and surgical debridement, morbidity and mortality due to Group A Streptococcal Necrotizing Fasciitis (GAS-NF), frequently associated with streptococcal toxic shock, remain as high as 50%. The pathways driving the sequelae of effector responses that include an uncoordinated activation of the host immune system and inflammatory cytokine storm are not completely elucidated. Our previous studies demonstrated a central role for the pro-inflammatory mediator, IL-1b, in modulating the severity and survival in a mouse model of GAS-NF. To fine-tune IL-1b levels while simultaneously attenuating GAS multiplication and toxin synthesis, here, we investigated the effect of MCC950 (MCC), a small molecule inhibitor for NLRP3 inflammasome activation as a host-directed adjunct to clindamycin (CLN) (10mg/kg each, everyday X 5 days) in a mouse model of GAS NF. Survival outcomes did not differ between CLN and CLNMCC groups. Yet, CLNMCC significantly reduced skin mRNA levels of IL-1a, IL-1b, IL-33, and TNF-a, protein levels of the active fragment of caspase-1 and increased the ratio of pro: active caspase-1 (but not ASC or NLRP3) that coincided with reduced levels of circulating neutrophils. CLNMCC treatment significantly reduced tissue damage, levels of plasma IL-10, IL-1β, and circulating CD11b+LY6G+ granulocytic myeloid subsets. These results indicate that NLRP3 inflammasome activation may be primarily involved in excess IL-1b release, neutrophil influx, and tissue damage during GAS-NF. The evidence of improved outcomes suggests, in part, a beneficial efficacy of MCC950 to target NLRP3 as a feasible adjunct to CLN and mitigate inflammation and pathology with no adverse effects or delayed toxicity

IntroductionStreptococcus pyogenes (Group A Streptococcus, GAS) bacteria cause a spectrum of human diseases ranging from self-limiting pharyngitis and mild uncomplicated skin infections (impetigo, erysipelas, cellulitis) to highly morbid and rapidly invasive life-threatening infections such as streptococcal toxic shock syndrome and necrotizing fasciitis (NF). HLA-Class II allelic polymorphisms are linked with differential outcomes and severity of GAS infections. The dysregulated immune response and peripheral cytokine storm elicited due to invasive GAS infections increase the risk for toxic shock and multiple organ failure in genetically susceptible individuals. We hypothesized that while the host immune mediators regulate the immune responses against peripheral GAS infections, these interactions may simultaneously trigger neuropathology and, in some cases, induce persistent alterations in the glial phenotypes. Here we studied the consequences of peripheral GAS skin infection on the brain in an HLA-II transgenic mouse model of GAS NF with and without treatment with an antibiotic, clindamycin (CLN).MethodsMice expressing the human HLA-II DR3 (DR3) or the HLA-II DR4 (DR4) allele were divided into three groups: i) uninfected controls, ii) subcutaneously infected with a clinical GAS strain isolated from a patient with GAS NF, and iii) GAS infected with CLN treatment (10mg/kg/5 days, intraperitoneal). The groups were monitored for 15 days post-infection. Skin GAS burden and lesion area, splenic and hippocampal mRNA levels of inflammatory markers, and immunohistochemical changes in hippocampal GFAP and Iba-1 immunoreactivity were assessed.ResultsSkin GAS burden and hippocampal mRNA levels of inflammatory markers S100A8/A9, IL-1β, IL-33, inflammasome-related caspase-1 (Casp1), and NLRP6 were elevated in infected DR3 but not DR4 mice. The levels of these markers were significantly reduced following CLN treatment in DR3 mice. Although GAS was not detectable in the brain, astrocyte and microglia activation were evident from increased GFAP and Iba-1 mRNA levels respectively, in DR3 and DR4 mice. However, CLN treatment significantly reduced GFAP immunoreactivity in DR3 mice and not DR4 mice.ConclusionOur data suggest a skin-brain axis during GAS NF demonstrating that peripherally induced pathological conditions regulate neuroimmune and gliotic events, and CLN may attenuate peripheral infection and subsequent neuroimmune changes in an HLA-II-dependent manner.