M. Kobritz1,2, M. Sfakianos1, G. Coppa1, M. Aziz1,2, P. Wang1,2 1Zucker School of Medicine at Hofstra/Northwell, Department Of Surgery, Manhasset, NY, USA 2The Feinstein Institutes for Medical Research, Center For Immunology And Inflammation, Manhasset, NY, USA
Introduction: Sepsis is a syndrome of dysregulated host response to infection leading to life-threatening organ dysfunction. Sepsis-induced intestinal dysfunction has been proposed as a key element in the progression to multisystem organ failure. Components of gut integrity that are dysregulated during sepsis include epithelial cell apoptosis driven by inflammatory mediators, increased permeability due to alterations in junctional proteins including junctional adhesion molecule A (JAM-A), and changes in the gut microbiome. Stimulator of interferon genes (STING) is an intracellular protein involved in the innate immune response, which is implicated in intestinal dysfunction in sepsis and inflammatory bowel disease. H151 is a novel small molecule inhibitor of STING, which shows promise as a therapeutic in animal models of inflammation and injury. However, H151’s effects on intestinal integrity are unknown. We hypothesize that H151 therapeutically reduces sepsis-induced acute intestinal dysfunction.
Methods: Murine intestinal epithelial cell line IEC6 cells were pre-treated with PBS or H151 (1.0, 2.0 uM). One hour after H151 treatment, cells were stimulated with lipopolysaccharide (LPS, 1 ug/mL). Four hours after LPS stimulation, cells and cell culture supernatant were collected. TNFa levels in the cell culture supernatant were assessed using ELISA. JAM-A levels in cell lysates were assessed by Western blot. Male, 8-week-old C57BL/6 mice underwent cecal ligation and puncture (CLP) to induce sepsis and were treated with H151 (10 mg/kg BW) or vehicle (10% Tween-80 in PBS) intraperitoneally. Twenty hours after CLP, mice underwent oral gavage with FITC-dextran (22 mg/mL) in sterile PBS. Five hours after gavage, serum was collected, and intestinal permeability was assessed using fluorimetry.
Results: TNFa in the cell culture supernatant increased 194-fold after stimulation with LPS as compared to controls (p<0.05). A dose-dependent decrease in TNFa was observed when cells were pre-treated with H151. In the cell culture supernatant of cells pre-treated with 1.0 and 2.0 uM H151, there was an 8.5% and 25.7% reduction in TNFa, respectively (p<0.05). Expression of JAM-A decreased by 47.8% in intestinal epithelial cells stimulated with LPS compared to controls, and JAM-A expression was recovered in cells pre-treated with H151. In vivo, relative fluorescence of FITC-dextran in the serum of mice subjected to CLP was increased 3.2-fold when compared to healthy controls, and a 41.1% decrease in fluorescence was observed in H151-treated mice when compared to CLP-vehicle.
Conclusion: Pre-treatment with H151 reduces inflammation and improves junctional adhesion protein dysregulation after LPS stimulation in vitro. Intestinal permeability is increased after CLP. H151, a novel STING inhibitor, reduces intestinal permeability when administered as a treatment at the time of CLP. Thus, targeting STING may ameliorate sepsis-induced acute intestinal dysfunction.