Active Release of eCIRP via Gasdermin D Channels to Induce Inflammation in Sepsis

Abstract

Extracellular cold-inducible RNA-binding protein (eCIRP) is an inflammatory mediator to cause inflammation and tissue injury in sepsis. Gasdermin D (GSDMD) is a protein that, when cleaved, forms pores in the cell membrane releasing intracellular contents into the extracellular milieu to exacerbate inflammation. We hypothesize that eCIRP is released actively from viable macrophages via GSDMD pores. We found that lipopolysaccharides (LPS) induced eCIRP secretion from macrophages into the extracellular space. LPS significantly increased the expression of caspase-11 and cleavage of the GSDMD as evidenced by increased N-terminal of GSDMD expression in RAW264.7 cells and mouse primary peritoneal macrophages. GSDMD inhibitor disulfiram decreased eCIRP release in vitro. Treatment with glycine to prevent pyroptosis-induced cell lysis did not significantly decrease eCIRP release from LPS-treated macrophages, indicating that eCIRP was actively released and was independent of pyroptosis. Downregulation of GSDMD gene expression by siRNA transfection suppressed eCIRP release in vitro following LPS stimulation. Moreover, GSDMD^−/− peritoneal macrophages and mice had decreased levels of eCIRP in the culture supernatants and blood treated with LPS in vitro and in vivo, respectively. GSDMD inhibitor disulfiram inhibited serum levels of eCIRP in endotoxemia and cecal ligation and puncture (CLP)-induced sepsis. We conclude that eCIRP release from living macrophages is mediated through GSDMD pores, suggesting that targeting GSDMD could be a novel and potential therapeutic approach to inhibit eCIRP-mediated inflammation in sepsis.

Introduction

In sepsis, inflammation is caused by pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) (1, 2). Both are recognized by immune cells through a group of pattern-recognition receptors (PRRs), followed by the activation of downstream pathways, leading to the production of inflammatory cytokines, chemokines, and vasoactive peptides (3, 4). Various DAMPs such as high mobility group box 1 (HMGB1), extracellular cold-inducible RNA-binding protein (eCIRP), heat shock proteins, S100 proteins, histones, and mitochondrial DNA have been identified as alarmin danger signals in triggering inflammatory responses in sepsis (5–8).

When cells are exposed to stressors such as lipopolysaccharides (LPS), hypothermia, hypoxia, and ultra-violet irradiation, the expression of CIRP is upregulated, and translocated from the nucleus to cytoplasmic stress granules, and then released to the extracellular space (9). eCIRP was discovered to act as a DAMP, causing inflammation and tissue injury in sepsis (7), acute lung injury (10), and ischemia/reperfusion injury (11, 12). Conversely, blockade of eCIRP has been shown to play a protective role and attenuated inflammation and organ injury in the septic mice as well as other inflammatory diseases (7, 12, 13). While the impact of eCIRP as a DAMP is well studied, precisely how eCIRP is released from cells remains elusive. Therefore, identifying the release mechanism of eCIRP is implicated in inflammation and sepsis.

Gasdermins D (GSDMD) is an effector protein that causes membrane permeabilization and pyroptosis (14–16). During infectious and sterile inflammation, both PAMPs and DAMPs are recognized by immune cells and initiate the activation of caspases, ultimately leading to cleavage of GSDMD to generate its active form, N-terminal fragment (GSDMD-NT), which oligomerizes in the cell membrane and forms membrane pores (17). GSDMD is activated by canonical and non-canonical inflammasome pathways, which involve caspase-1, and caspase-11, respectively (14, 15, 18, 19). Caspase-1 also processes pro-IL-1β to generate mature IL-1β, which is released by cell lysis during pyroptosis (20). The non-canonical inflammasome pathway is triggered by LPS in the cytoplasm of infected cells. Direct binding of LPS to the protein procaspase-11 causes the protein dimerization to become active caspase-11 (14, 19). The formation of membrane pores is followed by cell rupture, termed pyroptosis, with a burst release of pro-inflammatory cytokines and large intracellular components. However, in some circumstances, cells survive after GSDMD pores are repaired without membrane rupture, which are called hyperactivated cells (21–23). These pores in the plasma membrane act as channels for releasing low molecular weight cellular contents, such as IL-1β, IL-18, and ATP, into the extracellular space to initiate inflammation (16, 23, 24).

The recent discovery of the pore-forming protein GSDMD has provided a plausible mechanism for eCIRP active release from cells during inflammation and stress conditions. Herein, we hypothesized that eCIRP could be secreted by macrophages through GSDMD pores in sepsis. We showed that eCIRP was released from macrophages after being stimulated by LPS without cell lysis. Furthermore, LPS-induced eCIRP release was mediated through GSDMD-dependent pathway, which was prevented by GSDMD inhibitor disulfiram and gene knockdown by siRNA. In addition, a pharmacologic inhibitor of GSDMD disulfiram prevented the release of eCIRP and pro-inflammatory cytokines in septic mice. Finally, we found that GSDMD deficiency reduced eCIRP release in mouse models of endotoxemia and polymicrobial sepsis. Together, these data suggest GSDMD as a novel mechanism of eCIRP release from macrophages in sepsis.

Materials and methods

Mice

Wild-type (WT) C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA). Gasdermin D knockout mice (GSDMD^−/−) with C57BL/6 background were purchased from Jackson Laboratory (Bar Harbor, ME). Age (8-12 weeks) matched, male, healthy mice were used in all experiments. All mice were housed and kept at a temperature of 22 °C with free access to food and water throughout the experiment. The mice were kept on a 12 h light/dark cycle. All animal experiments were performed by the guidelines for using experimental animals by the National Institutes of Health (Bethesda, MD). All the mice experiments were approved by the Institutional Animal Care and Use Committees (IACUC) of the Feinstein Institutes for Medical Research.

Mouse models of sepsis and endotoxemia

Sepsis was induced in C57BL/6 mice by CLP as previously described (25). Mice were anesthetized with 2% isoflurane with oxygen at 1 L/min and then placed on an operating table in the supine position. A midline incision of the abdomen was made, and the abdomen was cut open. The cecum was exposed and ligated with 4-0 silk suture at 1 cm from the distal site. Two punctures were made with a 22-gauge needle at the end of the cecum, and a small amount of feces were extruded from the punctures. The cecum was returned to the abdominal cavity, and the abdomen was closed in layers. Resuscitative fluid (normal saline, 50 ml/kg) was administrated by subcutaneous injection. All operations were the same in the sham group except for no puncture and ligation of the cecum.

WT and GSDMD^−/− mice were intraperitoneally (i.p.) injected with LPS from E. coli serotype O55:B5 (Cat. No. L6529, Sigma-Aldrich, St. Louis, MO) at 10 mg/kg or PBS (vehicle). All animals survived to 20 h post-treatment, at which point they were humanely euthanized for plasma and tissue collection. All animals were included in the analyses, with the investigators blinded to both genotype and treatment groups.

Disulfiram administration

C57BL/6 WT mice were randomly divided into four groups: sham, sham + disulfiram, CLP or LPS + vehicle, and CLP or LPS + disulfiram. GSDMD inhibitor disulfiram (Cat. No. D2950000, Sigma-Aldrich) was dissolved in sterile corn oil and injected intraperitoneally (50 mg/kg) 0 h or 1 h before CLP operation. An equal volume of corn oil (vehicle) was intraperitoneally injected as a control. The dose of disulfiram was selected based on previous studies using disulfiram to treat septic mice (26). Blood and tissue samples were collected 20 hours post-CLP or LPS challenge to measure cytokines and protein expression. Plasma obtained after centrifugation at 2000 × g for 10 min was analyzed for inflammatory cytokines.

Isolation of mouse peritoneal macrophages

A method from the previous protocol was used for the isolation of mouse peritoneal macrophages (27). Briefly, the mice were sacrificed by CO₂ asphyxiation and sprayed with 70% ethanol. The peritoneal cavity was gently washed with phosphate-buffered saline (PBS, Invitrogen) supplemented with 2% fetal bovine serum (FBS, Invitrogen, Waltham, MA). The lavage was aspirated from the peritoneum using a 10 ml syringe after gently shaking the peritoneal cavity. The peritoneal exudate cells were centrifuged in a refrigerated (4 °C) centrifuge at 400 × g for 10 min. The supernatant was discarded, and the cell pellet was re-suspended in RPMI-1640 supplemented with 10% FBS and 100 U/ml penicillin, and the number of the cells was counted using a hemocytometer. Cells were seeded into 6-well tissue culture plates at a density of 1×10⁶ cells/well and cultured for 4 h at 37 °C to allow them to adhere to the substrate. Non-adherent cells were removed by gently washing with warm PBS two times. At this time, cells should be greater than 90% macrophages. Furthermore, the macrophages were allowed to adhere overnight (37 °C, 5% CO₂) and washed with a fresh medium to remove unattached cells before being used for experiments.

Disulfiram treatment of macrophages in vitro

RAW 264.7 cells were purchased from ATCC and cultured in DMEM medium supplemented with 10% heat-inactivated FBS, 1% penicillin-streptomycin, and 2 mM glutamine at 37 °C in an incubator supplemented with 5% CO₂. RAW 264.7 cells or primary peritoneal macrophages were plated into 12-well plates and pre-treated with or without GSDMD inhibitor disulfiram in the indicated dose (0.1, 1, 10, and 20 μM, 1 h prior), followed by stimulation with or without LPS (100 ng/ml) for 24 hours. After stimulation, cell culture supernatants were collected and cleared of debris by centrifugation at 400 × g for 10 min and stored at −20 °C.

Lactate dehydrogenase (LDH) assays and ELISA

For LDH assays, RAW 264.7 cells or primary peritoneal macrophages were seeded in 96 well plates. Cells were stimulated in opti-MEM-medium (Invitrogen) with or without different doses of LPS as indicated in the figure legends for 24 hours in the following day. Glycine (5 mM) was added at the same time as LPS. At the end of incubations, cell culture supernatants were collected and cleared by centrifugation at 400 × g for 10 min. LDH assays of cell supernatants were performed by CyQUANTTM LDH Cytotoxicity Assay kit (Cat. No. 2298167, Invitrogen) according to the manufacturer’s protocol. The absorbance was measured at 490 nm and 680 nm using BioTek Synergy Neo2 multi-mode reader (BioTek Instruments, Inc.). Percentages of cytotoxicity was calculated by using the following formula: % of cytotoxicity = (compound-treated LDH activity – spontaneous LDH activity)/(maximum LDH activity – spontaneous LDH activity) × 100. IL-6, TNF-α, and eCIRP levels were quantitatively measured from mouse plasma or cell supernatants by ELISA kits according to the manufacturer’s protocols. IL-6 (Cat. No. 555240) and TNF-α (Cat. No. 558534) ELISA kits were purchased from BD Biosciences. eCIRP ELISA kits (Cat. No. E12931m) were bought from WUHAN EIAAB science co., LTD.

Western blotting

RAW 264.7 cells or primary macrophages were seeded in 12-well plates as described above. At the end of the incubation, cell culture supernatants were collected and cleared of debris by centrifugation at 400 × g for 10 min. Supernatants were trichloroacetic acid (TCA, Cat. No. BP555-250, Fisher Scientific) precipitated with 10% TCA on ice and washed with acetone. Protein precipitates were dissolved with 1× tris buffer saline and 4× LDS sample buffer. Proteins were denatured by being heated for 5 min at 95 °C. Cells were washed with PBS twice and lysed in RIPA lysis buffer containing protease inhibitors (Pierce, Protease inhibitor tablet Cat. No. A32963, Thermo Scientific). Whole-cell lysates were centrifugated at 4 °C, 12000 rpm for 10 minutes. The supernatants were collected as proteins. Protein concentration in cell lysates was measured using the ABC protein assay kit (Cat. No. 5000002, Bio-Rad). Proteins were separated using NuPAGE 4–12% Bis-Tris gels (Invitrogen), transferred to PVDF membranes, and immunoblotted with the antibodies indicated in the figure legends according to the manufacturer’s recommendations. Antibodies used in this study are: GSDMD (Cat. No. 93709, 1:1000), cleaved GSDMD (Cat. No. 101375, 1:1000) and caspase-11 (Cat. No. 14340S) were from Cell Signaling Technology; CIRP (Cat. No. 10209-2-AP; 1:1000, Proteintech), β-actin (Cat. No. A5441, 1:10000, sigma), GAPDH (Cat. No. 60004-1-1g; 1:1000, Proteintech). After incubation of the blots with primary antibodies, membranes were washed 3 times, and then the blots were subsequently incubated with corresponding fluorescent secondary antibody (LI-COR, Lincoln, NE). Bands were detected using the Odyssey FC Dual-Mode Imaging system 2800 (LI-COR).

Gasdermin D siRNA transfection in macrophages

GSDMD siRNA (Cat. No. sc-145798) and negative control siRNA (Cat. No. sc-37007) were purchased from Santa Cruz Biotechnology. siRNA transfection reagent (Cat. No. sc-29528, Santa Cruz Biotechnology) was used for transfection of GSDMD siRNA into RAW 264.7 cells by following the protocol from the manufacturer. RAW 264.7 cells were seeded in a 12-well plate (2.5×10⁵ cells per well) in 1 ml antibiotic free DMEM medium supplement with 10 % FBS at 37 °C in a CO₂ incubator until the cells are 60-70 % confluent. GSDMD siRNA was mixed with siRNA transfection reagents and the mixture was incubated for 30 min at room temperature. The cell medium was changed to opti-medium before the transfection. Opti-medium containing the siRNA transfection mixture was added onto the washed cells. The cells were incubated for 6 hours at 37 °C in a CO₂ incubator. After that, normal DMEM medium containing 2 times serum and antibiotics concentration was added without removing the transfection medium. After culturing the cells for additional 18 hours, GSDMD expression in RAW 264.7 cells were checked by Western blot analysis. The sequence of GSDMD siRNA (m) is sense: GUCAAGUCUAGGCCAGAAATT; antisense: UUUCUGGCCUAGACUUGACTT.

Immunofluorescence staining

Before washing the RAW 264.7 cells, the cell nucleus was stained by using NucBlue™ Live cell stain ReadyProbes reagent (Hoechst33342, Invitrogen Cat. No. 2325910). Harvested cells were washed with cold PBS twice and the cell surface membrane was stained with MemBrite fix Cell Surface staining kit (Biotium, cat. No. 30093) according to the company’s instruction. Cells were rinsed with PBS and fixed with 4% methanol for 15 min at room temperature. After being washed with PBS 3 times, cells were blocked with PBS/5% BSA for 1 h at room temperature. Subsequently, cells were incubated with primary antibody against cleaved GSDMD (dilution 1:200; Cat. No. 101375, Cell Signaling Technology) overnight at 4 °C. Goat anti-rabbit IgG (dilution 1:250; Cat. No. D00304-15, LI-COR) served as a secondary antibody. Hoechst3334 was used for DNA dye. Cells were visualized by using Axio Observer.Z1/7 equipped with Zeiss LSM900 confocal microscopy system. The z-stack images of cells were acquired with Plan-Apochromat 63x/1.40 Oil DIC M27 objective lens. SR-4Y fast acquisition mode of Airyscan and 4× averaging was used. The images obtained by confocal microscope were merged and combined by FIJI Image J (28).

Homology modeling of CIRP and GSDMD

The amino acid sequences of mouse CIRP (P60824) and GSDMD (Q9D8T2) were retrieved from the Uniprot database. The models were generated using Iterative Threading ASSEmbly Refinement (I-TASSER) server (29) based on templates identified by the threading approach to maximize the percentage identity, sequence coverage and confidence. The CIRP structure has RNA binding domain (aa 6-84), disordered region (aa 70-172), and polar residues (aa 143-172). However, GSDMD has a pore-forming domain (aa 4-423), the homooligomeric ring-shaped pore complex contains 27-28 subunits when inserted in the membrane. The models were refined based on repetitive relaxations by short molecular dynamics simulations for mild (0.6 ps) and aggressive (0.8 ps) relaxations with 4 fs time step after structure perturbations. The model refinement enhanced certain parameters including, Rama favored residues and a decrease in poor rotamers. The docking of CIRP and GSDMD protein structure models was performed using ATTRACT tool (30), which uses an approach of conformational flexibility of binding partners. The docking process includes pre-calculation of potential energy on a grid, and then interactions are calculated by interpolation from nearest grid points. The protein-protein interactions between CIRP and GSDMD were calculated using the PDBePISA tool (31), which calculated the protein-protein interaction interface and thermodynamic properties of interaction.

Surface plasmon resonance assay

The surface plasmon resonance (SPR) assay was performed as described previously (32). To examine the direct interaction between rmCIRP and rmGSDMD, SPR (OpenSPR, Nicoya, Ontario, Canada) was performed between rmCIRP and rmGSDMD. rmCIRP was produced in-house as previously described (7) and rmGSDMD was purchased (Cat. No. MBS2105506, MyBioSource, San Diego, CA). rmCIRP was immobilized on carboxyl sensor and rmGSDMD was injected as an analyte in concentrations of 10 to 1000 nM. Binding reactions were performed in 50 mM Tris buffer, 150 mM NaCl, 0.05% P20, 0.2% Trehalose, 1% BSA, pH7.4. The carboxyl sensor was first cleaned by injection of 10 mM HCl 150 μl, followed by injection of 150 μl of the mixture of 1 aliquot of N-ethyl-N′-[3-diethylaminopropyl]-carbodiimide (EDC) and 1 aliquot of N-hydroxysuccinimide (NHS) to activate the sensor surface. An aliquot of 200 μl of 50 μg/ ml of the ligand diluted in 10 mM sodium acetate (pH5) was injected into flow cell-channel-2 of the sensor for immobilization. Next, 150 μl of 1 M ethylenediamine (pH8.5) was injected to deactivate the remaining active sites on channel 1&2. The flow cell-1 was used as a control to evaluate nonspecific binding. The binding analyses were performed at a flow rate of 40 μl per minute at 20°C. To evaluate the binding, the analyte ranging from 10 nM to 1μM were injected into flow cell-1 and flow cell-2, and the real-time interaction data were analyzed by TraceDrawer (Nicoya). The signals from the control channel (flow cell-1) were subtracted from the channel coated with the ligand (flow cell-2) for all samples. Data were globally fitted for 1:1 binding.

Statistical analysis

All statistical analyses were performed with GraphPad Prism version 7.0 software (GraphPad Software, La Jolla, CA). The Shapiro-Wilk test assessed the data for normality. Comparisons between two groups were performed with a two-tailed Student’s t-test. Comparisons between multiple groups were analyzed using a one-way or two-way analysis of variance and Tukey’s multiple comparisons test. The statistical significance was set at p value <0.05.

Results

LPS causes the active release of eCIRP from macrophages

In macrophage cell line RAW 264.7 cells, LPS significantly increased the release of eCIRP in a dose-dependent manner (Fig. 1A). To determine whether LPS causes the active release of eCIRP from live macrophages, we treated RAW 264.7 cells with glycine prior to the stimulation of the cells with LPS to prevent membrane rupture and pyroptosis-related cytolysis (21, 33). Glycine acts as an osmoprotectant to prevent pyroptosis-induced cell lysis (21). The effect of glycine was confirmed by inhibiting LDH release from LPS-treated macrophages considering LDH is released from cells when cells die, or their membranes are ruptured. We found that LPS alone increased LDH release into the culture supernatants of RAW 264.7 cells compared to the PBS-treated group. By contrast, glycine significantly decreased LPS-induced LDH release, indicating that glycine effectively abrogated pyroptosis-mediated cell damage (Fig. 1B). Interestingly, we found that eCIRP release from LPS-treated RAW 264.7 cells were marginally decreased and was not statistically significant under glycine-treated conditions, indicating that eCIRP was predominantly released via an active process, likely through the compromised (porous) cell membrane, apart from its passive release following cell lysis (Fig. 1C). Akin to these findings with RAW 264.7 cells, we found that treating primary mouse peritoneal macrophages with LPS significantly increased eCIRP release in a dose-dependent manner (Fig. 1D). As expected, treatment with glycine significantly reduced the LPS-induced LDH release from primary mouse peritoneal macrophages (Fig. 1E). Furthermore, we found that treatment of primary mouse peritoneal macrophages with glycine did not reduce the release of eCIRP in the culture supernatants compared to without glycine treated LPS stimulated cells (Fig. 1F). These data suggest that eCIRP is released from LPS-treated live macrophages via an active process.

Figure 1. LPS treatment causes active release of eCIRP from macrophages.

(A) RAW 264.7 cells were treated with different doses of LPS (10, 100, and 1000 ng/ml) for 24 hours. eCIRP present in the culture supernatants was quantified by Western blot. n=4-5 samples /group. *p<0.05 vs. (−) LPS. Samples tested passed the normality test. The blot presented was generated from a single experiment. The experiment was performed at least two times and the data presented are the sum of all values of these experiments. (B, C) RAW 264.7 cells were treated with or without LPS (indicated doses) or glycine (5 mM) for 24 hours. The release of (B) LDH in the cell supernatants was measured by CyQUANTTM LDH cytotoxicity assay kit, and (C) eCIRP levels in the culture supernatants were quantified by Western blot as shown in the gel image and quantitative bar diagram. n=5 samples/group. *p<0.05 vs. (−) LPS, (−) glycine; ^#p<0.05 vs. (+) LPS, (−) glycine. Samples tested passed the normality test. The blot presented was generated from a single experiment. The experiment was performed at least two times and the data presented are the sum of all values of these experiments. (D-F) LPS treatment leads to eCIRP release from live murine primary peritoneal macrophages. (D) Primary peritoneal macrophages isolated from C57BL/6 mice were treated with different doses of LPS (10, 100, and 1000 ng/ml) for 24 hours. eCIRP present in the extracellular supernatants was quantified by Western blot. n=4 samples/group. *p<0.05 vs. (−) LPS. Samples tested passed the normality test. The cells isolated from several mice were pooled and then plated into several wells per group serving as the technical replicates. The blot presented was generated from a single experiment. The experiment was performed at least two times and the data presented are the sum of all values of these experiments. (E, F) Mice primary peritoneal macrophages were treated with or without LPS (indicated doses) or glycine (5 mM) for 24 hours. (E) LDH release in the cell supernatants was measured by CyQUANTTM LDH cytotoxicity assay kit, and (F) eCIRP present in the cell culture supernatants was quantified by Western blot as shown in the blot image and quantitative bar diagram. n=5 samples for LPS-treated groups. *p<0.05 vs. (−) LPS, (−) glycine; #p<0.05 vs. (+) LPS, (−) glycine. Data are expressed as means ± SEM. Groups were compared by one-way ANOVA and Tukey’s multiple comparisons test. LPS vs LPS+Glycine did not show a statistically significant difference. n.s. = non-significant. Samples tested passed the normality test. The cells isolated from several mice were pooled and then plated into several wells per group serving as the technical replicates. The blot presented was generated from a single experiment. The experiment was performed at least two times and the data presented are the sum of all values of these experiments. PS, Ponceau S red staining.

LPS induces eCIRP release from macrophages through gasdermin D pores

GSDMD is activated by inflammatory caspases (caspase-1, −4, −5, and −11) in response to microbial infection and danger signals to generate an N-terminal cleavage product, GSDMD-NT, which binds to the cell membrane and oligomerizes in membranes to form membrane pores (34). Here, we found that treatment of RAW 264.7 cells with LPS significantly increased the expression of caspase-11 and the activation of the GSDMD as reflected by increased levels of GSDMD-NT (Fig. 2A). To determine if GSDMD mediates LPS-induced eCIRP release from macrophages, we used disulfiram to inhibit GSDMD pore formation. Disulfiram was identified as an inhibitor of GSDMD pore formation, thereby inhibiting pyroptosis and cytokines release without affecting GSDMD cleavage (26). Treatment of the RAW 264.7 macrophages with disulfiram significantly reduced GSDMD-mediated eCIRP release from LPS-stimulated macrophages in a dose-dependent manner (Fig. 2B). Akin to the RAW 264.7 cells, LPS treatment of primary murine peritoneal macrophages increased the generation of GSDMD-NT (Fig. 2C). Interestingly, the cells treated with disulfiram significantly decreased eCIRP release from primary mouse peritoneal macrophages treated with LPS (Fig. 2D). Using a human leukemia monocytic cell line, THP-1 cells, we confirmed that LPS induced eCIRP release occurred via GSDMD activation (Supplemental Fig. 1). Furthermore, using another bacterial product, peptidoglycan (PGN), from Gram-positive bacteria as a stimulant of mouse peritoneal macrophages, we found the increased formation of GSDMD-NT and the release of eCIRP from macrophages (Supplemental Fig. 2). These data indicate that LPS induces eCIRP’s active release from macrophages through GSDMD pores.

Figure 2. LPS-induced eCIRP release from macrophages occurs through gasdermin D.

RAW 264.7 cells were treated with or without LPS (100 ng/ml) for 24 hours. (A) After LPS stimulation, cell culture supernatants and cell lysates were collected. Caspase-11 expression and GSDMD cleavage protein (GSDMD-NT) were quantified by Western blot with β-actin as a loading control. Western blot images and quantitative bar diagrams are shown. n=5-6/group. *p<0.05 vs. (−) LPS. Groups were compared by a student’s t-test. The blot presented was generated from a single experiment. The experiment was performed at least two times and the data presented are the sum of all values of these experiments. (B) RAW 264.7 cells were pre-treated with or without disulfiram (indicated doses) for 1 h and then stimulated with LPS (100 ng/ml) for 24 h. eCIRP present in the culture supernatants was analyzed by Western blot and shown as blot image and quantitative bar diagram. n=5 samples/group. *p<0.05 vs. (−) LPS, (−) disulfiram; ^#p<0.05 vs. (+) LPS, (−) disulfiram. Data are expressed as means ± SEM. Samples tested passed the normality test. The blot presented was derived from a single experiment. The experiment was performed at least three times and the data presented are the sum of all values of these experiments. (C-D) LPS-induced eCIRP release occurs through the GSDMD pores in primary peritoneal macrophages. (C) Mice primary peritoneal macrophages were treated with or without LPS (100 ng/ml) for 24 hours. After LPS stimulation, culture supernatants and cell lysates were collected. Expression of caspase-11 and the cleavage of GSDMD (GSDMD-NT) were quantified by Western blot with β-actin as a loading control. Western blot images and quantitative bar diagram are shown. n=5 samples/group. *p<0.05 vs. (−) LPS. Groups were compared by a student’s t-test. The cells isolated from several mice were pooled and then plated into several wells per group serving as the technical replicates. The blot presented was derived from a single experiment. The experiment was performed at least two times and the data presented are the sum of all values of these experiments. (D) Mice primary peritoneal macrophages were pre-treated with or without disulfiram (indicated doses) for 1 h and then stimulated with LPS (100 ng/ml) for 24 h. eCIRP present in the extracellular supernatants was analyzed by Western Blot. Western blot images and quantitative bar diagram are shown. *p<0.05 vs. (−) LPS, (−) disulfiram; ^#p<0.05 vs. (+) LPS, (−) disulfiram. Data are expressed as means ± SEM. For A, C (n=4-6/group), groups were compared by student’s t-test and for B, D (n=5/group), groups were compared by one-way ANOVA and Tukey’s multiple comparisons test. Samples tested passed the normality test. The cells isolated from several mice were pooled and then plated into several wells per group serving as the technical replicates. The blot presented was generated from a single experiment. The experiment was performed at least three times and the data presented are the sum of all values of these experiments. PS, Ponceau S red staining.

LPS relocalizes GSDMD toward cell membranes

To determine the localization of GSDMD following LPS stimulation, we performed immunostaining in the macrophages. We found that GSDMD is predominantly located in the cytoplasmic and nuclear compartments in the PBS-treated cells, while in LPS-treated cells, GSDMD was colocalized in the plasma membrane. Of note, the antibody we used to stain GSDMD can stain both total and cleaved GSDMD (GSDMD-NT). However, given that only the cleaved form of GSDMD can bind and oligomerize to the membrane, the identification of GSDMD in the membrane of LPS-treated macrophages indicated that LPS activated GSDMD to localize into the plasma membrane (Fig. 3A). To validate whether CIRP is released through the GSDMD channels across the cell membrane, we evaluated the interaction between GSDMD and CIRP by using Iterative Threading ASSEmbly Refinement (I-TASSER) server based on templates identified by threading approach to maximize the percentage identity, sequence coverage, and confidence. As a result, we found that CIRP exhibited a transient interaction with GSDMD, and this interaction was found to occur at the GSDMD-NT domain (Fig. 3B). The CIRP-GSDMD complex showed an interface of surface area 972 Ȧ² and the free energy of binding (^ΔiG) −3.4 kcal/mol. The entropy change after dissociation (TΔS) was 12.7 kcal/mol. The CIRP and GSDMD interactions included 6 hydrogen bonds and 5 salt bridges, which indicated that polar amino acid residues formed salt bridges to stabilize the complex. However, the free energy of dissociation (ΔG^disso) was −5.9 kcal/mol. The negative value of ΔG^disso indicated that the interaction was very transient, after interaction with the globular domain of GSDMD, CIRP might dissociate from GSDMD. These data suggest that CIRP may bind with GSDMD transiently and then be released through the GSDMD pores. We also performed a surface plasmon resonance (SPR) or the Biacore assay with recombinant mouse (rm) CIRP and rmGSDMD to determine their real-time interaction. Our data showed that rmCIRP and rmGSDMD had moderate interaction with a K_D value of 4.87×10⁻⁷ M (Fig. 3C).

Figure 3. LPS relocalizes GSDMD toward cell membranes.

(A) RAW 264.7 cells were treated with or without LPS (100 ng/ml) for 24 hours. After LPS stimulation, the cells were fixed and then immunostained with cell surface membrane marker (green), cleaved form of GSDMD (GSDMD-NT) (red), and DNA (Hoechst 3334, blue), and the images were captured by confocal microscopy. (B) Computational model of GSDMD-NT’s (blue) interaction with CIRP (red), but not with GSDMD-CT (green) by using Iterative Threading ASSEmbly Refinement (I-TASSER) server. (C) rmCIRP immobilized on a sensor chip, and rmGSDMD of varying concentrations was injected as the analyte. rmCIRP binds to rmGSDMD with a K_D of 4.87×10⁻⁷ M. At least three (n=3) independent BIAcore experiments were performed generating similar K_D values.

Genetic ablation of GSDMD decreases eCIRP release in vitro

To further confirm the connection between GSDMD pores and eCIRP release, we adopted siRNA tool to transiently knockdown GSDMD expression and then assess eCIRP release after LPS stimulation in RAW 264.7 cells. We found that RAW 264.7 cells treated with GSDMD siRNA significantly decreased the expression of the full-length GSDMD compared to control siRNA treated macrophages (Fig. 4A). We also found that in PBS-treated RAW 264.7 cells, regardless of the control vs. GSDMD siRNA treated condition, eCIRP was not released. However, following LPS treatment in control siRNA-treated cells, there was a dramatic increase in eCIRP release compared to the PBS-treated cells. Interestingly, the LPS-treated RAW 264.7 cells transfected with GSDMD siRNA significantly decreased the release of eCIRP by 49% compared to LPS-treated control siRNA-transfected cells (Fig. 4B). We further isolated peritoneal macrophages from WT and GSDMD^−/− mice and confirmed the expression of full-length GSDMD under normal conditions. We found that GSDMD^−/− mice macrophages did not express GSDMD, while the WT macrophages expressed it adequately (Fig. 4C). In addition, we found that ex vivo treatment of macrophages of WT mice with LPS significantly increased eCIRP release. Conversely, the macrophages isolated from the GSDMD^−/− mice significantly decreased eCIRP release after treatment of macrophages with LPS (Fig. 4D, E). These data indicate that GSDMD knockout decreases eCIRP release in LPS-treated macrophages in vitro.

Figure 4. Knockdown of GSDMD expression decreases eCIRP release in vitro.

(A) RAW 264.7 cells were transfected with GSDMD siRNA (si-GSDMD) or negative control siRNA (NC). After transfection, GSDMD expression in RAW 264.7 cells were assessed by Western blot analysis. Western blot image and corresponding quantitative bar diagram are shown. n=7 samples/group, *p<0.05 vs. NC-siRNA. Groups were compared by a student’s t-test. The blot presented was generated from a single experiment. The experiment was performed at least three times and the data presented are the sum of all values of these experiments. (B) RAW 264.7 cells were transfected with si-GSDMD or negative control siRNA (NC), and then stimulated with LPS (100 ng/ml) for 24 h. eCIRP present in the extracellular supernatants was analyzed by Western blot. Western blot image and quantitative bar diagram are shown. n=7 samples/group, *p<0.05 vs.NC-siRNA, (−) LPS, ^#p<0.05 vs. NC-siRNA, (+) LPS. Samples tested passed the normality test. The blot presented was derived from a single experiment. The experiment was performed at least three times and the data presented are the sum of all values of these experiments. (C-E) Peritoneal macrophages isolated from WT or GSDMD^−/− mice were stimulated with PBS or LPS (100 ng/ml) for 24 hours. After LPS stimulation, the supernatants and cell lysates were collected. (C) GSDMD expression in the total cell lysates was detected by Western blot. (D, E) The levels of eCIRP in the supernatants were assessed by (D) Western blot and (E) ELISA. Western blot images and the bar diagram are shown. n=5-6 samples/group. *p<0.05 vs. WT, (−) LPS; ^#p<0.05 vs. WT, (+) LPS. Data are expressed as means ± SEM. The groups were compared by two-way ANOVA and SNK method. Samples tested passed the normality test. The cells isolated from several mice were pooled and then plated into several wells per group serving as the technical replicates. The blot presented was derived from a single experiment. The experiment was performed at least two times and the data presented are the sum of all values of these experiments. PS, Ponceau S red staining.

Genetic ablation or pharmacological inhibition of GSDMD inhibits the release of eCIRP in endotoxemia

To determine the in vivo impact of GSDMD on eCIRP release, we induced endotoxemia in mice by injecting LPS intraperitoneally. We found that WT mice after treatment with LPS significantly increased eCIRP levels in the blood compared with vehicle-treated mice. However, we found significantly reduced levels of serum eCIRP in GSDMD^−/− mice treated with LPS (Fig. 5A). We previously reported that following LPS treatment in normal mice or induction of bacterial sepsis by CLP in mice, the serum levels of eCIRP directly co-related with the serum levels of pro-inflammatory cytokines (7). To correlate the serum eCIRP levels with the levels of pro-inflammatory cytokines in endotoxemia, we assessed serum IL-6 and TNF-α. We found that serum IL-6 and TNF-α were increased in LPS treated mice, while those levels were significantly decreased in GSDMD^−/− mice treated with LPS (Fig. 5B–C). We also found that treatment of mice with GSDMD inhibitor, disulfiram markedly decreased plasma levels of eCIRP by 73% in LPS-induced endotoxemic mice (Fig. 5D). Similarly, we also found that the disulfiram-treated mice showed significantly decreased levels of serum IL-6 and TNF-α following endotoxemia compared to only LPS-treated mice (Fig. 5E–F). These data suggest that blocking GSDMD genetically or pharmacologically decreases the levels of serum eCIRP in endotoxemia.

Figure 5. Genetic ablation and pharmacological inhibition of GSDMD inhibit the release of eCIRP in LPS-injected mice.

(A-C) Endotoxemia was induced in WT or GSDMD^−/− mice by injecting LPS (10 mg/kg) intraperitoneally (i.p.) for 20 hours. PBS injected mice served as control group. Blood was collected from mice. The levels of (A) eCIRP, (B) IL-6, and (C) TNF-α in the plasma were assessed by ELISA. n=6-7 mice/group. *p<0.05 vs. WT, (−) LPS; #p<0.05 vs. WT, (+) LPS. The groups were compared by two-way ANOVA and SNK method. (D-F) Endotoxemia was induced in C57BL/6 mice by injecting LPS (10 mg/kg) intraperitoneally (i.p.). PBS-injected mice served as control group. Mice were injected i.p. with either disulfiram (50 mg/kg) or vehicle in equivalent volumes 1 h before LPS. Blood was harvested from mice 20 h after being challenged with LPS or PBS. The levels of (D) eCIRP, (E) IL-6, and (F) TNF-α in the plasma were assessed by ELISA. n=4-6 mice/group. *p<0.05 vs. (−) CLP, (−) disulfiram; ^#p<0.05 vs. (+) CLP, (−) disulfiram. Data are expressed as means ± SEM. Groups were compared by one-way ANOVA and Tukey’s multiple comparisons test. Samples tested passed the normality test. Each mouse per group serves as the biological replicates. The experiment was performed at least two times and the data presented are the sum of all values of these experiments.

Treatment with disulfiram decreases eCIRP release in septic mice

To determine the effects of GSDMD inhibitor disulfiram on eCIRP release in a clinically relevant model of sepsis, we induced polymicrobial sepsis in mice by CLP. We pre-treated or simultaneously treated the CLP mice with disulfiram, and then after 20 h of sepsis induction, we assessed eCIRP and pro-inflammatory cytokines levels in the blood. We found that the plasma levels of eCIRP were increased in CLP-induced septic mice compared to sham mice. At the same time, disulfiram pre-treatment significantly decreased the eCIRP release in the plasma in septic mice compared to vehicle-treated septic mice (Fig. 6A). In addition, we also found that disulfiram pretreatment reduced IL-6 and TNF-α release into the blood of septic mice compared to vehicle-treated mice (Fig. 6B, C). Akin to disulfiram pre-treatment findings, we also got similar results with treating the mice with disulfiram at the same time of CLP induction. Disulfiram treatment significantly decreased eCIRP, IL-6, and TNF-α release into the plasma of septic mice compared to vehicle-treated septic mice (Fig. 6D–F). These data suggest that inhibition of GSDMD pore formation by disulfiram decreases eCIRP release in sepsis (Fig. 7).

Figure 6. Inhibition of GSDMD by pre- and simultaneous treatment of disulfiram attenuates eCIRP release in CLP-induced septic mice.

Sepsis was induced in C57BL/6 mice by CLP. Sham-operated animals served as control mice. At 20 h of CLP or sham operation, blood was harvested from mice. (A-C) C57BL/6 mice were injected i.p. with either disulfiram (50 mg/kg) or vehicle in equivalent volumes 1 h before CLP. Plasma levels of (A) eCIRP, (B) IL-6, and (C) TNF-α were assessed by ELISA. (D-F) C57BL/6 mice were injected i.p. with either disulfiram (50 mg/kg) or vehicle in equivalent volumes 0 h before CLP. Plasma levels of (D) eCIRP, (E) IL-6, and (F) TNF-α were assessed by ELISA. n=6 mice/group. *p<0.05 vs. (−) CLP, (−) disulfiram; ^#p<0.05 vs. (+) CLP, (−) disulfiram. Data are expressed as means ± SEM. Groups were compared by one-way ANOVA and Tukey’s multiple comparisons test. Samples tested passed the normality test. Each mouse per group serves as the biological replicates. The experiment was performed at least two times and the data presented are the sum of all values of these experiments.

Figure 7. Gasdermin D pores release eCIRP from macrophage in Sepsis.

Bacterial sepsis or endotoxemia induces caspase-11 expression. Activated caspase-11 cleaves GSDMD, generating an active form, N-terminal fragment of GSDMD (GSDMD-NT), which oligomerizes in the cell membrane and forms membrane pores. During inflammation, CIRP is translocated from nuclear to cytoplasm, binds to GSDMD-NT in the cell membrane, and released through the GSDMD pores. Disulfiram, an inhibitor of GSDMD pore formation, attenuates eCIRP release in sepsis. GSDMD, gasdermin D.

Discussion

Extracellular CIRP acts as a DAMP to facilitate inflammation and tissue injury in sepsis (7, 9), but its release mechanism is not understood. Under normal conditions, CIRP is present in the nucleus, whereas hypoxia causes CIRP to translocate to the cytoplasm and then release into extracellular space (7). Stress causes intracellular CIRP methylation and subsequent translocation from the nucleus to the stress granules in the cytoplasm (35). We previously showed that CIRP is released via the lysosomal pathway with evidence of CIRP’s enrichment at the lysosomal compartment of macrophages under hypoxia (7). Akin to other DAMPs, passive release due to cellular lytic death might cause eCIRP release (9). Our current study is the first known report to demonstrate the mechanism of eCIRP’s active release from living cells by activating the pore-forming protein GSDMD. The recent identification of GSDMD secretomes (36–38) points to a critical question of what makes eCIRP different from other DAMPs released through GSDMD pathway. eCIRP can be distinguished from other DAMPs in several points. eCIRP is released through the GSDMD pore independent of cellular pyroptosis, differently from HMGB1, which is larger than eCIRP and has been shown to require larger pores typical of pyroptosis to be released (39). Moreover, we revealed that CIRP interacts with the N-terminal GSDMD. This binding was transient (moderate K_D), suggesting a grab-and-go mechanism. That means, when GSDMD is cleaved to be localized in the plasma membrane, it takes CIRP along with it in the membrane vicinity so that CIRP can be quickly released from cells. This unique phenomenon of eCIRP-GSDMD interaction facilitating eCIRP’s release or promoting GSDMD pore formation vice versa during LPS stimulation possibly depicts eCIRP’s striking features.

GSDMD is required for IL-β release from living neutrophils without pyroptosis (24). Our data demonstrated that LPS induced the cleavage of GSDMD as evidenced by increased N-terminal of GSDMD in RAW 264.7 cells, mouse primary peritoneal macrophages, and human macrophages. Disulfiram was recently identified as an inhibitor of GSDMD pore formation and pyroptosis in vitro and in vivo (26). Disulfiram blocked GSDMD pore formation and cytokines release without affecting caspase activation or GSDMD cleavage. In our study, disulfiram decreased eCIRP release to the cell culture medium in RAW 264.7 cells, mouse primary peritoneal macrophages, and human macrophages. Moreover, downregulation of GSDMD gene expression by siRNA transfection reduced eCIRP release induced by LPS in RAW 264.7 cells. GSDMD^−/− macrophages released less eCIRP to the culture medium after being challenged with LPS compared with PBS-treated WT and GSDMD^−/− macrophages. This could be a result of a decreased GSDMD pore formation in GSDMD deficient macrophages. To further investigate the in vivo effect of GSDMD on eCIRP release in endotoxemia and sepsis, we examined LPS-induced endotoxemia and CLP-induced sepsis in mice. CLP- or LPS-induced sepsis caused eCIRP release into the circulation compared to vehicle-treated mice, while disulfiram treatment reduced eCIRP release in septic mice. Moreover, the production of CIRP was significantly increased in LPS-treated WT mice compared to PBS-treated WT and GSDMD^−/− mice, while GSDMD^−/− mice showed less eCIRP expression in the plasma after being challenged with LPS. Taken together, our data provide important evidence corroborating eCIRP’s active release from macrophages via GSDMD pores in endotoxemia or sepsis.

Prior study demonstrates that GSDMD^−/− mice are less sick than the WT mice in CLP-induced sepsis (40). Here in our study, we did not repeat the assessments of various surrogate markers of sepsis other than our protein of interest eCIRP levels in serum and compared with the serum TNFα and IL-6, which positively correlated with serum eCIRP. In line with this data, we recently showed that blocking GSDMD activation using a pan-caspase inhibitor, zVAD-FMK or the GSDMD inhibitor disulfiram attenuated eCIRP’s effects on macrophage extracellular trap release (41), confirming eCIRP’s effects are mediated through GSDMD pathway. As such, it is conceivable that subverting the GSDMD system will show a phenotype of reducing the impact of eCIRP. Further study with GSDMD^−/− mice to see eCIRP’s effect would be interesting. The nuclear factor-kappa B (NF-κB) is a ubiquitous transcription factor involved in the expression of numerous genes related to inflammation and cellular functions (42). Disulphiram has been shown to downregulate the activity of NF-κB (43). Although there is no report demonstrating that CIRP’s transcription is directly regulated via NF-κB, due to the complex crosstalk of NF-κB with other signaling molecules, disulphiram-mediated inhibition of NF-κB could partially be responsible for CIRP’s transcription, and subsequently, its release. Moreover, since NF-κB regulates GSDMD expression (44), it is also possible that disulfiram’s negative effect on NF-κB for altering GSDMD expression may, at least in part, have impacted eCIRP’s release.

During inflammation, proteolytic cleavage of GSDMD is initiated by the activation of cells with PAMPs or DAMPs. GSDMD cleavage generates N-terminal fragments, which form pores on the cell membrane (17). GSDMD pores disrupt the integrity of the cell membrane and can cause pyroptotic cell death (14, 15). In contrast, under certain conditions, the cell can survive after GSDMD-mediated cell membrane pore formation as it is repaired by ubiquitous damaged cell membrane repair response (22). Thus, there is no membrane damage at the early stages of GSDMD-mediated pyroptosis, only disruption of the membrane permeability occurs. These cells still preserve their functions and can promote inflammation, which are called hyperactivated cells (23). GSDMD pores in the plasma membrane permit the passage of low molecular weight cellular contents, like IL-1β, to the extracellular space to aggravate inflammation (23). Of note, two known mechanisms explain how GSDMD is activated to form pores and worsen inflammation. The canonical pathway involves the assembly and activation of inflammasomes that include caspase-1, which then cleaves GSDMD into N- and C-terminal fragments(15). GSDMD-NT oligomerizes and inserts into the plasma membrane where it forms pores of 10-20 nm in inner diameter (17, 18, 21, 45). Alternatively, non-canonical GSDMD cleavage can be induced by caspase-11 in the mouse or caspase-4 and −5 in humans. The catalytic activity of caspase-11 is not stimulated by recruitment into inflammasomes but rather by its direct binding to LPS (46, 47). Upon LPS binding, active caspase-11 cleaves GSDMD like caspase-1 (46, 47) leading to pore formation and pyroptosis (14, 48, 49). In our study, while we did not assess the direct interaction between LPS and caspase-11, we revealed that upon stimulation of the macrophages with LPS, caspase-11 expression was significantly increased. Therefore, it may participate in GSDMD activation for eCIRP release.

Consistent with our previous finding, we showed that LPS significantly increased eCIRP release to the cell culture medium of RAW 264.7 cells and mouse primary peritoneal macrophages. LDH is a large protein to be released through the GSDMD pores and thus solely relies on cell lysis for its release. On the other hand, small-size molecules like IL-1β, are released through GSDMD pores (21, 23). Therefore, measuring LDH levels in the cell culture supernatants can be used to distinguish cell hyperactivation and pyroptosis (21). We found that LPS stimulation caused a small amount of LDH released from the cell compared to the control group. This result indicates that LPS induces a small percentage of cell death, and we assumed that these dead cells are likely to contribute at least in part to the eCIRP release. Then we used osmoprotectant glycine to prevent pyroptosis-induced cell lysis (21). Glycine treatment almost completely inhibited LDH release into the cell culture medium. However, LPS induced eCIRP release by the macrophages was largely unaffected by glycine. Thus, pre-pyroptotic GSDMD pore formation is sufficient to release eCIRP, a 17 kDa small protein. As such, we have demonstrated the active release of eCIRP through GSDMD pores by viable cells.

Exactly how CIRP translocates from the nucleus to the cytoplasm is still unclear. However, since GSDMD pore formation has been observed in the nuclear envelope membrane (50), it is reasonable to anticipate that GSDMD pores in the nuclear envelope can facilitate the translocation of nuclear CIRP to the cytoplasmic compartment. In summary, we have demonstrated eCIRP’s active release via GSDMD pores mediated in living macrophages in sepsis. This finding provides a better understanding of how eCIRP, a critical molecule for inflammation and tissue injury in sepsis and ischemia/reperfusion injury, is released. Targeting GSDMD pore formation could be a novel therapeutic approach to inhibit eCIRP-mediated inflammation and organ injury in sepsis.

Key points.

• eCIRP is released through GSDMD pores independent of pyroptotic cell death.

• GSDMD and eCIRP interact with each other and co-localize at the cell membrane.

• Targeting GSDMD by a pharmacological approach inhibits eCIRP release in sepsis.

Abbreviations

CLP

cecal ligation and puncture

DAMPs

damage-associated molecular patterns

eCIRP

extracellular cold-inducible RNA-binding protein

ELISA

enzyme-linked immunosorbent assay

GSDMD

gasdermin D

HMGB1

high mobility group box 1

I-TASSER

iterative threading ASSEmbly refinement

LDH

lactate dehydrogenase

LPS

lipopolysaccharides

PAMPs

pathogen-associated molecular patterns

PGN

peptidoglycan