Group A Streptococcus induces GSDMA-dependent pyroptosis in keratinocytes.

Gasdermins (GSDMs) are a family of pore-forming effectors that permeabilize the cell membrane during the cell death program pyroptosis1. GSDMs are activated by proteolytic removal of autoinhibitory carboxy-terminal domains, typically by caspase regulators1-9. However, no activator is known for one member of this family, GSDMA. Here we show that the major human pathogen group A Streptococcus (GAS) secretes a protease virulence factor, SpeB, that induces GSDMA-dependent pyroptosis. SpeB cleavage of GSDMA releases an active amino-terminal fragment that can insert into membranes to form lytic pores. GSDMA is primarily expressed in the skin10, and keratinocytes infected with SpeB-expressing GAS die of GSDMA-dependent pyroptosis. Mice have three homologues of human GSDMA, and triple-knockout mice are more susceptible to invasive infection by a pandemic hypervirulent M1T1 clone of GAS. These results indicate that GSDMA is critical in the immune defence against invasive skin infections by GAS. Furthermore, they show that GSDMs can act independently of host regulators as direct sensors of exogenous proteases. As SpeB is essential for tissue invasion and survival within skin cells, these results suggest that GSDMA can act akin to a guard protein that directly detects concerning virulence activities of microorganisms that present a severe infectious threat.

Constitutive secretion of pro-IL-18 allows keratinocytes to initiate inflammation during bacterial infection.

Group A Streptococcus (GAS, Streptococcus pyogenes) is a professional human pathogen that commonly infects the skin. Keratinocytes are one of the first cells to contact GAS, and by inducing inflammation, they can initiate the earliest immune responses to pathogen invasion. Here, we characterized the proinflammatory cytokine repertoire produced by primary human keratinocytes and surrogate cell lines commonly used in vitro. Infection induces several cytokines and chemokines, but keratinocytes constitutively secrete IL-18 in a form that is inert (pro-IL-18) and lacks proinflammatory activity. Canonically, IL-18 activation and secretion are coupled through a single proteolytic event that is regulated intracellularly by the inflammasome protease caspase-1 in myeloid cells. The pool of extracellular pro-IL-18 generated by keratinocytes is poised to sense extracellular proteases. It is directly processed into a mature active form by SpeB, a secreted GAS protease that is a critical virulent factor during skin infection. This mechanism contributes to the proinflammatory response against GAS, resulting in T cell activation and the secretion of IFN-γ. Under these conditions, isolates of several other major bacterial pathogens and microbiota of the skin were found to not have significant IL-18-maturing ability. These results suggest keratinocyte-secreted IL-18 is a sentinel that sounds an early alarm that is highly sensitive to GAS, yet tolerant to non-invasive members of the microbiota.

Inflammasome inhibition blocks cardiac glycoside cell toxicity.

Chronic heart failure and cardiac arrhythmias have high morbidity and mortality, and drugs for the prevention and management of these diseases are a large part of the pharmaceutical market. Among these drugs are plant-derived cardiac glycosides, which have been used by various cultures over millennia as both medicines and poisons. We report that digoxin and related compounds activate the NLRP3 inflammasome in macrophages and cardiomyocytes at concentrations achievable during clinical use. Inflammasome activation initiates the maturation and release of the inflammatory cytokine IL-1ß and the programmed cell death pathway pyroptosis in a caspase-1-dependent manner. Notably, the same fluxes of potassium and calcium cations that affect heart contraction also induce inflammasome activation in human but not murine cells. Pharmaceuticals that antagonize these fluxes, including glyburide and verapamil, also inhibit inflammasome activation by cardiac glycosides. Cardiac glycoside-induced cellular cytotoxicity and IL-1ß signaling are likewise antagonized by inhibitors of the NLRP3 inflammasome or the IL-1 receptor-targeting biological agent anakinra. Our results inform on the molecular mechanism by which the inflammasome integrates the diverse signals that activate it through secondary signals like cation flux. Furthermore, this mechanism suggests a contribution of the inflammasome to the toxicity and adverse events associated with cardiac glycosides use in humans and that targeted anti-inflammatories could provide an additional adjunct therapeutic countermeasure.

Molecular Genetic and Immune Functional Responses Distinguish Bone Marrow Mesenchymal Stromal Cells from Hepatic Stellate Cells.

Defining the immune physiology of culture-adapted mesenchymal stromal cells (MSCs) derived from distinct tissue compartments informs their potential utility as pharmaceuticals. Here, we have investigated the comparative immune plasticity of MSCs and hepatic stellate cells (HeSCs) isolated from human and murine bone marrow (BM) and liver, respectively. Although both BM-MSCs and HeSCs share mesenchymal phenotype and overall molecular genetic responses to inflammatory cues, HeSCs differ from BM-MSCs in a meaningful manner. We show that culture-adapted HeSCs express substantially higher levels of hepatocyte growth factor (HGF), matrix metalloproteinase-1, and chemokine (CC motif) ligand 2 (CCL2) than BM-MSCs. Both human BM-MSCs and HeSCs inhibit T-cell proliferation by a shared indoleamine 2,3-dioxygenase (IDO)-dependent mechanism. However, HeSCs are distinct from BM-MSCs by their significant differential expression of HGF, CCL2, IL-8, CCL11, and GMCSF when cocultured with and/or without activated peripheral blood mononuclear cells. We have investigated MSCs and HeSCs derived from murine systems to describe interspecies comparability. Murine BM-MSCs inhibit T-cell proliferation through inducible nitric oxide synthase (iNOS) but not IDO. However, murine HeSCs inhibit T-cell proliferation through a mechanism distinct from either IDO or iNOS. Altogether, these results suggest that although culture-adapted BM-MSCs and HeSCs display a similar phenotype, their secretome and immune plasticity are in part distinct likely mirroring their tissular origins. In addition, the discordance in immune biology between mouse and human sourced HeSC and BM-MSCs speaks to the importance of comparative biology when interrogating rodent systems for human translational insights. Stem Cells 2019;37:1075-1082.

Group A Streptococcal S Protein Utilizes Red Blood Cells as Immune Camouflage and Is a Critical Determinant for Immune Evasion.

Group A Streptococcus (GAS) is a human-specific pathogen that evades the host immune response through the elaboration of multiple virulence factors. Although many of these factors have been studied, numerous proteins encoded by the GAS genome are of unknown function. Herein, we characterize a biomimetic red blood cell (RBC)-captured protein of unknown function-annotated subsequently as S protein-in GAS pathophysiology. S protein maintains the hydrophobic properties of GAS, and its absence reduces survival in human blood. S protein facilitates GAS coating with lysed RBCs to promote molecular mimicry, which increases virulence in vitro and in vivo. Proteomic profiling reveals that the removal of S protein from GAS alters cellular and extracellular protein landscapes and is accompanied by a decrease in the abundance of several key GAS virulence determinants. In vivo, the absence of S protein results in a striking attenuation of virulence and promotes a robust immune response and immunological memory.