GITR exacerbates lysophosphatidylcholine-induced macrophage pyroptosis in sepsis via posttranslational regulation of NLRP3

Abstract

The NLRP3 inflammasome functions as an inflammatory driver, but its relationship with lipid metabolic changes in early sepsis remains unclear. Here, we found that GITR expression in monocytes/macrophages was induced by lysophosphatidylcholine (LPC) and was positively correlated with the severity of sepsis. GITR is a costimulatory molecule that is mainly expressed on T cells, but its function in macrophages is largely unknown. Our in vitro data showed that GITR enhanced LPC uptake by macrophages and specifically enhanced NLRP3 inflammasome-mediated macrophage pyroptosis. Furthermore, in vivo studies using either cecal ligation and puncture (CLP) or LPS-induced sepsis models demonstrated that LPC exacerbated sepsis severity/lethality, while conditional knockout of GITR in myeloid cells or NLRP3/caspase-1/IL-1β deficiency attenuated sepsis severity/lethality. Mechanistically, GITR specifically enhanced inflammasome activation by regulating the posttranslational modification (PTM) of NLRP3. GITR competes with NLRP3 for binding to the E3 ligase MARCH7 and recruits MARCH7 to induce deacetylase SIRT2 degradation, leading to decreasing ubiquitination but increasing acetylation of NLRP3. Overall, these findings revealed a novel role of macrophage-derived GITR in regulating the PTM of NLRP3 and systemic inflammatory injury, suggesting that GITR may be a potential therapeutic target for sepsis and other inflammatory diseases. GITR exacerbates LPC-induced macrophage pyroptosis in sepsis via posttranslational regulation of NLRP3. According to the model, LPC levels increase during the early stage of sepsis, inducing GITR expression on macrophages. GITR not only competes with NLRP3 for binding to the E3 ligase MARCH7 but also recruits MARCH7 to induce the degradation of the deacetylase SIRT2, leading to decreasing ubiquitination but increasing acetylation of NLRP3 and therefore exacerbating LPC-induced NLRP3 inflammasome activation, macrophage pyroptosis and systemic inflammatory injury.

Keywords: GITR; Macrophage; NLRP3 inflammasome; Posttranslational modification; Sepsis.

GITR exacerbates LPC-induced macrophage pyroptosis in sepsis via posttranslational regulation of NLRP3. According to the model, LPC levels increase during the early stage of sepsis, inducing GITR expression on macrophages. GITR not only competes with NLRP3 for binding to the E3 ligase MARCH7 but also recruits MARCH7 to induce the degradation of the deacetylase SIRT2, leading to decreasing ubiquitination but increasing acetylation of NLRP3 and therefore exacerbating LPC-induced NLRP3 inflammasome activation, macrophage pyroptosis and systemic inflammatory injury.

Fig. 1

GITR expression on monocytes is upregulated in sepsis and is positively correlated with serum LPC levels. A Low-density lipoprotein cholesterol (LDL-C) levels in the serum of patients with different SOFA scores. LPC levels in the serum of healthy donors vs. sepsis patients B or patients with different SOFA scores C were measured by ELISA. D The correlation between peripheral monocyte number and serum LPC levels. E The mRNA levels of the indicated inflammatory-related genes in peripheral blood mononuclear cells (PBMCs) from healthy donors (H1–H3) and sepsis patients (S1–S3) were measured by real-time PCR and visualized in heatmap. The percentages of GITR⁺CD11b⁺ cells were detected by flow cytometry in sepsis patients vs. healthy donors F or patients with different SOFA scores G, and their correlation with serum LDL-C H or LPC levels I was analyzed. B6 mice underwent cecal ligation and puncture (CLP) or sham laparotomy. At 12 h after the operation, the percentages of GITR⁺CD11b⁺ cells and LPC levels in PBMCs, spleens and lungs were analyzed by flow cytometry J and ELISA K, respectively

Fig. 2

GITR promotes LPC uptake and pyroptosis in macrophages. BMDMs from WT, Gitr^−/− or Casp1^−/− mice were treated with LPC (100 μM) for the indicated time periods and analyzed for the following assays. A, B The mRNA and protein levels of GITR (Tnfrsf18) in BMDMs were analyzed by real-time PCR and flow cytometry, respectively, before and 3 h, 6 h or 12 h after treatment. C The mRNA level of GITR after treatment with the JAK1/2 inhibitor ruxolitinib and the NF-κB inhibitor BAY-7085. At 3 h after LPC treatment, intracellular LPC concentrations in WT or Gitr^−/− BMDMs were measured by ELISA D, and LDH release E and IL-1β secretion F were analyzed in WT BMDMs at the indicated concentrations. For WT or Casp1^−/− BMDMs, G the cleavage of GSDMD, caspase-1 and IL-1β in the supernatants or cell lysates was detected by immunoblotting, and H LDH release in the supernatants was determined by an LDH kit. LDH release I and the secretion of IL-1β J, TNF-α K and IL-6 L in the supernatants of WT and Gitr^−/− BMDMs were measured by an LDH kit and ELISA

Fig. 3

GITR specifically promotes NLRP3 inflammasome activation. A–D BMDMs from Gitr^−/− (KO) vs. wild-type C57BL/6 (WT) mice A, C or immortalized murine BMDM cell line iBMDMs stably expressing GITR (OE) vs. control vectors (Vec) B, D were primed with LPS (200 ng/mL) for 3 h and then treated with nigericin or ATP for another 40 min. E–H WT or Gitr^−/− BMDMs were primed with LPS and then transfected with LPS E, flagellin F or poly(dA:dT) G for 6 h. I–J WT BMDMs were pretreated with MCC950 (10 μM) for 1 h, followed by LPC (100 μM) stimulation for 3 h. K WT or Gitr^−/− BMDMs were treated with LPC (100 μM) for 3 h. For all the cells mentioned above, caspase-1 or IL-1β cleavage in the supernatants or GSDMD cleavage in the cell lysates were detected by Western blotting, and IL-1β secretion in the supernatants was measured by ELISA

Fig. 4

GITR deficiency reduces the protein level of NLRP3 and inflammasome assembly. A–C WT and Gitr^−/− BMDMs were stimulated with LPS (200 ng/mL), and then Nlrp3 and Il1b mRNA levels were measured by real-time PCR A, B. The protein levels of NLPR3, ASC, caspase-1 and IL-1β were examined by Western blotting C at the indicated timepoints (0, 1, 2, 3 h) after LPS stimulation. D, F WT and Gitr^−/− BMDMs were mock treated or primed with LPS, followed by stimulation with or without nigericin. ASC oligomerization in cross-linked cytosolic pellets was detected by Western blotting D. Cell lysates were immunoprecipitated with anti-NLRP3 Abs, followed by immunoblot analysis of ASC and NLRP3 F. E ASC specks in LPS-primed WT or Gitr^−/− BMDMs with or without nigericin stimulation were visualized using confocal microscopy. Scale bar = 10 μM

Fig. 5

GITR inhibits the ubiquitination of NLRP3. A Immunoblot analysis of lysates from WT or Gitr^−/− BMDMs stimulated with LPS for 3 h, followed by IP with an anti-NLRP3 antibody and probing with an anti-ubiquitin antibody. B Immunoblot analyses of lysates from HEK293T cells transfected with GITR-Myc, NLRP3-Flag and HA-tagged ubiquitin (Ub-HA), followed by IP with anti-Flag, and probed with anti-HA. C Immunoblot analyses of lysates from HEK293T cells transfected with GITR-Myc, NLRP3-Flag, WT Ub-HA or K48 mutant Ub-HA, followed by IP with anti-Flag, and probed with anti-HA. D Immunoblot analyses of lysates from HEK293T cells transfected with GITR-Myc, Ub-HA, WT NLRP3-Flag/K324 mutant NLRP3-Flag/K430 mutant NLRP3-Flag/K689 mutant NLRP3-Flag/K799 mutant NLRP3-Flag, followed by IP with anti-Flag, and probed with anti-HA. E Flag-tagged NLRP3 or its truncated protein, GITR-Myc and MARCH7-HA were individually transfected into HEK293T cells. The cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with the indicated antibodies. F Immunoblot analysis of lysates from HEK293T cells transfected with GITR-Myc, NLRP3-Flag and MARCH7-HA, followed by IP with anti-HA and probing with anti-Flag and anti-Myc antibodies. G HEK293T cells were transfected with NLRP3-Flag, MARCH7-HA and the indicated amount (μg) of GITR-Myc. Co-IP analysis of Flag and Myc with anti-HA antibodies was subsequently performed for 48 h. H The control GST and GST-MARCH7 fusion proteins were purified with GST beads and mixed with purified His-GITR and Flag-NLRP3, after which the interactions were detected by Western blotting. I Immunoblot analysis of lysates from HEK293T cells transfected with GITR-Myc, NLRP3-Flag, Ub-HA and MARCH7-HA, followed by IP with anti-Flag, probed with anti-HA and Myc. J–K BMDMs were either mock-treated J or treated with LPC (100 μM) for 3 h K and fractionated using a subcellular protein fractionation kit, and the cellular localization of GITR/MARCH7/NLRP3 was detected by Western blotting. CE cytoplasmic extract, ME membrane extract, NE nuclear extract, CB chromatin-bound extract, and PE pellet extract

Fig. 6

GITR promotes the acetylation of NLRP3. A Immunoblot analysis of lysates from WT or Gitr^−/− BMDMs stimulated with LPS and then subjected to IP with an anti-NLRP3 antibody and probed with an anti-acetyl-k antibody. Immunoblot analysis of lysates from HEK293T cells transfected with GITR-Myc or NLRP3-Flag, followed by IP with anti-acetyl-k, probing with anti-Flag B, or IP with anti-Flag, and probing with anti-acetyl-k C. D Immunoblot analyses of lysates from HEK293T cells transfected with GITR-Myc, NLRP3-Flag, or HA-tagged ubiquitin (Ub-HA) followed by IP with anti-Flag and probing with anti-Acetyl-K. E WT and Gitr^−/− BMDMs were stimulated with LPS (100 ng/mL) or LPC (100 μM) for 3 h, after which the protein levels of SIRT2 were examined by Western blotting. F HEK293T cells were transfected with SIRT2-Flag (500 ng) and MARCH7-HA (0/200/400/800/1600 ng), and the expression of the proteins was detected by Western blotting. G Immunoblot analyses of lysates from HEK293T cells transfected with MARCH7-Myc, SIRT2-Flag and HA-tagged K48 ubiquitin (K48-HA) followed by IP with anti-Flag and probing with anti-HA. H HEK293T cells were transfected with SIRT2-Flag (500 ng) and GITR-Myc (0/100/200/400 ng), subjected to IP with anti-Flag, and probed with anti-Myc

Fig. 7

GITR aggravates NLRP3-dependent inflammatory injury and lethality in sepsis. The CLP operation or sham laparotomy was performed on the following mice. Survival curves A and representative H&E staining images of lung sections B of Gitr^fl/flLyz2-Cre and Gitr^fl/fl mice are shown. IL-1β levels in the spleen, lungs and liver were measured by ELISA C. D–F WT and Nlrp3^−/− mice were intraperitoneally injected with 0.5 mg of DTA-1 or IgG 6 h before CLP or sham operation, and survival curves D,E and representative H&E staining images of lung sections F are shown. Scale bar = 200 μM

Fig. 8

LPC aggravates inflammatory injury and lethality during sepsis in a GITR-dependent manner. Gitr^fl/flLyz2-cre (CKO) and Gitr^fl/fl (WT) mice were intraperitoneally injected with LPC (10 mg/kg) or control solvent, followed by an intraperitoneal injection of LPS (20 mg/kg) or solvent control after 6 hours, and the inflammatory injury or survival rate was analyzed at the indicated time points. A Schematic diagram of the sepsis model. B–F Survival curves B and representative H&E staining images of lung sections C of CKO and WT mice are shown. IL-1β levels in the liver, lungs and spleen were measured by ELISA D–F