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 GSDMDflox/flox-S100A8cre (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 GSDMDKO mice, as depicted in Fig. 4A and B. Furthermore, the GSDMDKO 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 GSDMDKO mice (Fig. 4C, SFig. 2A), further supporting the overall increase in G-MDSCs in the GSDMDKO mice. When MDSCs from GSDMDKO 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 GSDMDKO mice were depleted of another antibody (α-GR1), which blocked the tumor-promoting effect in the GSDMDKO mice (Fig. 4G and H). These findings suggest that the enhanced tumor growth in the GSDMDKO 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 GSDMDKO 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-MDSCsWT and G-MDSCsKO 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-MDSCsWT but not in the supernatant of G-MDSCsKO. Additionally, the secretion of IL-18 by G-MDSCsKO was lower than that of G-MDSCsWT, 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.