LPS Induces Active HMGB1 Release From Hepatocytes Into Exosomes Through the Coordinated Activities of TLR4 and Caspase-11/GSDMD Signaling

Abstract

High-mobility group box-1 (HMGB1), a ubiquitous nuclear protein, acts as a late mediator of lethality when released extracellularly during sepsis. The major source of circulating HMGB1 in sepsis is hepatocytes. However, the mechanism of HMGB1 release of hepatocytes during sepsis is not very clear. We have previously shown that bacterial endotoxin [lipopolysaccharide (LPS)] sensing pathways, including Toll-like receptor (TLR)4 and caspase-11, regulate hepatocyte HMGB1 release in response to LPS. Here, we report the novel function of caspase-11 and gasdermin D (GsdmD) in LPS-induced active HMGB1 released from hepatocytes. HMGB1 release during endotoxemia was caspase-11/GsdmD dependent via an active way in vivo and in vitro. Caspase-11/GsdmD was responsible for HMGB1 translocation from nucleus to the cytoplasm via calcium changing-induced phosphorylation of calcium-calmodulin kinase kinase (camkk)β during endotoxemia. Cleaved GsdmD accumulated on the endoplasmic reticulum, suggesting this may lead to calcium leak and intracellular calcium increase. Furthermore, we investigated that exosome was an important pathway for HMGB1 release from hepatocytes; this process was dependent on TLR4, independent of caspase-11 and GsdmD in vivo and in vitro. These findings provide a novel mechanism that TLR4 signaling results in an increase in caspase-11 expression, as well as increased exosome release, while caspase-11/GsdmD activation/cleavage leads to accumulation of HMGB1 in the cytoplasm through a process associated with the release of calcium from the endoplasmic reticulum and camkkβ activation.

Keywords: calcium; caspase-11; endotoxemia; extracellular vesicles; gasdermin D (GsdmD); innate immunity.

Figure 1

Lipopolysaccharide (LPS)-induced high-mobility group box-1 (HMGB1) release from hepatocytes is caspase-11 and gasdermin D (GsdmD) dependent. Immunoblots for HMGB1, β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), or tubulin in the supernatant and cell lysates (Cell-Ext) in (A) wild-type (WT) and caspase-11^−/− [caspase-11 knockout (KO)] and (D) WT and GsdmD^−/− (GsdmD KO) hepatocytes at 24 h after LPS (1 μg/ml). (B,E) Hepatocytes pretreated with siRNA to knock down caspase-11 or GsdmD prior to LPS treatment for 24 h as above. (C,F) Plasma HMGB1 levels in WT, caspase-11^−/−, or GsdmD^−/− mice at 4 h after intraperitoneal injection with LPS (5 mg/kg). Each point represents one mouse. *P < 0.05. n = 3.

Figure 2

Lipopolysaccharide (LPS) does not induce hepatocyte death in vitro or in vivo. (A) Immunofluorescence of liver from wild-type (WT) mice at 4 h after intraperitoneal injection with LPS (5 mg/kg). TMR = red; 4′,6-diamidino-2-phenylindole (DAPI) = blue; actin = white. (B) Plasma alanine aminotransferase (ALT) level in WT mice at 4 h after intraperitoneally injection with LPS (5 mg/kg). Each point represents one mouse. (C) WT hepatocytes were treated with LPS (1 μg/ml) for 24 h. Cytotoxicity was measured by using lactate dehydrogenase (LDH) release in the culture media. Data are expressed as mean ± SEM. (D) Immunofluorescence of Zombie-red staining (cell death) of WT hepatocytes 24 h after treatment with LPS (1 μg/ml). DAPI = blue. NS, no significant difference. n = 3.

Figure 3

Caspase-11 and gasdermin D (GsdmD) are required for high-mobility group box-1 (HMGB1) translocation to the cytosol in response to lipopolysaccharide (LPS). (A,C) Immunoblots for HMGB1, caspase-11, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), or tubulin in the cytoplasmic (Cyto) and nuclear (Nuc) lysates from wild-type (WT), caspase-11^−/− [Casp-11 knockout (KO)], or GsdmD^−/− (GsdmD KO) hepatocytes treated with LPS (1 μg/ml) for the time indicated. (B,D) Immunofluorescence of WT, caspase-11^−/−, or GsdmD^−/− hepatocytes treated with LPS (1 μg/ml) for the indicated time. HMGB1 = red; 4′,6-diamidino-2-phenylindole (DAPI) = blue; colocalization = magenta. n = 3.

Figure 4

Caspase-11 and gasdermin D (GsdmD) inhibited the phosphorylation of calcium-calmodulin kinase kinase (camkk)β. (A,B) Immunoblots for phospho-camkkβ (P-camkkβ) and total-camkkβ (T-camkkβ) in whole cell lysates from wild-type (WT), caspase-11^−/− [Casp-11 knockout (KO)], or GsdmD^−/− (GsdmD KO) hepatocytes were treated with lipopolysaccharide (LPS) (1 μg/ml) for the indicated times. (C) Intracellular Ca²⁺ measured by fluorescence intensity of Fura-2AM (F340/F380) in WT hepatocytes treated with or without LPS (1 μg/ml) for the indicated time. Data are expressed as mean ± SEM. (D) Intracellular Ca²⁺ measured by fluorescence intensity of Fura-2AM (F340/F380) in WT, caspase-11^−/−, or GsdmD^−/− hepatocytes treated with or without LPS (1 μg/ml) at indicated times. Data are expressed as relative levels compared with baseline controls and as mean ± SEM. ^*P < 0.05. n = 3.

Figure 5

Gasdermin D (GsdmD) is recruited to the endoplasmic reticulum (ER). (A) Morphological structure of isolated mouse ER as seen on standard transmission electron microscopy (TEM) (100,000 × magnification; scale bar, 100 nm). (B) Immunoblots for calnexin, ERp72, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), tubulin, and specificity protein 1 (SP1) in isolated ER, whole cell (Cell lysate), cytoplasm (Cyto), and nucleus (Nuc) of wild-type (WT) hepatocytes. (C) Immunoblots for GsdmD in ER (ER lysate) isolated from WT and caspase-11^−/− [Casp-11 knockout (KO)] hepatocytes after treatment with lipopolysaccharide (LPS) (1 μg/ml) for 8 h. n = 3.

Figure 6

Hepatocytes release high-mobility group box-1 (HMGB1) in exosomes. Immunoblots for CD81, TSG101, and HMGB1 in exosomes isolated from (A) cell culture media from wild-type (WT) hepatocytes treated with lipopolysaccharide (LPS) (1 μg/ml) for 24 h and (B) plasma from WT mice intraperitoneally injected with LPS (5 mg/kg) for 4 h. NanoSight™ analysis of exosomes isolated from (C) cell culture media and (D) mouse plasma. (E,F) Immunoblots for HMGB1 in the supernatant and cell lysates of hepatocytes treated with GW4869 or spiroepoxide for 2 h, then challenged with LPS (1 μg/ml) for 24 h. (G) Plasma HMGB1 level in WT mice injected intravenously with GW4869 (2.5 mg/kg) for 1 h prior to intraperitoneal injection of LPS (5 mg/kg) for 4 h. (H) Plasma HMGB1 level in WT mice pretreated for 48 h with scrambled (control) or Rab27a-targeted shRNA via intravenous injection followed by intraperitoneal injection with LPS (5 mg/kg) for 4 h. Each point represents one mouse. *P < 0.05. n = 3.

Figure 7

High-mobility group box-1 (HMGB1) release in exosomes is dependent on Toll-like receptor (TLR)4, caspase-11, and gasdermin D (GsdmD). (A,B) Immunoblots for HMGB1 in exosomes isolated from cell culture media from wild-type (WT), TLR4^−/− [TLR4 knockout (KO)], caspase-11^−/− (Casp-11 KO), or GsdmD^−/− (GsdmD KO) hepatocytes were treated with lipopolysaccharide (LPS) (1 μg/ml) for 24 h. (C–E) Immunoblots for HMGB1 in exosomes isolated from plasma of WT, TLR4^−/−, caspase-11^−/−, or GsdmD^−/− mice treated with LPS (5 mg/kg) for 4 h. Each lane represents one mouse. n = 3.

Figure 8

Exosome release from hepatocytes is dependent on Toll-like receptor (TLR)4 but independent of caspase-11 and gasdermin D (GsdmD). (A,B) Immunoblots of CD81 and TSG101 in exosomes isolated from cell culture media of wild-type (WT), TLR4^−/−, caspase-11^−/−, or GsdmD^−/− hepatocytes treated with lipopolysaccharide (LPS) (1 μg/ml) for 24 h. (C–E) Immunoblots for CD81 and TSG101 in exosomes isolated from plasma of WT, TLR4^−/−, caspase-11^−/−, or GsdmD^−/− mice treated with LPS (5 mg/kg) for 4 h. Each lane represents one mouse. (F) CD81 levels in exosomes isolated from plasma of WT, TLR4^−/−, caspase-11^−/−, or GsdmD^−/− mice treated with LPS (5 mg/kg) for 4 h. Each point represents one mouse. (G) A proposed model describing TLR4 signaling results in an increase in caspase-11 expression, as well as increased exosome release, while caspase-11/GsdmD activation/cleavage leads to accumulation of high-mobility group box-1 (HMGB1) in the cytoplasm through a process associated with the release of calcium from the endoplasmic reticulum and calcium-calmodulin kinase kinase (camkk)β activation. *P < 0.05. NS, no significant difference. n = 3.