Indirect regulation of HMGB1 release by gasdermin D

Abstract

The protein high-mobility group box 1 (HMGB1) is released into the extracellular space in response to many inflammatory stimuli, where it is a potent signaling molecule. Although research has focused on downstream HMGB1 signaling, the means by which HMGB1 exits the cell is controversial. Here we demonstrate that HMGB1 is not released from bone marrow-derived macrophages (BMDM) after lipopolysaccharide (LPS) treatment. We also explore whether HMGB1 is released via the pore-forming protein gasdermin D after inflammasome activation, as is the case for IL-1β. HMGB1 is only released under conditions that cause cell lysis (pyroptosis). When pyroptosis is prevented, HMGB1 is not released, despite inflammasome activation and IL-1β secretion. During endotoxemia, gasdermin D knockout mice secrete HMGB1 normally, yet secretion of IL-1β is completely blocked. Together, these data demonstrate that in vitro HMGB1 release after inflammasome activation occurs after cellular rupture, which is probably inflammasome-independent in vivo.

Fig. 1. LPS treatment alone does not lead to HMGB1 secretion in BMDM.

a BMDM were untreated or primed with LPS for 5 h (0.5 μg/ml) and then treated with or without nigericin (Ng; 20 μM) for the last 30 min. HMGB1 present in the extracellular media (supernatant) was quantified by ELISA. n = 3 (mean ± SEM). LPS treatment alone did not result in secretion of HMGB1 into the extracellular media. *p = 0.0009, n.s. not significant as determined by ANOVA with Tukey’s multiple comparison correction, two-sided. b BMDM were untreated, or treated with LPS for 5 h (0.5 μg ml⁻¹. Cells were lysed and immunoblotted for HMGB1 and GAPDH. Note that HMGB1 expression does not change in response to LPS treatment. c, d BMDM were primed with LPS for 5 h (0.5 μg ml⁻¹). The cells were fixed and immunostained for HMGB1 and LAMP1 or Rab7 and imaged by spinning disk confocal microscopy. Collapsed (Extended focus) images at ×63 magnification, size bar 20 μm. HMGB1 and LAMP1 or Rab7 do not colocalize in unstimulated or LPS-treated cells.

Fig. 2. Pyroptosis activators lead to HMGB1 release from macrophages.

BMDM were primed with LPS (0.5 μg ml⁻¹ for 4.5 h) and then treated with nigericin (Ng; 20 μM for 30 min) or potassium depletion (K⁺ dep; 2 h) as described in “Methods”. Untreated controls were not primed with LPS. a Immunoblot analysis of released HMGB1, GAPDH, and cleaved IL-1β (p17) in the cell culture media (supernatant). Dashed line indicates removed lanes. Both LPS + Ng and LPS + K⁺ dep result in the release of HMGB1, GAPDH, and processed IL-1β into the supernatant. Data depict n = 3 independent experiments. b LDH present in the extracellular media (supernatant) was quantified as a measure of pyroptosis-induced cytotoxicity. Data are expressed as supernatant LDH as a % of total LDH from lysates and supernatants, from n = 4 independent experiments. c Immunofluorescence analysis of HMGB1 (green) and ASC (red) in BMDM treated as indicated. ASC oligomerized into inflammasome specks (white arrows) in LPS + Ng treated cells. DAPI was used to identify cell nuclei; size bar 20 μm. Data with error bars are represented as mean ± SEM. Each panel is a representative experiment of at least three independent experiments. *p < 0.0001 and n.s. not significant as determined by ANOVA with Tukey’s multiple comparison correction, two-sided.

Fig. 3. Pyroptosis-induced HMGB1 release is gasdermin D-dependent.

BMDM prepared from wild-type mice were primed with LPS (0.5 μg ml⁻¹) for 4.5 h, then treated with or without nigericin (Ng; 20 μM) for 30 min. Necrosulfonamide (NSA) was added 3 h after LPS addition as indicated. Immunoblot analysis was used to detect released HMGB1 and cleaved IL-1β (p17) into the cell culture supernatant (a). LDH present in the extracellular media (supernatant) was quantified as a measure of pyroptosis-induced cytotoxicity. Data shown from n = 6 independent experiments (b). NSA inhibits HMGB1 and IL-1β release, as well as cytotoxicity. BMDM prepared from wild-type mice or gasdermin D knockout (Gsdmd^−/−) BMDM were primed with LPS for 4.5 h, then treated with nigericin (Ng; 20 μM) for 30 min. NSA was added 3 h after LPS addition as indicated. LDH present in the extracellular media was quantified as a measure of pyroptosis-induced cytotoxicity (c). Data in c indicate n = 3 independent experiments. Immunoblot analysis of released HMGB1 and cleaved IL-1β (p17) into the cell culture supernatant, showing the loss of HMGB1 or IL-1β release in response to LPS + Ng in Gsdmd^−/− BMDM (e). BMDM lysates from WT and Gsdmd^−/− animals were blotted for gasdermin D with GAPDH as a loading control (d). Immunofluorescence of WT and Gsdmd^−/− (f). BMDM primed with LPS and treated with or without nigericin (Ng). Green and red channels correspond to HMGB1 and red ASC oligomerized into inflammasome specks (white arrows), respectively. DAPI was used to identify cell nuclei. Note that ASC positive Gsdmd^−/− cells retain nuclear HMGB1. Data with error bars are represented as mean ± SEM. Each panel is a representative experiment of at least three independent experiments. *p < 0.0001, n.s. not significant as determined by ANOVA with Tukey’s multiple comparison correction, two-sided.

Fig. 4. HMGB1 is not secreted through the gasdermin D pore.

BMDM were primed with LPS (0.5 μg ml⁻¹; 4.5 h) and then treated with nigericin (Ng; 20 μM for 30 min) or potassium depletion (K⁺ dep; 2 h) in the absence or presence of 5 mM glycine as described in “Methods”. Untreated controls were not primed with LPS. a LDH present in the extracellular media (supernatant) was quantified as a measure of pyroptosis-induced cytotoxicity. Glycine effectively prevents cytotoxicity induced by LPS + Ng or K⁺ dep. Data shown from n = 3 independent experiments. b Immunoblot analysis of released HMGB1 and cleaved IL-1β (p17) into the cell culture media (supernatant) under the conditions tested in a. Glycine decreases HMGB1 release without significantly affecting IL-1β secretion. Quantitation of Western blots in b. Data in c and d shown from n = 3 independent experiments. BMDM were treated with or without LPS, then treated with PGPC (50 μg ml⁻¹) for 4 h, or were infected with Δoat S. aureus for 18 h at the MOI indicated, as described in the Methods. Cells treated with Δoat S. aureus were not primed with LPS. e LDH release under the indicated conditions. Data in e show n = 4 (untreated, PGPC, PGPC + LPS) or n = 3 (ΔOAT10, ΔOAT30, LPS + Ng) independent experiments. IL-1β (f) and HMGB1 (g) present in the extracellular media were quantified by ELISA. Both PGPC and Δoat S. aureus stimulate IL-1β release, but not HMGB1 secretion. Data shown in f from untreated (n = 7), PGPC, LPS + PGPC, ΔOAT10, ΔOAT30 (all n = 4), LPS + Ng (n = 5) independent experiments. Data shown in g from n = 3 independent experiments. h, i Immunofluorescence of HMGB1 and DAPI, indicating nuclear retention of HMGB1 under the above conditions, and loss of HMGB1 following LPS + Ng. Data with error bars are represented as mean ± SEM. Each panel is a representative experiment of at least three independent experiments. Adjusted p values, provided in the panels, and n.s. not significant as determined by ANOVA with Tukey’s multiple comparison correction, two-sided.

Fig. 5. HMGB1 is released through sufficiently large membrane pores.

BMDM were primed with LPS (0.5 μg ml⁻¹; 4.5 h) and then treated with nigericin (Ng; 20 μM for 30 min), potassium depletion (K⁺ dep; 2 h) or treated with pneumolysin (PL) alone. Untreated controls and pneumolysin-treated cells were not primed with LPS. Cells were treated with or without 5 mM glycine (Gly), necrosulfonamide (NSA) (5 μM), or PL (0.5 μg ml⁻¹). a Immunoblot analysis of released HMGB1 and cleaved IL-1β (p17) into the cell culture supernatant. PL causes release of HMGB1, but not IL-1 β processing. b LDH present in the extracellular media was quantified as a measure of cytotoxicity, with LPS + Ng and PL causing similar amounts of cell lysis. Data shown from n = 3 independent experiments. c Immunofluorescence of control and PL-treated BMDM. HMGB1 (green) and nuclei (DAPI; blue) indicate loss of nuclear HMGB1 in response to PL treatment. Data with error bars are represented as mean ± SEM. Each panel is a representative experiment of at least three independent experiments. *p < 0.0001 and n.s. not significant as determined by ANOVA with Tukey’s multiple comparison correction, two-sided.

Fig. 6. Endotoxemia leads to elevated plasma HMGB1 in the absence of gasdermin D.

Wild-type (WT) or gasdermin D knockout (Gsdmd^−/−) mice were treated with 20 mg kg⁻¹ of LPS i.p. or saline. After 6 h, plasma and spleens were collected for analysis. a Spleen lysates were immunobloted for NLRP3 and cleaved caspase-1 (p10) to assess for inflammasome activation, and quantified in b. Data indicate WT saline (n = 2), WT LPS (n = 3), Gsdmd^−/− saline (n = 3), Gsdmd^−/− LPS (n = 3) animals per group. NLRP3 upregulation and caspase-1 cleavage were similar in both LPS-treated WT and Gsdmd^−/− mice. Plasma from each mouse was collected and analyzed for c IL-1β and d HMGB1 by ELISA. For c and d, data indicate WT saline (n = 13), WT LPS (n = 8), Gsdmd^−/− saline (n = 9), Gsdmd^−/− LPS (n = 5) mice per group. Note that Gsdmd^−/− mice do not secrete IL-1β in response to LPS, but HMGB1 secretion is unchanged. Data with error bars are represented as mean ± SEM. Adjusted p values, provided in the panels, and n.s. not significant as determined by two-way ANOVA with Tukey’s multiple comparison correction, two-sided.