The progression of hepatocyte pyroptosis exacerbates radiation-induced liver disease via the gasdermin D/signal transducer and activator of transcription 5a/CXCL1 axis

The progression of hepatocyte pyroptosis exacerbates radiation-induced liver disease via the gasdermin D/signal transducer and activator of transcription 5a/CXCL1 axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The progression of hepatocyte pyroptosis exacerbates radiation-induced liver disease via the gasdermin D/signal transducer and activator of transcription 5a/CXCL1 axis Zhenwei Peng, aoran Dong, Guangyan Wei, Zhou Liang, Yuqin Di, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3161881/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Radiation-induced liver disease (RILD) severely impairs the outcome of patients exposed to irradiation (IR); however, the underlying mechanism is largely unknown. The N-terminus of gasdermin D (GSDMD-N) is mainly involved in driving the progression of pyroptosis, and its expression has been reported to be induced by IR in the gut and bone marrow, but its role in RILD remains unknown. By collecting liver samples from RILD patients and mice, we uncovered GSDMD-FL/N were significantly upregulated and positively correlated with RILD severity. Hepatocytes are found to be critical pyroptotic cells in RILD by combining single-cell RNA sequencing (scRNA-seq), immunofluorescence and fluorescence-activated cell sorting (FACS) analysis. Functional analysis and mechanistic studies were performed using hepatocyte-specific Gsdmd knockout ( Gsdmd ∆Hep ) mice and cell models and scRNA-seq analysis. Mechanistically, GSDMD was indispensable for triggering hepatocyte pyroptosis and initiating the activation of transcription factor stat5a, which subsequently facilitated the expression of CXCL1. As a pore-forming protein, increased GSDMD-N also mediated the secretion of CXCL1, which further recruited neutrophil into liver to accelerate the severity of RILD. We also discovered that pharmacological targeting GSDMD and its downstream CXCL1 effectively attenuated the progression of RILD. RILD progression requires increased GSDMD-FL/N in hepatocytes, causing pyroptosis and stat5a activation. Afterwards, driving CXCL1 express and release to recruit neutrophils into liver. Together, our study demonstrates that GSDMD as therapeutic targets to improve RILD. Biological sciences/Immunology/Cell death and immune response Health sciences/Oncology/Cancer/Cancer therapy/Radiotherapy radiation-induced liver disease GSDMD GSDMD-N pyroptosis neutrophil chemokine CXCL1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION The liver, a pivotal organ with multifarious functions, manifests as a radiosensitive entity warranting particular attention. (1) Exposure to ionizing irradiation, such as nuclear and radiological incidents, (2) total body irradiation (TBI) (3) before allogeneic stem cell transplantation, and radiotherapy for tumors (4–7) in the upper abdomen (liver, biliary, and pancreas) and thoracic (right lower lung, distal esophagus, and breast), can result in radiation-induced liver disease (RILD). RILD manifests as acute liver injury in the short-term after RT followed by chronic progression to liver fibrosis, (8, 9) while no clinic effective drugs to alleviate it. Patients will irreversibly develop into cirrhosis, liver failure and even death. Pathophysiologically, radiation results in the increase of inflammatory cytokines and recruitment of immune cells, ultimately leading to tissue fibrosis and liver dysfunction. (10) However, the underlying mechanism of immune regulation during RILD remains unclear. Pyroptosis, a form of inflammatory cell death, is crucial in the immune environment. During pyroptosis, inflammasome activation by canonical (NOD-like receptor [NLR], absent in melanoma 2 [AIM2], and caspase-1) or noncanonical (caspase-4/5 in humans or caspase-11 in mice) pathways leads to the cleavage of GSDMD. GSDMD-N then forms pores in the membrane and results in cell swelling, a burst of inflammatory cytokines and cytosolic content release and ultimately cell lysis. Therefore, GDSMD, as a key executioner of pyroptosis and cytokine release, is thought to be a crucial mediator in shaping the inflammatory environment involved in the progression of multiple liver diseases. (11–14) Recently, Xiao et al . (15) reported that TBI could induce GSDMD-FL/N increase in the gut and bone marrow of mice, which were positively correlated with the severity of bone loss, but the role of GSDMD in this process remains unclear. However, Wu et al . (16) revealed that there were no significant increase of GSDMD-FL/N in lung tissues after chest irradiation. These discoveries suggest that the existence and effects of GSDMD are complicated and vary in different radiation-induced tissue injuries. However, the occurrence of GSDMD-executed pyroptosis in RILD and its impact on the expression and secretion of inflammatory factors still remain elusive. Therefore, further investigation is warranted to elucidate the role of GSDMD-executed pyroptosis in RILD. Additionally, it is crucial to explore other uncharted functions of GSDMD in the context of RILD, considering that cleavage-independent functions of gasdermin proteins have been reported to influence the growth of pancreatic adenocarcinoma and the progression of asthma. (17, 18) In the present study, we unveil the intriguing upregulation of GSDMD-FL/N and the emergence of a pyroptotic phenotype in irradiated hepatocytes, shedding light on the molecular mechanisms underlying RILD. Through its targeted interaction with stat5a, GSDMD facilitated the phosphorylation of stat5a, leading to its translocation from the cytoplasm to the nucleus. This dynamic process played a critical role in mediating the expression of CXCL1, initiating a cascade of events within the liver microenvironment. GSDMD-N played a pivotal role in the secretion of CXCL1, attracting neutrophils to the liver and exacerbating the severity of RILD. Remarkably, we successfully mitigated neutrophil recruitment and alleviated RILD in mice by employing antibodies to block CXCL1. Furthermore, our promising findings indicate that pharmacological inhibition of GSDMD offers a potent approach to suppress the progression of RILD. Collectively, our discoveries highlight the exciting potential of targeting GSDMD as a therapeutic strategy to prevent RILD. MATERIALS AND MEHTHODS Animal studies Hepatocyte-specific Gsdmd knockout ( Gsdmd ∆Hep ) were generated by breeding Gsdmd Flox/Flox ( Gsdmd FL/FL ) with Alb-cre mice on the C57BL/6J background (GemPharmatech, Nanjing, China). All animals were acclimatized for 1 week before experiments and housed in a specific pathogen-free environment with a 12-hour light-dark cycle and permitted ad libitum consumption of water and a standard chow diet unless otherwise stated. This research was approved by the Institutional Care and Animal Use Committee of Sun Yat-sen University Cancer Center (20110M). Mouse model of RILD Using a previously established protocol, (19, 20) we generated a mouse model of hepatocellular carcinoma (HCC) by hydrodynamic injection of AKT/Ras and Sleeping Beauty transposon. Two weeks post-transfection, the mice were subjected to hepatic radiation, administered in three fractions of 4 Gy per day and subsequently analyzed pathologically. Male Gsdmd FL/FL and Gsdmd ∆Hep mice, aged between seven to ten weeks, were carefully anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg). A single fraction of 30 Gy hepatic radiation was administered to establish the short-term (ST) radiation-induced liver disease (RILD) model. To explore the long-term (LT) side effects of RILD, mice received five fractions of 6 Gy hepatic radiation every two days and were allowed to spontaneously recover for a period of 10 weeks. CXCL1 was blocked by administration of a mouse CXCL1 neutralizing antibody (100 µg/mouse, i.v. ) (R&D systems) 15 min before irradiation. For LT-RILD, mice were injected additionally with CXCL1 neutralizing antibody (25 µg/mouse, i.v. ) weekly after the last irradiation. Control mice were injected with the same dosage rat isotype-IgG control antibody. For ST-RILD, WT mice were given intraperitoneal injection of disulfiram (100 mg/kg, MCE) or vehicle 15 min before the first radiotherapy administrated and maintained until 7 days. For LT-RILD, in addition to the injection of 100 mg/kg disulfiram 15 min before radiotherapy, the injection of 50 mg/kg disulfiram was maintained every week after the end of radiotherapy. Disulfiram was dissolved in 10% DMSO and 100% corn oil, while control mice were given vehicle (containing only 10% DMSO and 100% corn oil). Adeno-associated virus (AAV9) vector that expressed GSDMD-FL (AAV9-GSDMD-FL) and control (AAV9-Control) were purchased from Genechem Company (Shanghai, China). The virus solution was diluted by PBS and then injected into Gsdmd ∆Hep mice through the tail vein at a dose of 1×10 11 v.g./mouse. The mice were subjected to 30 Gy hepatic irradiation and sacrificed at day 7. Human liver tissue and blood samples collection Liver tissues of HCC patients who underwent hepatectomy adjuvant radiotherapy were collected before and after radiotherapy ( n = 20) at the first affiliated hospital of Sun Yat-sen university. Blood specimens were collected from patients accepted hepatic irradiation from June 2020 to July 2021 in the radiotherapy department of the First Affiliated Hospital of Sun Yat-sen University. Patients with abnormal liver function before irradiation and those receiving other antitumor therapy were excluded (Figure S1 A). Finally, a total of 57 freshly blood was collected and serum was separated and stored at -80°C until further analysis. The human study was approved by the Institutional Ethics Committee of the first affiliated hospital of Sun Yat-sen university. The detailed methodology is described in the Supporting Experimental Procedures. RESULTS GSDMD and its pyroptosis-trigger GSDMD-N are upregulated in RILD To determine whether pyroptosis triggered by GSDMD-N was induced by irradiation, peritumor liver tissues from the same HCC patients ( n = 20) were collected before and after radiotherapy (detailed clinic information was shown in Table S1 ). Immunohistochemistry (IHC) showed that GSDMD-N was markedly upregulated in the liver of hepatocellular carcinoma (HCC) patients who received radiotherapy (Fig. 1 A). We then sought to dissect the relationship between GSDMD-N level and the severity of RILD. Ge et al. reported that GSDMD could be released from pyroptotic cardiomyocyte and can be measured in serum. (21) We next collected peripheral blood from patients after hepatic RT and detect the GSDMD-N level. The inclusion and exclusion criteria were shown in Figure S1 A. In short, we initially screened 184 patients with hepatic tumors accepted radiotherapy between June 2020 and July 2021. Patients with liver dysfunction before radiotherapy or accepted other anti-tumor therapies were excluded. Albumin-bilirubin (ALBI) score was used to assess liver toxicity induced by irradiation, (22–24) we found that patients with ALBI grade 2 had higher GSDMD-N levels than those with grade 1 (1855.0 ng/mL vs 331.3 ng/mL, p = 0.0177) (Fig. 1 B, Table S2 ). Further correlation analyses revealed that serum GSDMD-N levels were strongly positively correlated with the severity of liver injury (serum ALT, AST and ALP) and negatively correlated with liver function (serum ALB) in RILD patients (Figure S1 B). We next evaluated GSDMD expression in the liver tissue adjacent tumor in radiotherapy model of mouse HCC. Peritumor liver tissues also presented increased GSDMD-FL expression after hepatic radiation (Fig. 1 C). To confirm the expression pattern of GSDMD in RILD mice, we subjected mice to a universal short-term (ST) and long-term (LT) RILD modelling protocol (Figure S1 C). (25, 26) First, we observed that GSDMD-FL and GSDMD-N levels were robustly elevated in the irradiated murine liver tissue in both the ST-RILD and LT-RILD groups (Fig. 1 D). Moreover, hepatic GSDMD-N levels were closely correlated with the degree of liver injury (serum ALT and AST), liver function (serum ALB), and liver fibrosis (fibronectin, Figure S1 D-E). These results suggested that GSDMD and its pyroptosis-triggerred N-terminal fragment are upregulated in liver tissues during RILD. GSDMD-dependent hepatocyte pyroptosis occurs in RILD Previous studies have shown that GSDMD-dependent pyroptosis widely occurs in immune cells. (27, 28) To identify which immune cell type is involved in pyroptosis after irradiation in an unbiased and precise manner, we performed comprehensive scRNA-seq of whole liver tissue from control (ctrl) and ST-RILD mice. Within the immune compartments, we compared the expression of Gsdmd and other pyroptosis-associated gene markers, including Il1b , Il18, Casp1 , Caspase4/11 , Nlrp3 , Nod1 , and Nod2 (Fig. 1 E). Unexpectedly, Gsdmd was rarely elevated in hepatic immune cells after IR. Under physiological conditions, Gsdmd was slightly expressed in monocytes and Kupffer cells and barely expressed in neutrophils and T/B cells. Moreover, no noticeable increase in the expression of Gsdmd or other pyroptosis - associated genes were observed in these cells after IR. Since pyroptosis can also occur in hepatocytes rather than immune cells in steatohepatitis and CCL4-induced liver injury, (29) we assumed that hepatocytes might be the major pyroptotic cells in RILD, with strong activation of GSDMD. Indeed, a robust increase in GSDMD-FL expression was preferentially found in the parenchymal cells of RILD mouse livers (Fig. 1 F). Further immunofluorescence analysis was performed to clearly determine the GSDMD expression changes in hepatocytes and other cells. As we speculated, remarkable coexpression of GSDMD with hepatocellular nuclear factor 4-alpha (HNF4ɑ, a marker of hepatocytes) was observed in both ST-RILD and LT-RILD livers (Fig. 1 G), while CD11b + monocytes, CD3 + T cells, CK19 + cholangiocytes and α-SMA + hepatic stellate cells (HSCs) displayed low expression of GSDMD. To accurately confirm the occurrence of pyroptosis in hepatocyte, we isolated primary hepatocytes and other non-parenchymal cells (NPC) from the liver of ctrl and ST-RILD mice and compared their GSDMD-N levels by western blot (WB) analysis (Fig. 1 H). Hepatocytes also had the highest GSDMD-N level after irradiation compared with other cells, further indicating that hepatocytes are critical pyroptotic cells in RILD. Subsequently, to determine whether irradiation induced hepatocyte pyroptosis, primary hepatocytes were isolated from mice and irradiated with 8 Gy irradiation to establish ex vivo radiation models (Figure S1 F). Indeed, irradiation could aggravate hepatocyte injury, manifested as decreased ALB and increased ALT and AST levels 24 h after IR (Figure S1 G-H). Besides, the levels of the key pyroptotic proteins GSDMD-FL/N increased from 12h to 24 h after irradiation (Fig. 1 I). Compared to nonirradiated hepatocytes, irradiated hepatocytes also exhibited other late-stage features of pyroptosis: cell swelling with a balloon-shaped protrusion emerging from the plasma membrane by microscopy (Fig. 1 J), increased LDH levels in the supernatant (Fig. 1 K) and decreased cell viability (Fig. 1 L). These results confirmed that hepatocyte pyroptosis is activated after IR. To further investigate the requirement of GSDMD for hepatocyte pyroptosis, primary hepatocytes from Gsdmd FL/FL and Gsdmd ∆Hep mice were subjected to irradiation. Compared to Gsdmd FL/FL hepatocytes, Gsdmd ∆Hep hepatocytes were resistant to irradiation-mediated pyroptosis (Fig. 1 K-L). Altogether, these results show that GSDMD is required for triggering pyroptosis in irradiated hepatocytes. Collectively, these data demonstrated that GSDMD is required for driving hepatocyte pyroptosis in RILD. GSDMD deficiency ameliorates acute liver injury and chronic fibrosis in RILD To further investigate the role of GSDMD-dependent hepatocyte pyroptosis in RILD, we generated RILD models with Gsdmd FL/FL and Gsdmd ∆Hep mice. No significant difference in weight loss was found between these two groups of mice with ST-RILD (Figure S2 A), while Gsdmd ∆Hep mice with LT-RILD displayed less weight loss and regained body weight more quickly after irradiation, in contrast to the WT-IR mice (Figure S2 B). Moreover, in ST-RILD mice, liver tissues of Gsdmd FL/FL -IR mice manifested notably increased steatosis, while GSDMD deficiency markedly mitigated this pathological damage (Fig. 2 A). In LT-RILD mice, the livers of Gsdmd FL/FL -IR mice exhibited apparent scattered nodules (Fig. 2 B), notable hepatic stellate cell (HSC) activation (α-SMA + ), obvious extracellular matrix (ECM) deposition (collagen + and fibronectin + ) (Fig. 2 C-E), and enhanced expression of the profibrotic markers Col1a1 , Tgfb1 , and Timp1 (Fig. 2 F), indicating that chronic liver fibrosis was induced by irradiation. However, all of these fibrotic changes were notably suppressed in the context of GSDMD deficiency. In addition, in both ST- and LT-RILD mice, the absence of GSDMD also led to a striking decrease in serum ALT and AST and an improvement in ALB, reflecting enhanced liver function (Fig. 2 G). Collectively, these results suggest that GSDMD deficiency exerts a vital inhibitory effect on both early IR-induced hepatic injury and advanced liver fibrosis. To further determine the pathogenic role of GSDMD in RILD, Gsdmd ∆Hep mice were administered AAV9-GSDMD-FL to re-express GSDMD before IR (Figure S2 C-D). GSDMD-FL re-expression did not affect weight loss in Gsdmd ∆Hep ST-RILD mice (Figure S2 E) but resulted in a significant increase in hepatic histology damage and liver injury (Figure S2 F-G). These results confirmed that upregulation of GSDMD accelerated the progression of RILD. Similar protection from GSDMD deficiency was observed in an ex vivo hepatocyte irradiation model. Compared to Gsdmd FL/FL hepatocytes, Gsdmd ∆Hep hepatocytes released higher levels of ALB and lower levels of ALT and AST after IR (Fig. 2 H-I). Overall, these data suggested that GSDMD contributes to the progression of RILD probably depending on GSDMD driving hepatocyte pyroptosis. GSDMD deficiency inhibits the recruitment of neutrophils to the irradiated liver GSDMD influenced the progression of diseases by modulating inflammatory responses. (11, 21, 30, 31) Therefore, to explore whether GSDMD aggravates RILD by affecting the hepatic immune environment, we set out to obtain single-cell suspensions of liver tissue from Gsdmd FL/FL , Gsdmd FL/FL -IR, and Gsdmd ∆Hep -IR mice, followed by scRNA-seq to characterize immune cell profiles (Fig. 3 A). As shown in Fig. 3 B, hepatic immune cells were visualized by uniform manifold approximation and projection (UMAP). Compared to the Gsdmd FL/FL -Ctrl the hepatic immune cell composition of Gsdmd FL/FL -IR mice underwent significant changes, shown as a decrease in dendritic cells, T/B cells, and natural killer cells, a slight increase in Kupffer cells, and a striking elevation in neutrophils, suggesting an important role of neutrophils in RILD. Intriguingly, this IR-induced neutrophil infiltration of the liver was markedly reduced after GSDMD deficiency (Fig. 3 C), indicating that GSDMD might contribute to neutrophil recruitment under IR. Decreased liver infiltration of neutrophils in Gsdmd ∆Hep mice were then confirmed by flow cytometry analysis (Fig. 3 D). Moreover, myeloperoxidase (MPO)-positive active neutrophils were also significantly accumulated in both ST-RILD and LT-RILD Gsdmd FL/FL mouse livers, as detected by IHC, and were markedly reduced by knocking out Gsdmd (Fig. 3 E). Likewise, restoration of GSDMD expression by administration of AAV9-GSDMD-FL to Gsdmd ∆Hep -ST-RILD mice significantly increased hepatic neutrophil infiltration, with levels similar to those in Gsdmd FL/FL -ST-RILD mice (Fig. 3 F-G). Next, we wondered if GSDMD-mediated hepatocyte pyroptosis facilitated the recruitment of neutrophils. Purified neutrophils were then cocultured with non-irradiated or irradiated hepatocytes from Gsdmd FL/FL or Gsdmd ∆Hep mice, followed by counting of migratory neutrophils (Figure S3A-B). In the case of equivalent cells, the recruitment of neutrophils was significantly increased by coculture with Gsdmd FL/FL -IR hepatocytes but notably decreased when cocultured with Gsdmd ∆Hep -IR hepatocytes (Fig. 3 H). The activation of HSCs is critical for the progression of fibrosis in RILD while little is known about pyroptotic hepatocyte impact on HSCs. We designed both contact and non-contact co-culture experiments of hepatocytes with HSCs (Fig. 3SC). The results show that there was no difference between the two approaches in terms of having no effect on HSCs activation (Fig. 3 I-J). Collectively, our data illustrated that GSDMD-mediated hepatocyte pyroptosis facilitates neutrophil infiltration into the liver. Selectively CXCL1 neutralization effectively attenuated the progression of GSDMD driving RILD through suppressing neutrophil recruitment Given that neutrophils are not liver-resident immune cells and require chemokines for their trafficking, (30–34) we hypothesized that GSDMD might regulate neutrophil recruitment by affecting chemokine expression. Therefore, RNA-seq of Gsdmd FL/FL -IR and Gsdmd ∆Hep -IR mouse livers was performed to screen neutrophil trafficking-associated chemokines regulated by GSDMD. Notably, we found that CXCL1 was the most significantly downregulated chemokine in the livers of Gsdmd ∆Hep -IR mice compared with Gsdmd FL/FL -IR mice (Fig. 4 A). To validate whether GSDMD impacts CXCL1 expression, hepatic CXCL1 in Gsdmd FL/FL and Gsdmd ∆Hep mice before and after IR was examined at the mRNA and protein levels. We discovered that CXCL1 was maintained at a low expression level in both Gsdmd FL/FL and Gsdmd ∆Hep mice under steady-state conditions and was markedly increased in Gsdmd FL/FL mice after radiotherapy, while Gsdmd ∆Hep mice exhibited notable resistance to this upregulation (Fig. 4 B-C). Moreover, further re-expressing GSDMD-FL in Gsdmd ∆Hep mice effectively restored IR-induced hepatic CXCL1 upregulation (Fig. 4 D) implying CXCL1 should act as a downstream factor of GSDMD during the progression of RILD. To further investigate the role of CXCL1 in RILD through influencing neutrophil infiltration in vivo , Gsdmd FL/FL -IR mice were administered either a control immunoglobulin G (IgG) antibody or an anti-CXCL1 antibody (anti-CXCL1 Ab), which effectively neutralized CXCL1 in RILD models (Figure S4A-B). Flow cytometry analysis confirmed that CXCL1 inhibition resulted in twofold and fourfold decreases in liver neutrophil infiltration in the ST-RILD and LT-RILD mouse models, respectively (Fig. 4 E). Likewise, MPO + active neutrophils also decreased significantly after blocking CXCL1, as indicated by IHC (Figure S4C). Similar to the observation in Gsdmd ∆Hep mice, neutralizing CXCL1 had no effect on weight loss in ST-RILD mice but alleviated weight loss in the LT-RILD group (Figure S4D-E). Furthermore, compared to the IgG-treated mice, neutralization of CXCL1 resulted in reduced liver steatosis and congestion in ST-RILD mice and attenuated liver fibrosis in LT-RILD mice (Fig. 4 F-K). In addition, notable suppression of liver injury and a significant improvement in liver function in both the ST-RILD and LT-RILD mouse models were observed in mice treated with anti-CXCL1 Ab (Fig. 4 L). Taken together, these results suggest that selectively inhibiting CXCL1 can effectively suppress the progression of GSDMD driving RILD through the blockade of neutrophil recruitment. GSDMD regulates hepatocytes release CXCL1 to recruit neutrophil To further explore the mechanism of GSDMD regulating CXCL1 to recruit neutrophils during RILD, we first determine the cellular resource of CXCL1. Previous studies have shown that hepatic CXCL1 is mostly released from hepatocytes and HSCs in steatohepatitis and CCL4-induced liver fibrosis. (33,35) However, our immunofluorescence results demonstrated that CXCL1 was expressed rarely in HSCs (α-SMA + ) but mostly in hepatocytes (HNF4α + ) in RILD mice (Fig. 5 A). This observation was then validated in in vitro experiments. Double immunostaining of CXCL1 and GSDMD-FL revealed that radiation-induced upregulation of GSDMD-FL in hepatocytes was accompanied by a significant elevation of CXCL1 (Fig. 5 B). Because GSDMD cleavage mediated the release of cell contents and simultaneously initiated cell death, we investigated whether GSDMD-N mediated CXCL1 release before cell death after irradiation exposure. After 12h after irradiation, primary hepatocytes showed no significant increased PI uptake (Fig. 5 C). While, the CXCL1 levels in the supernatants increased after irradiation in Gsdmd FL/FL hepatocytes but decreased in Gsdmd ∆Hep hepatocytes (Fig. 5 D). Next, we investigated whether GSDMD-N facilitated the secretion of cytosolic CXCL1. As the last step of GSDMD cleave requires the “scissors”-caspase and caspase-1 activation is reported to be governed by AMI2 which sense double-strand DNA damage after irradiation, (36) we also detected irradiation can activate caspase-1 in hepatocytes (Fig. 5 E). To further define GSDMD-N function in controlling CXCL1 release, we used caspase-1 inhibitor Ac-YVAD-cmk to prevent the formation of GSDMD-N. Compared with IR-hepatocytes without inhibitor, Ac-YYAD-cmk effectively inhibit the release of CXCL1 into the supernatant but store in the cell (Fig. 5 F-G). Together, these results indicated irradiation-induced GSDMD-N promoted the secretion of CXCL1. To clarify that GSDMD regulate hepatocyte after irradiation can recruit neutrophils via CXCL1, WT hepatocytes administered an anti-CXCL1 Ab showed a significantly reduced ability to recruit neutrophils (Fig. 5 H). In contrast, the addition of the recombinant CXCL1 protein (CXCL1 Rp) to Gsdmd ∆Hep hepatocytes restored the chemotactic ability of neutrophils (Fig. 5 I). In vivo, restoration of GSDMD in Gsdmd ∆Hep mice by injecting AAV9-GSDMD-FL, administration of CXCL1-Ab also can inhibit the neutrophil infiltration into liver (Figure S5A-C). Afterwards, early radiation-induced liver injury was reversed (Figure S5D-F). Collectively, these data provide compelling evidence for the regulatory influence of GSDMD on hepatocytes, thereby driving the recruitment of neutrophil through the enhanced expression of CXCL1 upon irradiation. GSDMD deficiency inhibits the activation of the stat5a/CXCL1/neutrophil pathway Next, we explored the mechanism GSDMD regulate CXCL1. Through comprehensive analysis utilizing the Gene-Hancer database within the GeneCards Suite ( http://www.genecards.org/ ), we scrutinized the regulatory elements encompassing the promoter and enhancer regions of CXCL1. Leveraging integrative analysis with transcriptomic sequencing ( Gsdmd FL/F vs Gsdmd ∆Hep ), we unearthed seven putative transcription factors (Fig. 6 A), under the influence of GSDMD, that exhibit the potential to bind to the promoter or enhancer regions of CXCL1. Among the examined transcriptional regulators, stat5a exhibited the most prominent differential expression between Gsdmd FL/F and Gsdmd ∆Hep mice (Fig. 6 B). Herein, we sought to elucidate the role of stat5a as a regulator of CXCL1. First, IST5-002, an inhibitor that strongly suppressed the nuclear translocation of stat5a and P-stat5a in hepatocytes (Fig. 6 C), significantly reduced the production of CXCL1 in irradiated hepatocytes (Fig. 6 D). To investigate the regulatory role of stat5a in Cxcl1 transcription, we constructed luciferase reporter plasmids containing cloned stat5a binding sites within the Cxcl1 promoter region. We observed an interferon gamma-activated site (GAS)-like core sequence (TTCT/CNA/GGAA) in promoter of mouse Cxcl1 gene. Strikingly, upon irradiation, we observed a substantial increase in luciferase activity specifically at the stat5a binding sites. Notably, this effect was markedly diminished when the stat5a binding site was mutated, highlighting the critical involvement of stat5a in driving cxcl1 transcription in response to irradiation (Fig. 6 E and Figure S6A). In addition, the application of IST5-002 to irradiated hepatocytes effectively inhibited the migration of neutrophils (Fig. 6 F). These results indicated that stat5a regulated the recruitment of neutrophils in a CXCL1-dependent manner. In vitro investigations revealed that irradiation-treated hepatocytes exhibited enhanced stat5a activation, as evidenced by the elevated protein levels of phosphorylated stat5a (P-stat5a) (Fig. 6 G). Notably, the absence of Gsdmd also attenuated the nuclear translocation of P-stat5a induced by irradiation, as observed through nuclear protein analysis (Fig. 6 H). Moreover, co-immunoprecipitation (coIP) assay confirmed that GSDMD and stat5a could directly interact with each other (Fig. 6 I). Subsequently, we conducted chromatin immunoprecipitation (ChIP) assays, which revealed that depletion of GSDMD significantly impeded the binding of stat5a to the promoter region of the CXCL1 gene (Fig. 6 J). This finding underscores the critical involvement of stat5a in GSDMD-mediated CXCL1 expression. Intriguingly, we also observed that GSDMD deficiency markedly suppressed stat5a activation in livers affected by RILD (Fig. 6 K and L), whereas re-expression of GSDMD-FL reinstated the levels of P-stat5a in Gsdmd ∆Hep mice (Fig. 6 M). Collectively, these findings highlight the role of GSDMD in regulating CXCL1 expression through enhancing stat5a phosphorylation and nuclear translocation. In response to extracellular stress signals, such as irradiation, several downstream signaling pathways, including extracellular signal-regulated kinases (ERKs), p38 MAP kinase, and c-Jun N-terminal kinase (JNK), are recruited and activated. (20, 37, 38) To elucidate the key pathways involved in the upregulation of GSDMD expression, we conducted further investigations. Comparative analysis revealed that, in irradiated hepatocytes, only the phosphorylation of ERK was significantly elevated when compared to the vehicle group. Moreover, the inhibition of ERK using GDC-0994 resulted in a substantial decrease in GSDMD expression (Figure S6 B-D). These findings highlight the predominant role of the ERK pathway in mediating the irradiation-induced increase in GSDMD expression. The therapeutic potential of the GSDMD inhibitor disulfiram in preventing RILD To determine whether GSDMD-mediated pyroptosis acts as a key therapeutic target, disulfiram (DSF), a drug approved by the FDA for inhibiting pyroptosis, was administered to RILD mice. In RILD, Gsdmd FL/FL mice were treated with vehicle ctrl or DSF according to the protocol shown in Figure S7A. Indeed, the administration of DSF resulted in a significant reduction in GSDMD-FL/N in irradiated livers, as assessed by WB and IHC (Fig. 7 A and Figure S7B). Moreover, DSF treatment reduced the activation of stat5a, the expression of CXCL1, and subsequent neutrophil infiltration (Fig. 7 A, C-D), further confirming the molecular mechanisms of GSDMD in RILD, as we discussed above. Of note, in contrast to treatment with the vehicle control, DSF administration significantly curbed acute liver injury in ST-RILD mice and reduced weight loss and liver fibrosis in LT-RILD mice (Fig. 7 E-I, Figure S7C-D), which was accompanied by an improvement in liver function, as determined by serum ALT, AST, and ALB levels (Fig. 7 J). The pyroptosis-inhibitory and radioprotective roles of DSF were also noted in irradiated hepatocytes ex vivo . DSF treatment markedly reduced GSDMD-FL/N expression and LDH levels (Fig. 7 K-L). In addition, stat5a activation and CXCL1 release were both inhibited in DSF-treated hepatocytes (Fig. 7 K, M). Ultimately, DSF-treated hepatocytes were protected from radiation toxicities (Figure S7E). These findings suggest that DSF, a pyroptosis inhibitor, is a promising therapeutic strategy for the management of RILD in cancer patients. DISCUSSION RILD not only harms the public’s health but greatly hinders radiotherapy’s application. (8, 9) Unfortunately, underlying mechanisms and therapeutic agents were both unrevealed in RILD. In this study, we elucidated that irradiation triggers elevated expression of GSDMD in hepatocytes, thereby exacerbating RILD progression. With the use of genetically and pharmacologically modified models, our data found that IR mediated upregulation of GSDMD activated stat5a to drive CXCL1 expression while GSDMD-N, a mature form of GSDMD, formed a pore to release the accumulated CXCL1 in the hepatocytes exposed to IR. As a neutrophil chemokine, CXCL1 from hepatocytes recruits neutrophil into liver and promotes RILD progression. Therefore, GSDMD emerges as a pivotal determinant in the regulation of RILD, underscoring its potential as a promising therapeutic target to impede the progression of this condition. GSDMD is composed of ≈ 480 amino acids and widely expresses in many organs. (11, 21, 39) Previous studies have revealed that GSDMD plays an important role in maintaining homeostasis, (40) eliminating pathogen infection, (41–43) and regulating the development of diseases. (13, 34, 44, 45) GSDMD is inactive in steady state, and its function mainly depend on GSDMD-N, which performs a pore-forming function through oligomerization in the plasma membrane. Membrane perforation leads to the release of many cytokines, which activate downstream pathways. Wang et al . (40) reported that GSDMD-N pores promote mucin granule secretion in the gut epithelium by mediating Ca 2+ entry and help maintain gut homeostasis. In bacterial infection, GSDMD-N drives pyroptosis of infected cells and leads to the release of intracellular bacteria followed by neutrophil-mediated killing. (41, 46) Moreover, the release of IL-33, IL-1β and IL-18 through the pores formed by GSDMD-N also aggravates the progression of allergic airway inflammation, (45) septic shock, (47) and colitis. (48) Hence, GSDMD-FL/N are regarded as critical proteins regulating homeostasis and the outcome of disease. A previous study reported that TBI increased gut and bone marrow expressed GSDMD-FL/N, which correlated positively with the severity of bone loss. (15) However, the effect of GSDMD-N executed pyroptosis and mediated inflammatory factors release in RILD remains unclear. Here, we revealed that IR mobilize GSDMD-N expression and the pyroptosis in hepatocytes. Of note, the level of GSDMD-N positively correlated with the severity of RILD, and inhibition of GSDMD could effectively alleviate the progression of RILD. GSDMD-N mediated pyroptosis has been well documented in different cell types and promotes the release of multiple cytokines. Recent studies have demonstrated that GSDMD-N promotes macrophages pyroptosis and release regulator factors, which induces coagulation, leading to disseminated intravascular coagulation in sepsis. (49, 50) GSDMD-driven pyroptosis in myeloid cells also plays a pivotal role in the pathogenesis of hepatic ischemia–reperfusion injury. (13) Although the role of pyroptosis in immune cells has been extensively studied, its implications in hepatocytes and the resulting impact on the immune microenvironment remain largely unexplored. Wang et al. reported that a natural compound could alleviate CCL4-induced liver injury by inhibiting the pyroptosis of hepatocytes, but the underlying mechanism is uncertain. (51) In this study, we first revealed that the critical pyroptotic cell type was hepatocyte in RILD. In addition, we also demonstrated that the impact of GSDMD-N in remodeling immune environment via its pore-forming activity. In brief, upon IR, CXCL1 were released from hepatocytes via GSDMD-N cleaved by caspase-1 and recruited neutrophils to change the immune environment of liver. Our study also has found cleavage-independent function of GSDMD, where it activates the transcription factor stat5a to drive CXCL1 expression. In support of this cleavage-independent function, other gasdermin family proteins were reported to have cleavage-independent function to regulate the downstream genes. Huang et al . reported that GSDME mediates the transcription factor to enter the nucleus where it promotes the expression of downstream genes. (17) GSDMB is also found to be localized in the nucleus and regulates the expression of genes by acting as a transcriptional coactivation or enhancer. (18) These findings highlight the need for further investigation to fully understand the diverse roles of GSDMD. Our study also aims to evaluate the efficacy of a protective drug for RILD. Disulfiram is known to be approved by FDA to inhibit GSDMD-N mediated pore formation. In addition, disulfiram also can suppress the expression of GSDMD-FL in Nigericin-primed THP-1 cells. (52) Administration of disulfiram significantly decreased the severity of RILD. In addition, other side effects induced by antitumor therapy, including chemotherapy, (53, 54) were reported to be related to GSDMD-mediated pyroptosis, which indicates that pyroptosis is a potential therapeutic target in alleviating the side effects of other anti-tumor therapies in cancer patients. Moreover, DSF has been reported to be effective against diverse cancer types in preclinical studies through p97/Nuclear protein localization protein 4 homolog (NPL4) pathway. (55–57) These results suggest that DSF may not only alleviate RILD, but also further enhance the tumor control rate. STAT5a is a member of STAT family, a group of latent transcription factors that are activated in response to various cytokine signaling pathways. The predominant mechanism by which stat5 influences neutrophils, primarily through the action of granulocyte colony-stimulating factor (G-CSF), is by modulating the transcription of genes associated with cell survival and proliferation( 58 ). In contrast, STAT3, another member of the STAT family, has been reported to be essential for the chemotaxis of mature neutrophils towards CXCR2 ligands, including CXCL2( 59 , 60 ). Our luciferase activity assays and CXCL1 ELISA measurement provide evidence that stat5a is indispensable for the transcription of cxcl1 following irradiation. Additionally, we discovered that stat5a plays a crucial role in neutrophil chemotaxis. This collective evidence demonstrates that, as a transcription factor, stat5a also mediates the recruitment of neutrophils through CXCL1. In conclusion, our study demonstrated that irradiation induced GSDMD activation in hepatocytes, which plays a crucial role in RILD progression. GSDMD induces pyroptotic phenotype and increased synthesis and release of CXCL1 in hepatocytes. Mechanistically, GSDMD promotes stat5a into the nucleus which subsequently increased CXCL1 synthesis and GSDMD-N supports CXCL1 release from forming pores. Afterwards, as a chemokine, CXCL1 acts to recruit neutrophils into liver to ultimately aggravate the severity of RILD. Both gene knockout and pharmacological inhibition of GSDMD effectively ameliorate RILD. These findings provide a novel target for the development of effective strategies for treating patients with RILD. Declarations Acknowledgements NO Authors contributions Zhenwei Peng, Yi Zhou, Xiongjun Wang contributed to design and data interpretation. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3161881","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":221715469,"identity":"c1bb8c36-415b-4af9-a748-c4e7e807b4f1","order_by":0,"name":"Zhenwei 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Left, representative immunohistochemistry-stained images; Right, statistical analyses of the level of GSDMD-N from all HCC patients of Student’s \u003cem\u003et\u003c/em\u003e test. (Scale bar, 200 ×, 50 μm). (B) Serum level of GSDMD-N in patients with ALBI grade 2 (\u003cem\u003en\u003c/em\u003e= 42) and patients with grade 1 (\u003cem\u003en\u003c/em\u003e= 15). Student’s \u003cem\u003et\u003c/em\u003e test. (C) IHC staining of GSDMD in liver of mice in hydrodynamic injection liver tumor radiotherapy model and normal liver tissues are circled by dashed lines (Scale bar, 10×, 2 μm; 400×, 50 μm). Mice (\u003cem\u003en\u003c/em\u003e= 5/group) were irradiated with 4 Gy hepatic radiation per day for three fractions after 4 weeks hydrodynamic transfection and sacrificed after 2 weeks. (D) \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003en\u003c/em\u003e= 3-5/group) treated without or with one dose of 30 Gy hepatic irradiation and sacrificed at day 7 in ST-RILD. The expression level of GSDMD-FL and GSDMD-N were analyzed by Immunoblot (Left). Right, the same as left, except the mice accepted 6 Gy hepatic irradiation every two days and sacrificed at 10 weeks after the last radiotherapy in LT-RILD. (E) \u003cem\u003eGsdmd\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e and other pyroptosis-related genes expression in each immune cluster of Ctrl and IR groups. Y axis indicates log-normalized expression. (F) Representative low-magnification (Scale bars, 100×, 200 μm) and blow-up magnification (Scale bars, 400×, 50 μm) images of GSDMD-FL staining in Ctrl, ST-RILD and LT-RILD mice (\u003cem\u003en\u003c/em\u003e= 5/group). Left, representative images; Right, statistical analyses of the level of GSDMD-FL in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e mice treated with or without IR. Student’s \u003cem\u003et\u003c/em\u003e test. (G) Immunofluorescence visualization of GSDMD-FL (green), DAPI (blue), CD11b (red), CD3 (red), CK19 (red), ɑ-SMA (red) in liver sections from Ctrl and IR-treated mice (ST- and LT-RILD, \u003cem\u003en\u003c/em\u003e= 5/group). Up, representative images (Scale bars, 200×, 100 μm); Down, statistical analysis of the change of the cells co-stained with GSDMD-FL. Student’s \u003cem\u003et\u003c/em\u003e test. (H) GSDMD-N protein expressions from hepatocyte, monocyte-granulocyte, HSC, T cell and sinusoidal endothelial cell (SEC) were detected by immunoblot analysis. (I) Immunoblot analysis of GSDMD-FL and GSDMD-N expression from WT primary mouse hepatocytes exposed to 8 Gy irradiation at different times. (J-L) \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e hepatocytes 24 h after 8 Gy irradiation, cell morphology was observed under microscopy (black indicates bubble-like morphology) (J), and cell viability was assessed by LDH assay (K) and propidium iodide (PI) staining (L). Student’s \u003cem\u003et\u003c/em\u003e test and one-way ANOVA plus Dunnett’s multiple comparisons test. Data are mean ± SEM. *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3161881/v1/b1caf1e69131dc493de7f96f.png"},{"id":40904647,"identity":"1318306b-11bb-4e58-a7fc-0bdd8809a473","added_by":"auto","created_at":"2023-08-01 19:18:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2537509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGSDMD deficiency ameliorates acute liver injury and chronic fibrosis in RILD. \u003c/strong\u003e(A) Representative high magnification images of Hematoxylin Eosin staining (HE) staining in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003en\u003c/em\u003e= 4-5/group) treated with or without one single 30 Gy hepatic radiation. (Scale bars, 200×, 200 μm, 400×, 50 μm). (B-D) Representative liver macroscopic image (B), ɑ-SMA staining (C) and sirius-red staining (D) in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e or\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice in LT-RILD. Scale bars, 400×, 50 μm; 200×, 100 μm. Statistical analysis of ɑ-SMA and collagen area were present in the right, 5 fields were observed per mice (\u003cem\u003en\u003c/em\u003e= 4-5/group). One-way ANOVA plus Dunnett’s multiple comparisons test. (E) Immunoblot analysis of liver protein expression of COL1A1, Fibronectin and ɑ-SMA from \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003en\u003c/em\u003e= 1-4/group). (F) Key hepatic profibrogenic genes of \u003cem\u003eCol1a1\u003c/em\u003e, \u003cem\u003eTgfb1\u003c/em\u003e and \u003cem\u003eTimp1\u003c/em\u003e in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003en\u003c/em\u003e= 3-5/group) treated with IR. One-way ANOVA plus Dunnett’s multiple comparisons test. (G) Serum levels of liver function markers ALT, AST and ALB were determined in Ctrl-, ST- and LT-RILD groups (\u003cem\u003en\u003c/em\u003e= 3-5/group). One-way ANOVA plus Dunnett’s multiple comparisons test. (H) Immunoblot analysis was performed to measure ALB from irradiated or non-irradiated \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e primary mouse hepatocytes. (I) Supernatant levels of liver function markers ALT and AST were determined in hepatocytes accept no IR and IR. One-way ANOVA plus Dunnett’s multiple comparisons test. Data are expressed as mean ± SEM. *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3161881/v1/9d82df6e26cb41dbfbc32289.png"},{"id":40904642,"identity":"c99651be-885f-4069-a52e-fccef2dce10f","added_by":"auto","created_at":"2023-08-01 19:18:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1495544,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGSDMD regulates the recruitment of neutrophils to the irradiated liver. \u003c/strong\u003e(A-B) Workflow and Proportion plot of immune cells. (A) Three livers from \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e -Ctrl, \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e-ST and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e-ST group were used. 7 days after the last 30 Gy irradiation, the livers were digested and 10 × Genomics scRNA-seq was used to profile the cells; (B) UMAP plot of 20,833 liver single cells from three group mice, colored by the group. (C) Colors indicate the cell type annotated using Single R and manual annotation and validated by cell-type-specific gene expression. (D) Flow Cytometric analysis of liver-infiltrated neutrophils (FVD\u003csup\u003e-\u003c/sup\u003eCD45\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e) of \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003en\u003c/em\u003e= 3-5/group). Student’s \u003cem\u003et\u003c/em\u003e test. (E) Neutrophil infiltration (\u003cem\u003en\u003c/em\u003e= 3-5/group) was analyzed by immunohistology staining (left, scale bars, 200×, 50 μm) with antibodies against MPO, and following morphometric quantification (right). One-way ANOVA plus Dunnett’s multiple comparisons test. (F-G) Detection of the neutrophil infiltration in liver of \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice treated with or without AAV9-GSDMD-FL adeno-associated virus (\u003cem\u003en\u003c/em\u003e= 3-5/group). (F) Representative plots (left) and statical analysis (right) of the percentage of CD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e cells with the flow cytometry. (G) MPO staining images (left) and quantification (right). Student’s \u003cem\u003et\u003c/em\u003e test. (H) Quantification of neutrophil migration in response to \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e-hepatocytes pretreated with irradiation or not. One-way ANOVA plus Dunnett’s multiple comparisons test. (I-J) ɑ-SMA expression of HSCs were analyzed with immunoblot in non-contact manner (I) and contact manner (J) co-culture hepatocytes with HSCs. Data are expressed as mean ± SEM. *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3161881/v1/14255df1ed083d7706f49a50.png"},{"id":40904640,"identity":"6c8ce037-3a79-4801-881e-5c80e36d7c2a","added_by":"auto","created_at":"2023-08-01 19:18:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3155142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelective CXCL1 neutralization alleviates the severity of RILD through hampering neutrophil recruitment. \u003c/strong\u003e(A) Volcano plot depiction of neutrophil chemokine genes between \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003en\u003c/em\u003e= 5). (B) Detection of the hepatic mRNA level of \u003cem\u003eCxcl1\u003c/em\u003e in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003en\u003c/em\u003e= 4-5) accepted or were free of 30 Gy or 6 Gy×5 IR. One-way ANOVA plus Dunnett’s multiple comparisons test. (C) Representative immunohistochemistry images of CXCL1 in liver tissue of Ctrl and IR group from \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice (Scale bars, 40×, 200 μm; 400×, 50 μm，left). Statical analysis of CXCL1 expression were calculated from five fields per liver (\u003cem\u003en\u003c/em\u003e= 5, right). One-way ANOVA plus Dunnett’s multiple comparisons test. (D) Representative liver CXCL1 staining from Ctrl- and IR-mice treated with AAV9-control and AAV9-GSDMD-FL adeno virus (Scale bars, 40×, 200 μm, \u003cem\u003en\u003c/em\u003e= 4). (E) Detection of the neutrophil infiltration in liver of mice treated with isotype IgG antibody and anti-CXCL1 antibody. The percentage of cells with CD11b\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e with flow cytometry (left) and its statical analysis were presented in both ST- and LT-RILD cohort (right, \u003cem\u003en\u003c/em\u003e= 3-5 mice/group). Student’s \u003cem\u003et\u003c/em\u003e test. (F) Representative liver HE images from ST-RILD mice (Scale bars, 200×, 50 μm). (G-I) Representative liver macroscopic images (G), ɑ-SMA staining (H) and sirius-red staining (I) in cohort of LT-RILD mice. (Scale bars, 400×, 50 μm, \u003cem\u003en\u003c/em\u003e= 3-5 mice/group). Student’s \u003cem\u003et\u003c/em\u003e test. (J) Hepatic mRNA levels of \u003cem\u003eCol1a1\u003c/em\u003e, \u003cem\u003eTgfb1\u003c/em\u003e and \u003cem\u003eTimp1 \u003c/em\u003efrom LT-RILD mice. Student’s \u003cem\u003et\u003c/em\u003e test. (K) Immunoblot analysis of COL1A1, Fibronectin and ɑ-SMA. (L) Serum levels of ALT, AST and ALB. \u003cem\u003en\u003c/em\u003e= 3-5 mice/group. One-way ANOVA plus Dunnett’s multiple comparisons test. *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3161881/v1/41b5dff2c56333f2ba86461d.png"},{"id":40904643,"identity":"b074592a-8feb-4b5b-b412-f01c0be4eed9","added_by":"auto","created_at":"2023-08-01 19:18:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1668510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGSDMD regulates hepatocytes release CXCL1 to recruit neutrophil. \u003c/strong\u003e(A) Immunofluorescence visualization of CXCL1 (green), HNF4α (red), ɑ-SMA (red) in liver sections from Ctrl and IR-treated mice (\u003cem\u003en\u003c/em\u003e= 5). (Scale bars, 200×, 100 μm). Student’s \u003cem\u003et\u003c/em\u003e test. (B) Double-immunostaining for CXCL1 and GSDMD-FL in primary hepatocytes 24 h after irradiation. Confocal analysis demonstrates coexpression of CXCL1 and GSDMD-FL in hepatocytes receiving radiation. Scale bars, 200×, 50 μm. (C) Cell viability was assessed by Annexin-V\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e+\u003c/sup\u003e. Student’s \u003cem\u003et\u003c/em\u003e test. (D) ELISA assay of CXCL1 release in culture supernatants from \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e hepatocyte accepted irradiation or not. One-way ANOVA plus Dunnett’s multiple comparisons test. (E) Immunoblot analysis of caspase-1 and cleaved-caspase-1 expression from WT primary mouse hepatocytes exposed to 8 Gy irradiation at 12h. (F) Immunoblot analysis of WT hepatocytes treated with 20 μM Ac-YVAD-cmk for 24h, and exposed to 8 Gy irradiation. (G) ELISA assay of CXCL1 release in culture supernatants from hepatocytes pretreated without or with 20 μM Ac-YVAD-cmk. Student’s \u003cem\u003et\u003c/em\u003e test. (H-I) Quantification of neutrophil migration. Quantification of neutrophil migration in response to irradiated \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e hepatocytes pretreated with anti-CXCL1 antibody or not (H); Quantification of neutrophil migration in response to irradiated \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e hepatocytes pretreated with CXCL1 RP or not (I). Student’s \u003cem\u003et\u003c/em\u003e test. Data are expressed as mean ± SEM. **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3161881/v1/b9425155c8c3b327e96816c6.png"},{"id":40904644,"identity":"04c9fa13-e428-4397-9016-167623bde033","added_by":"auto","created_at":"2023-08-01 19:18:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":780297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGSDMD deficiency inhibits the activation of the stat5a/CXCL1/neutrophil pathway. \u003c/strong\u003e(A) Identification of promoters and enhancers of CXCL1 with integrative analysis of transcriptomic sequencing and database within the GeneCards Suite (\u003ca href=\"http://wwww.genecards.org/\"\u003ehttp://wwww.genecards.org/\u003c/a\u003e). (B) Heatmap of expression of the indicated genes from (A). (C-D) Primary hepatocytes were pretreated with or without 25 μM stat5a inhibitor IST5-002 for 6 h prior to 8 Gy irradiation. After 24 h, the translocation inhibit ability were determined by the WB assay (C); the culture medium level of CXCL1 was assayed by ELISA (D). One-way ANOVA plus Dunnett’s multiple comparisons test. (E) Luciferase activity assay was performed to measure the binding of stat5a at the WT or the promoter region with mutations (Mut) of \u003cem\u003eCxcl1\u003c/em\u003e gene with or without IR in HEK 293T cell. One-way ANOVA plus Dunnett’s multiple comparisons test. (F) Quantification of neutrophil migration in response to irradiated hepatocytes pretreated with IST5-002 or not. One-way ANOVA plus Dunnett’s multiple comparisons test. (G) Immunoblot analysis of stat5a and P-stat5a from \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep \u003c/em\u003e\u003c/sup\u003eprimary hepatocyte exposed to 8 Gy irradiation. (H) Protein level of stat5a and P-stat5a in cytoplasmic extracts and nuclear extracts from 8 Gy irradiated \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e primary hepatocytes were determined by Immunoblot. (I) Co-IP analysis for physical interaction of GSDMD and stat5a in 293T cells.\u0026nbsp; (J) ChIP assays were performed to detect the promoter sites of stat5a binding to CXCL1. IgG as a negative control for ChIP assay. One-way ANOVA plus Dunnett’s multiple comparisons test. (K-L) Immunoblot analysis of stat5a and P-stat5a from \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and\u003cem\u003e Gsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice (Ctrl, ST- and LT-RILD group). (M) Immunoblot analysis of stat5a and P-stat5a of IR-\u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice administered with AAV9-Control and AAV9-GSDMD-FL. Data are expressed as mean ± SEM. *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3161881/v1/8acc3662d1903f3480c62603.png"},{"id":40904645,"identity":"ad64fde9-43a7-468b-bd52-fd94bc87a011","added_by":"auto","created_at":"2023-08-01 19:18:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5729795,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe therapeutic potential of the GSDMD inhibitor disulfiram in preventing RILD. \u003c/strong\u003e(A) Hepatic protein levels of GSDMD-FL, GSDMD-N, stat5a and P-stat5a between DSF and vehicle groups were evaluated by Immunoblot. (B) Representative staining of CXCL1 in liver tissue from vehicle and DSF-treated mice (Scale bars, 40×, 200 μm). \u003cem\u003en\u003c/em\u003e= 3-5 mice/group. One-way ANOVA plus Dunnett’s multiple comparisons test. (C-D) The neutrophil infiltration was evaluated by flow cytometry (C) and MPO immunostaining (D) in vehicle and DSF-treated mice (Scale bars, 200×, 50 μm). \u003cem\u003en\u003c/em\u003e= 3-5 mice/group. One-way ANOVA plus Dunnett’s multiple comparisons test. (E) Representative images of HE staining in vehicle or DSF-treated ST-RILD mice. Scale bars, 100×, 50 μm. \u003cem\u003en\u003c/em\u003e= 3-5 mice/group. (F-H) Representative liver macroscopic images (F), ɑ-SMA staining (G) and sirus-red staining (H) in cohort of LT-RILD mice (Scale bars, 200×, 50 μm). \u003cem\u003en\u003c/em\u003e= 3-5 mice/group. Student’s \u003cem\u003et\u003c/em\u003etest. (I) Immunoblot analysis of COL1A1, Fibronectin and ɑ-SMA. (J) Serum levels of ALT, AST, and ALB. \u003cem\u003en\u003c/em\u003e= 3-5 mice/group. One-way ANOVA plus Dunnett’s multiple comparisons test. (K) Immunoblot analysis of GSDMD-FL/N, ALB, stat5a and P-stat5a. (L-M) Supernatants from vehicle or disulfiram pre-treated primary hepatocyte were collected for LDH (L) and CXCL1 (M) assay. Data are expressed as mean ± SEM. One-way ANOVA plus Dunnett’s multiple comparisons test. **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3161881/v1/e1aacc17f276855d19f76100.png"},{"id":41672628,"identity":"fa71933e-b07f-4ce9-8956-74fdaa8214ad","added_by":"auto","created_at":"2023-08-17 04:18:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4854435,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3161881/v1/42338950-da5d-42cf-94b4-834a8208940e.pdf"},{"id":40905831,"identity":"02d387fb-8cda-4f49-93a0-5cca6c89f642","added_by":"auto","created_at":"2023-08-01 19:26:26","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2831914,"visible":true,"origin":"","legend":"Supplementary file","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-3161881/v1/1c6917247121fe83b1c2d5cd.docx"},{"id":40904649,"identity":"92aadbc3-5dd5-461f-8798-a08ee9a46909","added_by":"auto","created_at":"2023-08-01 19:18:26","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":8872709,"visible":true,"origin":"","legend":"","description":"","filename":"UnprocessedWesternblotimages.docx","url":"https://assets-eu.researchsquare.com/files/rs-3161881/v1/b8409b6e2275cc498360b6b0.docx"}],"financialInterests":"(Not answered)","formattedTitle":"The progression of hepatocyte pyroptosis exacerbates radiation-induced liver disease via the gasdermin D/signal transducer and activator of transcription 5a/CXCL1 axis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe liver, a pivotal organ with multifarious functions, manifests as a radiosensitive entity warranting particular attention.\u003csup\u003e(1)\u003c/sup\u003e Exposure to ionizing irradiation, such as nuclear and radiological incidents,\u003csup\u003e(2)\u003c/sup\u003e total body irradiation (TBI)\u003csup\u003e(3)\u003c/sup\u003e before allogeneic stem cell transplantation, and radiotherapy for tumors\u003csup\u003e(4\u0026ndash;7)\u003c/sup\u003e in the upper abdomen (liver, biliary, and pancreas) and thoracic (right lower lung, distal esophagus, and breast), can result in radiation-induced liver disease (RILD). RILD manifests as acute liver injury in the short-term after RT followed by chronic progression to liver fibrosis,\u003csup\u003e(8, 9)\u003c/sup\u003e while no clinic effective drugs to alleviate it. Patients will irreversibly develop into cirrhosis, liver failure and even death. Pathophysiologically, radiation results in the increase of inflammatory cytokines and recruitment of immune cells, ultimately leading to tissue fibrosis and liver dysfunction.\u003csup\u003e(10)\u003c/sup\u003e However, the underlying mechanism of immune regulation during RILD remains unclear.\u003c/p\u003e \u003cp\u003ePyroptosis, a form of inflammatory cell death, is crucial in the immune environment. During pyroptosis, inflammasome activation by canonical (NOD-like receptor [NLR], absent in melanoma 2 [AIM2], and caspase-1) or noncanonical (caspase-4/5 in humans or caspase-11 in mice) pathways leads to the cleavage of GSDMD. GSDMD-N then forms pores in the membrane and results in cell swelling, a burst of inflammatory cytokines and cytosolic content release and ultimately cell lysis. Therefore, GDSMD, as a key executioner of pyroptosis and cytokine release, is thought to be a crucial mediator in shaping the inflammatory environment involved in the progression of multiple liver diseases.\u003csup\u003e(11\u0026ndash;14)\u003c/sup\u003e Recently, Xiao \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e(15)\u003c/sup\u003e reported that TBI could induce GSDMD-FL/N increase in the gut and bone marrow of mice, which were positively correlated with the severity of bone loss, but the role of GSDMD in this process remains unclear. However, Wu \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e(16)\u003c/sup\u003e revealed that there were no significant increase of GSDMD-FL/N in lung tissues after chest irradiation. These discoveries suggest that the existence and effects of GSDMD are complicated and vary in different radiation-induced tissue injuries. However, the occurrence of GSDMD-executed pyroptosis in RILD and its impact on the expression and secretion of inflammatory factors still remain elusive. Therefore, further investigation is warranted to elucidate the role of GSDMD-executed pyroptosis in RILD. Additionally, it is crucial to explore other uncharted functions of GSDMD in the context of RILD, considering that cleavage-independent functions of gasdermin proteins have been reported to influence the growth of pancreatic adenocarcinoma and the progression of asthma.\u003csup\u003e(17, 18)\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn the present study, we unveil the intriguing upregulation of GSDMD-FL/N and the emergence of a pyroptotic phenotype in irradiated hepatocytes, shedding light on the molecular mechanisms underlying RILD. Through its targeted interaction with stat5a, GSDMD facilitated the phosphorylation of stat5a, leading to its translocation from the cytoplasm to the nucleus. This dynamic process played a critical role in mediating the expression of CXCL1, initiating a cascade of events within the liver microenvironment. GSDMD-N played a pivotal role in the secretion of CXCL1, attracting neutrophils to the liver and exacerbating the severity of RILD. Remarkably, we successfully mitigated neutrophil recruitment and alleviated RILD in mice by employing antibodies to block CXCL1. Furthermore, our promising findings indicate that pharmacological inhibition of GSDMD offers a potent approach to suppress the progression of RILD. Collectively, our discoveries highlight the exciting potential of targeting GSDMD as a therapeutic strategy to prevent RILD.\u003c/p\u003e"},{"header":"MATERIALS AND MEHTHODS","content":"\u003cp\u003e \u003cb\u003eAnimal studies\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHepatocyte-specific \u003cem\u003eGsdmd\u003c/em\u003e knockout (\u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e) were generated by breeding \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFlox/Flox\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e) with Alb-cre mice on the C57BL/6J background (GemPharmatech, Nanjing, China). All animals were acclimatized for 1 week before experiments and housed in a specific pathogen-free environment with a 12-hour light-dark cycle and permitted ad libitum consumption of water and a standard chow diet unless otherwise stated. This research was approved by the Institutional Care and Animal Use Committee of Sun Yat-sen University Cancer Center (20110M).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMouse model of RILD\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUsing a previously established protocol,\u003csup\u003e(19, 20)\u003c/sup\u003e we generated a mouse model of hepatocellular carcinoma (HCC) by hydrodynamic injection of AKT/Ras and Sleeping Beauty transposon. Two weeks post-transfection, the mice were subjected to hepatic radiation, administered in three fractions of 4 Gy per day and subsequently analyzed pathologically.\u003c/p\u003e \u003cp\u003eMale \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice, aged between seven to ten weeks, were carefully anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg). A single fraction of 30 Gy hepatic radiation was administered to establish the short-term (ST) radiation-induced liver disease (RILD) model. To explore the long-term (LT) side effects of RILD, mice received five fractions of 6 Gy hepatic radiation every two days and were allowed to spontaneously recover for a period of 10 weeks. CXCL1 was blocked by administration of a mouse CXCL1 neutralizing antibody (100 \u0026micro;g/mouse, \u003cem\u003ei.v.\u003c/em\u003e) (R\u0026amp;D systems) 15 min before irradiation. For LT-RILD, mice were injected additionally with CXCL1 neutralizing antibody (25 \u0026micro;g/mouse, \u003cem\u003ei.v.\u003c/em\u003e) weekly after the last irradiation. Control mice were injected with the same dosage rat isotype-IgG control antibody. For ST-RILD, WT mice were given intraperitoneal injection of disulfiram (100 mg/kg, MCE) or vehicle 15 min before the first radiotherapy administrated and maintained until 7 days. For LT-RILD, in addition to the injection of 100 mg/kg disulfiram 15 min before radiotherapy, the injection of 50 mg/kg disulfiram was maintained every week after the end of radiotherapy. Disulfiram was dissolved in 10% DMSO and 100% corn oil, while control mice were given vehicle (containing only 10% DMSO and 100% corn oil). Adeno-associated virus (AAV9) vector that expressed GSDMD-FL (AAV9-GSDMD-FL) and control (AAV9-Control) were purchased from Genechem Company (Shanghai, China). The virus solution was diluted by PBS and then injected into \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice through the tail vein at a dose of 1\u0026times;10\u003csup\u003e11\u003c/sup\u003e v.g./mouse. The mice were subjected to 30 Gy hepatic irradiation and sacrificed at day 7.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHuman liver tissue and blood samples collection\u003c/b\u003e \u003c/p\u003e \u003cp\u003eLiver tissues of HCC patients who underwent hepatectomy adjuvant radiotherapy were collected before and after radiotherapy (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) at the first affiliated hospital of Sun Yat-sen university.\u003c/p\u003e \u003cp\u003eBlood specimens were collected from patients accepted hepatic irradiation from June 2020 to July 2021 in the radiotherapy department of the First Affiliated Hospital of Sun Yat-sen University. Patients with abnormal liver function before irradiation and those receiving other antitumor therapy were excluded (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Finally, a total of 57 freshly blood was collected and serum was separated and stored at -80\u0026deg;C until further analysis. The human study was approved by the Institutional Ethics Committee of the first affiliated hospital of Sun Yat-sen university.\u003c/p\u003e \u003cp\u003eThe detailed methodology is described in the Supporting Experimental Procedures.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eGSDMD and its pyroptosis-trigger GSDMD-N are upregulated in RILD\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether pyroptosis triggered by GSDMD-N was induced by irradiation, peritumor liver tissues from the same HCC patients (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) were collected before and after radiotherapy (detailed clinic information was shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Immunohistochemistry (IHC) showed that GSDMD-N was markedly upregulated in the liver of hepatocellular carcinoma (HCC) patients who received radiotherapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We then sought to dissect the relationship between GSDMD-N level and the severity of RILD. Ge \u003cem\u003eet al.\u003c/em\u003e reported that GSDMD could be released from pyroptotic cardiomyocyte and can be measured in serum.\u003csup\u003e(21)\u003c/sup\u003e We next collected peripheral blood from patients after hepatic RT and detect the GSDMD-N level. The inclusion and exclusion criteria were shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA. In short, we initially screened 184 patients with hepatic tumors accepted radiotherapy between June 2020 and July 2021. Patients with liver dysfunction before radiotherapy or accepted other anti-tumor therapies were excluded. Albumin-bilirubin (ALBI) score was used to assess liver toxicity induced by irradiation,\u003csup\u003e(22\u0026ndash;24)\u003c/sup\u003e we found that patients with ALBI grade 2 had higher GSDMD-N levels than those with grade 1 (1855.0 ng/mL vs 331.3 ng/mL, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0177) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Further correlation analyses revealed that serum GSDMD-N levels were strongly positively correlated with the severity of liver injury (serum ALT, AST and ALP) and negatively correlated with liver function (serum ALB) in RILD patients (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next evaluated GSDMD expression in the liver tissue adjacent tumor in radiotherapy model of mouse HCC. Peritumor liver tissues also presented increased GSDMD-FL expression after hepatic radiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To confirm the expression pattern of GSDMD in RILD mice, we subjected mice to a universal short-term (ST) and long-term (LT) RILD modelling protocol (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC).\u003csup\u003e(25, 26)\u003c/sup\u003e First, we observed that GSDMD-FL and GSDMD-N levels were robustly elevated in the irradiated murine liver tissue in both the ST-RILD and LT-RILD groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Moreover, hepatic GSDMD-N levels were closely correlated with the degree of liver injury (serum ALT and AST), liver function (serum ALB), and liver fibrosis (fibronectin, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD-E). These results suggested that GSDMD and its pyroptosis-triggerred N-terminal fragment are upregulated in liver tissues during RILD.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGSDMD-dependent hepatocyte pyroptosis occurs in RILD\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have shown that GSDMD-dependent pyroptosis widely occurs in immune cells.\u003csup\u003e(27, 28)\u003c/sup\u003e To identify which immune cell type is involved in pyroptosis after irradiation in an unbiased and precise manner, we performed comprehensive scRNA-seq of whole liver tissue from control (ctrl) and ST-RILD mice. Within the immune compartments, we compared the expression of \u003cem\u003eGsdmd\u003c/em\u003e and other pyroptosis-associated gene markers, including \u003cem\u003eIl1b\u003c/em\u003e, \u003cem\u003eIl18, Casp1\u003c/em\u003e, \u003cem\u003eCaspase4/11\u003c/em\u003e, \u003cem\u003eNlrp3\u003c/em\u003e, \u003cem\u003eNod1\u003c/em\u003e, and \u003cem\u003eNod2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Unexpectedly, \u003cem\u003eGsdmd\u003c/em\u003e was rarely elevated in hepatic immune cells after IR. Under physiological conditions, \u003cem\u003eGsdmd\u003c/em\u003e was slightly expressed in monocytes and Kupffer cells and barely expressed in neutrophils and T/B cells. Moreover, no noticeable increase in the expression of \u003cem\u003eGsdmd\u003c/em\u003e or other pyroptosis\u003cem\u003e-\u003c/em\u003eassociated genes were observed in these cells after IR. Since pyroptosis can also occur in hepatocytes rather than immune cells in steatohepatitis and CCL4-induced liver injury,\u003csup\u003e(29)\u003c/sup\u003e we assumed that hepatocytes might be the major pyroptotic cells in RILD, with strong activation of GSDMD. Indeed, a robust increase in GSDMD-FL expression was preferentially found in the parenchymal cells of RILD mouse livers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Further immunofluorescence analysis was performed to clearly determine the GSDMD expression changes in hepatocytes and other cells. As we speculated, remarkable coexpression of GSDMD with hepatocellular nuclear factor 4-alpha (HNF4ɑ, a marker of hepatocytes) was observed in both ST-RILD and LT-RILD livers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), while CD11b\u003csup\u003e+\u003c/sup\u003e monocytes, CD3\u003csup\u003e+\u003c/sup\u003e T cells, CK19\u003csup\u003e+\u003c/sup\u003e cholangiocytes and α-SMA\u003csup\u003e+\u003c/sup\u003e hepatic stellate cells (HSCs) displayed low expression of GSDMD. To accurately confirm the occurrence of pyroptosis in hepatocyte, we isolated primary hepatocytes and other non-parenchymal cells (NPC) from the liver of ctrl and ST-RILD mice and compared their GSDMD-N levels by western blot (WB) analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Hepatocytes also had the highest GSDMD-N level after irradiation compared with other cells, further indicating that hepatocytes are critical pyroptotic cells in RILD.\u003c/p\u003e \u003cp\u003eSubsequently, to determine whether irradiation induced hepatocyte pyroptosis, primary hepatocytes were isolated from mice and irradiated with 8 Gy irradiation to establish \u003cem\u003eex vivo\u003c/em\u003e radiation models (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF). Indeed, irradiation could aggravate hepatocyte injury, manifested as decreased ALB and increased ALT and AST levels 24 h after IR (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG-H). Besides, the levels of the key pyroptotic proteins GSDMD-FL/N increased from 12h to 24 h after irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Compared to nonirradiated hepatocytes, irradiated hepatocytes also exhibited other late-stage features of pyroptosis: cell swelling with a balloon-shaped protrusion emerging from the plasma membrane by microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ), increased LDH levels in the supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK) and decreased cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). These results confirmed that hepatocyte pyroptosis is activated after IR. To further investigate the requirement of GSDMD for hepatocyte pyroptosis, primary hepatocytes from \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice were subjected to irradiation. Compared to \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e hepatocytes, \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e hepatocytes were resistant to irradiation-mediated pyroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-L). Altogether, these results show that GSDMD is required for triggering pyroptosis in irradiated hepatocytes.\u003c/p\u003e \u003cp\u003eCollectively, these data demonstrated that GSDMD is required for driving hepatocyte pyroptosis in RILD.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGSDMD deficiency ameliorates acute liver injury and chronic fibrosis in RILD\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the role of GSDMD-dependent hepatocyte pyroptosis in RILD, we generated RILD models with \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice. No significant difference in weight loss was found between these two groups of mice with ST-RILD (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA), while \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice with LT-RILD displayed less weight loss and regained body weight more quickly after irradiation, in contrast to the WT-IR mice (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Moreover, in ST-RILD mice, liver tissues of \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e-IR mice manifested notably increased steatosis, while GSDMD deficiency markedly mitigated this pathological damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In LT-RILD mice, the livers of \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e-IR mice exhibited apparent scattered nodules (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), notable hepatic stellate cell (HSC) activation (α-SMA\u003csup\u003e+\u003c/sup\u003e), obvious extracellular matrix (ECM) deposition (collagen\u003csup\u003e+\u003c/sup\u003e and fibronectin\u003csup\u003e+\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E), and enhanced expression of the profibrotic markers \u003cem\u003eCol1a1\u003c/em\u003e, \u003cem\u003eTgfb1\u003c/em\u003e, and \u003cem\u003eTimp1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), indicating that chronic liver fibrosis was induced by irradiation. However, all of these fibrotic changes were notably suppressed in the context of GSDMD deficiency. In addition, in both ST- and LT-RILD mice, the absence of GSDMD also led to a striking decrease in serum ALT and AST and an improvement in ALB, reflecting enhanced liver function (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Collectively, these results suggest that GSDMD deficiency exerts a vital inhibitory effect on both early IR-induced hepatic injury and advanced liver fibrosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further determine the pathogenic role of GSDMD in RILD, \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice were administered AAV9-GSDMD-FL to re-express GSDMD before IR (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC-D). GSDMD-FL re-expression did not affect weight loss in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e ST-RILD mice (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eE) but resulted in a significant increase in hepatic histology damage and liver injury (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eF-G). These results confirmed that upregulation of GSDMD accelerated the progression of RILD.\u003c/p\u003e \u003cp\u003eSimilar protection from GSDMD deficiency was observed in an \u003cem\u003eex vivo\u003c/em\u003e hepatocyte irradiation model. Compared to \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e hepatocytes, \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e hepatocytes released higher levels of ALB and lower levels of ALT and AST after IR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I). Overall, these data suggested that GSDMD contributes to the progression of RILD probably depending on GSDMD driving hepatocyte pyroptosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGSDMD deficiency inhibits the recruitment of neutrophils to the irradiated liver\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGSDMD influenced the progression of diseases by modulating inflammatory responses.\u003csup\u003e(11, 21, 30, 31)\u003c/sup\u003e Therefore, to explore whether GSDMD aggravates RILD by affecting the hepatic immune environment, we set out to obtain single-cell suspensions of liver tissue from \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e-IR, and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e-IR mice, followed by scRNA-seq to characterize immune cell profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, hepatic immune cells were visualized by uniform manifold approximation and projection (UMAP). Compared to the \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e-Ctrl the hepatic immune cell composition of \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e-IR mice underwent significant changes, shown as a decrease in dendritic cells, T/B cells, and natural killer cells, a slight increase in Kupffer cells, and a striking elevation in neutrophils, suggesting an important role of neutrophils in RILD. Intriguingly, this IR-induced neutrophil infiltration of the liver was markedly reduced after GSDMD deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), indicating that GSDMD might contribute to neutrophil recruitment under IR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDecreased liver infiltration of neutrophils in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice were then confirmed by flow cytometry analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Moreover, myeloperoxidase (MPO)-positive active neutrophils were also significantly accumulated in both ST-RILD and LT-RILD \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e mouse livers, as detected by IHC, and were markedly reduced by knocking out \u003cem\u003eGsdmd\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Likewise, restoration of GSDMD expression by administration of AAV9-GSDMD-FL to \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e-ST-RILD mice significantly increased hepatic neutrophil infiltration, with levels similar to those in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e-ST-RILD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G).\u003c/p\u003e \u003cp\u003eNext, we wondered if GSDMD-mediated hepatocyte pyroptosis facilitated the recruitment of neutrophils. Purified neutrophils were then cocultured with non-irradiated or irradiated hepatocytes from \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice, followed by counting of migratory neutrophils (Figure S3A-B). In the case of equivalent cells, the recruitment of neutrophils was significantly increased by coculture with \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e-IR hepatocytes but notably decreased when cocultured with \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e-IR hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). The activation of HSCs is critical for the progression of fibrosis in RILD while little is known about pyroptotic hepatocyte impact on HSCs. We designed both contact and non-contact co-culture experiments of hepatocytes with HSCs (Fig.\u0026nbsp;3SC). The results show that there was no difference between the two approaches in terms of having no effect on HSCs activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-J).\u003c/p\u003e \u003cp\u003eCollectively, our data illustrated that GSDMD-mediated hepatocyte pyroptosis facilitates neutrophil infiltration into the liver.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSelectively CXCL1 neutralization effectively attenuated the progression of GSDMD driving RILD through suppressing neutrophil recruitment\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven that neutrophils are not liver-resident immune cells and require chemokines for their trafficking,\u003csup\u003e(30\u0026ndash;34)\u003c/sup\u003e we hypothesized that GSDMD might regulate neutrophil recruitment by affecting chemokine expression. Therefore, RNA-seq of \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e-IR and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e-IR mouse livers was performed to screen neutrophil trafficking-associated chemokines regulated by GSDMD. Notably, we found that CXCL1 was the most significantly downregulated chemokine in the livers of \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e-IR mice compared with \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e-IR mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To validate whether GSDMD impacts CXCL1 expression, hepatic CXCL1 in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice before and after IR was examined at the mRNA and protein levels. We discovered that CXCL1 was maintained at a low expression level in both \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice under steady-state conditions and was markedly increased in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e mice after radiotherapy, while \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice exhibited notable resistance to this upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). Moreover, further re-expressing GSDMD-FL in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice effectively restored IR-induced hepatic CXCL1 upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) implying CXCL1 should act as a downstream factor of GSDMD during the progression of RILD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the role of CXCL1 in RILD through influencing neutrophil infiltration \u003cem\u003ein vivo\u003c/em\u003e, \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e-IR mice were administered either a control immunoglobulin G (IgG) antibody or an anti-CXCL1 antibody (anti-CXCL1 Ab), which effectively neutralized CXCL1 in RILD models (Figure S4A-B). Flow cytometry analysis confirmed that CXCL1 inhibition resulted in twofold and fourfold decreases in liver neutrophil infiltration in the ST-RILD and LT-RILD mouse models, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Likewise, MPO\u003csup\u003e+\u003c/sup\u003e active neutrophils also decreased significantly after blocking CXCL1, as indicated by IHC (Figure S4C).\u003c/p\u003e \u003cp\u003eSimilar to the observation in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice, neutralizing CXCL1 had no effect on weight loss in ST-RILD mice but alleviated weight loss in the LT-RILD group (Figure S4D-E). Furthermore, compared to the IgG-treated mice, neutralization of CXCL1 resulted in reduced liver steatosis and congestion in ST-RILD mice and attenuated liver fibrosis in LT-RILD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-K). In addition, notable suppression of liver injury and a significant improvement in liver function in both the ST-RILD and LT-RILD mouse models were observed in mice treated with anti-CXCL1 Ab (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003eTaken together, these results suggest that selectively inhibiting CXCL1 can effectively suppress the progression of GSDMD driving RILD through the blockade of neutrophil recruitment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGSDMD regulates hepatocytes release CXCL1 to recruit neutrophil\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further explore the mechanism of GSDMD regulating CXCL1 to recruit neutrophils during RILD, we first determine the cellular resource of CXCL1. Previous studies have shown that hepatic CXCL1 is mostly released from hepatocytes and HSCs in steatohepatitis and CCL4-induced liver fibrosis.\u003csup\u003e(33,35)\u003c/sup\u003e However, our immunofluorescence results demonstrated that CXCL1 was expressed rarely in HSCs (α-SMA\u003csup\u003e+\u003c/sup\u003e) but mostly in hepatocytes (HNF4α\u003csup\u003e+\u003c/sup\u003e) in RILD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This observation was then validated in \u003cem\u003ein vitro\u003c/em\u003e experiments. Double immunostaining of CXCL1 and GSDMD-FL revealed that radiation-induced upregulation of GSDMD-FL in hepatocytes was accompanied by a significant elevation of CXCL1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Because GSDMD cleavage mediated the release of cell contents and simultaneously initiated cell death, we investigated whether GSDMD-N mediated CXCL1 release before cell death after irradiation exposure. After 12h after irradiation, primary hepatocytes showed no significant increased PI uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). While, the CXCL1 levels in the supernatants increased after irradiation in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e hepatocytes but decreased in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Next, we investigated whether GSDMD-N facilitated the secretion of cytosolic CXCL1. As the last step of GSDMD cleave requires the \u0026ldquo;scissors\u0026rdquo;-caspase and caspase-1 activation is reported to be governed by AMI2 which sense double-strand DNA damage after irradiation,\u003csup\u003e(36)\u003c/sup\u003e we also detected irradiation can activate caspase-1 in hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). To further define GSDMD-N function in controlling CXCL1 release, we used caspase-1 inhibitor Ac-YVAD-cmk to prevent the formation of GSDMD-N. Compared with IR-hepatocytes without inhibitor, Ac-YYAD-cmk effectively inhibit the release of CXCL1 into the supernatant but store in the cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-G). Together, these results indicated irradiation-induced GSDMD-N promoted the secretion of CXCL1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo clarify that GSDMD regulate hepatocyte after irradiation can recruit neutrophils via CXCL1, WT hepatocytes administered an anti-CXCL1 Ab showed a significantly reduced ability to recruit neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). In contrast, the addition of the recombinant CXCL1 protein (CXCL1 Rp) to \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e hepatocytes restored the chemotactic ability of neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). In vivo, restoration of GSDMD in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice by injecting AAV9-GSDMD-FL, administration of CXCL1-Ab also can inhibit the neutrophil infiltration into liver (Figure S5A-C). Afterwards, early radiation-induced liver injury was reversed (Figure S5D-F). Collectively, these data provide compelling evidence for the regulatory influence of GSDMD on hepatocytes, thereby driving the recruitment of neutrophil through the enhanced expression of CXCL1 upon irradiation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGSDMD deficiency inhibits the activation of the stat5a/CXCL1/neutrophil pathway\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we explored the mechanism GSDMD regulate CXCL1. Through comprehensive analysis utilizing the Gene-Hancer database within the GeneCards Suite (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genecards.org/\u003c/span\u003e\u003cspan address=\"http://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), we scrutinized the regulatory elements encompassing the promoter and enhancer regions of CXCL1. Leveraging integrative analysis with transcriptomic sequencing (\u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/F\u003c/em\u003e\u003c/sup\u003e vs \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e), we unearthed seven putative transcription factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), under the influence of GSDMD, that exhibit the potential to bind to the promoter or enhancer regions of CXCL1. Among the examined transcriptional regulators, stat5a exhibited the most prominent differential expression between \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/F\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Herein, we sought to elucidate the role of stat5a as a regulator of CXCL1. First, IST5-002, an inhibitor that strongly suppressed the nuclear translocation of stat5a and P-stat5a in hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), significantly reduced the production of CXCL1 in irradiated hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). To investigate the regulatory role of stat5a in \u003cem\u003eCxcl1\u003c/em\u003e transcription, we constructed luciferase reporter plasmids containing cloned stat5a binding sites within the \u003cem\u003eCxcl1\u003c/em\u003e promoter region. We observed an interferon gamma-activated site (GAS)-like core sequence (TTCT/CNA/GGAA) in promoter of mouse Cxcl1 gene. Strikingly, upon irradiation, we observed a substantial increase in luciferase activity specifically at the stat5a binding sites. Notably, this effect was markedly diminished when the stat5a binding site was mutated, highlighting the critical involvement of stat5a in driving \u003cem\u003ecxcl1\u003c/em\u003e transcription in response to irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and Figure S6A). In addition, the application of IST5-002 to irradiated hepatocytes effectively inhibited the migration of neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). These results indicated that stat5a regulated the recruitment of neutrophils in a CXCL1-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn vitro investigations revealed that irradiation-treated hepatocytes exhibited enhanced stat5a activation, as evidenced by the elevated protein levels of phosphorylated stat5a (P-stat5a) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Notably, the absence of Gsdmd also attenuated the nuclear translocation of P-stat5a induced by irradiation, as observed through nuclear protein analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Moreover, co-immunoprecipitation (coIP) assay confirmed that GSDMD and stat5a could directly interact with each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Subsequently, we conducted chromatin immunoprecipitation (ChIP) assays, which revealed that depletion of GSDMD significantly impeded the binding of stat5a to the promoter region of the CXCL1 gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). This finding underscores the critical involvement of stat5a in GSDMD-mediated CXCL1 expression. Intriguingly, we also observed that GSDMD deficiency markedly suppressed stat5a activation in livers affected by RILD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK and L), whereas re-expression of GSDMD-FL reinstated the levels of P-stat5a in \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). Collectively, these findings highlight the role of GSDMD in regulating CXCL1 expression through enhancing stat5a phosphorylation and nuclear translocation.\u003c/p\u003e \u003cp\u003eIn response to extracellular stress signals, such as irradiation, several downstream signaling pathways, including extracellular signal-regulated kinases (ERKs), p38 MAP kinase, and c-Jun N-terminal kinase (JNK), are recruited and activated.\u003csup\u003e(20, 37, 38)\u003c/sup\u003e To elucidate the key pathways involved in the upregulation of GSDMD expression, we conducted further investigations. Comparative analysis revealed that, in irradiated hepatocytes, only the phosphorylation of ERK was significantly elevated when compared to the vehicle group. Moreover, the inhibition of ERK using GDC-0994 resulted in a substantial decrease in GSDMD expression (Figure S6 B-D). These findings highlight the predominant role of the ERK pathway in mediating the irradiation-induced increase in GSDMD expression.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe therapeutic potential of the GSDMD inhibitor disulfiram in preventing RILD\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether GSDMD-mediated pyroptosis acts as a key therapeutic target, disulfiram (DSF), a drug approved by the FDA for inhibiting pyroptosis, was administered to RILD mice. In RILD, \u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003eFL/FL\u003c/em\u003e\u003c/sup\u003e mice were treated with vehicle ctrl or DSF according to the protocol shown in Figure S7A. Indeed, the administration of DSF resulted in a significant reduction in GSDMD-FL/N in irradiated livers, as assessed by WB and IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and Figure S7B). Moreover, DSF treatment reduced the activation of stat5a, the expression of CXCL1, and subsequent neutrophil infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, C-D), further confirming the molecular mechanisms of GSDMD in RILD, as we discussed above. Of note, in contrast to treatment with the vehicle control, DSF administration significantly curbed acute liver injury in ST-RILD mice and reduced weight loss and liver fibrosis in LT-RILD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-I, Figure S7C-D), which was accompanied by an improvement in liver function, as determined by serum ALT, AST, and ALB levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe pyroptosis-inhibitory and radioprotective roles of DSF were also noted in irradiated hepatocytes \u003cem\u003eex vivo\u003c/em\u003e. DSF treatment markedly reduced GSDMD-FL/N expression and LDH levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK-L). In addition, stat5a activation and CXCL1 release were both inhibited in DSF-treated hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK, M). Ultimately, DSF-treated hepatocytes were protected from radiation toxicities (Figure S7E). These findings suggest that DSF, a pyroptosis inhibitor, is a promising therapeutic strategy for the management of RILD in cancer patients.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eRILD not only harms the public\u0026rsquo;s health but greatly hinders radiotherapy\u0026rsquo;s application.\u003csup\u003e(8, 9)\u003c/sup\u003e Unfortunately, underlying mechanisms and therapeutic agents were both unrevealed in RILD. In this study, we elucidated that irradiation triggers elevated expression of GSDMD in hepatocytes, thereby exacerbating RILD progression. With the use of genetically and pharmacologically modified models, our data found that IR mediated upregulation of GSDMD activated stat5a to drive CXCL1 expression while GSDMD-N, a mature form of GSDMD, formed a pore to release the accumulated CXCL1 in the hepatocytes exposed to IR. As a neutrophil chemokine, CXCL1 from hepatocytes recruits neutrophil into liver and promotes RILD progression. Therefore, GSDMD emerges as a pivotal determinant in the regulation of RILD, underscoring its potential as a promising therapeutic target to impede the progression of this condition.\u003c/p\u003e \u003cp\u003eGSDMD is composed of \u0026asymp;\u0026thinsp;480 amino acids and widely expresses in many organs. \u003csup\u003e(11, 21, 39)\u003c/sup\u003e Previous studies have revealed that GSDMD plays an important role in maintaining homeostasis,\u003csup\u003e(40)\u003c/sup\u003e eliminating pathogen infection,\u003csup\u003e(41\u0026ndash;43)\u003c/sup\u003e and regulating the development of diseases.\u003csup\u003e(13, 34, 44, 45)\u003c/sup\u003e GSDMD is inactive in steady state, and its function mainly depend on GSDMD-N, which performs a pore-forming function through oligomerization in the plasma membrane. Membrane perforation leads to the release of many cytokines, which activate downstream pathways. Wang \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e(40)\u003c/sup\u003e reported that GSDMD-N pores promote mucin granule secretion in the gut epithelium by mediating Ca\u003csup\u003e2+\u003c/sup\u003e entry and help maintain gut homeostasis. In bacterial infection, GSDMD-N drives pyroptosis of infected cells and leads to the release of intracellular bacteria followed by neutrophil-mediated killing.\u003csup\u003e(41, 46)\u003c/sup\u003e Moreover, the release of IL-33, IL-1β and IL-18 through the pores formed by GSDMD-N also aggravates the progression of allergic airway inflammation,\u003csup\u003e(45)\u003c/sup\u003e septic shock,\u003csup\u003e(47)\u003c/sup\u003e and colitis.\u003csup\u003e(48)\u003c/sup\u003e Hence, GSDMD-FL/N are regarded as critical proteins regulating homeostasis and the outcome of disease. A previous study reported that TBI increased gut and bone marrow expressed GSDMD-FL/N, which correlated positively with the severity of bone loss.\u003csup\u003e(15)\u003c/sup\u003e However, the effect of GSDMD-N executed pyroptosis and mediated inflammatory factors release in RILD remains unclear. Here, we revealed that IR mobilize GSDMD-N expression and the pyroptosis in hepatocytes. Of note, the level of GSDMD-N positively correlated with the severity of RILD, and inhibition of GSDMD could effectively alleviate the progression of RILD.\u003c/p\u003e \u003cp\u003eGSDMD-N mediated pyroptosis has been well documented in different cell types and promotes the release of multiple cytokines. Recent studies have demonstrated that GSDMD-N promotes macrophages pyroptosis and release regulator factors, which induces coagulation, leading to disseminated intravascular coagulation in sepsis.\u003csup\u003e(49, 50)\u003c/sup\u003e GSDMD-driven pyroptosis in myeloid cells also plays a pivotal role in the pathogenesis of hepatic ischemia\u0026ndash;reperfusion injury.\u003csup\u003e(13)\u003c/sup\u003e Although the role of pyroptosis in immune cells has been extensively studied, its implications in hepatocytes and the resulting impact on the immune microenvironment remain largely unexplored. Wang \u003cem\u003eet al.\u003c/em\u003e reported that a natural compound could alleviate CCL4-induced liver injury by inhibiting the pyroptosis of hepatocytes, but the underlying mechanism is uncertain.\u003csup\u003e(51)\u003c/sup\u003e In this study, we first revealed that the critical pyroptotic cell type was hepatocyte in RILD. In addition, we also demonstrated that the impact of GSDMD-N in remodeling immune environment via its pore-forming activity. In brief, upon IR, CXCL1 were released from hepatocytes via GSDMD-N cleaved by caspase-1 and recruited neutrophils to change the immune environment of liver.\u003c/p\u003e \u003cp\u003eOur study also has found cleavage-independent function of GSDMD, where it activates the transcription factor stat5a to drive CXCL1 expression. In support of this cleavage-independent function, other gasdermin family proteins were reported to have cleavage-independent function to regulate the downstream genes. Huang \u003cem\u003eet al\u003c/em\u003e. reported that GSDME mediates the transcription factor to enter the nucleus where it promotes the expression of downstream genes.\u003csup\u003e(17)\u003c/sup\u003e GSDMB is also found to be localized in the nucleus and regulates the expression of genes by acting as a transcriptional coactivation or enhancer.\u003csup\u003e(18)\u003c/sup\u003e These findings highlight the need for further investigation to fully understand the diverse roles of GSDMD.\u003c/p\u003e \u003cp\u003eOur study also aims to evaluate the efficacy of a protective drug for RILD. Disulfiram is known to be approved by FDA to inhibit GSDMD-N mediated pore formation. In addition, disulfiram also can suppress the expression of GSDMD-FL in Nigericin-primed THP-1 cells.\u003csup\u003e(52)\u003c/sup\u003e Administration of disulfiram significantly decreased the severity of RILD. In addition, other side effects induced by antitumor therapy, including chemotherapy,\u003csup\u003e(53, 54)\u003c/sup\u003e were reported to be related to GSDMD-mediated pyroptosis, which indicates that pyroptosis is a potential therapeutic target in alleviating the side effects of other anti-tumor therapies in cancer patients. Moreover, DSF has been reported to be effective against diverse cancer types in preclinical studies through p97/Nuclear protein localization protein 4 homolog (NPL4) pathway.\u003csup\u003e(55\u0026ndash;57)\u003c/sup\u003e These results suggest that DSF may not only alleviate RILD, but also further enhance the tumor control rate.\u003c/p\u003e \u003cp\u003eSTAT5a is a member of STAT family, a group of latent transcription factors that are activated in response to various cytokine signaling pathways. The predominant mechanism by which stat5 influences neutrophils, primarily through the action of granulocyte colony-stimulating factor (G-CSF), is by modulating the transcription of genes associated with cell survival and proliferation(\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). In contrast, STAT3, another member of the STAT family, has been reported to be essential for the chemotaxis of mature neutrophils towards CXCR2 ligands, including CXCL2(\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Our luciferase activity assays and CXCL1 ELISA measurement provide evidence that stat5a is indispensable for the transcription of \u003cem\u003ecxcl1\u003c/em\u003e following irradiation. Additionally, we discovered that stat5a plays a crucial role in neutrophil chemotaxis. This collective evidence demonstrates that, as a transcription factor, stat5a also mediates the recruitment of neutrophils through CXCL1.\u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrated that irradiation induced GSDMD activation in hepatocytes, which plays a crucial role in RILD progression. GSDMD induces pyroptotic phenotype and increased synthesis and release of CXCL1 in hepatocytes. Mechanistically, GSDMD promotes stat5a into the nucleus which subsequently increased CXCL1 synthesis and GSDMD-N supports CXCL1 release from forming pores. Afterwards, as a chemokine, CXCL1 acts to recruit neutrophils into liver to ultimately aggravate the severity of RILD. Both gene knockout and pharmacological inhibition of GSDMD effectively ameliorate RILD. These findings provide a novel target for the development of effective strategies for treating patients with RILD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNO\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhenwei Peng,\u0026nbsp;Yi Zhou,\u0026nbsp;Xiongjun Wang\u0026nbsp;contributed to design and data interpretation.\u0026nbsp;Zhenwei Peng,\u0026nbsp;Yi Zhou,\u0026nbsp;Xiongjun Wang\u0026nbsp;contributed to revise article critically for important intellectual content and final approve the version to be published. Aoran Dong,\u0026nbsp;Guangyan Wei, Yuqin Di and Zhou Liang\u0026nbsp;contributed to\u0026nbsp;data generation and manuscript draft. Yuhao Tang, Yunyan.Ling, Shuping Li and Yong Chen contributed to data generation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e: nothing to report.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi T, Cao Y, Li B, Dai R. The biological effects of radiation-induced liver damage and its natural protective medicine. Prog Biophys Mol Biol 2021;167:87\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar P, Wang P, Farese AM, MacVittie TJ, Kane MA. Metabolomics of Multiorgan Radiation Injury in Non-human Primate Model Reveals System-wide Metabolic Perturbations. Health Phys 2021;121:395\u0026ndash;405.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong JYC, Filippi AR, Dabaja BS, Yahalom J, Specht L. Total Body Irradiation: Guidelines from the International Lymphoma Radiation Oncology Group (ILROG). 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Blood 2010;115:3354\u0026ndash;3363.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"radiation-induced liver disease, GSDMD, GSDMD-N, pyroptosis, neutrophil, chemokine CXCL1","lastPublishedDoi":"10.21203/rs.3.rs-3161881/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3161881/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRadiation-induced liver disease (RILD) severely impairs the outcome of patients exposed to irradiation (IR); however, the underlying mechanism is largely unknown. The N-terminus of gasdermin D (GSDMD-N) is mainly involved in driving the progression of pyroptosis, and its expression has been reported to be induced by IR in the gut and bone marrow, but its role in RILD remains unknown. By collecting liver samples from RILD patients and mice, we uncovered GSDMD-FL/N were significantly upregulated and positively correlated with RILD severity. Hepatocytes are found to be critical pyroptotic cells in RILD by combining single-cell RNA sequencing (scRNA-seq), immunofluorescence and fluorescence-activated cell sorting (FACS) analysis. Functional analysis and mechanistic studies were performed using hepatocyte-specific \u003cem\u003eGsdmd\u003c/em\u003e knockout (\u003cem\u003eGsdmd\u003c/em\u003e\u003csup\u003e\u003cem\u003e∆Hep\u003c/em\u003e\u003c/sup\u003e) mice and cell models and scRNA-seq analysis. Mechanistically, GSDMD was indispensable for triggering hepatocyte pyroptosis and initiating the activation of transcription factor stat5a, which subsequently facilitated the expression of CXCL1. As a pore-forming protein, increased GSDMD-N also mediated the secretion of CXCL1, which further recruited neutrophil into liver to accelerate the severity of RILD. We also discovered that pharmacological targeting GSDMD and its downstream CXCL1 effectively attenuated the progression of RILD. RILD progression requires increased GSDMD-FL/N in hepatocytes, causing pyroptosis and stat5a activation. Afterwards, driving CXCL1 express and release to recruit neutrophils into liver. Together, our study demonstrates that GSDMD as therapeutic targets to improve RILD.\u003c/p\u003e","manuscriptTitle":"The progression of hepatocyte pyroptosis exacerbates radiation-induced liver disease via the gasdermin D/signal transducer and activator of transcription 5a/CXCL1 axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-01 19:18:20","doi":"10.21203/rs.3.rs-3161881/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e5460e50-8fac-4555-ad81-b043a4355ee5","owner":[],"postedDate":"August 1st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":23572090,"name":"Biological sciences/Immunology/Cell death and immune response"},{"id":23572091,"name":"Health sciences/Oncology/Cancer/Cancer therapy/Radiotherapy"}],"tags":[],"updatedAt":"2023-08-17T04:10:39+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-01 19:18:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3161881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3161881","identity":"rs-3161881","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}