Ubiquitin-independent degradation of Bim blocks macrophage pyroptosis in sepsis-related tissue injury

Ubiquitin-independent degradation of Bim blocks macrophage pyroptosis in sepsis-related tissue injury | 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 Ubiquitin-independent degradation of Bim blocks macrophage pyroptosis in sepsis-related tissue injury Lei Li, Peilin Shi, Yingying Du, Bo Yang, Qiujing Guan, Yiming Jing, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3556715/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2024 Read the published version in Cell Death & Disease → Version 1 posted 10 You are reading this latest preprint version Abstract Pyroptosis, a typical inflammatory cell death mode, has been increasingly demonstrated to have therapeutic value in inflammatory diseases such as sepsis. However, the mechanisms and therapeutic targets of sepsis remain elusive. Here, we reported that REGγ inhibition promoted pyroptosis by regulating members of the gasdermin family in macrophages. Mechanistically, REGγ directly degraded Bim, a factor of the BCL-2 family that can inhibit the accumulation of GSDMD/E, ultimately preventing the occurrence of pyroptosis. Furthermore, cecal ligation and puncture (CLP)-induced sepsis model mice showed downregulation of REGγ at both the RNA and protein levels. Gasdermin-mediated pyroptosis was augmented in REGγ-knockout mice, and these mice exhibited more severe sepsis-related tissue injury. More importantly, we found that REGγ expression was downregulated in clinical sepsis samples, such as those from patients with Pseudomonas aeruginosa (PA) infection. Finally, PA-infected mice showed decreased REGγ levels in the lung. In summary, our study reveals that the REGγ-Bim-GSDMD/E pathway is a novel regulatory mechanism of pyroptosis in sepsis-related tissue injury. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Additional Declarations (Not answered) Supplementary Files OriginalWesternBlots.pdf supplementaryfigures.pdf TableS1.xlsx TableS2.xlsx MovieS1.mp4 Supplementary movie S1 MovieS2.mp4 Supplementary movie S2 Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2024 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 21 Dec, 2023 Review # 1 received at journal 20 Dec, 2023 Review # 2 received at journal 07 Dec, 2023 Reviewer # 2 agreed at journal 06 Dec, 2023 Reviewer # 1 agreed at journal 03 Dec, 2023 Reviewers invited by journal 29 Nov, 2023 Submission checks completed at journal 08 Nov, 2023 First submitted to journal 07 Nov, 2023 Unknown event 06 Nov, 2023 Editor assigned by journal 04 Nov, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-3556715","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":254342326,"identity":"c7f99258-66e6-459f-84ad-e23ca9a4b4c1","order_by":0,"name":"Lei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYBACPnbGxscMBnA+M2EtbMyMzcakamFgk0biE6WFua26oMCGgZ/97DEJhgrrxAb2swcIOazt9gyDNAbJnrw0CYYz6YkNPHkJhLXwGBxmMLjBYybB2HY4sUGCx4CglmKQFnuwln9EamEG2yIB0tJAnJZmaaBfeCTO5BhbJBxLN27jycGvhZ+9/eHngj82cvztZwxvfKixlu1nP4NfCwzwgMkEkL1EqR8Fo2AUjIJRgBcAAOS/M09Nx/2MAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3114-1653","institution":"Institute of Biomedical Sciences, School of Life Sciences, East China Normal University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Li","suffix":""},{"id":254342327,"identity":"fe639399-e157-41ee-9d20-0b161a2c419e","order_by":1,"name":"Peilin Shi","email":"","orcid":"","institution":"Institute of Biomedical Sciences, School of Life Sciences, East China Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Peilin","middleName":"","lastName":"Shi","suffix":""},{"id":254342328,"identity":"a5bb9327-f313-4dfd-b9a3-12e834754829","order_by":2,"name":"Yingying Du","email":"","orcid":"","institution":"Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences, School of Life Sciences, East China Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Du","suffix":""},{"id":254342329,"identity":"38d5106b-669b-4170-a40e-2b330925e897","order_by":3,"name":"Bo Yang","email":"","orcid":"","institution":"Institute of Biomedical Sciences, School of Life Sciences, East China Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Yang","suffix":""},{"id":254342330,"identity":"d65787d9-2360-47de-afc0-462e7fa15cc4","order_by":4,"name":"Qiujing Guan","email":"","orcid":"","institution":"Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences, School of Life Sciences, East China Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiujing","middleName":"","lastName":"Guan","suffix":""},{"id":254342331,"identity":"eb5ab8fd-06a1-46cc-878a-7eca3f331390","order_by":5,"name":"Yiming Jing","email":"","orcid":"","institution":"Institute of Biomedical Sciences, School of Life Sciences, East China Normal University,","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yiming","middleName":"","lastName":"Jing","suffix":""},{"id":254342332,"identity":"c7aa84e7-0be3-4cf6-9af3-892ea4f5e91e","order_by":6,"name":"Hao Tang","email":"","orcid":"","institution":"Changzheng Hospital, Naval Military Medical University, Shanghai China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Tang","suffix":""},{"id":254342333,"identity":"e842bda4-a381-49d0-8712-98abe51e8937","order_by":7,"name":"Shihui Shen","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shihui","middleName":"","lastName":"Shen","suffix":""},{"id":254342334,"identity":"573faea2-bdbd-4c71-8b09-d1bd5a8c4eb9","order_by":8,"name":"Chunrong Wu","email":"","orcid":"","institution":"Shanghai Fifth People's Hospital, Fudan University, China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chunrong","middleName":"","lastName":"Wu","suffix":""},{"id":254342335,"identity":"363e4286-8dfd-44fa-93c2-9c76c0ea72e3","order_by":9,"name":"Yunyan Zhang","email":"","orcid":"","institution":"Changzheng Hospital, Naval Military Medical University, Shanghai China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yunyan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2023-11-04 09:45:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3556715/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3556715/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-024-07072-z","type":"published","date":"2024-09-30T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":47454114,"identity":"aeff5ed2-d47a-425a-aa1a-b7482edf1fc7","added_by":"auto","created_at":"2023-12-01 17:29:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1917074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eREGγ deficiency promoted pyroptotic cell death. \u003c/strong\u003e(\u003cstrong\u003eA) \u003c/strong\u003eMorphological changes were observed with fluorescence microscopy by Hoechst 33342/PI staining after the 8 hours treatment with 40 μM cisplatin in HeLa shN/shREGγ cells (blue fluorescence: hoechst 33342, red fluorescence: PI). Scale bar, 20 μm. (\u003cstrong\u003eB) \u003c/strong\u003eA time-course flow-cytometry analysis of Annexin V–APC and propidium iodide-stained HeLa cells. (\u003cstrong\u003eC) \u003c/strong\u003eCell morphology of HeLa shN cells (left) and shREGγ cells (right) after stimulation with 40 μM cisplatin under microscope. Scale bar, 20 μm. (\u003cstrong\u003eD) \u003c/strong\u003eHeLa cells were treated with 40 μM cisplatin at different time and then analyzed for the expression of GSDME-mediated pyroptosis pathway by western blot. FL: full length, NT: N-terminal. The asterisk indicates the band being analyzed. (\u003cstrong\u003eE) \u003c/strong\u003eLDH release into HeLa culture medium under cisplatin stimulation was measured. Data are means ± SD taken from three technical replicates. \u003cstrong\u003e(F) \u003c/strong\u003eBMDMs were treated with 1 μg/mL LPS priming before 5 mM ATP for 6 hours and 12 hours. LDH release into cell culture medium was measured. Data are means ± SD taken from three technical replicates. (\u003cstrong\u003eG) \u003c/strong\u003eFlow cytometry of Annexin V-APC and propidium iodide-stained BMDMs. (\u003cstrong\u003eH) \u003c/strong\u003eBMDMs were pre-priming with 1 μg/mL LPS at different time points before stimulated with 5 mM ATP. GSDMD-mediated pyroptosis pathway were analyzed by western blot. (\u003cstrong\u003eI) \u003c/strong\u003emRNA levels of GSDME were assessed by qPCR in HeLa cells. (\u003cstrong\u003eJ) \u003c/strong\u003emRNA levels of GSDMD were assessed by qPCR in BMDMs. Data are means ± SD taken from three technical replicates. ns, not significant, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. Differences were measured by two-way ANOVA. All data are representative of three independent experiments.\u003c/p\u003e","description":"","filename":"figure01.png","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/dd43d035ed72cef652f27cc6.png"},{"id":47454116,"identity":"1a0f19ab-0ba4-43d5-980a-1e8f75b7a925","added_by":"auto","created_at":"2023-12-01 17:29:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1302193,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of Bim alleviated the promotive effect of REGγ deficiency on pyroptosis. \u003c/strong\u003e(\u003cstrong\u003eA) \u003c/strong\u003eBim expression in HeLa was assessed by western blot and semiquantitative analysis. (\u003cstrong\u003eB) \u003c/strong\u003eBim 29 expression in BMDMs was assessed by western blot and semiquantitative analysis. (\u003cstrong\u003eC) \u003c/strong\u003emRNA level of Bim in HeLa cells was analyzed by qPCR. (\u003cstrong\u003eD) \u003c/strong\u003emRNA level of Bim in BMDMs was analyzed by qPCR. (\u003cstrong\u003eE) \u003c/strong\u003eLDH release were measured of HeLa cells treated with cisplatin. #1/2 means cells were transfected with \u003cem\u003eBim-\u003c/em\u003especific sgRNAs. (\u003cstrong\u003eF) \u003c/strong\u003eCell morphology of HeLa cells after stimulation with 40 μM cisplatin under microscope. Scale bar, 20 μm (up). Cell death mode was shown with flow cytometry of Annexin V-APC and propidium iodide-stained HeLa cells (down). (\u003cstrong\u003eG) \u003c/strong\u003eHeLa cells were treated with 40 μM cisplatin and then analyzed by western blot. NC means cells were transfected with sgRNA negative control. #1/2 means cells were transfected with \u003cem\u003ea Bim-\u003c/em\u003especific sgRNAs. The asterisk indicates the band being analyzed. Data are means ± SD taken from three technical replicates. ns, not significant, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. Differences were measured by two-way ANOVA. All data are representative of three independent experiments.\u003c/p\u003e","description":"","filename":"figure02.png","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/4d54924ace32d77feadb3dfd.png"},{"id":47455446,"identity":"de096edf-7a7f-4af4-a4a8-b720ad263701","added_by":"auto","created_at":"2023-12-01 17:45:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":517621,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eREGγ mediated ubiquitin-ATP-independent degradation of Bim. \u003c/strong\u003e(\u003cstrong\u003eA) \u003c/strong\u003eSchematic diagram of amino acid sequence for three main isoforms of Bim, including BimEL, BimL and BimS. (\u003cstrong\u003eB) \u003c/strong\u003eThe physical interactions between REGγ and Bim were defined by reciprocal co- immunoprecipitation followed by western blot analysis. 293T cells were transiently expressed 4 μg of Flag-REGγ, 2 μg of HA- BimEL (and BimL, BimS), or 2 μg of the control HA-vector. The asterisk indicates the location of flag beads. (\u003cstrong\u003eC) \u003c/strong\u003eHeLa shN cells and HeLa shREGγ cells were treated with 100 μg/mL cycloheximide (CHX) for the indicated times followed by western blot analysis. (\u003cstrong\u003eD) \u003c/strong\u003eThe degradation rate of Bim is visualized by a statistical line graph. Data are means ± SD taken from three technical replicates. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. Differences were measured by two-way ANOVA. (\u003cstrong\u003eE) \u003c/strong\u003eFlag-REGγ was transfected into 293T KO (\u003cem\u003eREGγ \u003c/em\u003eknockout) cells, accompanied by HA-Bim. The expression of Bim plasmids assessed by western blot. (\u003cstrong\u003eF) \u003c/strong\u003eBim was 30 translated in \u003cem\u003evitro \u003c/em\u003ein TNT® Quick Coupled Transcription/Translation System. And then incubated at 37 °C with purified protein REGγ and 20S proteasome. Fold expression of Bim was assessed by western blot and semiquantitative results. (\u003cstrong\u003eG) \u003c/strong\u003eQuantitated results in (F) show Bim degradation changes. Data are means ± SD taken from three technical replicates. ns, not significant, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. Differences were measured by ordinary one-way ANOVA. All data are representative of three independent experiments\u003c/p\u003e","description":"","filename":"figure03.png","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/468fe3eb5deef9532fe5b378.png"},{"id":47455053,"identity":"6e2d49e2-2b8d-4f69-af0b-022300053b98","added_by":"auto","created_at":"2023-12-01 17:37:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2887831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCecal ligation and puncture (CLP)-induced sepsis downregulated the expression of REGγ. \u003c/strong\u003e(\u003cstrong\u003eA) \u003c/strong\u003eSchematic diagram of mice CLP-induced sepsis model. (\u003cstrong\u003eB) \u003c/strong\u003eThe wet-dry ratio of major organs from each group. ns, not significant, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. Differences were measured by two-way ANOVA. (\u003cstrong\u003eC) \u003c/strong\u003eH\u0026amp;E staining of liver, kidney, lung, heart, and spleen tissue from 6-10 weeks C56BL/6 male wild-type mice and CLP-mice. Scale bar, 100 μm. (\u003cstrong\u003eD to F) \u003c/strong\u003eBlood serum ALT, UREA and CK levels from each group, respectively. Each spot represents a sample from an individual mouse, n=3-10. Data are means ± SD, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. Differences were measured by T test. (\u003cstrong\u003eG) \u003c/strong\u003eMice were sacrificed after 12 or 24 hours. BMDMs were extract from sham group and CLP group for western blot analysis. mRNA level of REGγ was assessed by qPCR. Short stands for short exposure and long stands for long exposure. The asterisk indicates the band being analyzed. Data are means ± SD, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. (\u003cstrong\u003eH) \u003c/strong\u003eExpression of transcription factors in BMDMs were assessed by qPCR. Data are means ± SD, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. Differences were measured by ordinary one-way ANOVA. \u003cstrong\u003e(I) \u003c/strong\u003eImmunofluorescence of REGγ in the lung in CLP-induced sepsis mice. Scale bar, 100 μm.\u003c/p\u003e","description":"","filename":"figure04.png","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/36fb4b9988a1fe40e44562be.png"},{"id":47455052,"identity":"f1ac1bc4-c97a-4a10-92a9-a95df4b9b55f","added_by":"auto","created_at":"2023-12-01 17:37:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1060291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eREGγ expression decreased in clinical samples of patients with sepsis. (A-B) \u003c/strong\u003eThe 31 mRNA level of REGγ in the peripheral blood samples from healthy controls (Healthy) and sepsis patients (SS). (\u003cstrong\u003eA) \u003c/strong\u003eT0 represents the blood samples collected immediately after receiving patients, T1 represents the blood samples collected 1 hour after receiving patients, and T2 represents the blood samples collected 3 hours after receiving patients. Data was derived from the GEO database GSE69063. (\u003cstrong\u003eB) \u003c/strong\u003eSS-Day0 represents the blood samples collected on the first day after receiving patients, SS-Day7 represents the blood samples collected on the seventh day after receiving patients. The data was derived from the GEO database GSE32707. (\u003cstrong\u003eC) \u003c/strong\u003eBlood REGγ level from patients with melioidosis infection (SS-M) and other infections (SS-O) caused by sepsis as well as patients under recovery (recovery). The data was derived from the GEO database GSE100159. All data are means ± SD, ns, not significant, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. Differences were measured by ordinary one-way ANOVA. (\u003cstrong\u003eD) \u003c/strong\u003eHuman peripheral blood mononuclear cells (PBMCs) were extracted from blood samples of patients with sepsis, which were then subjected to western blot. (\u003cstrong\u003eE) \u003c/strong\u003ePseudomonas aeruginosa (PA) was injected intraperitoneally into wild-type mice. Lung and colon tissue were extract from each group for western blot analysis (left). High means high PA injected concentration 1×106 CFU/mL, low means lower PA injected concentration 2×105 CFU/mL. Lung and colon tissue from CLP WT-mice were extract from each group for western blot analysis (right). \u003cstrong\u003e(F) \u003c/strong\u003eH\u0026amp;E staining of lung tissues from 6-10 weeks C56BL/6 male wild-type mice with PA infection. Scale bar, 100 μm. \u003cstrong\u003e(G) \u003c/strong\u003eImmunofluorescence of REGγ in the lung tissue in PA-infected mice. Scale bar, 100 μm.\u003c/p\u003e","description":"","filename":"figure05.png","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/53dad1c09e6986bcbc75f766.png"},{"id":47454121,"identity":"49d98ef9-a188-4aa1-9e60-7b6504f866a2","added_by":"auto","created_at":"2023-12-01 17:29:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3244972,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCLP-induced sepsis is exacerbated by the null expression of REGγ. \u003c/strong\u003e(\u003cstrong\u003eA) \u003c/strong\u003eSurvival curves of CLP-induced sepsis mice within 10 days compared with sham group, n=3-12. (\u003cstrong\u003eB) \u003c/strong\u003eMouse weight when sacrificed is shown relative to weight before CLP operation. Each spot represents a 32 sample from an individual mouse. Differences were measured by T test. (\u003cstrong\u003eC) \u003c/strong\u003emRNA level of IL-1β, IL-18 and TNFα of primary BMDMs from wide-type mice and \u003cem\u003eREGγ-/- \u003c/em\u003emice with CLP-induced sepsis. Differences were measured by two-way ANOVA. (\u003cstrong\u003eD) \u003c/strong\u003eBlood serum ALT, UREA and CK levels from each group, respectively. Each spot represents a sample from an individual mouse, n=3- 10. Differences were measured by two-way ANOVA. (\u003cstrong\u003eE) \u003c/strong\u003eH\u0026amp;E staining of heart, liver, spleen, lung and kidney tissue from 6-10 weeks C56BL/6 male wild-type mice and CLP-mice. Scale bar, 100 μm. (\u003cstrong\u003eF) \u003c/strong\u003eStatistics for thymus weights relative to the weight of mice from each group. Differences were measured by two-way ANOVA. (\u003cstrong\u003eG) \u003c/strong\u003eWestern blot analysis of BMDMs from each group. The asterisk indicates the band being analyzed. ns, not significant, *\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":"figure06.png","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/0e84da370ddcfe78aa96dde6.png"},{"id":47455056,"identity":"cb235e39-f556-4ea6-85f2-11ff415ea311","added_by":"auto","created_at":"2023-12-01 17:37:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2894291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel of REGγ-Bim-pyrptosis pathway in macrophage to induce sepsis. \u003c/strong\u003eWhen cells are stimulated by pyroptosis inducing factors, the transcription factors of REGγ such as \u003cem\u003eFos \u003c/em\u003eare downregulated, inducing the decreased expression of REGγ. Normally, REGγ is able to degrade pro-apoptotic protein Bim through a non-ubiquitin-dependent pathway, thus impeding the cell death process. However, the degradation of Bim is blocked when the expression of REGγ is inhibited, which promotes the accumulation of Bim. Accumulated Bim promotes the continuously activation of the caspase family, followed by the cleavage of gasdermin, and finally lead to the pyroptosis. In macrophages, excessive pyroptosis induces excessive release of inflammatory factors, which could ultimately aggravate sepsis in mice. In conclusion, the inhibition of REGγ aggravates pyroptosis- mediated sepsis.\u003c/p\u003e","description":"","filename":"figure07.png","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/6e43b9c96cc7ed5148428c19.png"},{"id":65671461,"identity":"fcf55495-132f-439d-941d-0e690bab63e0","added_by":"auto","created_at":"2024-10-01 07:12:31","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3219086,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Articlefile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1_covered_730faa48-780d-4a76-9142-8a08918d13b9.pdf"},{"id":47455729,"identity":"e4fa9474-cd2b-48c6-a503-e363c103f460","added_by":"auto","created_at":"2023-12-01 17:53:41","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":463270,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalWesternBlots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/ab0ee0e571bf4b67966bc765.pdf"},{"id":47454120,"identity":"e39c2455-1a5c-4a6a-ba23-369344074aba","added_by":"auto","created_at":"2023-12-01 17:29:41","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":480369,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/3b4f3908f23a066eda7ba181.pdf"},{"id":47454123,"identity":"267ae874-aa7d-41cf-b4f5-9034622e0c48","added_by":"auto","created_at":"2023-12-01 17:29:42","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12272,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/5f34202acfed197b92d37b5f.xlsx"},{"id":47454119,"identity":"b4052cc7-45d6-4c3c-b178-0c1e25791f1a","added_by":"auto","created_at":"2023-12-01 17:29:41","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10249,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/783c5a6a820989c71316910c.xlsx"},{"id":47454122,"identity":"b41277e5-614a-4d0f-8c4d-75d546ee5d2a","added_by":"auto","created_at":"2023-12-01 17:29:41","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3375655,"visible":true,"origin":"","legend":"Supplementary movie S1","description":"","filename":"MovieS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/db63d96f97baed613f242cba.mp4"},{"id":47454126,"identity":"1cc943bc-3e3f-44e8-9573-0aca99870020","added_by":"auto","created_at":"2023-12-01 17:29:42","extension":"mp4","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14991113,"visible":true,"origin":"","legend":"Supplementary movie S2","description":"","filename":"MovieS2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-3556715/v1/71a17b0ac1586d4afa12f616.mp4"}],"financialInterests":"(Not answered)","formattedTitle":"\u003cp\u003eUbiquitin-independent degradation of Bim blocks macrophage pyroptosis in sepsis-related tissue injury\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3556715/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3556715/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Pyroptosis, a typical inflammatory cell death mode, has been increasingly demonstrated to have therapeutic value in inflammatory diseases such as sepsis. However, the mechanisms and therapeutic targets of sepsis remain elusive. Here, we reported that REGγ inhibition promoted pyroptosis by regulating members of the gasdermin family in macrophages. Mechanistically, REGγ directly degraded Bim, a factor of the BCL-2 family that can inhibit the accumulation of GSDMD/E, ultimately preventing the occurrence of pyroptosis. Furthermore, cecal ligation and puncture (CLP)-induced sepsis model mice showed downregulation of REGγ at both the RNA and protein levels. Gasdermin-mediated pyroptosis was augmented in REGγ-knockout mice, and these mice exhibited more severe sepsis-related tissue injury. More importantly, we found that REGγ expression was downregulated in clinical sepsis samples, such as those from patients with Pseudomonas aeruginosa (PA) infection. Finally, PA-infected mice showed decreased REGγ levels in the lung. In summary, our study reveals that the REGγ-Bim-GSDMD/E pathway is a novel regulatory mechanism of pyroptosis in sepsis-related tissue injury.","manuscriptTitle":"Ubiquitin-independent degradation of Bim blocks macrophage pyroptosis in sepsis-related tissue injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-01 17:29:36","doi":"10.21203/rs.3.rs-3556715/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2023-12-21T12:26:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-12-20T06:31:36+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2023-12-08T04:17:39+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-12-07T00:37:23+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2023-12-04T00:41:07+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2023-11-29T10:19:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-11-08T11:03:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2023-11-07T15:58:57+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2023-11-06T12:40:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-11-04T09:42:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b50e9750-e24b-490c-9a9c-fb09a653cabd","owner":[],"postedDate":"December 1st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-01T07:12:11+00:00","versionOfRecord":{"articleIdentity":"rs-3556715","link":"https://doi.org/10.1038/s41419-024-07072-z","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2024-09-30 04:00:00","publishedOnDateReadable":"September 30th, 2024"},"versionCreatedAt":"2023-12-01 17:29:36","video":"","vorDoi":"10.1038/s41419-024-07072-z","vorDoiUrl":"https://doi.org/10.1038/s41419-024-07072-z","workflowStages":[]},"version":"v1","identity":"rs-3556715","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3556715","identity":"rs-3556715","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}