{"paper_id":"427badfe-21e6-4265-810c-b3c6e42eee68","body_text":"Deletion of gasdermin D promotes granulocytic myeloid-derived suppressor cells differentiation by decreased release of mitochondrial DNA to promote tumor escape | 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 Research Article Deletion of gasdermin D promotes granulocytic myeloid-derived suppressor cells differentiation by decreased release of mitochondrial DNA to promote tumor escape Min Gu, Weiwei Chen, Shizhen Ding, Zhijie Lin, Li Qian, Weiming Xiao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4760791/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jul, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 11 You are reading this latest preprint version Abstract Gasdermin D (GSDMD), an effector molecule of cell apoptosis, is known to be activated in various cells during inflammation. However, the patterns of GSDMD activation in immune-regulatory cells such as myeloid-derived suppressor cells (MDSCs) remain unclear. In this study, we found that neutrophils in colorectal cancer (CRC) tissues exhibited reduced GSDMD transcription, as evidenced by a single-cell RNA-sequencing result. Additionally, HLA-DR- CD11b + cells from the peripheral blood of CRC patients exhibited a significant reduction in GSDMD activation. Mice with ubiquitous GSDMD deficiency bred in a clean environment exhibited a notable increase in G-MDSCs. These GSDMD-/- MDSCs enhanced immunosuppressive activity by both inhibiting effector T-cell activity and promoting regulatory T-cell induction. This enhancement was also observed in GSDMDflox/flox-S100A8cre mice, in which GSDMD was specifically deleted in MDSCs. The tumor-promoting effects in the GSDMD-/- and GSDMDflox/flox-S100A8cre mice were abrogated following MDSC depletion, as shown by the use of an anti-DR5 antibody. In the absence of GSDMD, G-MDSCs showed reduced inflammasome activation and decreased production of IL-1β and IL-18. Furthermore, a significant reduction in interferon-related factor 8/7 (IRF8/7) was observed in GSDMD-/- G-MDSCs via bulk RNA sequencing analysis. After treatment with LPS/nigericin, these cells maintained mitochondrial integrity, thus impairing the mtDNA release and the downstream c-GAS/STING/TBK1/IRF8/7 signaling axis activation. Reduced IRF8/7 levels were responsible for increased differentiation of GSDMD-/- G-MDSCs. Finally, treatment with a GSDMD recombinant lentivirus injected into in situ tumors significantly inhibited tumor growth and reduced G-MDSC levels, suggesting that a GSDMD-based vaccine could simultaneously exert anti-carcinoma and anti-MDSC effects. GSDMD MDSCs mtDNA IRF8/7 tumor escape Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Myeloid-derived suppressor cells (MDSCs) are induced during the development of cancer or in cases of chronic inflammation through the abnormal differentiation of myeloid precursor cells. There are two subsets of MDSCs: granulocytic/polymorphonuclear MDSCs (G-MDSCs/PMN-MDSCs) and monocytic MDSCs (M-MDSCs). MDSCs effectively inhibit the activity of T, B, and NK cells through the use of surface molecules such as PD-L1 or DR5, as well as the secretion of reactive oxygen species (ROS), arginase 1 (ARG1), nitric oxide, TGF-β1, IL-10, and PGE2 [ 1 , 2 ]. In addition to their immunosuppressive effects, MDSCs can facilitate the migration of tumor cells into the bloodstream and prepare the premetastatic niche through the release of neutrophil extracellular traps (NETs), thereby enhancing the colonization of circulating tumor cells and promoting tumor metastasis [ 3 ]. Various factors produced by tumors or the bone marrow stroma in response to chronic inflammation, including GM-CSF, G-CSF, M-CSF, and VEGF, play important roles in the expansion of immature myeloid cells [ 4 , 5 ]. Transcription factors such as STAT3, STAT5, IRF8, IRF7, and NOTCH contribute to the abnormal activation of MDSCs [ 6 ]. Gasdermin D (GSDMD) is a crucial molecule in pyroptosis and is expressed in various tissues and immune cells. GSDMD is composed of an N-terminal domain (NTD) of 242 amino acids and a C-terminal domain (CTD) consisting of a 43-amino acid linker and 199 amino acids. Upon activation of the inflammasome, GSDMD is cleaved and forms pyroptotic pores through the oligomerization of GSDMD-NTD, leading to the induction of pyroptosis and the release of IL-1β [ 7 ]. Abnormal activation of GSDMD is observed in inflammatory diseases such as sepsis [ 8 ], viral infections [ 9 ], neurodegenerative diseases [ 10 ], nonalcoholic steatohepatitis (NASH) [ 11 ], inflammatory bowel disease (IBD) [ 12 ], and malignant tumors [ 13 ]. However, the presence and activation state of GSDMD in immunosuppressive cells, specifically MDSCs, within the tumor microenvironment have not been investigated. In this study, we demonstrated that decreased activation of GSDMD promotes the induction of G-MDSCs, thereby enhancing tumor growth. We also investigated the underlying molecular mechanisms of G-MDSC development. Materials and Methods 2.1. Biopsies Biopsies were obtained from colorectal cancer (CRC) patients, comprising both male and female individuals aged between 50 and 75 years, with a mean age of 62.5 years. Clinical features of these patients were shown in supplementary table. Control normal tissues located 5 cm around the tumor sites were also collected. All tumor samples were independently analyzed by two pathologists. This study was approved by the Ethics Committee of the Affiliated Hospital of Yangzhou University (2021-YKL4-28-004), and informed consent was obtained from all participants. 2.2. Mice and cell lines Wild-type C57BL/6 mice were obtained from the Comparative Medical Center of Yangzhou University (Yangzhou, China). The gsdmd -knockout (KO) mice (strain no. T010437), casp1 −/− mice (strain no. T002799), g sdmd flox/flox mice (strain no. T059954), and s100a8 cre mice (strain no. T005636) were obtained from GemPharmatech (Nanjing, China). The gsdmd flox/flox mice were crossed with s100a8 Cre mice to obtain gsdmd flox/flox s100a8 cre conditional knockout (cKO) mice. A colon carcinoma cell line (MC38) and a melanoma cell line (B16F10) with a C57BL/6J mouse background were obtained from the American Type Culture Collection (ATCC). The tumor cell lines were cultured in Dulbecco's modified Eagle’s medium (Gibco, Massachusetts, USA). 2.3. Western blot After protein lysates were generated using RIPA buffer (Thermo Fisher Scientific, USA), protein concentrations were determined using a BCA protein assay kit (TransGen Biotech, Beijing, China). Equal amounts of protein (30 µg) were separated by SDS‒PAGE on a 10% gel and transferred to PVDF membranes (Millipore, USA). The membranes were blocked at room temperature for 1 hour. Primary antibodies were incubated overnight at 4°C, followed by washing and incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, USA) for 2 hours. The protein bands were then visualized using an imaging system. 2.4. Flow cytometry Single-cell suspensions were collected and stained with fluorescently conjugated antibodies for 30 minutes at 4°C. Intracellular staining was performed by permeabilizing cells with 0.5% saponin in phosphate-buffered saline (PBS) for 20 minutes, followed by incubation with fluorescein-conjugated antibodies specific to the target protein for 30 minutes at 4°C. After incubation, the cells were washed and analyzed by flow cytometry using a FACSVerse (BD Biosciences, New Jersey, USA). 2.5. Detection of cell proliferation Cell proliferation was assessed using a Cell Counting Kit-8 (Biosharp, Anhui, China) according to the manufacturer's instructions. Cells were seeded in 96-well plates at a density of 5,000 cells per well and allowed to adhere overnight. After 24 hours, 10 µL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for 2 hours. The absorbance was measured at 450 nm using a microplate reader. 2.6. LDH release assay Lactate dehydrogenase (LDH) release was quantified using an LDH cytotoxicity assay kit (Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocol. 2.7. Tumor transplantation B16F10 or MC38 cells were harvested, washed with PBS, and resuspended in Hank's balanced salt solution (HBSS) at a concentration of 1x10 7 /mL. Cancer cells (100 µL) were either subcutaneously injected into the backs of the mice or into the lateral tail vein. Mice were euthanized on day 21 postinjection, and tumor tissues or lungs with metastatic melanoma were harvested for further analysis. 2.8. In vivo depletion of MDSCs Anti-mouse antibodies were injected intraperitoneally in 200 µl of PBS. Antibodies against DR5 (MD5.1) or Gr-1 (RB6-8C5) were purchased from BioXcell (USA), with each dose containing 50 µg of α-DR5 or α-Gr-1. Mice were administered 4 doses every 3 days. 2.9. Lentivirus transfection Lentiviral vectors expressing GSDMD, IRF8, or IRF7 were obtained from Gene Create (Gene Create, Wuhan, China). Murine splenic MDSCs were isolated using the MDSC Cell Isolation Kit (Miltenyi Biotec, Germany). MDSCs were infected with lentivirus in the presence of 8 µg/mL polybrene (Sigma‒Aldrich, USA), and stable cells were selected using puromycin (2 µg/mL) at 48 hours post infection. The efficiency of transduction was assessed via fluorescence microscopy after 72 hours of infection. 2.10. Bulk RNA sequencing MDSCs were isolated from mouse spleens using the Miltenyi Biotec MDSC Isolation Kit (Miltenyi Biotec, Germany), with each sample containing 1 × 10 6 cells. Total RNA was harvested using 1 mL of TRIzol reagent. The quality and integrity of the RNA were assessed on 1% agarose gels, and concentrations were determined using a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). The mRNA was then isolated, fragmented, and converted into double-stranded cDNA. Subsequent library preparation and sequencing were performed on the Illumina HiSeq X Ten platform (Illumina, CA, USA). The data analysis included gene expression profiling, differential gene expression, and gene enrichment studies. 2.11. Immunohistochemistry and immunofluorescence After the paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through a graded alcohol series, antigen retrieval was performed using 10 mM sodium citrate buffer (pH 6.0) in a microwave for 10 minutes. The sections were then blocked with 5% BSA and incubated sequentially with primary and secondary antibodies. For immunohistochemistry, signal detection was carried out using a DAB substrate kit (BOSTER, Wuhan, China), and the sections were counterstained with hematoxylin (Servicebio, Beijing, China). For immunofluorescence, nuclei were stained with DAPI, and slides were mounted with anti-fade mounting medium (Solarbio, Beijing, China). Images were acquired using a fluorescence microscope. 2.12. Statistical analysis All the data are presented as the means ± SDs. One-way ANOVA followed by Tukey’s post- hoc test was used to determine significant differences between multiple groups, while two-tailed Student's t tests were used for comparisons between two groups. Statistical significance is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Availability of data and materials The data generated in this study are available within the article and its supplementary data files. Expression profile data analyzed in this study were obtained from Gene Expression Omnibus (GEO) at GSE161277. Results 3.1. Low GSDMD activation in MDSCs from colorectal cancer patients Since immunohistochemical staining of GSDMD cannot reflect its activation status (Production of N-terminal) on tissue sections, we isolated MDSCs (HLA-DR - CD11b + ) from the peripheral blood of CRC patients to examine two fragments of activated GSDMD via western blotting. While cleaved fragments of GSDMD were present in HLA-DR - CD11b + cells from healthy controls, they were nearly absent in MDSCs from CRC patients ( Fig. 1A and B ). These findings align with the single-cell RNA-seq results of Zheng et al. [14], where reduced GSDMD transcription was observed in neutrophils but not in monocytes within the tumor microenvironment ( Fig. 1C-E ). 3.2. Enrichment of G-MDSCs in GSDMD-deficient mice We subsequently generated GSDMD-KO mice and reared them in a clean environment rather than under specific pathogen-free conditions. Enlarged spleens were observed in the GSDMD-KO mice ( Fig. 2A and B ). The frequencies and numbers of MDSCs (CD11b + Gr-1 + ) were increased in the spleen, bone marrow, and peripheral blood of the GSDMD -/- mice ( Fig. 2C ). Among these CD11b + GR1 + cells, a greater proportion of Ly6G + Ly6C - cells was observed, whereas the proportion of Ly6G - Ly6C + cells did not significantly change in the KO mice. This indicates the enrichment of G-MDSCs in the spleen ( Fig. 2D ), bone marrow, and peripheral blood ( Fig. 2E ) of GSDMD-deficient mice. These GSDMD -/- MDSCs exhibited elevated expression levels of ARG1, inducible nitric oxide synthase (iNOS), c/EBPβ ( Fig. 2F ), IL-10 ( Fig. 2G ) and PD-L1 ( Fig. 2H ), suggesting their potent immunosuppressive function. When GSDMD -/- MDSCs were cocultured with CD8 + T cells at different ratios ex vivo, there was a significant decrease in IFN-γ production by CD8 + T cells ( Fig. 2I ). GSDMD -/- MDSCs also inhibited IFN-γ production by CD4 + T cells ( Fig. 2J ) and effectively induced the differentiation of regulatory T cells (CD4 + CD25 + Foxp3 + ) ( Fig. 2K ). Furthermore, when bone marrow cells from WT or KO mice were treated with GM-CSF/IL-6, an increase in CD11b + Gr-1 + cells was observed in the culture of WT-BM cells, in contrast to the culture of KO-BM cells. This finding suggested that the GSDMD -/- CD11b + Gr-1 + cells had already differentiated into MDSCs under physiological conditions ( Fig. 2L ). Moreover, the expansion of GSDMD -/- G-MDSCs increased ( Fig. 2M ), and early/late apoptosis decreased ( Fig. 2N ). Additionally, GSDMD -/- G-MDSCs displayed decreased LDH release following stimulation with LPS/nigericin, indicating their reduced sensitivity to pyroptosis ( Fig. 2O ). Thus, the loss of GSDMD could promote the generation of G-MDSCs. 3.3. Intrinsic deficiency of GSDMD facilitates the induction of G-MDSCs S100A8 and S100A9 are ubiquitously expressed in MDSCs [15]. Subsequently, variations in MDSCs were assessed in GSDMD flox/flox -S100A8 cre (cKO) mice. As anticipated, there was an increase in CD11b + Gr1 + cells in the cKO mice ( Fig. 3A ). Additionally, compared with those of GSDMD-floxed mice, the numbers of G-MDSCs in the spleen and blood of cKO mice were greater ( Fig. 3B, SFig. 1A ), while no significant changes in M-MDSCs were detected. GSDMD -/- G-MDSCs from cKO mice also exhibited increased CCK8 and Ki67 expression, indicating their proliferative potential ( Fig. 3C ). Furthermore, these GSDMD -/- G-MDSCs exhibited decreased apoptosis and reduced LPS/nigericin-stimulated LDH release ( Fig. 3D ). When bone marrow cells from cKO mice were exposed to GM-CSF/IL-6, comparable numbers of CD11b + Gr1 + cells were observed before and after treatment, providing support for the preferential differentiation of GSDMD -/- MDSCs in vivo ( Fig. 3E ). G-MDSCs from cKO mice also exhibited increased expression of IL-10, PD-L1, ARG1, iNOS, and C/EBPβ ( Fig. 3F-H, SFig. 1B ). Moreover, compared with G-MDSCs from GSDMD-floxed mice, G-MDSCs from cKO mice more efficiently inhibited IFN-γ production in CD8 + T cells ( Fig. 3I ). These GSDMD-/- G-MDSCs also induced the generation of regulatory T (CD4 + CD25 + Foxp3 + ) cells ex vivo ( Fig. 3J ). Therefore, the intrinsic deficiency of GSDMD in S100A8 + cells appears to facilitate the differentiation of G-MDSCs. 3.4. GSDMD -/- G-MDSCs promoted tumor growth in vivo The tumor-promoting activity of GSDMD -/- G-MDSCs was initially observed in mice transplanted with MC38 cells. Accelerated tumor growth was observed in the GSDMD KO mice, as depicted in Fig. 4A and B . Furthermore, the GSDMD KO mice exhibited an increase in G-MDSCs and a decrease in CD8 + T cells, CD8 + NKG2D + T cells, and NK cells within the tumors. The frequencies of G-MDSCs were also greater in the blood and spleen of the tumor-bearing GSDMD KO mice ( Fig. 4C, SFig. 2A ), further supporting the overall increase in G-MDSCs in the GSDMD KO mice. When MDSCs from GSDMD KO mice were pre-depleted with the DR5 antibody, tumor growth was substantially suppressed ( Fig. 4D and E ), accompanied by the restoration of MDSCs in the tumor and spleen ( Fig. 4F, SFig. 2B ). Similar results were observed when G-MDSCs in the GSDMD KO mice were depleted of another antibody (α-GR1), which blocked the tumor-promoting effect in the GSDMD KO mice ( Fig. 4G and H ). These findings suggest that the enhanced tumor growth in the GSDMD KO mice is dependent on MDSCs. Enhanced tumor growth (MC38) was observed in GSDMD △S100A8 mice, as shown in Fig. 4I and J . Transplanted tumor tissues exhibited increased numbers of G-MDSCs with high expression of IL-10 and TGF-β1, as depicted in Fig. 4K and SFig. 2C , with a corresponding decrease in CD4 + T, CD8 + T, and NK cells recruited into tumors in GSDMD △S100A8 mice, as shown in Fig. 4L . Moreover, tumor-infiltrated lymphocytes from GSDMD △S100A8 mice exhibited a sharp decrease in IFN-γ production, as shown in Fig. 4L . Similarly, the growth of melanoma (B16F10) cells was promoted in GSDMD △S100A8 mice, accompanied by an increase in G-MDSCs and a decrease in CD8 + T cells in tumor tissues, as indicated in Fig. 4M , N, and SFig. 2D . Notably, when GSDMD △S100A8 mice were depleted of MDSCs using the DR5 antibody, tumor growth (MC38) was completely restored, as depicted in Fig. 4O and P . Furthermore, tumor growth was significantly enhanced when mice were adoptively transfused with GSDMD -/- G-MDSCs, as presented in Fig. 4Q . Higher levels of GSDMD -/- G-MDSCs, along with fewer IFN-γ-producing CD4 + T, CD8 + T, and NK cells, were observed in the tumor tissues, as demonstrated in Fig. 4R and SFig. 2E-F, indicating the profound immunosuppressive activity of these GSDMD -/- G-MDSCs. These findings confirmed that GSDMD -/- G-MDSCs facilitated tumor growth in vivo. 3.5. Reduced inflammasome activation in GSDMD -/- G-MDSCs Given that GSDMD is a key effector molecule for pyroptosis, the expression of GSDMD, IL-18, and IL-1β was examined in normal and GSDMD -/- G-MDSCs. As expected, G-MDSCs from KO mice lacked the expression of full-length or cleaved GSDMD. However, these GSDMD -/- G-MDSCs exhibited decreased production of IL-18 and IL-1β in both resting and stimulated states with LPS/nigericin, indicating impaired pyroptosis of the GSDMD -/- G-MDSCs, as shown in Fig. 5A . Furthermore, the expression of molecules involved in inflammasome activation, such as NLRP3, AIM2, ASC, full-length caspase-1, and cleaved caspase-1, was downregulated in G-MDSCs from GSDMD KO mice, particularly after stimulation with LPS/nigericin, as illustrated in Fig. 5B . In terms of the secretion of IL-1β in cell supernatants, there were no differences between G-MDSCs WT and G-MDSCs KO under physiological conditions. However, after stimulation with LPS/nigericin, an increase in the level of IL-1β was observed in the cell supernatant of G-MDSCs WT but not in the supernatant of G-MDSCs KO . Additionally, the secretion of IL-18 by G-MDSCs KO was lower than that of G-MDSCs WT , both under physiological conditions and after stimulation with LPS/nigericin, as displayed in Fig. 5C . Similarly, G-MDSCs from GSDMD conditional knockout (cKO) mice exhibited similar decreases in NLRP3 inflammasome activation, IL-1β production, and IL-18 production, as shown in Fig. 5D and E, respectively. In summary, GSDMD deficiency in G-MDSCs resulted in a nearly complete loss of inflammasome activation. ROS are considered secondary signals for NLRP3 activation [16]. The levels of cellular ROS (cROS) and mitochondrial ROS (mROS) were decreased in G-MDSCs from knockout or conditional knockout (cKO) mice ( Fig. 5F ). Similarly, peripheral blood CD11b + Gr-1 + cells from CRC patients also exhibited low levels of cROS and mROS ( Fig. 5G, SFig. 3 ), indicating a correlation between low inflammasome activation and increased MDSC activity. Additionally, CD11b + cells from WT or KO mice were transfected with the GSDMD recombinant lentivirus ( Fig. 5H ). Interestingly, the reintroduction of GSDMD significantly suppressed the induction of both subsets of MDSCs ( Fig. 5I ) and increased MDSC death ( Fig. 5J ). These GSDMD-rescued MDSCs, including G-MDSCs and M-MDSCs from the KO mice, exhibited reduced production of IL-10 and PD-L1 ( Fig. 5K ), suggesting that GSDMD plays a key role in modulating G/M-MDSC activity. 3.6. A decrease in IRF8/7 contributes to the induction of GSDMD -/- G-MDSCs To understand how GSDMD deficiency influences the induction of G-MDSCs, we performed bulk RNA sequencing on CD11b + Gr1 + cells from normal and GSDMD knockout mice. The analysis revealed a total of 1052 upregulated genes and 613 downregulated genes ( Fig. 6A ). Gene Ontology (GO) analysis indicated that genes involved in the innate immune response were the most downregulated ( SFig. 4A ), while Kyoto Encyclopedia of Genes and Genomes (KEGG)informatics revealed that genes involved in the cell cycle were the most upregulated ( SFig. 4B ). The volcano plot showed differentially expressed genes (DEGs), including interferon-activated gene 205 (Ifi205) and Cyclin-A2 (CCNA2) ( Fig. 6B ). Gene set enrichment analysis (GSEA) confirmed the downregulation of IFN-α/γ signaling ( Fig. 6C ) and the cytosolic DNA sensing pathway ( Fig. 6D ) in GSDMD -/- MDSCs. Variations in genes related to the cell cycle and IFN regulatory factors (IRFs) are shown in the heatmap ( Fig. 6E ). The protein levels of IRF8, IRF7, and STAT1, which are involved in G-MDSC development, were remarkably decreased in the GSDMD -/- G-MDSCs, especially after stimulation with LPS/nigericin ( Fig. 6F ). These GSDMD -/- G-MDSCs showed increased expression of expansion-associated molecules such as c-Myc/p-c-Myc and PI3K (p110), as well as antiapoptotic Bcl-2 and Bcl-xL molecules. The key MDSC-suppressive molecule STAT3 was also upregulated ( Fig. 6G ). These findings indicate that GSDMD -/- G-MDSCs exhibit enhanced cell proliferation, a reduced cytosolic DNA response, and an impaired IFN-regulatory factor response. IRF8 is essential for the development of monocytes/macrophages and dendritic cells [17]. Downregulation of IRF8 is a typical feature of MDSC development [18]. When an IRF8 recombinant lentivirus was transfected into GSDMD -/- CD11b + Gr1 + cells ( Fig. 6H ), the differentiation of GSDMD -/- G-MDSCs was repressed ( Fig. 6I ), and the expression of PD-L1 and IL-10 decreased ( Fig. 6J ). IRF7 deficiency causes a significant increase in G-MDSCs but has no obvious influence on the suppressive activity of G-MDSCs [19]. When GSDMD -/- CD11b + Gr1 + cells were transfected with an IRF7 recombinant lentivirus ( Fig. 6K ), the induction of G-MDSCs was inhibited ( Fig. 6L ). Despite the decrease in PD-L1 in G-MDSCs, no decrease in IL-10 was detected in IRF7-rescued G-MDSCs ( Fig. 6M ). In summary, the loss of GSDMD results in decreased IRF8/IRF7 expression for the differentiation of G-MDSCs. 3.7. Downregulated mtDNA-STING-IRF8/7 signaling in GSDMD -/- MDSCs The N-terminus of GSDMD can permeabilize the inner and outer membranes of mitochondria [20], leading to mitochondrial DNA (mtDNA) leakage [21]. Combined with the RNA-seq results, we wondered whether GSDMD deficiency led to the disappearance of mtDNA leakage and resulted in low stimulation of the cGAS/STING/IRF3 signaling pathway. As a consequence, low production of IFN-α/β further decreased the induction of IFN-inducible molecules (IRF8/7/9/4) ( Fig. 6F ). Cytosolic mtDNA (D-loop1, D-loop3, and MT-ND) was detected by quantitative PCR. There were no changes in the remaining G-MDSCs between the WT and KO mice, but the amount of cytosolic mtDNA in the GSDMD -/- G-MDSCs decreased under LPS/nigericin stimulation ( Fig. 7A ). Immunofluorescence staining of mtDNA revealed fewer double-positive GSDMD -/- G-MDSCs than normal G-MDSCs after treatment with LPS/nigericin ( Fig. 7B and C ). Thus, GSDMD -/- G-MDSCs sharply reduced the release of mtDNA into the cytoplasm during pyroptosis. As expected, LPS/nigericin-treated GSDMD -/- G-MDSCs exhibited obviously decreased expression of cGAS, IRF3, IRF8, and IRF7. Although comparable levels of total STING and TBK1 were detected in both G-MDSCs, the levels of phosphorylated STING and TBK1 were much lower in the GSDMD -/- G-MDSCs than in the normal G-MDSCs ( Fig. 7D ), confirming the substantial downregulation of the cGAS/STING/TBK1/IRF3 signaling pathway in the GSDMD-deficient G-MDSCs. Given the activation of NF-κB by TBK1, a decreased level of NF-κB p65/p-p65 was also confirmed in the GSDMD -/- G-MDSCs ( SFig. 5 ). Finally, after GSDMD -/- CD11b + Gr1 + cells were cultured with recombinant IFN-β ex vivo, the induction and activity of G-MDSCs were repressed in a dose-dependent manner. A STING agonist also exerted similar effects on G-MDSC differentiation and activity ( Fig. 7E ). Thus, we demonstrated that downregulation of the cGAS/STING/TBK1/IRF3 signaling pathway contributed to the induction of GSDMD -/- G-MDSCs. 3.8. Upregulation of GSDMD inhibits tumor growth involved with G-MDSCs Then, we analyzed whether injecting the GSDMD recombinant lentivirus into tumor tissues would mediate the antitumor effect. After MC38 cells were injected into the backs of the mice for 10 days to induce tumor formation, the administration of the GSDMD recombinant lentivirus to the tumors inhibited tumor growth. The same inhibitory effect was also observed in tumor-bearing mice after treatment with the STING agonist diABZI ( Fig. 8A and B ). Due to the small size of the tumors, we were unable to obtain sufficient single cells from the diABZI-treated tumors. After treatment with the GSDMD lentivirus (pGSDMD), the number of MDSCs in tumor tissues substantially decreased ( Fig. 8C ). The number of G-MDSCs in pGSDMD-injected tumors also decreased, accompanied by reduced expression of PD-L1, IL-10, and TGF-β1 ( Fig. 8D ). Moreover, increases in the numbers of CD8 + T and NK cells with enhanced production of IFN-γ were observed in pGSDMD-injected tumor tissues ( Fig. 8E ). The antitumor effect mediated by pGSDMD was confirmed in mice transplanted with melanoma cells (B16BL6) ( Fig. 8F and G ). A decrease in the number of MDSCs ( Fig. 8H ), particularly G-MDSCs, was also observed in pGSDMD-injected melanoma cells. These G-MDSCs also downregulated the expression of IL-10 and TGF-β1, with no variations in PD-L1 ( Fig. 8I ). Therefore, intratumor injection of the GSDMD recombinant lentivirus suppressed tumor growth, which was associated with a reduction in G-MDSCs. Discussion The tumor-promoting activity of MDSCs has been widely recognized. This study demonstrated that HLA-DR - CD11b + myeloid cells in CRC patients exhibited decreased GSDMD activation and loss of GSDMD-induced G-MDSCs through low levels of IRF8 expression. IRF8 governs myeloid cell development [17] and is a key negative regulator of MDSCs [18]. These GSDMD -/- G-MDSCs also showed enhanced immune-suppressive activity, as evidenced by increased production of ARG1, iNOS, and C/EBPβ. Although ubiquitous GSDMD-deficient mice and conditional GSDMD-knockout mice exhibited an increase in G-MDSCs, transfection of GSDMD into MDSCs inhibited the induction of G-MDSCs and M-MDSCs (Fig. 5). The downregulation of IRF8 in the GSDMD -/- G-MDSCs resulted from attenuated mitochondrial damage and decreased activation of the mtDNA-cGAS-STING signaling axis (Fig. 9). When tumors were injected with the GSDMD recombinant lentivirus, tumor growth was suppressed, and the infiltration of G-MDSCs decreased. Therefore, stimulating GSDMD activation can inhibit the differentiation of G-MDSCs in the tumor microenvironment (TME), thereby controlling tumor progression. Immunosuppressive MDSCs are highly enriched in the TME and are recognized as a significant barrier to effective immunotherapy. The observation of low GSDMD activation in MDSCs derived from tumors suggests that these cells have enhanced survival mechanisms. ROS present in the TME play a role in suppressing the activity of effector T cells and NK cells while promoting the induction of regulatory T cells and B cells [22, 23]. Although ROS are detrimental to neighboring cells, MDSCs are able to survive by upregulating the expression of antioxidant factors, such as nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor that regulates genes involved in reducing oxidative stress [24]. Additionally, ROS serve as a second signal for the activation of the NLRP3 inflammasome [16]. The decrease in cytoplasmic ROS levels observed in MDSCs from patients with CRC suggests that MDSCs with robust resistance to oxidative stress are able to inhibit GSDMD-mediated cellular activation, ultimately promoting the survival and induction of MDSCs. Mitochondrial damage occurs as soon as GSDMD is cleaved, preceding damage to the plasma membrane. The N-terminal pore-forming fragment of GSDMD (GSDMD-NT) can rapidly damage both the inner and outer mitochondrial membranes (OMMs), leading to a reduction in mitochondrial numbers, loss of transmembrane potential, and the release of mitochondrial proteins and DNA from the matrix and intermembrane space [20, 21]. In this study, we confirmed that GSDMD -/- G-MDSCs exhibited lower mtDNA levels in the cytoplasm than did normal G-MDSCs, particularly when exposed to LPS/nigericin stimulation. The downregulation of the cGAS/STING/TBK1 signaling axis in GSDMD -/- G-MDSCs, as well as the impaired induction of G-MDSCs by IFN-β and a STING agonist (diABZI) ex vivo, indicated that the decreased release of mtDNA was responsible for the attenuation of interferon regulatory factors 8 and 7 (IRF8 and IRF7). Notably, when the GSDMD recombinant lentivirus was injected into tumor tissues, it affected both the carcinoma cells and the stromal cells. Activation of GSDMD also induces pyroptosis in tumor cells [25, 26]. Therefore, the administration of a GSDMD-based vaccine directly into the tumor tissue would have a dual effect: eliminating both tumor cells and MDSCs simultaneously. Additionally, enhanced GSDMD activation in myeloid cells theoretically promotes the maturation of dendritic cells and the polarization of M1 macrophages through the upregulation of the cGAS/STING/TBK1 signaling pathway [27, 28]. In mouse models bearing MC38 or B16BL6 cell-transplanted tumors, the STING activator diABZI demonstrated the greatest antitumor effects [29, 30], indicating that diABZI not only enhances dendritic cell maturation and M1 macrophage polarization but also inhibits MDSC induction. In summary, this study demonstrated that MDSCs in the TME decrease GSDMD activation, which positively promotes their survival and immunosuppressive activity. Furthermore, the loss of GSDMD in MDSCs is dependent on the downregulation of the mtDNA/cGAS/STING/TBK1/IRF8/7 signaling axis. Additionally, in situ injection of a GSDMD recombinant lentivirus into tumors has shown profoundly antitumor effects. These findings confirm that a GSDMD-containing vaccine could be developed for the interventional treatment of tumors. Declarations Availability of data and materials The datasets generated and/or analysed during the current study are available in the GEO repository. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE161277 Conflict of Interest The authors have no relevant financial or non-financial interests to disclose. Authors' contributions All authors contributed to the study conception and design. Gong W: Writing - review & editing, Writing - original draft, Conceptualization. Lu G: Writing - original draft, Conceptualization. Gu M: Software, Data curation, Validation, Methodology. Chen W: Data curation, Validation, Methodology. Ding S: Data curation, Validation, Methodology. Lin Z: Data curation, Validation, Methodology. Qian L: Supervision. Xiao W: Supervision. Jia X: Supervision. Funding This work was supported by the National Natural Science Foundation of China (grant Nos. 82241043 and 82100870), the Society Development Science Foundation of Jiangsu Province, China (grant Nos. BE2022775). Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the Affiliated Hospital of Yangzhou University (2021-YKL4-28-004). Consent to participate Informed consent was obtained from all individual participants included in the study. References Hegde S, Leader AM, Merad M. MDSC: Markers, development, states, and unaddressed complexity. Immunity . 2021;54(5):875-884. Barry ST, Gabrilovich DI, Sansom OJ, Campbell AD, Morton JP. Therapeutic targeting of tumour myeloid cells. Nat Rev Cancer . 2023;23(4):216-237. Ortiz-Espinosa S, Morales X, Senent Y, Diego Alignani , Beatriz Tavira , Irati Macaya, et al. Complement C5a induces the formation of neutrophil extracellular traps by myeloid-derived suppressor cells to promote metastasis. Cancer Lett . 2022;529:70-84. Wu Y, Yi M, Niu M, Mei Q, Wu K. Myeloid-derived suppressor cells: an emerging target for anticancer immunotherapy. 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Gasdermin D permeabilization of mitochondrial inner and outer membranes accelerates and enhances pyroptosis. Immunity . 2023;56(11):2523-2541.e8. Zhang YF, Zhou L, Mao HQ, Yang FH, Chen Z, Zhang L. Mitochondrial DNA leakage exacerbates odontoblast inflammation through gasdermin D-mediated pyroptosis. Cell Death Discov . 2021;7(1):381. Shah R, Ibis B, Kashyap M, Boussiotis VA. The role of ROS in tumor infiltrating immune cells and cancer immunotherapy. Metabolism . 2024;151:155747. Ohl K, Tenbrock K. Reactive Oxygen Species as Regulators of MDSC-Mediated Immune Suppression. Front Immunol . 2018;9:2499. Huang J, Zhao Y, Zhao K, Yin K, Wang S. Function of reactive oxygen species in myeloid-derived suppressor cells. Front Immunol . 2023;14:1226443. Published 2023 Aug 14. Lv T, Xiong X, Yan W, Liu M, Xu H, He Q. Targeting of GSDMD sensitizes HCC to anti-PD-1 by activating cGAS pathway and downregulating PD-L1 expression. J Immunother Cancer . 2022;10(6):e004763. Xing Y, Zhang F, Ji P, Mengying Wei, Chunhui Yin, Angang Yang, et al. Efficient Delivery of GSDMD-N mRNA by Engineered Extracellular Vesicles Induces Pyroptosis for Enhanced Immunotherapy. Small . 2023;19(20):e2204031. Kalantari P, Shecter I, Hopkins J, Andrea Pilotta Gois, Yoelkys Morales, Bijan F Harandi, et al. The balance between gasdermin D and STING signaling shapes the severity of schistosome immunopathology. Proc Natl Acad Sci U S A . 2023;120(13):e2211047120. Banerjee I, Behl B, Mendonca M, Gaurav Shrivastava, Ashley J Russo, Antoine Menoret, et al. Gasdermin D Restrains Type I Interferon Response to Cytosolic DNA by Disrupting Ionic Homeostasis. Immunity . 2018;49(3):413-426.e5. Wu YT, Fang Y, Wei Q, Heping Shi, Huiling Tan, Yafang Deng, et al. Tumor-targeted delivery of a STING agonist improvescancer immunotherapy. Proc Natl Acad Sci U S A . 2022;119(49):e2214278119. Berger G, Knelson EH, Jimenez-Macias JL, Michal O Nowicki, Saemi Han, Eleni Panagioti, et al. STING activation promotes robust immune response and NK cell-mediated tumor regression in glioblastoma models. Proc Natl Acad Sci U S A . 2022;119(28):e2111003119. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfigures.pdf Supplementarytable.docx Cite Share Download PDF Status: Published Journal Publication published 25 Jul, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted Editorial decision: Revision requested 06 Sep, 2024 Reviews received at journal 04 Sep, 2024 Reviews received at journal 04 Sep, 2024 Reviewers agreed at journal 22 Aug, 2024 Reviewers agreed at journal 21 Aug, 2024 Reviewers agreed at journal 20 Aug, 2024 Reviewers agreed at journal 20 Aug, 2024 Reviewers invited by journal 20 Aug, 2024 Editor assigned by journal 19 Jul, 2024 Submission checks completed at journal 19 Jul, 2024 First submitted to journal 18 Jul, 2024 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-4760791\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":332554958,\"identity\":\"ae76bdce-c999-48a6-9b96-6ab35c919cb3\",\"order_by\":0,\"name\":\"Min Gu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Yangzhou University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Min\",\"middleName\":\"\",\"lastName\":\"Gu\",\"suffix\":\"\"},{\"id\":332554959,\"identity\":\"d11a1568-6281-42f8-a48d-97132776d779\",\"order_by\":1,\"name\":\"Weiwei Chen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Yangzhou University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Weiwei\",\"middleName\":\"\",\"lastName\":\"Chen\",\"suffix\":\"\"},{\"id\":332554960,\"identity\":\"804b75d2-151e-42d1-8d9c-58c63fc39e9f\",\"order_by\":2,\"name\":\"Shizhen Ding\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Yangzhou University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Shizhen\",\"middleName\":\"\",\"lastName\":\"Ding\",\"suffix\":\"\"},{\"id\":332554962,\"identity\":\"b7c3ba77-2bde-49e6-9328-b9b4022696a9\",\"order_by\":3,\"name\":\"Zhijie Lin\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Yangzhou University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zhijie\",\"middleName\":\"\",\"lastName\":\"Lin\",\"suffix\":\"\"},{\"id\":332554964,\"identity\":\"0dfcaca4-5c52-4b74-a59d-31c1d29c1acd\",\"order_by\":4,\"name\":\"Li Qian\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Yangzhou University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Li\",\"middleName\":\"\",\"lastName\":\"Qian\",\"suffix\":\"\"},{\"id\":332554966,\"identity\":\"34e06600-abec-4a26-8747-dcdd204666ae\",\"order_by\":5,\"name\":\"Weiming Xiao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Yangzhou University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Weiming\",\"middleName\":\"\",\"lastName\":\"Xiao\",\"suffix\":\"\"},{\"id\":332554968,\"identity\":\"e686637d-92a9-4568-8a50-ecb2caf0f980\",\"order_by\":6,\"name\":\"Xiaoqin Jia\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Yangzhou University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xiaoqin\",\"middleName\":\"\",\"lastName\":\"Jia\",\"suffix\":\"\"},{\"id\":332554969,\"identity\":\"0f724670-9031-4571-9557-ba702c1ff0e4\",\"order_by\":7,\"name\":\"Guotao Lu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Yangzhou University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Guotao\",\"middleName\":\"\",\"lastName\":\"Lu\",\"suffix\":\"\"},{\"id\":332554971,\"identity\":\"896aa78b-8e5d-4845-95f8-44bd1b4591d1\",\"order_by\":8,\"name\":\"Weijuan Gong\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYFCCAyDCBsLmIUFLGklawOAwCVoMDh6/+Ljg1/nE+TMSGB+8bWOQNyeo5cCZYuOZfbcTG2ckMBvObWMw3NlAQIvZgTNp0rw9txObJRLYpHnbGBIMDhCn5Vxim0QC+28itRw/Js3z40BiD9AWZqK02B84w2zM25BsPIPnYbPknHMShhsIaZGccfzhY54/drLz25MPfnhTZiNP0BYGiTMGDIxtDI4NDIwNIC4h9UDA3/6AgeEPgz0RSkfBKBgFo2CkAgAwkkWc4PsTWwAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Yangzhou University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Weijuan\",\"middleName\":\"\",\"lastName\":\"Gong\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-07-18 07:44:33\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4760791/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4760791/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1007/s00262-025-04104-1\",\"type\":\"published\",\"date\":\"2025-07-25T15:58:01+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":62730686,\"identity\":\"5bc9230b-2379-4677-a918-e15c84f0e2eb\",\"added_by\":\"auto\",\"created_at\":\"2024-08-18 23:20:02\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":632818,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMDSCs from CRC patients decrease GSDMD activation. \\u003cstrong\\u003eA,\\u003c/strong\\u003e Levels of full-length GSDMD/Caspase1, cleaved GSDMD/Caspase1, and arginase-1 (Arg-1) in MDSCs from the peripheral blood of CRC patients. \\u003cstrong\\u003eB, \\u003c/strong\\u003eComparison of the above bands. \\u003cstrong\\u003eC, \\u003c/strong\\u003eMarking monocytes and neutrophils based on the scRNA sequencing results [14]. Comparison of GSDMD mRNA levels in neutrophils (\\u003cstrong\\u003eD\\u003c/strong\\u003e) or monocytes (\\u003cstrong\\u003eE\\u003c/strong\\u003e) between CRC patients and healthy controls (HCs).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4760791/v1/362ab2253e8b5349ef1c4590.png\"},{\"id\":62730685,\"identity\":\"10444657-3f9d-49e2-95d1-dfe0c1437dea\",\"added_by\":\"auto\",\"created_at\":\"2024-08-18 23:20:02\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":995920,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe increase of G-MDSCs in the GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice. \\u003cstrong\\u003eA,\\u003c/strong\\u003e Enlarged spleens of GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice. \\u003cstrong\\u003eB,\\u003c/strong\\u003e Spleen weights of the GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice. \\u003cstrong\\u003eC, \\u003c/strong\\u003eDistributions of CD11b\\u003csup\\u003e+ \\u003c/sup\\u003eGr-1\\u003csup\\u003e+ \\u003c/sup\\u003ecells in the bone marrow, spleen and peripheral blood. Variations in G-MDSCs and M-MDSCs in the spleen (\\u003cstrong\\u003eD\\u003c/strong\\u003e), bone marrow, and blood (\\u003cstrong\\u003eE\\u003c/strong\\u003e) of GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice. \\u003cstrong\\u003eF,\\u003c/strong\\u003e Increased production of Arg-1, iNOS, and c/EBPβ in G-MDSCs from GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice. Increased expression of IL-10 (\\u003cstrong\\u003eG\\u003c/strong\\u003e) and PD-L1 (\\u003cstrong\\u003eH\\u003c/strong\\u003e) in G-MDSCs from GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice. G-MDSCs\\u003csup\\u003e \\u003c/sup\\u003efrom GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice decreased the percentage of CD8\\u003csup\\u003e+\\u003c/sup\\u003e T cells (\\u003cstrong\\u003eI\\u003c/strong\\u003e) and IFN-γ production by CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells (\\u003cstrong\\u003eJ\\u003c/strong\\u003e).\\u003cstrong\\u003e K,\\u003c/strong\\u003e G-MDSCs\\u003csup\\u003e \\u003c/sup\\u003efrom GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice promoted the induction of regulatory T cells.\\u003cstrong\\u003e L,\\u003c/strong\\u003e Bone marrow cells were cultured with GM-CSF and IL-6, and CD11b\\u003csup\\u003e+ \\u003c/sup\\u003eGr-1\\u003csup\\u003e+ \\u003c/sup\\u003ecells were detected by flow cytometry. \\u003cstrong\\u003eM, \\u003c/strong\\u003eMTT and CCK8 assays were used to determine the proliferative capacity of G-MDSCs. \\u003cstrong\\u003eN, \\u003c/strong\\u003eThe spontaneous apoptosis of G-MDSCs. \\u003cstrong\\u003eO, \\u003c/strong\\u003eLDH release of G-MDSCs after stimulation with LPS and nigericin. Each experiment was repeated at least twice. 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\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4760791/v1/f260b7fa9776972869ed8316.png\"},{\"id\":62731067,\"identity\":\"ae8dbe79-58fd-4cdd-a1c0-841a6d409809\",\"added_by\":\"auto\",\"created_at\":\"2024-08-18 23:28:02\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":720612,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe increase of G-MDSCs in the GSDMD\\u003csup\\u003eflox/flox\\u003c/sup\\u003e-S100A8\\u003csup\\u003ecre\\u003c/sup\\u003e mice. \\u003cstrong\\u003eA, \\u003c/strong\\u003eDetection of CD11b\\u003csup\\u003e+ \\u003c/sup\\u003eGr-1\\u003csup\\u003e+ \\u003c/sup\\u003ecells by flow cytometry. \\u003cstrong\\u003eB,\\u003c/strong\\u003e The increase of G-MDSCs in the spleen and blood of GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice. \\u003cstrong\\u003eC,\\u003c/strong\\u003e Proliferation of G-MDSCs was detected by the CCK8 or Ki67 assay. \\u003cstrong\\u003eD,\\u003c/strong\\u003e Apoptosis and LPS/nigericin-stimulated LDH release of G-MDSCs. \\u003cstrong\\u003eE,\\u003c/strong\\u003e MDSC differentiation induced by GM-CSF/IL-6. IL-10 (\\u003cstrong\\u003eF\\u003c/strong\\u003e) and PD-L1 (\\u003cstrong\\u003eG\\u003c/strong\\u003e) expression in G-MDSCs from GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice. \\u003cstrong\\u003eH, \\u003c/strong\\u003eProduction of Arg-1, iNOS, and c/EBPβ in G-MDSCs from GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice. \\u003cstrong\\u003eI,\\u003c/strong\\u003e G-MDSCs from cKO mice downregulated IFN-γ production by CD8\\u003csup\\u003e+\\u003c/sup\\u003e T cells. \\u003cstrong\\u003eJ, \\u003c/strong\\u003eG-MDSCs from GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mic-induced regulatory T cells. Each experiment was performed at least three times. 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\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4760791/v1/c3cdc93ab569dea80f371496.png\"},{\"id\":62730178,\"identity\":\"84ea9ba7-b3ec-4739-80d9-dcc969ede3b7\",\"added_by\":\"auto\",\"created_at\":\"2024-08-18 23:12:02\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1122813,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe tumor-promoting activity of GSDMD\\u003csup\\u003e-/- \\u003c/sup\\u003eG-MDSCs. \\u003cstrong\\u003eA,\\u003c/strong\\u003e Growth curve of MC38 cell-transplanted tumors in WT or KO mice. \\u003cstrong\\u003eB,\\u003c/strong\\u003e Morphology and weight of MC38 cell-formed tumors. \\u003cstrong\\u003eC,\\u003c/strong\\u003e Increases in G-MDSCs in the tumor, spleen and blood of tumor-bearing GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice (upper panel) and variations in CD8\\u003csup\\u003e+\\u003c/sup\\u003e T cells, CD8\\u003csup\\u003e+\\u003c/sup\\u003e NKG2D\\u003csup\\u003e+ \\u003c/sup\\u003eT cells, and NK cells in tumors. Transplanted MC38 tumors from WT, KO, and α-DR5-treated KO mice (\\u003cstrong\\u003eD\\u003c/strong\\u003e) and statistical analysis of tumor weight (\\u003cstrong\\u003eE\\u003c/strong\\u003e). \\u003cstrong\\u003eF, \\u003c/strong\\u003eDistributions of G-MDSCs in transplanted tumors and spleens. Morphology (\\u003cstrong\\u003eG\\u003c/strong\\u003e) and growth curve (\\u003cstrong\\u003eH\\u003c/strong\\u003e) of MC38-transplanted tumors in GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice treated with α-Gr-1. Morphology (\\u003cstrong\\u003eI\\u003c/strong\\u003e) and weight (\\u003cstrong\\u003eJ\\u003c/strong\\u003e) of MC38 cell-formed tumors in GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice. \\u003cstrong\\u003eK, \\u003c/strong\\u003eVariations in the frequencies and surface markers of G-MDSCs from tumor tissues of GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice. \\u003cstrong\\u003eL, \\u003c/strong\\u003eVariations in CD4\\u003csup\\u003e+ \\u003c/sup\\u003eT, CD8+ T, and NK cells from tumor tissues of GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice and IFN-γ production. \\u003cstrong\\u003eM, \\u003c/strong\\u003eMorphology and weight of B16F10 cell-formed tumors in GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice. \\u003cstrong\\u003eN,\\u003c/strong\\u003e G-MDSCs from B16F10-formed tumors of GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice. Morphology (\\u003cstrong\\u003eO\\u003c/strong\\u003e) and weight (\\u003cstrong\\u003eP\\u003c/strong\\u003e) of MC38-formed tumors in GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice treated with α-DR5. Morphology (Q), weight, and frequency of G-MDSCs (\\u003cstrong\\u003eR\\u003c/strong\\u003e) in MC38-derived tumors from mice with adoptive transfer of MDSCs or GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003eMDSCs. Each experiment was performed at least twice. 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\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4760791/v1/ea0d1de14dc3f803451237ac.png\"},{\"id\":62730175,\"identity\":\"43bba825-bc1d-46cb-865d-db6bb0c93b81\",\"added_by\":\"auto\",\"created_at\":\"2024-08-18 23:12:02\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1024626,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDownregulated inflammasome activation in GSDMD\\u003csup\\u003e-/- \\u003c/sup\\u003eG-MDSCs.\\u003cstrong\\u003e A, \\u003c/strong\\u003eFull-length and cleaved GSDMD, IL-18, and IL-1β in physiologic and LPS/nigericin-stimulated G-MDSCs from GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice. \\u003cstrong\\u003eB, \\u003c/strong\\u003eNLRP3, AIM2, ASC, and Caspase 1 levels in the above GSDMD\\u003csup\\u003e-/- \\u003c/sup\\u003eG-MDSCs. \\u003cstrong\\u003eC, \\u003c/strong\\u003eConcentrations of IL-18 and IL-1β in the supernatants of G-MDSCs from GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice. \\u003cstrong\\u003eD,\\u003c/strong\\u003e Decreased activation of NLRP3 in G-MDSCs from GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice. \\u003cstrong\\u003eE,\\u003c/strong\\u003e IL-18 and IL-1β levels in the supernatants of G-MDSCs from GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice. Decreased levels of cROS and mROS in G-MDSCs from GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice, GSDMD△S100A8 mice (\\u003cstrong\\u003eF\\u003c/strong\\u003e) and CRC patients (\\u003cstrong\\u003eG\\u003c/strong\\u003e). Rescue of GSDMD expression (\\u003cstrong\\u003eH\\u003c/strong\\u003e) inhibited the induction of G-MDSCs and M-MDSCs (\\u003cstrong\\u003eI\\u003c/strong\\u003e). Rescue of GSDMD expression reversed LDH release (\\u003cstrong\\u003eJ\\u003c/strong\\u003e) and suppressed IL-10/PD-L1 expression (\\u003cstrong\\u003eK\\u003c/strong\\u003e). Each experiment was repeated at least twice. 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\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4760791/v1/b2c657c250d9129615f54b15.png\"},{\"id\":62730185,\"identity\":\"60d2a7d8-9dc0-4087-986d-522dee81a0d4\",\"added_by\":\"auto\",\"created_at\":\"2024-08-18 23:12:03\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1204042,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eVariations in genes involved in the cell cycle and IRFs indicated by bulk RNA-seq. \\u003cstrong\\u003eA,\\u003c/strong\\u003e Total differentially expressed genes (DEGs). \\u003cstrong\\u003eB, \\u003c/strong\\u003eHeatmap of DEGs. Downregulation of IFN-α/γ response genes (\\u003cstrong\\u003eC\\u003c/strong\\u003e) and cytosolic DNA-sensing genes (\\u003cstrong\\u003eD\\u003c/strong\\u003e) by GSEA. \\u003cstrong\\u003eE,\\u003c/strong\\u003e Heatmap of DEGs related to the cell cycle and IRFs. \\u003cstrong\\u003eF,\\u003c/strong\\u003e Protein levels of IRF8, IRF7 and STAT1 in GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e G-MDSCs. \\u003cstrong\\u003eG, \\u003c/strong\\u003eEnhanced expression of c-Myc, p-c-Myc, PI3K (p110), Bcl-xL, Bcl-2, and p-STAT3 in GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e G-MDSCs. Rescue of IRF8 expression inhibited the induction of G-MDSCs (\\u003cstrong\\u003eH-J\\u003c/strong\\u003e). Rescue of IRF7 expression inhibited the induction of G-MDSCs (\\u003cstrong\\u003eK-Ms\\u003c/strong\\u003e). Each experiment was repeated at least twice. 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\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4760791/v1/542af987d807bfc33ffc2a3b.png\"},{\"id\":62730180,\"identity\":\"6b5a4614-c544-4bff-b35d-f42a9425f90f\",\"added_by\":\"auto\",\"created_at\":\"2024-08-18 23:12:02\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":958140,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eReduced mtDNA release results in low IRF7/8 levels in GSDMD\\u003csup\\u003e-/- \\u003c/sup\\u003eMDSCs. \\u003cstrong\\u003eA,\\u003c/strong\\u003e Cytosolic DNA detected by PCR. Double staining of mitochondria and DNA (\\u003cstrong\\u003eB\\u003c/strong\\u003e) and fluorescence data (\\u003cstrong\\u003eC\\u003c/strong\\u003e). \\u003cstrong\\u003eD,\\u003c/strong\\u003e Protein levels of cGAS/STING/TBK1/IRF7/8/3. \\u003cstrong\\u003eE, \\u003c/strong\\u003eInhibition of GSDMD\\u003csup\\u003e-/- \\u003c/sup\\u003eG-MDSCs by IFN-β and a STING agonist (diABZI). Each experiment was performed three times. 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\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4760791/v1/892efa89a24e3f15cb59face.png\"},{\"id\":62730179,\"identity\":\"aee12553-ce7c-45f9-ad6e-01e7e69a2d0c\",\"added_by\":\"auto\",\"created_at\":\"2024-08-18 23:12:02\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":898523,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eInjection of the GSDMD recombinant virus impaired tumor growth. After 10 days of subcutaneous transplantation of MC38 cells, the recombinant GSDMD-containing lentivirus (\\u003cem\\u003eMOI\\u003c/em\\u003e=100), nigericin (12.5mg/kg), or diABZI (3mg/kg) was injected intratumorally into the mice once every 3 days. After 15 days, the mice were killed, and the tumor tissues were isolated. Morphology (\\u003cstrong\\u003eA\\u003c/strong\\u003e) and weight (\\u003cstrong\\u003eB\\u003c/strong\\u003e) of transplanted MC38 tumors. \\u003cstrong\\u003eC,\\u003c/strong\\u003e Infiltrated MDSCs in tumors were detected by flow cytometry. \\u003cstrong\\u003eD, \\u003c/strong\\u003eFrequencies of G-MDSCs and PD-L1, IL-10, and TGF-β1 in G-MDSCs from tumors from mice given different treatments. \\u003cstrong\\u003eE,\\u003c/strong\\u003e Frequencies of CD8\\u003csup\\u003e+\\u003c/sup\\u003e T, IFN-γ\\u003csup\\u003e+\\u003c/sup\\u003e CD8\\u003csup\\u003e+\\u003c/sup\\u003e T, NK, and IFN-γ\\u003csup\\u003e+ \\u003c/sup\\u003eNK cells in the tumors of the above mice. Morphology (\\u003cstrong\\u003eF\\u003c/strong\\u003e) and weight (\\u003cstrong\\u003eG\\u003c/strong\\u003e) of transplanted B16F10 tumors. \\u003cstrong\\u003eH,\\u003c/strong\\u003e Infiltrated MDSCs from B16F10 tumors. \\u003cstrong\\u003eI, \\u003c/strong\\u003eFrequencies of G-MDSCs and PD-L1, IL-10, and TGF-β1 in G-MDSCs from B16F10 tumors from mice subjected to different treatments.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4760791/v1/59b007445a832bb1919159db.png\"},{\"id\":62730687,\"identity\":\"a50b326a-bdfb-4fb7-842a-e708bf2d0d70\",\"added_by\":\"auto\",\"created_at\":\"2024-08-18 23:20:02\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":202058,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eA working model showing that GSDMD deficiency induces G-MDSCs.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4760791/v1/4a0fad8f528d3cd2d82473e9.png\"},{\"id\":88507169,\"identity\":\"b8c5185e-e250-4b08-9ceb-745467739d1d\",\"added_by\":\"auto\",\"created_at\":\"2025-08-07 07:36:54\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":10331911,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4760791/v1/f24a0195-bd60-41d6-81f7-05598b5b2ae2.pdf\"},{\"id\":62730181,\"identity\":\"0e5b569a-aa31-44e4-87fd-d3aa3051b767\",\"added_by\":\"auto\",\"created_at\":\"2024-08-18 23:12:02\",\"extension\":\"pdf\",\"order_by\":11,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1209771,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementaryfigures.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4760791/v1/96bbb2ec090e4d6d9f1e02ac.pdf\"},{\"id\":62730689,\"identity\":\"828b3410-08b4-4a9c-abce-4f801bc67685\",\"added_by\":\"auto\",\"created_at\":\"2024-08-18 23:20:02\",\"extension\":\"docx\",\"order_by\":12,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":18058,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementarytable.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4760791/v1/5ebc8e4b5dd6927abb2122d4.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Deletion of gasdermin D promotes granulocytic myeloid-derived suppressor cells differentiation by decreased release of mitochondrial DNA to promote tumor escape\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eMyeloid-derived suppressor cells (MDSCs) are induced during the development of cancer or in cases of chronic inflammation through the abnormal differentiation of myeloid precursor cells. There are two subsets of MDSCs: granulocytic/polymorphonuclear MDSCs (G-MDSCs/PMN-MDSCs) and monocytic MDSCs (M-MDSCs). MDSCs effectively inhibit the activity of T, B, and NK cells through the use of surface molecules such as PD-L1 or DR5, as well as the secretion of reactive oxygen species (ROS), arginase 1 (ARG1), nitric oxide, TGF-β1, IL-10, and PGE2 [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. In addition to their immunosuppressive effects, MDSCs can facilitate the migration of tumor cells into the bloodstream and prepare the premetastatic niche through the release of neutrophil extracellular traps (NETs), thereby enhancing the colonization of circulating tumor cells and promoting tumor metastasis [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Various factors produced by tumors or the bone marrow stroma in response to chronic inflammation, including GM-CSF, G-CSF, M-CSF, and VEGF, play important roles in the expansion of immature myeloid cells [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Transcription factors such as STAT3, STAT5, IRF8, IRF7, and NOTCH contribute to the abnormal activation of MDSCs [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eGasdermin D (GSDMD) is a crucial molecule in pyroptosis and is expressed in various tissues and immune cells. GSDMD is composed of an N-terminal domain (NTD) of 242 amino acids and a C-terminal domain (CTD) consisting of a 43-amino acid linker and 199 amino acids. Upon activation of the inflammasome, GSDMD is cleaved and forms pyroptotic pores through the oligomerization of GSDMD-NTD, leading to the induction of pyroptosis and the release of IL-1β [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. Abnormal activation of GSDMD is observed in inflammatory diseases such as sepsis [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e], viral infections [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e], neurodegenerative diseases [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e], nonalcoholic steatohepatitis (NASH) [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e], inflammatory bowel disease (IBD) [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e], and malignant tumors [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. However, the presence and activation state of GSDMD in immunosuppressive cells, specifically MDSCs, within the tumor microenvironment have not been investigated. In this study, we demonstrated that decreased activation of GSDMD promotes the induction of G-MDSCs, thereby enhancing tumor growth. We also investigated the underlying molecular mechanisms of G-MDSC development.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1. Biopsies\\u003c/h2\\u003e \\u003cp\\u003eBiopsies were obtained from colorectal cancer (CRC) patients, comprising both male and female individuals aged between 50 and 75 years, with a mean age of 62.5 years. Clinical features of these patients were shown in supplementary table. Control normal tissues located 5 cm around the tumor sites were also collected. All tumor samples were independently analyzed by two pathologists. This study was approved by the Ethics Committee of the Affiliated Hospital of Yangzhou University (2021-YKL4-28-004), and informed consent was obtained from all participants.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. Mice and cell lines\\u003c/h2\\u003e \\u003cp\\u003eWild-type C57BL/6 mice were obtained from the Comparative Medical Center of Yangzhou University (Yangzhou, China). The \\u003cem\\u003egsdmd\\u003c/em\\u003e-knockout (KO) mice (strain no. T010437), \\u003cem\\u003ecasp1\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e mice (strain no. T002799), g\\u003cem\\u003esdmd\\u003c/em\\u003e\\u003csup\\u003eflox/flox\\u003c/sup\\u003e mice (strain no. T059954), and \\u003cem\\u003es100a8\\u003c/em\\u003e\\u003csup\\u003ecre\\u003c/sup\\u003e mice (strain no. T005636) were obtained from GemPharmatech (Nanjing, China). The \\u003cem\\u003egsdmd\\u003c/em\\u003e\\u003csup\\u003eflox/flox\\u003c/sup\\u003e mice were crossed with \\u003cem\\u003es100a8\\u003c/em\\u003e\\u003csup\\u003eCre\\u003c/sup\\u003e mice to obtain \\u003cem\\u003egsdmd\\u003c/em\\u003e\\u003csup\\u003eflox/flox\\u003c/sup\\u003e\\u003cem\\u003es100a8\\u003c/em\\u003e\\u003csup\\u003ecre\\u003c/sup\\u003e conditional knockout (cKO) mice. A colon carcinoma cell line (MC38) and a melanoma cell line (B16F10) with a C57BL/6J mouse background were obtained from the American Type Culture Collection (ATCC). The tumor cell lines were cultured in Dulbecco's modified Eagle\\u0026rsquo;s medium (Gibco, Massachusetts, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3. Western blot\\u003c/h2\\u003e \\u003cp\\u003eAfter protein lysates were generated using RIPA buffer (Thermo Fisher Scientific, USA), protein concentrations were determined using a BCA protein assay kit (TransGen Biotech, Beijing, China). Equal amounts of protein (30 \\u0026micro;g) were separated by SDS‒PAGE on a 10% gel and transferred to PVDF membranes (Millipore, USA). The membranes were blocked at room temperature for 1 hour. Primary antibodies were incubated overnight at 4\\u0026deg;C, followed by washing and incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, USA) for 2 hours. The protein bands were then visualized using an imaging system.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4. Flow cytometry\\u003c/h2\\u003e \\u003cp\\u003eSingle-cell suspensions were collected and stained with fluorescently conjugated antibodies for 30 minutes at 4\\u0026deg;C. Intracellular staining was performed by permeabilizing cells with 0.5% saponin in phosphate-buffered saline (PBS) for 20 minutes, followed by incubation with fluorescein-conjugated antibodies specific to the target protein for 30 minutes at 4\\u0026deg;C. After incubation, the cells were washed and analyzed by flow cytometry using a FACSVerse (BD Biosciences, New Jersey, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5. Detection of cell proliferation\\u003c/h2\\u003e \\u003cp\\u003eCell proliferation was assessed using a Cell Counting Kit-8 (Biosharp, Anhui, China) according to the manufacturer's instructions. Cells were seeded in 96-well plates at a density of 5,000 cells per well and allowed to adhere overnight. After 24 hours, 10 \\u0026micro;L of CCK-8 solution was added to each well, and the plates were incubated at 37\\u0026deg;C for 2 hours. The absorbance was measured at 450 nm using a microplate reader.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6. LDH release assay\\u003c/h2\\u003e \\u003cp\\u003eLactate dehydrogenase (LDH) release was quantified using an LDH cytotoxicity assay kit (Beyotime Biotechnology, Shanghai, China) following the manufacturer\\u0026rsquo;s protocol.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.7. Tumor transplantation\\u003c/h2\\u003e \\u003cp\\u003eB16F10 or MC38 cells were harvested, washed with PBS, and resuspended in Hank's balanced salt solution (HBSS) at a concentration of 1x10\\u003csup\\u003e7\\u003c/sup\\u003e/mL. Cancer cells (100 \\u0026micro;L) were either subcutaneously injected into the backs of the mice or into the lateral tail vein. Mice were euthanized on day 21 postinjection, and tumor tissues or lungs with metastatic melanoma were harvested for further analysis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.8. In vivo depletion of MDSCs\\u003c/h2\\u003e \\u003cp\\u003eAnti-mouse antibodies were injected intraperitoneally in 200 \\u0026micro;l of PBS. Antibodies against DR5 (MD5.1) or Gr-1 (RB6-8C5) were purchased from BioXcell (USA), with each dose containing 50 \\u0026micro;g of α-DR5 or α-Gr-1. Mice were administered 4 doses every 3 days.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.9. Lentivirus transfection\\u003c/h2\\u003e \\u003cp\\u003eLentiviral vectors expressing GSDMD, IRF8, or IRF7 were obtained from Gene Create (Gene Create, Wuhan, China). Murine splenic MDSCs were isolated using the MDSC Cell Isolation Kit (Miltenyi Biotec, Germany). MDSCs were infected with lentivirus in the presence of 8 \\u0026micro;g/mL polybrene (Sigma‒Aldrich, USA), and stable cells were selected using puromycin (2 \\u0026micro;g/mL) at 48 hours post infection. The efficiency of transduction was assessed via fluorescence microscopy after 72 hours of infection.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.10. Bulk RNA sequencing\\u003c/h2\\u003e \\u003cp\\u003eMDSCs were isolated from mouse spleens using the Miltenyi Biotec MDSC Isolation Kit (Miltenyi Biotec, Germany), with each sample containing 1 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e cells. Total RNA was harvested using 1 mL of TRIzol reagent. The quality and integrity of the RNA were assessed on 1% agarose gels, and concentrations were determined using a Qubit\\u0026reg; 2.0 Fluorometer (Life Technologies, CA, USA). The mRNA was then isolated, fragmented, and converted into double-stranded cDNA. Subsequent library preparation and sequencing were performed on the Illumina HiSeq X Ten platform (Illumina, CA, USA). The data analysis included gene expression profiling, differential gene expression, and gene enrichment studies.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.11. Immunohistochemistry and immunofluorescence\\u003c/h2\\u003e \\u003cp\\u003eAfter the paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through a graded alcohol series, antigen retrieval was performed using 10 mM sodium citrate buffer (pH 6.0) in a microwave for 10 minutes. The sections were then blocked with 5% BSA and incubated sequentially with primary and secondary antibodies. For immunohistochemistry, signal detection was carried out using a DAB substrate kit (BOSTER, Wuhan, China), and the sections were counterstained with hematoxylin (Servicebio, Beijing, China). For immunofluorescence, nuclei were stained with DAPI, and slides were mounted with anti-fade mounting medium (Solarbio, Beijing, China). Images were acquired using a fluorescence microscope.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.12. Statistical analysis\\u003c/h2\\u003e \\u003cp\\u003eAll the data are presented as the means\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SDs. One-way ANOVA followed by Tukey\\u0026rsquo;s post- hoc test was used to determine significant differences between multiple groups, while two-tailed Student's \\u003cem\\u003et\\u003c/em\\u003e tests were used for comparisons between two groups. Statistical significance is indicated as follows: *, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05; **, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01; ***, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001.\\u003c/p\\u003e \\u003c/div\\u003e\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe data generated in this study are available within the article and its supplementary data files. Expression profile data analyzed in this study were obtained from Gene Expression Omnibus (GEO) at GSE161277.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e3.1. Low GSDMD activation in MDSCs\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003efrom\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;colorectal cancer patients\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSince immunohistochemical staining of GSDMD cannot reflect its activation status (Production of N-terminal) on tissue sections, we isolated MDSCs (HLA-DR\\u003csup\\u003e-\\u0026nbsp;\\u003c/sup\\u003eCD11b\\u003csup\\u003e+\\u003c/sup\\u003e) from the peripheral blood of CRC patients to examine two fragments of activated GSDMD\\u0026nbsp;via\\u0026nbsp;western\\u0026nbsp;blotting. While cleaved fragments of GSDMD were present in HLA-DR\\u003csup\\u003e-\\u0026nbsp;\\u003c/sup\\u003eCD11b\\u003csup\\u003e+\\u003c/sup\\u003e cells from healthy controls, they were nearly absent in MDSCs from CRC patients (\\u003cstrong\\u003eFig. 1A\\u0026nbsp;\\u003c/strong\\u003eand\\u003cstrong\\u003e\\u0026nbsp;B\\u003c/strong\\u003e). These findings align with the single-cell RNA-seq results\\u0026nbsp;of\\u0026nbsp;Zheng et al.\\u0026nbsp;[14], where reduced GSDMD transcription was observed in neutrophils but not in monocytes within the tumor microenvironment (\\u003cstrong\\u003eFig. 1C-E\\u003c/strong\\u003e).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.2. Enrichment\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eof G-MDSCs in GSDMD-deficient mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe subsequently generated GSDMD-KO mice and reared them in a clean environment rather than under specific pathogen-free conditions. Enlarged spleens were observed in\\u0026nbsp;the\\u0026nbsp;GSDMD-KO mice (\\u003cstrong\\u003eFig. 2A\\u003c/strong\\u003e and\\u0026nbsp;\\u003cstrong\\u003eB\\u003c/strong\\u003e). The frequencies and numbers of MDSCs (CD11b\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eGr-1\\u003csup\\u003e+\\u003c/sup\\u003e) were increased in the spleen, bone marrow, and peripheral blood of\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e mice (\\u003cstrong\\u003eFig. 2C\\u003c/strong\\u003e). Among these CD11b\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eGR1\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003ecells, a\\u0026nbsp;greater\\u0026nbsp;proportion of Ly6G\\u003csup\\u003e+\\u003c/sup\\u003e Ly6C\\u003csup\\u003e-\\u003c/sup\\u003e cells was observed, whereas\\u0026nbsp;the proportion of\\u0026nbsp;Ly6G\\u003csup\\u003e-\\u003c/sup\\u003e Ly6C\\u003csup\\u003e+\\u003c/sup\\u003e cells\\u0026nbsp;did not significantly change in the\\u0026nbsp;KO mice. This indicates the enrichment of G-MDSCs in the spleen (\\u003cstrong\\u003eFig. 2D\\u003c/strong\\u003e), bone marrow, and peripheral blood (\\u003cstrong\\u003eFig. 2E\\u003c/strong\\u003e) of GSDMD-deficient mice. These GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eMDSCs exhibited elevated expression levels of ARG1, inducible nitric oxide synthase (iNOS), c/EBP\\u0026beta; (\\u003cstrong\\u003eFig. 2F\\u003c/strong\\u003e), IL-10 (\\u003cstrong\\u003eFig. 2G\\u003c/strong\\u003e) and PD-L1 (\\u003cstrong\\u003eFig. 2H\\u003c/strong\\u003e), suggesting their potent\\u0026nbsp;immunosuppressive\\u0026nbsp;function.\\u003c/p\\u003e\\n\\u003cp\\u003eWhen GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eMDSCs were cocultured with CD8\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eT cells at different ratios ex vivo, there was a significant decrease in IFN-\\u0026gamma; production by CD8\\u003csup\\u003e+\\u003c/sup\\u003e T cells (\\u003cstrong\\u003eFig. 2I\\u003c/strong\\u003e). GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eMDSCs also inhibited IFN-\\u0026gamma; production by CD4\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eT cells (\\u003cstrong\\u003eFig. 2J\\u003c/strong\\u003e) and effectively induced the differentiation of regulatory T cells (CD4\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eCD25\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eFoxp3\\u003csup\\u003e+\\u003c/sup\\u003e) (\\u003cstrong\\u003eFig. 2K\\u003c/strong\\u003e). Furthermore, when bone marrow cells from WT or KO mice were treated with GM-CSF/IL-6, an increase in CD11b\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eGr-1\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003ecells was observed in the culture of WT-BM cells, in contrast to the culture of KO-BM cells. This\\u0026nbsp;finding suggested\\u0026nbsp;that\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e CD11b\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eGr-1\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003ecells had already differentiated into MDSCs under physiological conditions (\\u003cstrong\\u003eFig. 2L\\u003c/strong\\u003e).\\u0026nbsp;Moreover, the expansion of\\u0026nbsp;GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs\\u0026nbsp;increased\\u0026nbsp;(\\u003cstrong\\u003eFig. 2M\\u003c/strong\\u003e),\\u0026nbsp;and early/late apoptosis\\u0026nbsp;decreased\\u0026nbsp;(\\u003cstrong\\u003eFig. 2N\\u003c/strong\\u003e). Additionally, GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs displayed decreased LDH release following stimulation with LPS/nigericin, indicating their reduced sensitivity to pyroptosis (\\u003cstrong\\u003eFig. 2O\\u003c/strong\\u003e). Thus, the loss of GSDMD could promote the generation of G-MDSCs.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.3. Intrinsic deficiency of GSDMD facilitates the induction of G-MDSCs\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eS100A8 and S100A9 are ubiquitously expressed in MDSCs\\u0026nbsp;[15]. Subsequently, variations\\u0026nbsp;in\\u0026nbsp;MDSCs were assessed in GSDMD\\u003csup\\u003eflox/flox\\u003c/sup\\u003e-S100A8\\u003csup\\u003ecre\\u003c/sup\\u003e (cKO) mice. As anticipated, there was an increase in CD11b\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eGr1\\u003csup\\u003e+\\u003c/sup\\u003e cells in\\u0026nbsp;the\\u0026nbsp;cKO mice (\\u003cstrong\\u003eFig. 3A\\u003c/strong\\u003e). Additionally,\\u0026nbsp;compared with those of GSDMD-floxed mice, the numbers\\u0026nbsp;of G-MDSCs in the spleen and blood\\u0026nbsp;of cKO mice were greater\\u0026nbsp;(\\u003cstrong\\u003eFig. 3B, SFig. 1A\\u003c/strong\\u003e), while no significant changes in M-MDSCs\\u0026nbsp;were detected. GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e G-MDSCs\\u0026nbsp;from\\u0026nbsp;cKO mice also\\u0026nbsp;exhibited increased\\u0026nbsp;CCK8 and Ki67\\u0026nbsp;expression, indicating their proliferative potential (\\u003cstrong\\u003eFig. 3C\\u003c/strong\\u003e). Furthermore, these GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs\\u0026nbsp;exhibited\\u0026nbsp;decreased apoptosis and reduced LPS/nigericin-stimulated LDH release (\\u003cstrong\\u003eFig. 3D\\u003c/strong\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003eWhen bone marrow cells from cKO mice were exposed to GM-CSF/IL-6, comparable\\u0026nbsp;numbers\\u0026nbsp;of CD11b\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eGr1\\u003csup\\u003e+\\u003c/sup\\u003e cells were observed before and after treatment, providing support for the preferential differentiation of GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e MDSCs in vivo (\\u003cstrong\\u003eFig. 3E\\u003c/strong\\u003e). G-MDSCs from cKO mice also exhibited increased\\u0026nbsp;expression\\u0026nbsp;of IL-10, PD-L1, ARG1, iNOS, and C/EBP\\u0026beta; (\\u003cstrong\\u003eFig. 3F-H, SFig. 1B\\u003c/strong\\u003e). Moreover,\\u0026nbsp;compared with\\u0026nbsp;G-MDSCs from\\u0026nbsp;GSDMD-floxed mice, G-MDSCs from\\u0026nbsp;cKO mice more efficiently inhibited IFN-\\u0026gamma; production in CD8\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eT cells (\\u003cstrong\\u003eFig. 3I\\u003c/strong\\u003e). These GSDMD-/- G-MDSCs also induced the generation of regulatory T (CD4\\u003csup\\u003e+\\u003c/sup\\u003e CD25\\u003csup\\u003e+\\u003c/sup\\u003e Foxp3\\u003csup\\u003e+\\u003c/sup\\u003e) cells ex vivo (\\u003cstrong\\u003eFig. 3J\\u003c/strong\\u003e). Therefore, the intrinsic deficiency of GSDMD in S100A8\\u003csup\\u003e+\\u003c/sup\\u003e cells appears to facilitate the differentiation of G-MDSCs.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.4. GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs promoted tumor growth in vivo\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe tumor-promoting activity of GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs was initially observed in mice transplanted with MC38 cells. Accelerated tumor growth was observed in\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice, as depicted in\\u0026nbsp;\\u003cstrong\\u003eFig. 4A\\u003c/strong\\u003e and\\u003cstrong\\u003e\\u0026nbsp;B\\u003c/strong\\u003e. Furthermore,\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice exhibited an increase in G-MDSCs and a decrease in CD8\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eT cells, CD8\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eNKG2D\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eT cells, and NK cells within the tumors.\\u0026nbsp;The frequencies\\u0026nbsp;of G-MDSCs were also\\u0026nbsp;greater\\u0026nbsp;in the blood and spleen of\\u0026nbsp;the\\u0026nbsp;tumor-bearing GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice (\\u003cstrong\\u003eFig. 4C, SFig. 2A\\u003c/strong\\u003e), further supporting the overall increase in G-MDSCs in\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice. When MDSCs\\u0026nbsp;from\\u0026nbsp;GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice were pre-depleted\\u0026nbsp;with\\u0026nbsp;the DR5 antibody, tumor growth was substantially suppressed (\\u003cstrong\\u003eFig. 4D\\u0026nbsp;\\u003c/strong\\u003eand\\u003cstrong\\u003e\\u0026nbsp;E\\u003c/strong\\u003e), accompanied by the restoration of MDSCs in the tumor and spleen (\\u003cstrong\\u003eFig. 4F, SFig. 2B\\u003c/strong\\u003e). Similar results were observed when G-MDSCs in\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice were depleted\\u0026nbsp;of\\u0026nbsp;another antibody (\\u0026alpha;-GR1),\\u0026nbsp;which blocked\\u0026nbsp;the tumor-promoting effect in\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice (\\u003cstrong\\u003eFig. 4G\\u0026nbsp;\\u003c/strong\\u003eand\\u003cstrong\\u003e\\u0026nbsp;H\\u003c/strong\\u003e). These findings suggest that the enhanced tumor growth in\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice is dependent on MDSCs.\\u003c/p\\u003e\\n\\u003cp\\u003eEnhanced tumor growth (MC38) was observed in GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice, as shown in\\u0026nbsp;\\u003cstrong\\u003eFig. 4I\\u0026nbsp;\\u003c/strong\\u003eand\\u003cstrong\\u003e\\u0026nbsp;J\\u003c/strong\\u003e. Transplanted tumor tissues\\u0026nbsp;exhibited\\u0026nbsp;increased\\u0026nbsp;numbers of\\u0026nbsp;G-MDSCs with high expression of IL-10 and TGF-\\u0026beta;1, as depicted in\\u0026nbsp;\\u003cstrong\\u003eFig. 4K\\u003c/strong\\u003e and\\u0026nbsp;\\u003cstrong\\u003eSFig. 2C\\u003c/strong\\u003e, with a corresponding decrease in CD4\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eT, CD8\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eT, and NK cells recruited into tumors in GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice, as shown in\\u0026nbsp;\\u003cstrong\\u003eFig. 4L\\u003c/strong\\u003e. Moreover, tumor-infiltrated lymphocytes\\u0026nbsp;from\\u0026nbsp;GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice exhibited a sharp decrease in IFN-\\u0026gamma; production, as\\u0026nbsp;shown\\u0026nbsp;in\\u0026nbsp;\\u003cstrong\\u003eFig. 4L\\u003c/strong\\u003e.\\u0026nbsp;Similarly, the\\u0026nbsp;growth of melanoma (B16F10)\\u0026nbsp;cells\\u0026nbsp;was promoted in GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice, accompanied by\\u0026nbsp;an increase in\\u0026nbsp;G-MDSCs and\\u0026nbsp;a decrease in\\u0026nbsp;CD8\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eT cells in tumor tissues, as indicated in\\u0026nbsp;\\u003cstrong\\u003eFig. 4M\\u003c/strong\\u003e,\\u003cstrong\\u003e\\u0026nbsp;N,\\u0026nbsp;\\u003c/strong\\u003eand \\u003cstrong\\u003eSFig.\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e2D\\u003c/strong\\u003e. Notably, when GSDMD\\u003csup\\u003e△S100A8\\u003c/sup\\u003e mice were depleted of MDSCs using the DR5 antibody, tumor growth (MC38) was completely restored, as depicted in\\u0026nbsp;\\u003cstrong\\u003eFig. 4O\\u0026nbsp;\\u003c/strong\\u003eand\\u003cstrong\\u003e\\u0026nbsp;P\\u003c/strong\\u003e. Furthermore, tumor growth was significantly enhanced when mice were adoptively transfused with GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e G-MDSCs, as presented in\\u0026nbsp;\\u003cstrong\\u003eFig. 4Q\\u003c/strong\\u003e. Higher levels of GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e G-MDSCs, along with\\u0026nbsp;fewer\\u0026nbsp;IFN-\\u0026gamma;-producing CD4\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eT, CD8\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eT, and NK cells, were observed in the tumor tissues, as demonstrated in\\u0026nbsp;\\u003cstrong\\u003eFig. 4R\\u003c/strong\\u003e and\\u003cstrong\\u003e\\u0026nbsp;SFig. 2E-F,\\u0026nbsp;\\u003c/strong\\u003eindicating the\\u0026nbsp;profound\\u0026nbsp;immunosuppressive activity of these GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e G-MDSCs.\\u0026nbsp;These\\u0026nbsp;findings\\u0026nbsp;confirmed\\u0026nbsp;that GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs facilitated tumor growth in vivo.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.5. Reduced inflammasome activation\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ein\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e G-MDSCs\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eGiven that GSDMD is a key effector molecule for pyroptosis, the expression of GSDMD, IL-18, and IL-1\\u0026beta; was examined in normal and GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e G-MDSCs. As expected, G-MDSCs\\u0026nbsp;from\\u0026nbsp;KO mice lacked the expression of full-length or cleaved GSDMD. However, these GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs exhibited decreased production of IL-18 and IL-1\\u0026beta; in both resting and stimulated states with LPS/nigericin, indicating impaired pyroptosis of\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs, as shown in\\u0026nbsp;\\u003cstrong\\u003eFig. 5A\\u003c/strong\\u003e. Furthermore,\\u0026nbsp;the expression of\\u0026nbsp;molecules involved in inflammasome activation, such as NLRP3, AIM2, ASC, full-length\\u0026nbsp;caspase-1,\\u0026nbsp;and\\u0026nbsp;cleaved caspase-1,\\u0026nbsp;was\\u0026nbsp;downregulated in G-MDSCs\\u0026nbsp;from\\u0026nbsp;GSDMD\\u003csup\\u003eKO\\u003c/sup\\u003e mice, particularly after stimulation with LPS/nigericin, as illustrated in\\u0026nbsp;\\u003cstrong\\u003eFig. 5B\\u003c/strong\\u003e. In terms of the secretion of IL-1\\u0026beta; in cell supernatants, there were no\\u0026nbsp;differences\\u0026nbsp;between G-MDSCs\\u003csup\\u003eWT\\u003c/sup\\u003e and G-MDSCs\\u003csup\\u003eKO\\u003c/sup\\u003e under physiological conditions. However, after stimulation with LPS/nigericin, an\\u0026nbsp;increase in the\\u0026nbsp;level of IL-1\\u0026beta; was observed in the cell supernatant of G-MDSCs\\u003csup\\u003eWT\\u003c/sup\\u003e but not in the supernatant of G-MDSCs\\u003csup\\u003eKO\\u003c/sup\\u003e. Additionally, the secretion of IL-18 by G-MDSCs\\u003csup\\u003eKO\\u003c/sup\\u003e was lower than that of G-MDSCs\\u003csup\\u003eWT\\u003c/sup\\u003e, both under physiological conditions and after stimulation with LPS/nigericin, as displayed in\\u0026nbsp;\\u003cstrong\\u003eFig. 5C\\u003c/strong\\u003e.\\u0026nbsp;Similarly, G-MDSCs from GSDMD conditional knockout (cKO) mice exhibited similar\\u0026nbsp;decreases\\u0026nbsp;in NLRP3 inflammasome activation, IL-1\\u0026beta; production, and IL-18 production, as shown in Fig. 5D and E, respectively. In summary,\\u0026nbsp;GSDMD\\u0026nbsp;deficiency\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003ein G-MDSCs resulted in a nearly complete loss of inflammasome activation.\\u003c/p\\u003e\\n\\u003cp\\u003eROS\\u0026nbsp;are\\u0026nbsp;considered secondary\\u0026nbsp;signals\\u0026nbsp;for NLRP3 activation\\u0026nbsp;[16]. The levels of cellular ROS (cROS) and mitochondrial ROS (mROS) were decreased in G-MDSCs\\u0026nbsp;from\\u0026nbsp;knockout or conditional knockout (cKO) mice (\\u003cstrong\\u003eFig. 5F\\u003c/strong\\u003e). Similarly, peripheral blood\\u0026nbsp;CD11b\\u003csup\\u003e+\\u003c/sup\\u003eGr-1\\u003csup\\u003e+\\u003c/sup\\u003e cells from CRC patients also exhibited low levels of cROS and mROS (\\u003cstrong\\u003eFig. 5G, SFig. 3\\u003c/strong\\u003e), indicating a correlation between low inflammasome activation and increased MDSC activity. Additionally, CD11b\\u003csup\\u003e+\\u003c/sup\\u003e cells from WT or KO mice were transfected with the GSDMD\\u0026nbsp;recombinant lentivirus (\\u003cstrong\\u003eFig. 5H\\u003c/strong\\u003e). Interestingly, the reintroduction of GSDMD significantly suppressed the induction of both subsets of MDSCs (\\u003cstrong\\u003eFig. 5I\\u003c/strong\\u003e) and increased MDSC death (\\u003cstrong\\u003eFig. 5J\\u003c/strong\\u003e). These GSDMD-rescued MDSCs, including G-MDSCs and M-MDSCs\\u0026nbsp;from the\\u0026nbsp;KO mice, exhibited reduced production of IL-10 and PD-L1 (\\u003cstrong\\u003eFig. 5K\\u003c/strong\\u003e), suggesting that GSDMD plays a key role in modulating G/M-MDSC\\u0026nbsp;activity.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.6. A decrease\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;in IRF8/7 contributes to the induction of GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo understand how GSDMD deficiency influences the induction of G-MDSCs, we performed bulk RNA sequencing on CD11b\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eGr1\\u003csup\\u003e+\\u003c/sup\\u003e cells from normal and GSDMD knockout mice. The analysis revealed a total of 1052 upregulated genes and 613 downregulated genes (\\u003cstrong\\u003eFig. 6A\\u003c/strong\\u003e). Gene Ontology (GO)\\u0026nbsp;analysis indicated that genes involved in the innate immune response were the most downregulated (\\u003cstrong\\u003eSFig. 4A\\u003c/strong\\u003e), while\\u0026nbsp;Kyoto Encyclopedia of Genes and Genomes (KEGG)informatics revealed that genes involved in the cell cycle were the most upregulated (\\u003cstrong\\u003eSFig. 4B\\u003c/strong\\u003e). The volcano plot showed differentially\\u0026nbsp;expressed\\u0026nbsp;genes (DEGs), including interferon-activated gene 205 (Ifi205) and Cyclin-A2 (CCNA2) (\\u003cstrong\\u003eFig. 6B\\u003c/strong\\u003e). Gene set enrichment analysis (GSEA) confirmed the downregulation of IFN-\\u0026alpha;/\\u0026gamma; signaling (\\u003cstrong\\u003eFig. 6C\\u003c/strong\\u003e) and\\u0026nbsp;the\\u0026nbsp;cytosolic DNA sensing pathway (\\u003cstrong\\u003eFig. 6D\\u003c/strong\\u003e) in GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eMDSCs.\\u003c/p\\u003e\\n\\u003cp\\u003eVariations in genes related to the cell cycle and IFN regulatory factors (IRFs)\\u0026nbsp;are\\u0026nbsp;shown in the heatmap (\\u003cstrong\\u003eFig. 6E\\u003c/strong\\u003e).\\u0026nbsp;The protein\\u0026nbsp;levels of IRF8, IRF7, and STAT1, which are involved in G-MDSC development, were remarkably decreased in\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs, especially after stimulation with LPS/nigericin (\\u003cstrong\\u003eFig. 6F\\u003c/strong\\u003e). These\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eGSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs showed increased expression of expansion-associated molecules such as c-Myc/p-c-Myc and PI3K\\u0026nbsp;(p110), as well as\\u0026nbsp;antiapoptotic\\u0026nbsp;Bcl-2 and Bcl-xL molecules. The key MDSC-suppressive molecule STAT3 was also upregulated (\\u003cstrong\\u003eFig. 6G\\u003c/strong\\u003e). These findings indicate that GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs exhibit enhanced cell proliferation,\\u0026nbsp;a\\u0026nbsp;reduced cytosolic DNA response, and\\u0026nbsp;an\\u0026nbsp;impaired IFN-regulatory factor response.\\u003c/p\\u003e\\n\\u003cp\\u003eIRF8 is essential for the development of monocytes/macrophages and dendritic cells\\u0026nbsp;[17]. Downregulation of IRF8 is a typical feature of MDSC development\\u0026nbsp;[18]. When an IRF8\\u0026nbsp;recombinant lentivirus was transfected into GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eCD11b\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eGr1\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003ecells (\\u003cstrong\\u003eFig. 6H\\u003c/strong\\u003e), the differentiation of GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e G-MDSCs was repressed (\\u003cstrong\\u003eFig. 6I\\u003c/strong\\u003e), and the expression\\u0026nbsp;of PD-L1 and IL-10\\u0026nbsp;decreased\\u0026nbsp;(\\u003cstrong\\u003eFig. 6J\\u003c/strong\\u003e). IRF7 deficiency causes a significant\\u0026nbsp;increase in\\u0026nbsp;G-MDSCs\\u0026nbsp;but has\\u0026nbsp;no obvious influence on the suppressive activity of G-MDSCs\\u0026nbsp;[19]. When GSDMD\\u003csup\\u003e-/- CD11b\\u003c/sup\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e\\u003csup\\u003eGr1\\u003c/sup\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003ecells were transfected with an IRF7\\u0026nbsp;recombinant lentivirus (\\u003cstrong\\u003eFig. 6K\\u003c/strong\\u003e), the induction of G-MDSCs was inhibited (\\u003cstrong\\u003eFig. 6L\\u003c/strong\\u003e). Despite\\u0026nbsp;the decrease in\\u0026nbsp;PD-L1\\u0026nbsp;in\\u0026nbsp;G-MDSCs, no decrease in IL-10 was\\u0026nbsp;detected\\u0026nbsp;in IRF7-rescued G-MDSCs (\\u003cstrong\\u003eFig. 6M\\u003c/strong\\u003e). In summary, the loss of GSDMD results in decreased IRF8/IRF7 expression for the differentiation of G-MDSCs.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.7. Downregulated mtDNA-STING-IRF8/7 signaling in GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003eMDSCs\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe N-terminus\\u0026nbsp;of GSDMD can permeabilize the inner and outer membranes of mitochondria\\u0026nbsp;[20], leading to mitochondrial DNA (mtDNA) leakage\\u0026nbsp;[21].\\u0026nbsp;Combined\\u0026nbsp;with the RNA-seq results, we wondered whether GSDMD deficiency led to the disappearance of mtDNA leakage and resulted in low stimulation of the cGAS/STING/IRF3 signaling pathway. As a consequence, low production of IFN-\\u0026alpha;/\\u0026beta; further\\u0026nbsp;decreased\\u0026nbsp;the induction of IFN-inducible molecules (IRF8/7/9/4) (\\u003cstrong\\u003eFig. 6F\\u003c/strong\\u003e). Cytosolic\\u0026nbsp;mtDNA\\u0026nbsp;(D-loop1, D-loop3, and MT-ND)\\u0026nbsp;was\\u0026nbsp;detected by quantitative PCR. There were no changes in the\\u0026nbsp;remaining\\u0026nbsp;G-MDSCs between\\u0026nbsp;the\\u0026nbsp;WT and KO mice, but\\u0026nbsp;the amount of\\u0026nbsp;cytosolic\\u0026nbsp;mtDNA\\u0026nbsp;in\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs decreased under\\u0026nbsp;LPS/nigericin stimulation\\u0026nbsp;(\\u003cstrong\\u003eFig. 7A\\u003c/strong\\u003e).\\u0026nbsp;Immunofluorescence staining\\u0026nbsp;of mtDNA\\u0026nbsp;revealed\\u0026nbsp;fewer double-positive GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs than normal G-MDSCs\\u0026nbsp;after treatment\\u0026nbsp;with LPS/nigericin\\u0026nbsp;(\\u003cstrong\\u003eFig. 7B\\u0026nbsp;\\u003c/strong\\u003eand\\u003cstrong\\u003e\\u0026nbsp;C\\u003c/strong\\u003e). Thus, GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs sharply reduced the release of mtDNA into the cytoplasm\\u0026nbsp;during\\u0026nbsp;pyroptosis.\\u003c/p\\u003e\\n\\u003cp\\u003eAs expected, LPS/nigericin-treated GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs\\u0026nbsp;exhibited\\u0026nbsp;obviously decreased\\u0026nbsp;expression\\u0026nbsp;of cGAS, IRF3, IRF8,\\u0026nbsp;and\\u0026nbsp;IRF7. Although comparable levels of total STING and TBK1 were\\u0026nbsp;detected\\u0026nbsp;in both G-MDSCs,\\u0026nbsp;the levels of\\u0026nbsp;phosphorylated STING and TBK1\\u0026nbsp;were\\u0026nbsp;much lower in\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs than\\u0026nbsp;in the\\u0026nbsp;normal G-MDSCs (\\u003cstrong\\u003eFig. 7D\\u003c/strong\\u003e), confirming the substantial downregulation of the cGAS/STING/TBK1/IRF3 signaling pathway in\\u0026nbsp;the\\u0026nbsp;GSDMD-deficient G-MDSCs. Given the activation of NF-\\u0026kappa;B by TBK1, a decreased level of NF-\\u0026kappa;B p65/p-p65 was also confirmed in\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs (\\u003cstrong\\u003eSFig. 5\\u003c/strong\\u003e). Finally, after GSDMD\\u003csup\\u003e-/- CD11b\\u003c/sup\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e\\u003csup\\u003eGr1\\u003c/sup\\u003e\\u003csup\\u003e+\\u003c/sup\\u003e\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003ecells were cultured with recombinant IFN-\\u0026beta; ex vivo, the induction and activity of G-MDSCs were repressed in a dose-dependent manner. A STING agonist also exerted similar effects\\u0026nbsp;on\\u0026nbsp;G-MDSC differentiation and activity (\\u003cstrong\\u003eFig. 7E\\u003c/strong\\u003e). Thus, we demonstrated that downregulation of the cGAS/STING/TBK1/IRF3 signaling pathway contributed to the induction of GSDMD\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003eG-MDSCs.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.8. Upregulation of GSDMD inhibits tumor growth involved with G-MDSCs\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThen,\\u0026nbsp;we analyzed whether injecting the GSDMD\\u0026nbsp;recombinant lentivirus into tumor tissues would mediate\\u0026nbsp;the antitumor\\u0026nbsp;effect. After MC38 cells were injected into the\\u0026nbsp;backs\\u0026nbsp;of\\u0026nbsp;the\\u0026nbsp;mice for 10\\u0026nbsp;days\\u0026nbsp;to\\u0026nbsp;induce tumor formation, the\\u0026nbsp;administration of the GSDMD\\u0026nbsp;recombinant lentivirus\\u0026nbsp;to the\\u0026nbsp;tumors\\u0026nbsp;inhibited\\u0026nbsp;tumor growth. The same inhibitory effect was also observed in tumor-bearing\\u0026nbsp;mice after\\u0026nbsp;treatment\\u0026nbsp;with\\u0026nbsp;the\\u0026nbsp;STING agonist diABZI (\\u003cstrong\\u003eFig. 8A\\u0026nbsp;\\u003c/strong\\u003eand\\u003cstrong\\u003e\\u0026nbsp;B\\u003c/strong\\u003e). Due to the small size of the tumors, we were unable to obtain sufficient single cells from the diABZI-treated tumors. After treatment with the GSDMD\\u0026nbsp;lentivirus (pGSDMD),\\u0026nbsp;the number of\\u0026nbsp;MDSCs in tumor tissues substantially decreased (\\u003cstrong\\u003eFig. 8C\\u003c/strong\\u003e).\\u0026nbsp;The number of\\u0026nbsp;G-MDSCs in pGSDMD-injected tumors also decreased, accompanied by reduced\\u0026nbsp;expression\\u0026nbsp;of PD-L1, IL-10, and TGF-\\u0026beta;1 (\\u003cstrong\\u003eFig. 8D\\u003c/strong\\u003e).\\u0026nbsp;Moreover, increases in\\u0026nbsp;the numbers of\\u0026nbsp;CD8\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003eT and NK cells with enhanced production of IFN-\\u0026gamma; were observed in pGSDMD-injected tumor tissues (\\u003cstrong\\u003eFig. 8E\\u003c/strong\\u003e). The\\u0026nbsp;antitumor\\u0026nbsp;effect mediated by pGSDMD was confirmed in mice transplanted with melanoma cells (B16BL6) (\\u003cstrong\\u003eFig. 8F\\u0026nbsp;\\u003c/strong\\u003eand\\u003cstrong\\u003e\\u0026nbsp;G\\u003c/strong\\u003e).\\u0026nbsp;A\\u0026nbsp;decrease\\u0026nbsp;in the number\\u0026nbsp;of MDSCs (\\u003cstrong\\u003eFig. 8H\\u003c/strong\\u003e), particularly G-MDSCs, was also observed in pGSDMD-injected melanoma\\u0026nbsp;cells. These G-MDSCs also downregulated\\u0026nbsp;the expression\\u0026nbsp;of IL-10 and TGF-\\u0026beta;1, with no variations in PD-L1 (\\u003cstrong\\u003eFig. 8I\\u003c/strong\\u003e). Therefore,\\u0026nbsp;intratumor\\u0026nbsp;injection of the GSDMD\\u0026nbsp;recombinant lentivirus\\u0026nbsp;suppressed\\u0026nbsp;tumor growth, which\\u0026nbsp;was\\u0026nbsp;associated with\\u0026nbsp;a\\u0026nbsp;reduction\\u0026nbsp;in\\u0026nbsp;G-MDSCs.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe tumor-promoting activity of MDSCs has been widely recognized. This study demonstrated that HLA-DR\\u003csup\\u003e-\\u0026nbsp;\\u003c/sup\\u003eCD11b\\u003csup\\u003e+\\u003c/sup\\u003e myeloid cells in CRC patients exhibited decreased GSDMD activation and loss of GSDMD-induced G-MDSCs through low levels of IRF8 expression. IRF8 governs myeloid cell development\\u0026nbsp;[17]\\u0026nbsp;and is a key negative regulator of MDSCs\\u0026nbsp;[18]. These GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e G-MDSCs also showed enhanced immune-suppressive activity, as evidenced by increased production of ARG1,\\u0026nbsp;iNOS, and C/EBP\\u0026beta;. Although ubiquitous GSDMD-deficient mice and conditional GSDMD-knockout mice\\u0026nbsp;exhibited\\u0026nbsp;an increase in G-MDSCs, transfection of GSDMD into MDSCs inhibited the induction of G-MDSCs and M-MDSCs (Fig. 5). The downregulation of IRF8 in\\u0026nbsp;the\\u0026nbsp;GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003e G-MDSCs resulted from attenuated\\u0026nbsp;mitochondrial\\u0026nbsp;damage and decreased activation of the mtDNA-cGAS-STING signaling axis (Fig. 9). When tumors were injected with the GSDMD\\u0026nbsp;recombinant lentivirus, tumor growth was suppressed, and the\\u0026nbsp;infiltration of G-MDSCs\\u0026nbsp;decreased. Therefore, stimulating GSDMD activation can inhibit the differentiation of G-MDSCs in the tumor microenvironment (TME), thereby controlling tumor progression.\\u003c/p\\u003e\\n\\u003cp\\u003eImmunosuppressive MDSCs are highly enriched in the TME and are recognized as a significant barrier to effective immunotherapy. The observation of low GSDMD activation in MDSCs derived from tumors suggests that these cells have enhanced survival mechanisms. ROS present in the TME play a role in suppressing the activity of effector T cells and NK cells while promoting the induction of regulatory T cells and B cells\\u0026nbsp;[22, 23]. Although ROS are detrimental to neighboring cells, MDSCs are able to survive by upregulating the expression of antioxidant factors, such as nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor that regulates genes involved in reducing oxidative stress\\u0026nbsp;[24]. Additionally, ROS serve as a second signal for the activation of the NLRP3 inflammasome\\u0026nbsp;[16]. The decrease in cytoplasmic ROS levels observed in MDSCs from patients with CRC suggests that MDSCs with robust resistance to oxidative stress are able to inhibit GSDMD-mediated cellular activation, ultimately promoting the survival and induction of MDSCs.\\u003c/p\\u003e\\n\\u003cp\\u003eMitochondrial\\u0026nbsp;damage occurs as soon as GSDMD is cleaved, preceding damage to the plasma membrane. The N-terminal pore-forming fragment of GSDMD (GSDMD-NT) can rapidly damage both the inner and outer mitochondrial membranes (OMMs),\\u0026nbsp;leading to a reduction in mitochondrial numbers, loss of transmembrane potential, and the release of mitochondrial proteins and DNA from the matrix and intermembrane space\\u0026nbsp;[20, 21]. In this study, we confirmed that GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003eG-MDSCs exhibited lower mtDNA levels in the cytoplasm\\u0026nbsp;than did\\u0026nbsp;normal G-MDSCs, particularly when exposed to LPS/nigericin stimulation. The downregulation of the cGAS/STING/TBK1 signaling axis in GSDMD\\u003csup\\u003e-/-\\u003c/sup\\u003eG-MDSCs, as well as the impaired induction of G-MDSCs by IFN-\\u0026beta; and a STING agonist (diABZI) ex vivo, indicated that the decreased release of mtDNA was responsible for the attenuation of interferon regulatory factors 8 and 7 (IRF8 and IRF7).\\u003c/p\\u003e\\n\\u003cp\\u003eNotably,\\u0026nbsp;when the GSDMD\\u0026nbsp;recombinant lentivirus was injected into tumor tissues, it affected both the carcinoma cells and the stromal cells. Activation of GSDMD also induces pyroptosis in tumor cells\\u0026nbsp;[25, 26]. Therefore, the administration of a GSDMD-based vaccine directly into the\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003etumor tissue would have a dual effect: eliminating both tumor cells and MDSCs simultaneously. Additionally, enhanced GSDMD activation in myeloid cells theoretically\\u0026nbsp;promotes\\u0026nbsp;the maturation of dendritic cells and\\u0026nbsp;the\\u0026nbsp;polarization of M1 macrophages through the upregulation of the cGAS/STING/TBK1 signaling\\u0026nbsp;pathway\\u0026nbsp;[27, 28]. In mouse models bearing MC38 or B16BL6 cell-transplanted tumors, the STING activator diABZI demonstrated the greatest\\u0026nbsp;antitumor\\u0026nbsp;effects\\u0026nbsp;[29, 30], indicating that diABZI not only enhances dendritic cell maturation and M1 macrophage polarization but also inhibits MDSC induction.\\u003c/p\\u003e\\n\\u003cp\\u003eIn summary, this study demonstrated that MDSCs in the TME decrease GSDMD activation, which positively promotes their survival and immunosuppressive activity. Furthermore, the loss of GSDMD in MDSCs is dependent on the downregulation of the mtDNA/cGAS/STING/TBK1/IRF8/7 signaling axis. Additionally, in situ injection of a GSDMD recombinant lentivirus into tumors has shown profoundly antitumor effects. These findings confirm that a GSDMD-containing vaccine could be developed for the interventional treatment of tumors.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets generated and/or analysed during the current study are available in the GEO repository. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE161277\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of Interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors have no relevant financial or non-financial interests to disclose.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026apos; contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll authors contributed to the study conception and design. Gong W: Writing - review \\u0026amp; editing, Writing - original draft, Conceptualization. Lu G: Writing - original draft, Conceptualization. Gu M: Software, Data curation, Validation, Methodology. Chen W: Data curation, Validation, Methodology. Ding S: Data curation, Validation, Methodology. Lin Z: Data curation, Validation, Methodology. Qian L: Supervision. Xiao W: Supervision. Jia X: Supervision.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by the National Natural Science Foundation of China (grant Nos. 82241043 and 82100870), the Society Development Science Foundation of Jiangsu Province, China (grant Nos. BE2022775).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the Affiliated Hospital of Yangzhou University (2021-YKL4-28-004).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eInformed consent was obtained from all individual participants included in the study.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eHegde S, Leader AM, Merad M. MDSC: Markers, development, states, and unaddressed complexity. \\u003cem\\u003eImmunity\\u003c/em\\u003e. 2021;54(5):875-884. \\u003c/li\\u003e\\n\\u003cli\\u003eBarry ST, Gabrilovich DI, Sansom OJ, Campbell AD, Morton JP. Therapeutic targeting of tumour myeloid cells. \\u003cem\\u003eNat Rev Cancer\\u003c/em\\u003e. 2023;23(4):216-237. \\u003c/li\\u003e\\n\\u003cli\\u003eOrtiz-Espinosa S, Morales X, Senent Y, Diego Alignani , Beatriz Tavira , Irati Macaya, et al. 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Gasdermin D Restrains Type I Interferon Response to Cytosolic DNA by Disrupting Ionic Homeostasis. \\u003cem\\u003eImmunity\\u003c/em\\u003e. 2018;49(3):413-426.e5. \\u003c/li\\u003e\\n\\u003cli\\u003eWu YT, Fang Y, Wei Q, Heping Shi, Huiling Tan, Yafang Deng, et al. Tumor-targeted delivery of a STING agonist improvescancer immunotherapy. \\u003cem\\u003eProc Natl Acad Sci U S A\\u003c/em\\u003e. 2022;119(49):e2214278119. \\u003c/li\\u003e\\n\\u003cli\\u003eBerger G, Knelson EH, Jimenez-Macias JL, Michal O Nowicki, Saemi Han, Eleni Panagioti, et al. STING activation promotes robust immune response and NK cell-mediated tumor regression in glioblastoma models. \\u003cem\\u003eProc Natl Acad Sci U S A\\u003c/em\\u003e. 2022;119(28):e2111003119. \\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"cancer-immunology-immunotherapy\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"ciim\",\"sideBox\":\"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)\",\"snPcode\":\"262\",\"submissionUrl\":\"https://submission.nature.com/new-submission/262/3\",\"title\":\"Cancer Immunology, Immunotherapy\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"GSDMD, MDSCs, mtDNA, IRF8/7, tumor escape\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4760791/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4760791/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eGasdermin D (GSDMD), an effector molecule of cell apoptosis, is known to be activated in various cells during inflammation. However, the patterns of GSDMD activation in immune-regulatory cells such as myeloid-derived suppressor cells (MDSCs) remain unclear. In this study, we found that neutrophils in colorectal cancer (CRC) tissues exhibited reduced GSDMD transcription, as evidenced by a single-cell RNA-sequencing result. Additionally, HLA-DR- CD11b\\u0026thinsp;+\\u0026thinsp;cells from the peripheral blood of CRC patients exhibited a significant reduction in GSDMD activation. Mice with ubiquitous GSDMD deficiency bred in a clean environment exhibited a notable increase in G-MDSCs. These GSDMD-/- MDSCs enhanced immunosuppressive activity by both inhibiting effector T-cell activity and promoting regulatory T-cell induction. This enhancement was also observed in GSDMDflox/flox-S100A8cre mice, in which GSDMD was specifically deleted in MDSCs. The tumor-promoting effects in the GSDMD-/- and GSDMDflox/flox-S100A8cre mice were abrogated following MDSC depletion, as shown by the use of an anti-DR5 antibody. In the absence of GSDMD, G-MDSCs showed reduced inflammasome activation and decreased production of IL-1β and IL-18. Furthermore, a significant reduction in interferon-related factor 8/7 (IRF8/7) was observed in GSDMD-/- G-MDSCs via bulk RNA sequencing analysis. After treatment with LPS/nigericin, these cells maintained mitochondrial integrity, thus impairing the mtDNA release and the downstream c-GAS/STING/TBK1/IRF8/7 signaling axis activation. Reduced IRF8/7 levels were responsible for increased differentiation of GSDMD-/- G-MDSCs. Finally, treatment with a GSDMD recombinant lentivirus injected into in situ tumors significantly inhibited tumor growth and reduced G-MDSC levels, suggesting that a GSDMD-based vaccine could simultaneously exert anti-carcinoma and anti-MDSC effects.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Deletion of gasdermin D promotes granulocytic myeloid-derived suppressor cells differentiation by decreased release of mitochondrial DNA to promote tumor escape\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-08-18 23:11:57\",\"doi\":\"10.21203/rs.3.rs-4760791/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-09-06T06:50:45+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-09-04T14:09:36+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-09-04T05:05:14+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"121425434025521462757564417870461139375\",\"date\":\"2024-08-22T08:12:29+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"264296903553452042551958986996199276964\",\"date\":\"2024-08-21T06:55:14+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"157921008301132584761895791712924035027\",\"date\":\"2024-08-21T01:41:11+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"159857479408844332862256562865756863034\",\"date\":\"2024-08-20T21:58:23+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-08-20T08:04:06+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-07-19T04:27:15+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-07-19T04:27:00+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Cancer Immunology, Immunotherapy\",\"date\":\"2024-07-18T07:43:17+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"cancer-immunology-immunotherapy\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"ciim\",\"sideBox\":\"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)\",\"snPcode\":\"262\",\"submissionUrl\":\"https://submission.nature.com/new-submission/262/3\",\"title\":\"Cancer Immunology, Immunotherapy\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"fc97220d-f1b1-4be3-b9ee-8eb089198c3f\",\"owner\":[],\"postedDate\":\"August 18th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-08-07T07:29:39+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-4760791\",\"link\":\"https://doi.org/10.1007/s00262-025-04104-1\",\"journal\":{\"identity\":\"cancer-immunology-immunotherapy\",\"isVorOnly\":false,\"title\":\"Cancer Immunology, Immunotherapy\"},\"publishedOn\":\"2025-07-25 15:58:01\",\"publishedOnDateReadable\":\"July 25th, 2025\"},\"versionCreatedAt\":\"2024-08-18 23:11:57\",\"video\":\"\",\"vorDoi\":\"10.1007/s00262-025-04104-1\",\"vorDoiUrl\":\"https://doi.org/10.1007/s00262-025-04104-1\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4760791\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4760791\",\"identity\":\"rs-4760791\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}