Fig 1.
Immunosuppression in sepsis is associated with the dysfunction of immune cells.
(A) Proportion of patients infected with each pathogen. Patients with polymicrobial infections are counted in each relevant category. The denominator is the total number of patients with secondary infections (n = 69). (B) Proportion of infected patients by site; Patients with multiple sites of secondary infection were counted in each relevant category. The denominator is the total number of patients with secondary infections (n = 69). (C) Mortality rates of sepsis patients with primary (n = 417) and secondary infections (n = 69). (D) Comparison of monocyte counts in sepsis patients with primary and secondary infections. (E) mRNA expression levels of IL-6 and IL-1β measured by RT-PCR in PBMCs isolated from healthy individuals (n = 14) and sepsis survivors (n = 9), with or without LPS stimulation for 4 hours; Fig 1E: Citation to Use: Created in BioRender. An, Q. (2026) https://BioRender.com/swt26rj. (F) THP-1 cells (a human acute monocytic leukemia cell line) were differentiated into macrophages by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA), followed by LPS stimulation for 3, 6, 12, and 24 hours, and IL-6 and IL-1β mRNA expression levels were measured by RT-PCR. (G) Peritoneal macrophages (PMs) were isolated from wild-type (WT) C57BL/6 mice, followed by LPS stimulation for 6, 12, and 24 hours, and IL-6 and IL-1β mRNA expression levels were measured by RT-PCR. (H) A cell model of secondary infection induced by LPS in sepsis; Fig 1H: Citation to Use: Created in BioRender. An, Q. (2026) https://BioRender.com/tfpzsz9. (I, J) Stimulation methods of the LPS-induced secondary infection cell model applied to THP-1 and PMs, with IL-6 and IL-1β mRNA expression levels measured by RT-PCR. (K) PMs treated with stimulation methods from the LPS-induced secondary infection cell model, followed by transcriptomic sequencing to generate a heatmap of inflammatory gene expression. All experiments were repeated at least 3 times with similar results. The data are expressed as means ± SDs. Statistical analyses were performed using two-way ANOVA followed by Tukey’s multiple comparisons test (E), and one-way ANOVA for panels (F, G, I, J, K). Data were considered statistically significant when *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. https://doi.org/10.6084/m9.figshare.30581252.
Fig 2.
H2AK119ub plays a critical role in immunosuppression during secondary infections in sepsis.
(A) Stimulation methods of the LPS-induced secondary infection cell model were applied to PMs derived from WT mice. Western blotting (WB) analysis was used to detect H3K27ac, H3K27me3, H3K9me3, H3K79me3, H3K36me3, and H2AK119ub levels. (B) WB analysis of H2AK119ub levels in peripheral blood PBMCs from healthy individuals and sepsis survivors. (C) PMs derived from WT mice were treated with different concentrations of the histone H2A ubiquitination inhibitor PRT4165 for 4 hours, followed by LPS stimulation for 4 hours. WB analysis was used to detect H2AK119ub and IL-1β levels. (D, E) Before the establishment of the LPS-induced secondary infection model in PMs from WT mice, PMs were treated with 10 μM PRT4165 for 4 hours. IL-6 and IL-1β mRNA expression levels were measured by RT-PCR, and the level of IL-6 protein in the cell supernatant was measured by ELISA. (F) Schematic representation of intraperitoneal injection of PRT4165 in WT mice. The experimental group (n=3) received intraperitoneal injections of PRT4165 (20 mg/kg) for 3 consecutive days, followed by intraperitoneal injection of an E. coli suspension (3 × 10⁸ cfu/mL, 1 mL/mouse) dissolved in PBS. The control group (n=3) received intraperitoneal injection of the E. coli suspension only. Peritoneal lavage fluid was collected 4 hours later. Fig 2F: Citation to Use: Created in BioRender. An, Q. (2026) https://BioRender.com/ss4krtx. (G, H) The collected peritoneal lavage fluid was centrifuged, and the cell pellet was analyzed for IL-1β and IL-6 mRNA expression levels using RT-PCR. The supernatant was analyzed for IL-1β and IL-6 protein levels using ELISA. All experiments were repeated at least 3 times with similar results. The data are expressed as means ± SDs. Statistical analysis was performed using two-sided Student’s t-test (B, G, H) and one-way ANOVA (A, D, E). Data were considered statistically significant when *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.https://doi.org/10.6084/m9.figshare.30581426.
Fig 3.
MYSM1 interacts with H2AK119ub and reduces the level of H2AK119ub at the IL-6 and IL-1β promoters.
(A) MYSM1 expression in PBMCs from control and experimental groups exposed to Staphylococcus aureus SEI antigen for 6 hours, obtained from the GSE11281 dataset; (B) In the LPS-induced secondary infection cell model of sepsis using PMs derived from WT mice, MYSM1 mRNA expression levels were measured by RT-PCR. (C) In the LPS-induced secondary infection cell model of sepsis using PMs derived from WT mice, immunoprecipitation (Co-IP) and western blot (WB) analysis were performed to detect the interaction between H2AK119ub and MYSM1. (D, E) In the LPS-induced secondary infection cell model of sepsis using PMs derived from WT and MYSM1ex2-Δ/Δ mice, H2AK119ub and IL-1β levels were detected by WB, IL-6 protein in the cell supernatant was measured by ELISA. (F, G) Cut&Tag-qPCR analysis of H2AK119ub enrichment at the IL-6 and IL-1β promoter regions in PMs derived from WT and MYSM1ex2-Δ/Δ mice in an LPS-induced secondary infection sepsis model. (H) Mechanistic diagram illustrating that MYSM1 removes H2AK119 ubiquitination at the IL-6 and IL-1β promoter regions through its deubiquitinase activity, thereby suppressing the expression of the inflammatory cytokine IL-6 and IL-1β. Fig 3H: Citation to Use: Created in BioRender. An, Q. (2026) https://BioRender.com/eqf1660. All experiments were repeated at least 3 times with similar results. The data are expressed as means ± SDs. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test (E, F, G), two-sided Student’s t-test (A), and one-way ANOVA (B). Data were considered statistically significant when *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. https://doi.org/10.6084/m9.figshare.30581489.
Fig 4.
Deletion of the N-terminal domain of MYSM1 upregulates H2AK119ub, promoting the inflammatory response.
(A) Schematic representation of the LPS-induced secondary infection cell model of sepsis using PMs derived from WT and MYSM1ex2-Δ/Δ mice; Fig 4A: Citation to Use: Created in BioRender. An, Q. (2026) https://BioRender.com/lq1wp3y. (B, C, D) In the LPS-induced secondary infection model of sepsis using PMs derived from WT and MYSM1ex2-Δ/Δ mice, IL-6 protein levels in the cell supernatant were detected by ELISA, and IL-6 and IL-1β mRNA expression levels in cell extracts were measured by RT-PCR. (E, F) Before inducing the secondary infection model of sepsis using PMs derived from WT and MYSM1ex2-Δ/Δ mice with LPS, PMs were pretreated with 10 μM PRT4165 for 4 hours. The expression of IL-6 mRNA in the cell pellet was detected by RT-PCR, and the level of IL-6 protein in the cell supernatant was measured by ELISA. All experiments were repeated at least 3 times with similar results. The data are expressed as means ± SDs. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test (B, C, D, E, F). Data were considered statistically significant when *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. https://doi.org/10.6084/m9.figshare.30581501.
Fig 5.
Deletion of the N-terminal domain of MYSM1 enhances early inflammatory response in secondary infections in septic mice.
(A) According to the schematic diagram of the animal model 1, WT and MYSM1ex2-Δ/Δ mice were intraperitoneally injected with LPS solution and E. coli suspension (3 × 108 CFU/mL). Tissue collection and analysis were performed 4 hours post-injection; Fig 5A: Citation to Use: Created in BioRender. An, Q. (2026) https://BioRender.com/68gegzc. (B) Peritoneal lavage fluid was collected from WT (n = 3) and MYSM1ex2-Δ/Δ mice (n = 3), and the mRNA expression levels of IL-1β and IL-6 were determined by RT-PCR. (C) Lung tissues were collected from WT (n = 4) and MYSM1ex2-Δ/Δ mice (n = 4). The protein expression of F4/80, LY6G, and IL-1β in the lungs was examined by immunohistochemical analysis, and the histological features were evaluated using hematoxylin and eosin (H&E) staining. (D) Spleen tissues were collected from WT (n = 4) and MYSM1ex2-Δ/Δ mice (n = 4), The protein expression of F4/80 and IL-1β in the spleen was examined by immunohistochemical analysis, and the histological morphology was analyzed using hematoxylin and eosin (H&E) staining. The data were expressed as means ± SDs. The statistical analysis was carried out using two-sided Student’s t-test (B, C). The data were considered statistically significant when *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. https://doi.org/10.6084/m9.figshare.30581516.
Fig 6.
Deletion of the N-terminal domain of MYSM1 improves late survival and prognosis after secondary infection in septic mice.
(A) Experimental mice were intraperitoneally injected according to the schematic diagram of the animal model 2. Subsequently, the spleens were isolated, and morphological observations were conducted and photographed; Fig 6A: Citation to Use: Created in BioRender. An, Q. (2026) https://BioRender.com/z5mdalr. (B) Spleens were isolated and weighed from the LPS pretreatment + E. coli infection group of WT (n = 8) and MYSM1ex2-Δ/Δ (n = 11) mice, and the spleen index was analyzed for WT and MYSM1ex2-Δ/Δ mice; Fig 6B: Citation to Use: Created in BioRender. An, Q. (2026) https://BioRender.com/4kjxje4. (C) Survival curves were recorded and analyzed for the LPS pretreatment + E. coli infection group of WT (n = 12) and MYSM1ex2-Δ/Δ (n = 12) mice; Fig 6C: Citation to Use: Created in BioRender. Xjl, X. (2026) https://BioRender.com/okvc1xv. (D) Survival curve of WT (n = 12) and MYSM1ex2-Δ/Δ (n = 12) mice pretreated with PRT4165 (20 mg/kg, once daily for 3 consecutive days) prior to intraperitoneal challenge with LPS or E. coli. Fig 6D: Citation to Use: Created in BioRender. An, Q. (2026) https://BioRender.com/kxjpyt0. (E) Bacterial load in the lung tissues was measured for the LPS pretreatment + E. coli infection group of WT (n = 4) and MYSM1ex2-Δ/Δ (n = 4) mice. (F) Protein levels of F4/80, LY6G, and IL-1β in the lungs of the LPS pretreatment + E. coli infection group of WT (n = 4) and MYSM1ex2-Δ/Δ (n = 4) mice were determined by immunohistochemical analysis. Histological features of the lung tissues were analyzed using hematoxylin and eosin (H&E) staining. The data were expressed as means ± SDs. Statistical analysis was performed using two-sided Student’s t-test (B, E, F) or Kaplan–Meier survival analysis (C, D). The data were considered statistically significant when *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. https://doi.org/10.6084/m9.figshare.30581531.
Fig 7.
Model of MYSM1-mediated epigenetic regulation in sepsis.
MYSM1 controls H2AK119ub deposition at IL-6 and IL-1β promoters, shaping immune dynamics during secondary infection. Fig 7: Citation to Use: Created in BioRender. An, Q. (2026) https://BioRender.com/ey2ee9y. https://doi.org/10.6084/m9.figshare.30581543.