Abstract
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.
Highlights
Pyroptosis is a programmed process of lytic, proinflammatory cell death[1] involved in a host of disorders including sepsis, stroke, intestinal inflammation, and Tcell depletion during HIV infection[2,3,4,5]
To understand the structural requirements for cytoprotection during pyroptosis, we systematically tested a panel of amino acids and related small molecules for their ability to prevent pyroptotic lysis (Supplemental Fig. S1)
Consistent with prior studies[14], we observed that glycine prevented lactate dehydrogenase (LDH) release from Salmonella-infected and lethal toxin-treated macrophages (Fig. 1a, b and Fig. S2A), without affecting the detection of LDH released from detergent-treated cells (Fig. S3A)
Summary
Pyroptosis is a programmed process of lytic, proinflammatory cell death[1] involved in a host of disorders including sepsis, stroke, intestinal inflammation, and Tcell depletion during HIV infection[2,3,4,5]. Pyroptosis contributes to pathological inflammation and cell death, it is an essential protective host response to infection[6]. Pyroptosis is mediated by proteases in the caspase-1 family, which are activated by the innate immune signaling platforms termed inflammasomes. Caspase-1 cleaves gasdermin D, releasing the N-terminal pore-forming domain, which inserts into the plasma membrane[8]. Gasdermin D pores mediate osmotic cell swelling, rupture of the plasma membrane, and release of intracellular contents including the enzyme lactate dehydrogenase (LDH)[9,10]. The importance of pyroptotic death in the pathogenesis of disease is highlighted by protection of gasdermin D knockout mice from conditions including septic lethality[11] and autoinflammatory disease[12,13]
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