P0078 The role of epithelial STING in a genetic IBD Model of autophagy deficiency and its interplay with the Integrated stress response

Abstract Background Stimulator of interferon genes (STING) regulates intestinal homeostasis and inflammation by detecting cytoplasmic double-stranded DNA, driving type-1 interferon (IFN) responses.1 The autophagy-related gene ATG16L1, a major risk factor for inflammatory bowel disease (IBD), links to STING overactivation in intestinal epithelial cells (IEC), causing inflammation via IFN1, NF-κB, and TNFα signaling.2 Excessive STING activity is associated with autoinflammatory disorders1, whereas a full-body Sting knockout (Tmem173gt/gt) protects against dextran sodium sulfate (DSS)-induced colitis.3 STING and ATG16L1, key regulators of cellular stress and inflammation, may interact with the integrated stress response (ISR). Activating transcription factor 4 (ATF4), a central ISR mediator, is induced by intestinal inflammation4 and could be affected by STING activation through mitochondrial stress and autophagy dysfunction caused by ATG16L1 deficiency. This study investigates the so far unknown role of STING in IEC and its interplay with autophagy deficiency and ATF4-mediated ISR. Methods Murine IEC (ModeK cells) were treated with DNA fractions, pro-inflammatory stimuli, and STING agonists to analyse responses via WB and RT-qPCR in vitro. In vivo, we used epithelial STING deficient mice (Tmem173dIEC) for clinical and histopathological analysis in DSS-induced colitis and steady-state, the latter also with combined Atg16l1 deficiency. Mice were analyzed at 12 and 52 weeks of age. Protein and RNA analyses of crypt cells were performed via WB and RT-qPCR. Results Unlike Tmem173gt/gt mice, Tmem173dIEC mice were not protected from DSS-induced colitis, suggesting that epithelial STING alone is less critical to immune responses. However, aged untreated Tmem173dIEC mice showed reduced epithelial inflammation and cell death compared to wildtype mice. In Atg16l1-deficient ModeK cells, silencing Sting reduced pro-inflammatory responses to STING agonists and pro-inflammatory stimulants. To assess if STING deficiency ameliorates inflammation in Atg161ldIEC mice, we created Atg161ldIEC/Tmem173dIEC mice, observing reduced relative liver and spleen weights in young animals. We observed increased Atf4 expression in DSS-treated colitis, while Atf4 was absent in Sting-deficient ModeK cells, indicating STING regulates proinflammatory cytokine induction via ATF4. Silencing Atf4 decreased the activation of Sting, supporting a bidirectional link between STING signalling and the ISR, which will be further explored in aged and DSS-treated Tmem173dIEC and Atg161ldIEC/Tmem173dIEC mice. Conclusion We show that STING essentially coordinates pro-inflammatory cytokine induction in response to Atg16l1 deficiency in intestinal epithelial cells via a mechanism involving ATF4. References 1. Wottawa F, Bordoni D, Baran N, Rosenstiel P, Aden K. The role of cGAS/STING in intestinal immunity. Eur J Immunol. 2021;51(4):785-797. doi:10.1002/eji.202048777 2. Aden K, Tran F, Ito G, Sheibani-Tezerji R, Lipinski S, Kuiper JW, et al. ATG16L1 orchestrates interleukin-22 signaling in the intestinal epithelium via cGAS–STING. J Exp Medicine. 2018;215(11):2868-2886. doi:10.1084/jem.20171029 3. Martin GR, Blomquist CM, Henare KL, Jirik FR. Stimulator of interferon genes (STING) activation exacerbates experimental colitis in mice. Sci Rep. 2019;9(1):14281. doi:10.1038/s41598-019-50656-5 4. Pakos-Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman AM. The integrated stress response. Embo Rep. 2016;17(10):1374-1395. doi:10.15252/embr.201642195 4