Muscimol inhibits plasma membrane rupture and ninjurin-1 oligomerization during pyroptosis.

Pyroptosis is a cell death process that causes inflammation and contributes to numerous diseases. Pyroptosis is mediated by caspase-1 family proteases that cleave the pore-forming protein gasdermin D, causing plasma membrane rupture and release of pathogenic cellular contents. We previously identified muscimol as a small molecule that prevents plasma membrane rupture during pyroptosis via an unidentified mechanism. Here, we show that muscimol has reversible activity to prevent cellular lysis without affecting earlier pyroptotic events. Although muscimol is a well-characterized agonist for neuronal GABAA receptors, muscimol protection is not altered by GABAA receptor antagonists or recapitulated by other GABAA agonists, suggesting that muscimol acts via a novel mechanism. We find that muscimol blocks oligomerization of ninjurin-1, which is required for plasma membrane rupture downstream of gasdermin D pore formation. Our structure-activity relationship studies reveal distinct molecular determinants defining inhibition of pyroptotic lysis compared to GABAA binding. In addition, we demonstrate that muscimol reduces lethality during LPS-induced septic shock. Together, these findings demonstrate that ninjurin-1-mediated plasma membrane rupture can be pharmacologically modulated and pave the way toward identification of therapeutic strategies for pathologic conditions associated with pyroptosis.

Monitoring Calcium Fluxes and Lysosome Exocytosis During Pyroptosis.

Inflammasome-mediated activation of inflammatory caspases (caspase-1, caspase-4, caspase-5, caspase-11) initiates a cascade of cellular events that lead to proinflammatory cell death, or pyroptosis. Proteolytic cleavage of gasdermin D results in the formation of transmembrane pores that allow the release of mature cytokines IL-1ß and IL-18. Gasdermin pores also allow calcium influx through the plasma membrane, triggering the fusion of lysosomal compartments with the cell surface and release of their contents into the extracellular milieu in a process termed lysosome exocytosis. This chapter outlines methods for measuring calcium flux, lysosome exocytosis, and membrane disruption after inflammatory caspase activation.

Failure of CD4 T Cell-Deficient Hosts To Control Chronic Nontyphoidal Salmonella Infection Leads to Exacerbated Inflammation, Chronic Anemia, and Altered Myelopoiesis.

Immunocompromised patients are more susceptible to recurrent nontyphoidal Salmonella (NTS) bacteremia. A key manifestation of HIV infection is the loss of CD4 T cells, which are crucial for immunity to Salmonella infection. We characterized the consequences of CD4 T cell depletion in mice where virulent Salmonella establish chronic infection, similar to chronic NTS disease in humans. Salmonella-infected, CD4-depleted 129X1/SvJ mice remained chronically colonized for at least 5 weeks, displaying increased splenomegaly and more severe splenitis than infected mice with CD4 T cells. Mature erythrocytes, immature erythroid cells, and phagocytes accounted for the largest increase in splenic cellularity. Anemia, which is associated with increased mortality in Salmonella-infected humans, was exacerbated by CD4 depletion in infected mice and was accompanied by increased splenic sequestration of erythrocytes and fewer erythropoietic elements in the bone marrow, despite significantly elevated levels of circulating erythropoietin. Splenic sequestration of red blood cells, the appearance of circulating poikilocytes, and elevated proinflammatory cytokines suggest inflammation-induced damage to erythrocytes contributes to anemia and splenic retention of damaged cells in infected animals. Depleting CD4 T cells led to increased myeloid cells in peripheral blood, spleen, and bone marrow, as well as expansion of CD8 T cells, which has been observed in CD4-depleted humans. This work describes a mouse model of Salmonella infection that recapitulates several aspects of human disease and will allow us to investigate the interplay of innate and adaptive immune functions with chronic inflammation, anemia, and susceptibility to Salmonella infection.

Diverse small molecules prevent macrophage lysis during pyroptosis.

Pyroptosis is a programmed process of proinflammatory cell death mediated by caspase-1-related proteases that cleave the pore-forming protein, gasdermin D, causing cell lysis and release of inflammatory intracellular contents. The amino acid glycine prevents pyroptotic lysis via unknown mechanisms, without affecting caspase-1 activation or pore formation. Pyroptosis plays a critical role in diverse inflammatory diseases, including sepsis. Septic lethality is prevented by glycine treatment, suggesting that glycine-mediated cytoprotection may provide therapeutic benefit. In this study, we systematically examined a panel of small molecules, structurally related to glycine, for their ability to prevent pyroptotic lysis. We found a requirement for the carboxyl group, and limited tolerance for larger amino groups and substitution of the hydrogen R group. Glycine is an agonist for the neuronal glycine receptor, which acts as a ligand-gated chloride channel. The array of cytoprotective small molecules we identified resembles that of known glycine receptor modulators. However, using genetically deficient Glrb mutant macrophages, we found that the glycine receptor is not required for pyroptotic cytoprotection. Furthermore, protection against pyroptotic lysis is independent of extracellular chloride conductance, arguing against an effect mediated by ligand-gated chloride channels. Finally, we conducted a small-scale, hypothesis-driven small-molecule screen and identified unexpected ion channel modulators that prevent pyroptotic lysis with increased potency compared to glycine. Together, these findings demonstrate that pyroptotic lysis can be pharmacologically modulated and pave the way toward identification of therapeutic strategies for pathologic conditions associated with pyroptosis.

Temporal and anatomical host resistance to chronic Salmonella infection is quantitatively dictated by Nramp1 and influenced by host genetic background.

The lysosomal membrane transporter, Nramp1, plays a key role in innate immunity and resistance to infection with intracellular pathogens such as non-typhoidal Salmonella (NTS). NTS-susceptible C57BL/6 (B6) mice, which express the mutant Nramp1D169 allele, are unable to control acute infection with Salmonella enterica serovar Typhimurium following intraperitoneal or oral inoculation. Introducing functional Nramp1G169 into the B6 host background, either by constructing a congenic strain carrying Nramp1G169 from resistant A/J mice (Nramp-Cg) or overexpressing Nramp1G169 from a transgene (Nramp-Tg), conferred equivalent protection against acute Salmonella infection. In contrast, the contributions of Nramp1 for controlling chronic infection are more complex, involving temporal and anatomical differences in Nramp1-dependent host responses. Nramp-Cg, Nramp-Tg and NTS-resistant 129×1/SvJ mice survived oral Salmonella infection equally well for the first 2-3 weeks, providing evidence that Nramp1 contributes to the initial control of NTS bacteremia preceding establishment of chronic Salmonella infection. By day 30, increased host Nramp1 expression (Tg>Cg) provided greater protection as indicated by decreased splenic bacterial colonization (Tg

Coordinated host responses during pyroptosis: caspase-1-dependent lysosome exocytosis and inflammatory cytokine maturation.

Activation of caspase-1 leads to pyroptosis, a program of cell death characterized by cell lysis and inflammatory cytokine release. Caspase-1 activation triggered by multiple nucleotide-binding oligomerization domain-like receptors (NLRs; NLRC4, NLRP1b, or NLRP3) leads to loss of lysosomes via their fusion with the cell surface, or lysosome exocytosis. Active caspase-1 increased cellular membrane permeability and intracellular calcium levels, which facilitated lysosome exocytosis and release of host antimicrobial factors and microbial products. Lysosome exocytosis has been proposed to mediate secretion of IL-1ß and IL-18; however, blocking lysosome exocytosis did not alter cytokine processing or release. These studies indicate two conserved secretion pathways are initiated by caspase-1, lysosome exocytosis, and a parallel pathway resulting in cytokine release, and both enhance the antimicrobial nature of pyroptosis.

Proteomic profiling of lipopolysaccharide-activated macrophages by isotope coded affinity tagging.

Lipopolysaccharide (LPS), a glycolipid component of the outer membranes of Gram-negative bacteria, initiates proinflammatory, proapoptotic, and antiapoptotic pathways upon binding to macrophage TLR4. Macrophages that are exposed to LPS become activated and exhibit altered morphology and response to infection. We performed isotope coded affinity tagging (ICAT), multidimensional liquid chromatography, and mass spectrometry to identify proteins that are differently expressed between naive and LPS-activated macrophages. We performed replicate ICAT analyses on RAW 264.7 cultured mouse macrophages as well as C57BL/6 bone marrow derived mouse macrophages. We identified and obtained relative abundances for 1064 proteins, of which we identified 36 as having significantly different expression levels upon activation by LPS. We also compared our results with a two color microarray gene expression assay performed by the Institute for Systems Biology and observed approximately 75% agreement between mRNA transcription and protein expression regarding up- or down-regulation of gene products. We used Western blot analysis to confirm the findings of ICAT and mRNA for one protein, sequestosome 1, the cellular concentration of which was observed to increase upon activation by LPS.

Quantification of green fluorescent protein in cellular supernatant by capillary electrophoresis with laser-induced fluorescence detection for measurement of cell death.

A common method for quantifying cell death is measuring the concentration of lactate dehydrogenase (LDH) released by cells as their membranes become unstable. In cells expressing green fluorescent protein (GFP), degradation of the cell membrane also results in the release of GFP into the surrounding supernatant. In this study, we used capillary electrophoresis with laser-induced fluorescence detection to measure the levels of GFP in supernatants of UBIGFP/BL6 primary macrophages that had been infected with Salmonella typhimurium, treated with staurosporine, or exposed to H(2)O(2), all known inducers of cell death. We also used a standard LDH assay to measure the release of LDH into supernatants. We observed the rate of cell death quantified by release of GFP and LDH into supernatant to be essentially identical, demonstrating that GFP release is at least as good as an indicator of macrophage cell death as the established LDH release method.

CD8(+) T cells restrict Yersinia pseudotuberculosis infection: bypass of anti-phagocytosis by targeting antigen-presenting cells.

All Yersinia species target and bind to phagocytic cells, but uptake and destruction of bacteria are prevented by injection of anti-phagocytic Yop proteins into the host cell. Here we provide evidence that CD8(+) T cells, which canonically eliminate intracellular pathogens, are important for restricting Yersinia, even though bacteria are primarily found in an extracellular locale during the course of disease. In a model of infection with attenuated Y. pseudotuberculosis, mice deficient for CD8(+) T cells were more susceptible to infection than immunocompetent mice. Although exposure to attenuated Y. pseudotuberculosis generated T(H)1-type antibody responses and conferred protection against challenge with fully virulent bacteria, depletion of CD8(+) T cells during challenge severely compromised protective immunity. Strikingly, mice lacking the T cell effector molecule perforin also succumbed to Y. pseudotuberculosis infection. Given that the function of perforin is to kill antigen-presenting cells, we reasoned that cell death marks bacteria-associated host cells for internalization by neighboring phagocytes, thus allowing ingestion and clearance of the attached bacteria. Supportive of this model, cytolytic T cell killing of Y. pseudotuberculosis-associated host cells results in engulfment by neighboring phagocytes of both bacteria and target cells, bypassing anti-phagocytosis. Our findings are consistent with a novel function for cell-mediated immune responses protecting against extracellular pathogens like Yersinia: perforin and CD8(+) T cells are critical for hosts to overcome the anti-phagocytic action of Yops.

Chlamydia trachomatis infection alters the development of memory CD8+ T cells.

The obligate intracellular bacterium Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States and the leading cause of preventable blindness worldwide. Prior exposure to C. trachomatis has been shown to provide incomplete protection against subsequent infection. One possible explanation for the limited immunity afforded by prior C. trachomatis infection is poor activation of Chlamydia-specific memory CD8+ T cells. In this study, we examined the development of CD8+ memory T cell responses specific for the Chlamydia Ag CrpA. The percentage of CrpA63-71-specific T cells expressing an effector memory T cell phenotype (IL-7R+ CD62low) was dramatically diminished in mice immunized with C. trachomatis, compared with mice immunized with vaccinia virus expressing the CrpA protein. These alterations in memory T cell development were correlated with a significant reduction in the capacity of convalescent mice to mount an enhanced recall response to Chlamydia Ags, compared with the primary response. CrpA-specific memory T cells primed during VacCrpA infection also failed to respond to a challenge with Chlamydia. We therefore investigated whether C. trachomatis infection might have a global inhibitory effect on CD8+ T cell activation by coinfecting mice with C. trachomatis and Listeria monocytogenes and we found that the activation of Listeria-specific naive and memory CD8+ T cells was reduced in the presence of C. trachomatis. Together, these results suggest that Chlamydia is able to alter the development of CD8+ T cell responses during both primary and secondary infection, perhaps accounting for the incomplete protection provided by prior Chlamydia infection.

An inclusion membrane protein from Chlamydia trachomatis enters the MHC class I pathway and stimulates a CD8+ T cell response.

During its developmental cycle, the intracellular bacterial pathogen Chlamydia trachomatis remains confined within a protective vacuole known as an inclusion. Nevertheless, CD8(+) T cells that recognize Chlamydia Ags in the context of MHC class I molecules are primed during infection. MHC class I-restricted presentation of these Ags suggests that these proteins or domains from them have access to the host cell cytoplasm. Chlamydia products with access to the host cell cytoplasm define a subset of molecules uniquely positioned to interface with the intracellular environment during the pathogen's developmental cycle. In addition to their use as candidate Ags for stimulating CD8(+) T cells, these proteins represent novel candidates for therapeutic intervention of infection. In this study, we use C. trachomatis-specific murine T cells and an expression-cloning strategy to show that CT442 from Chlamydia is targeted by CD8(+) T cells. CT442, also known as CrpA, is a 15-kDa protein of undefined function that has previously been shown to be associated with the Chlamydia inclusion membrane. We show that: 1) CD8(+) T cells specific for an H-2D(b)-restricted epitope from CrpA are elicited at a significant level (approximately 4% of splenic CD8(+) T cells) in mice in response to infection; 2) the response to this epitope correlates with clearance of the organism from infected mice; and 3) immunization with recombinant vaccinia virus expressing CrpA elicits partial protective immunity to subsequent i.v. challenge with C. trachomatis.

T cell responses to Chlamydia trachomatis.

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States, as well as the leading cause of preventable blindness worldwide. Immunity to C. trachomatis requires a variety of cell types, each employing an array of effector functions. Recent work has demonstrated that both CD4+ and CD8+ T lymphocytes play a major role in protective immunity to C. trachomatis, predominantly through their secretion of interferon-gamma. This review describes the generation of acquired immunity to C. trachomatis and focuses on how T cells contribute to both protection and immunopathology.