FC089: The Absence of Sting Prevents Peritoneal Damage in a Murine Model of Peritoneal Fibrosis
Authors: Marchant Vanessa , García-Giménez Jorge , González Guadalupe , Rubio-Soto Irene , Kopytina Valeria , Tejera Antonio , Tejedor Lucia , Marquez-Exposito Laura , López-Cabrera Manuel , Mario Ramos Cortassa Adrián , Ruiz-Ortega Marta 
Published: May 2022

Abstract

BACKGROUND AND AIMS
Peritoneal dialysis (PD) is the current replacement therapy for end-stage renal disease (ESRD) patients until renal transplantation can be achieved. However, chronic exposure to non-physiological PD fluids (PDF) and occasional infection episodes may lead to peritoneal damage and membrane transport failure. Chronic inflammation, together with mesothelial-to-mesenchymal transition (MMT) and submesothelial fibrosis, is a hallmark of PDF-associated peritoneal damage whose intrinsic mechanisms are still expected to be disclosed [1–2]. Activation of the innate immunity is the cornerstone for infectious agents’ elimination, also enrolled in response to damage-associated molecular patterns (DAMPs) released from stressed or injured host cells. Leakage of DNA from stressed mitochondria and damaged genomic DNA may act as DAMPs that are recognized by cytosolic double-strand DNA sensors, like the stimulator of interferon gene (STING), to mount an inflammatory response [3]. Excessive activation of STING is involved in autoinflammatory, autoimmune, and increasingly related to several chronic inflammatory conditions [4]. Thus, this study aimed to investigate the role of STING in experimental peritoneal damage.

METHOD
We induced peritoneal fibrosis in wild-type and STING-KO mice by intraperitoneal administration of 0.1% chlorhexidine gluconate (CHX) daily for 4 weeks. No treated WT and STING-KO mice were used as control. Then, mice were euthanized, and peritoneal tissue and peritoneal lavage fluids were recollected for the following experiments. STING mRNA (Tmem173) and protein levels in control and CHX-treated wild-type were measured by RT-qPCR, western blot and immunohistochemistry. Peritoneal levels of the STING downstream proteins TBK1 and IRF3 and their phosphorylated isoforms were assayed by western blot. Peritoneal membrane thickness was measured in formalin-fixed paraffin-embedded tissues stained with Masson's trichrome stain. Fibronectin protein levels were evaluated by western blot and MMT, fibrosis, and inflammation markers were determined by RT-qPCR. Inflammatory cells present in peritoneal effluents were assayed by flow cytometry.

RESULTS
We found that in the peritoneum of CHX-treated wild-type mice both Tmem173 expression and STING protein levels were increased. Interestingly, STING-positive cells were located in areas of peritoneal thickness and immune infiltration. Moreover, the protein levels of IRF3, p-IRF3, TBK1 and p-TBK1 were increased, indicating that the STING pathway is activated in experimental peritoneal damage. In STING-KO mice, CHX-induced peritoneal damage was prevented. STING-KO mice under CHX treatment showed decreased peritoneal membrane thickness and attenuated CHX-induced expression of fibrosis and MMT markers, such as Tgb1, Snai1, Cdh2, Col1a1, Fn1 and fibronectin protein when compared with CHX-treated WT mice. The absence of STING also prevented the gene expression of cytokines (Il1b, Il6 and Ifng) and chemokines (Ccl5, Ccl2 and Ccl19) and macrophages infiltration into the peritoneum. Inflammatory cell recruitment into the peritoneal cavity was also decreased in STING-KO mice, including CD3+CD4+ lymphocytes and CD11b+F4/80+ macrophages.

CONCLUSION
In summary, STING absence prevents peritoneal membrane thickness, fibrosis, and inflammation induced by CHX in a mice model. These results suggest that STING plays a key role in peritoneal membrane injury. Therefore, STING may become a new potential therapeutic target in PD-associated peritoneal damage.