Targeting STING to disrupt macrophage-mediated adhesion in encapsulating peritoneal sclerosis

Abstract

Encapsulating peritoneal sclerosis (EPS) is a life-threatening fibrotic condition characterized by severe abdominal adhesions, chronic inflammation, and significant morbidity. The lack of effective treatments for EPS stems from a limited understanding of its underlying mechanisms. In this study, we developed a modified mouse model of PD-induced EPS and investigated the role of the STING signaling pathway in disease progression. Our findings reveal that STING activation in peritoneal mesothelial cells significantly increases the secretion of the macrophage chemokine CCL2, leading to enhanced macrophage infiltration and the formation of pathological adhesions. Notably, pharmacological inhibition of STING using the inhibitor H151 effectively reduced macrophage infiltration and fibrosis, demonstrating its therapeutic potential in alleviating EPS. These results identify the STING pathway as a critical mediator of EPS pathogenesis and suggest that STING inhibitors could offer a promising therapeutic strategy to prevent or reverse EPS, particularly in clinical settings such as peritoneal dialysis.

Fig. 1. Modifying an EPS mouse model to closely resemble PD-induced abdominal adhesions.

A Schematic of the experimental regimen for establishing the mouse model of EPS. B Gross macroscopic examination of the abdominal cavity. Control mice displayed smooth, well-expanded mesenteries and sharp liver edges; PD group and LPS group showed no observable changes; SHS-treated mice showed blunted liver edges (yellow arrow), but little interorgan adhesions. In the PD + LPS + SHS group, there were extensive adhesions between abdominal organs (blue arrow) and thickened liver margins caused by surface fibrosis. C Quantification of adhesion scores across Control group (n = 7), PD group (n = 6), LPS group (n = 7), SHS group (n = 16), and PD + SHS + LPS group (n = 25). D Dynamic body weight changes in mice over the course of the study. E Kaplan–Meier survival curve showing survival rates in each group. EPS mice obtained a survival rate of 83.3% (25/30) and a success rate of 100% (25/25). F Ultrasonographic comparison highlights the anatomical similarities between the murine EPS model and the human condition. a–d Ultrasound imaging of control mice revealed a smooth parietal peritoneum and normal gastrointestinal motility (a). In contrast, the EPS mice showed peritoneal thickening and calcification (b, arrows), adhesions between the parietal peritoneum and intestinal loops (c, arrows), and a characteristic “cauliflower-like” central clumping of small bowel loops (d). Supplementary Video 1 provides dynamic ultrasonography of the abdominal cavity in both control and EPS mice. e–h In human patients, control subjects demonstrated a smooth peritoneal surface (e), while EPS patients exhibited peritoneal thickening (f, arrows), calcification (g, arrows), and significant adhesions between the parietal peritoneum and intestinal loops (h, arrows). ***p < 0.001 by Student’s t test or ANOVA test. Fig. 1A, and the ultrasound, human, mouse drawing elements in Fig. 1F were created in BioRender. Sun, J. (2025) https://BioRender.com/vbajaol.

Fig. 2. EPS mouse model exhibits fibrosis, inflammation, and increased vascular density in peritoneum, consisting with the pathological characteristics in human.

A Cross-sectional images of the abdominal cavity in EPS mice, showing widespread, diffuse adhesions forming clot-like structures throughout the peritoneal cavity. Hematoxylin and eosin (HE) and Masson’s trichrome staining of the parietal (B) and visceral (C) peritoneum, demonstrating significant peritoneal thickening and fibrotic deposition in EPS mice. D Immunohistochemical analysis of the parietal peritoneum showing increased expression of extracellular matrix (ECM) markers, including COL1A1, fibronectin (FN), and α-smooth muscle actin (α-SMA), indicative of active fibrosis. E Immunohistochemical analysis of inflammatory markers IL-1β, IL-6, and TNF-α in the parietal peritoneum, showing significant upregulation of these cytokines in EPS mice, highlighting the inflammatory component of the disease. F Immunofluorescent staining of CD31 in the visceral peritoneum (omentum) showing denser angiogenesis in EPS mice compared to controls. Quantification of peritoneal thickness in the parietal (G) and visceral (H) peritoneum, revealing significant thickening in EPS mice (n = 6 per group). I ELISA analysis of peritoneal lavage fluid, confirming an increase in IL-6 levels in EPS mice compared to controls (n = 6 per group). Data are presented as mean ± SEM.*** p < 0.001, two-tailed Student’s t-test.

Fig. 3. Histological features and dynamic formation of intraperitoneal adhesions in EPS mice.

A Representative images of H&E and Masson’s trichrome staining showing the dynamic pathological progression of adhesion formation in EPS mice. Mice were sacrificed at days 1, 7, 14, and 21 to capture different stages of adhesion development (AW: abdominal wall; BW: bowel wall). B Immunohistochemistry staining of adhesion cross-sections showing the distribution of key inflammatory cell types involved in adhesion formation. CD3 was used to identify lymphocytes, Ly6G for neutrophils, and F4/80 for macrophages. C Quantitative analysis of immune cell infiltrations in Fig. 3B (n = 6 per group). D A graphical diagram depicting the dynamic formation of interorgan adhesions, illustrating the transition from early inflammatory cell infiltration to fibrous scar tissue formation over time. Data are presented as mean ± SEM. *** p < 0.001, two-tailed Student’s t-test.

Fig. 4. Transcriptomic analysis reveals increased fibrosis and inflammatory infiltration in the peritoneum of EPS mice.

A Heatmap of RNA-seq data comparing gene expression in the visceral peritoneum of Control (n = 3) and EPS (n = 3) mice. B Gene Ontology (GO) biological process enrichment analysis of differentially expressed genes (DEGs) significantly upregulated in EPS mice compared to controls. C GSEA) showing enhanced myeloid leukocyte activation and regulation of leukocyte adhesion in EPS mice. D UMAP plot illustrating cell clusters from two publicly available single-cell RNA sequencing (scRNA-seq) datasets of mouse omentum. Mes: mesothelial cells; Endo: Endothelial cells; Fib: Fibroblasts; Neutro: Neutrophils; Macro: Macrophages; T: T cells. E Venn diagram showing the overlap between cell type-specific marker genes from scRNA-seq datasets and DEGs identified in our bulk RNA-seq data. F Deconvolution analysis using scRNA-seq datasets to compare cellular proportions in the Control and EPS groups, highlighting an increased proportion of fibroblasts among parenchymal cells and macrophages among immune cells in EPS mice (n = 3 per group). G Flow cytometry analysis of visceral peritoneum from intestine, confirming a significant increase in immune cell infiltration, particularly macrophages, in EPS mice compared to controls (n = 5 per group). H Correlation analysis between COL1A1 expression and the infiltration of various inflammatory cell types, showing a strong positive correlation between macrophage infiltration and extracellular matrix (ECM) progression. I Correlation analysis between the macrophages proportion detected by flow cytometry with COL1A1 expression in the peritoneum by immunohistochemistry and peritoneal thickness. Data are presented as mean ± SEM. * p < 0.05, *** p < 0.001, two-tailed Student’s t-test.

Fig. 5. Activation of the cGAS-STING pathway in mesothelial cells regulates macrophage chemotaxis.

A KEGG analysis showing significant enrichment of upregulated genes in cell chemotaxis and cytoplasmic DNA-sensing pathways in EPS mice. GSVA scores demonstrating a positive correlation between the cytoplasmic DNA-sensing pathway and cytokine chemotaxis (B) and inflammatory response pathways (C). D ELISA results showing elevated CCL2 levels in the peritoneal lavage fluid of EPS mice (n = 6 per group). E Immunofluorescence indicating increased CCL2 expression in the EPS parietal peritoneum, primarily co-localized with Cytokeratin 7+ mesothelial cells. F Immunofluorescence showing increased STING expression in the EPS parietal peritoneum, also co-localized with Cytokeratin 7+ mesothelial cells. G Western blot confirming the activation of cGAS-STING and downstream proteins in the EPS peritoneum (n = 6 per group). H Cytoplasmic DNA leakage observed in mesothelial cells stimulated with LPS + SHS. I, J Western blot and immunofluorescence showing STING activation and downstream signaling in mesothelial cells under EPS stimulation, with H151 reducing this activation (n = 4 independent experiments). K qPCR showing upregulation of inflammatory-related genes in mesothelial cells under EPS stimulation, with or without H151 pretreatment (n = 3 independent experiments). L qPCR demonstrating increased CCL2 gene expression in mesothelial cells under EPS stimulation, with H151 reducing the effect (n = 3 independent experiments). M Immunofluorescent staining of CCL2 in EPS-stimulated mesothelial cells. N ELISA showing elevated CCL2 levels in mesothelial cell supernatant under EPS stimulation, with H151 reducing CCL2 secretion (n = 3 independent experiments). O Schematic of the trans-well assay to assess macrophage migration. Mesothelial cells were seeded in the lower chamber, stimulated with EPS, and co-cultured with macrophages from the upper chamber (Created in BioRender. Sun, J. (2025) https://BioRender.com/vbajaol). P Bright-field image of macrophage migration after co-culturing with EPS-stimulated mesothelial cells. Q Quantification of macrophage migration under different conditions, with H151 alleviating macrophage migration (n = 3 independent experiments). R, S Representative image and quantification of macrophage migration under anti-CCL2 treatment, (n = 3 independent experiments). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed Student’s t-test.

Fig. 6. Inhibition of the cGAS-STING activation effectively ameliorated abdominal adhesion in EPS.

A The schematic diagram of administering the STING inhibitor H151 (Created in BioRender. Sun, J. (2025) https://BioRender.com/vbajaol). Western blot (B) and immunofluorescence (C) demonstrating that H151 effectively reduced the activation of STING and its downstream proteins in peritoneal tissues (n = 6 per group). D ELISA exhibited that H151 partially lowered the CCL2 concentration in peritoneal lavage fluids (n = 6 per group). E–F Immunohistochemistry (IHC) showed H151 effectively reduced macrophage infiltration (n = 6 per group). G Correlation analysis between the CCL2 concentration in peritoneal lavage fluid and the F4/80 infiltration from (F) among the 3 groups. Gross macroscopic viewing of abdomen (H) and the macroscopic adhesion score (I) demonstrated from a macro perspective that H151 effectively alleviated abdominal adhesion (n = 6 per group). J–L Histopathological assessment of intra-abdominal adhesions and fibrous deposition on peritoneal surface. a MASSON staining of the abdominal cross-sections presented the adhesion condition in different groups, and the MASSON⁺ area (b) was calculated and the statistic diagram is shown in Figure K (n = 6 per group). c Masson’s trichrome staining illustrates the thickness of the parietal peritoneum among the three groups, with (L) showing the corresponding statistical bar graph (n = 6 per group). d–f Immunohistochemical images presenting the expression of three classic ECM-related proteins in parietal peritoneum: COL1A1, Fibronectin, and α-SMA. M Immunohistochemical analysis of inflammatory markers IL-1β, IL-6, and TNF-α in the parietal peritoneum. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t-test.

Fig. 7. IRF3 as the key transcription factor promoting CCL2 secretion in mesothelial cells.

A Bar plot showing the activity scores of differentially expressed transcription factors in Saline and EPS group, based on bulk RNA-seq data. B Volcano plot displaying target genes of IRF3 that are differentially expressed between Saline and EPS group. Red dots indicate genes activated by IRF3, while blue dots represent genes inhibited by IRF3. C Predicted IRF3 binding motifs identified using the JASPAR database. D Schematic illustration showing the IRF3 binding motif within the promoter region of the CCL2 gene. E–F ChIP assays and ChIP-qPCR analysis of IRF3 binding to CCL2 in mesothelial cells treated with or without EPS (SHS + LPS) and H151 (n = 3 independent experiments). Gene silencing of IRF3 significantly reduced both the transcriptional expression (G) and secretion (H) of CCL2 induced by SHS + LPS. I–J Trans-well experiments demonstrating IRF3 silencing suppressed EPS-induced macrophage migration. Data are presented as mean ± SEM. ***p < 0.001, two-tailed Student’s t-test.

Fig. 8. STING and CCL2 are upregulated in parietal peritoneum of EPS patients.

HE staining and immunofluorescence of peritoneal tissue from clinical patients showed the increased expression of STING (A) and CCL2 (B) in EPS patients compared to healthy control (n = 6 per group). C–H Immunofluorescence of peritoneal dialysis effluent smear showed the increased expression of CCL2 (C), STING (D), and p-IRF3 (E) in EPS patients compared to short-term PD patients (SPD) (n = 6 per group). With the corresponding quantitative statistics of fluorescence co-localization showing in F, G and H. I–J ELISA measurements revealed a significant increased CCL2 concentrations (E) and cGAMP concentrations (F) in the peritoneal dialysis effluent in EPS patients compared to control (n = 6 per group). K Correlation analysis presented a significant positive correlation between cGAMP and CCL2 (n = 12). Data are presented as mean ± SEM. *p < 0.05, ***p < 0.001, two-tailed Student’s t-test.