Diverse small molecules prevent macrophage lysis during pyroptosis

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.

Fig. 1. Specific small molecules with structural similarity to glycine inhibit pyroptotic lysis.

Bone marrow-derived macrophages were treated with Salmonella (a, d) or anthrax lethal toxin (b, e) in the presence of glycine or related small molecules that vary at the amino position (titrated from 1.7–60 mM). LDH released during pyroptotic lysis was measured and compared to LDH released in the absence of inhibitor (% inhibition). Representative data (mean ± SD, n = 3) from three or more independent experiments are shown. c To determine whether glycine or β-alanine block caspase-1 activation, macrophages were treated with PBS, Salmonella or anthrax lethal toxin in the presence of medium alone, 5 mM glycine or 15 mM β-alanine. Active caspase-1 was identified by FAM-YVAD-FMK staining. Cumulative data from two independent experiments (mean ± SD, n = 7 high power fields with 328–574 total cells queried per condition) are shown

Fig. 2. Substitution of amino acid side chains influences cytoprotection.

Bone marrow-derived macrophages were treated with Salmonella (a) or anthrax lethal toxin (b) in the presence of glycine or related amino acids with R group substitutions (titrated from 1.7–60 mM). LDH released during pyroptotic lysis was measured and compared to LDH released in the absence of inhibitor (% inhibition). Representative data (mean ± SD, n = 3) from three or more independent experiments are shown. c Cytoprotective amino acids do not block caspase-1 activation. Macrophages were treated with PBS, Salmonella or anthrax lethal toxin in the presence of medium alone, glycine (5 mM), 1-aminocyclopropane carboxylic acid (1-ACPC, 15 mM), l-alanine (15 mM), d-alanine (45 mM), or d-serine (15 mM). Active caspase-1 was identified by FAM-YVAD-FMK staining. Cumulative data from two independent experiments (mean ± SD, n = 7 high power fields with 357–588 total cells queried per condition) are shown

Fig. 3. The carboxyl group is essential for protecting cells from pyroptotic lysis.

a Bone marrow-derived macrophages were infected with Salmonella in the presence of glycine, methylamine, ethylamine, or ethanolamine (titrated from 1.7–15 mM). b Macrophages were treated with anthrax lethal toxin in the presence of glycine or ethanolamine. LDH released during pyroptotic lysis was measured and compared to LDH released in the absence of inhibitor (% inhibition). Representative data (mean ± SD, n = 3) from three or more independent experiments are shown

Fig. 4. Glycine receptor agonists and antagonists both inhibit pyroptotic lysis.

a Macrophages were pretreated with increasing concentrations of propofol for 30 min prior to addition of the GlyR agonists, glycine, β-alanine, or taurine (all 5 mM). Salmonella-induced LDH release was measured and compared to LDH released by pyroptotic cells in the absence of all inhibitors (% inhibition). Representative data (mean ± SD, n = 3) from three or more independent experiments are shown. *P < 0.05, **P < 0.01 (unpaired t-test) indicates significance compared to 0 mM propofol. b Macrophages were infected with Salmonella in the presence of GlyR antagonists, strychnine, or brucine (titrated from 0.06 to 5 mM). Representative data (mean ± SD, n = 3) from three or more independent experiments are shown. c GlyR antagonists do not block caspase-1 activation. Macrophages were treated with PBS or Salmonella in the presence of medium alone, 1.7 mM strychnine, or 1.7 mM brucine. Active caspase-1 was identified by FAM-YVAD-FMK staining. Cumulative data from two independent experiments (mean ± SD, n = 7 high power fields with 413–578 total cells queried per condition) are shown

Fig. 5. GlyR is not required for glycine-mediated inhibition of pyroptotic lysis.

a Bone marrow-derived macrophages from wild type and GlyR β subunit-deficient (Glrb spa/spa) mice were infected with Salmonella in the presence or absence of 5 mM glycine or 1.7 mM strychnine and LDH release was measured after 60 min. b Wild-type macrophages were treated with Salmonella or anthrax lethal toxin in buffer containing either chloride or gluconate anions and LDH release was measured. Representative data (mean ± SD, n = 3) from three or more independent experiments are shown. *P < 0.05, **P < 0.01, n.s. nonsignificant (unpaired t-test)

Fig. 6. Diverse ligand-gated ion channel modulators prevent pyroptotic lysis.

Bone marrow-derived macrophages were treated with Salmonella (a, c, f) or anthrax lethal toxin (b, d, g) in the presence of various molecules known to bind GlyR and/or GABA_A receptor (titrated from 0.06 to 5 mM; 0.007 to 0.6 mM pregnenolone sulfate). LDH released during pyroptotic lysis was measured and compared to LDH released in the absence of inhibitor (% inhibition). Representative data (mean ± SD, n = 3) from three or more independent experiments are shown. e, h Macrophages were treated with PBS, Salmonella (ST), or anthrax lethal toxin (LT) in the presence of medium alone, 0.2 mM pregnenolone sulfate (e) or 1.7 mM muscimol (h). Active caspase-1 was identified by FAM-YVAD-FMK staining. Cumulative data from two independent experiments (mean ± SD, n = 7 high power fields with 421–578 total cells queried per condition) are shown