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Objective Toll-like receptors (TLRs) 7 and 9 are important innate signaling molecules with opposing roles in the development and progression of systemic lupus erythematosus (SLE). While multiple studies support the notion of a dependency on TLR-7 for disease development, genetic ablation of TLR-9 results in severe disease with glomerulonephritis (GN) by a largely unknown mechanism. This study was undertaken to examine the suppressive role of TLR-9 in the development of severe lupus in a mouse model. Methods We crossed Sle1 lupus-prone mice with TLR-9-deficient mice to generate Sle1TLR-9-/- mice. Mice ages 4.5-6.5 months were evaluated for severe autoimmunity by assessing splenomegaly, GN, immune cell populations, autoantibody and total Ig profiles, kidney dendritic cell (DC) function, and TLR-7 protein expression. Mice ages 8-10 weeks were used for functional B cell studies, Ig profiling, and determination of TLR-7 expression. Results Sle1TLR-9-/- mice developed severe disease similar to TLR-9-deficient MRL and Nba2 models. Sle1TLR-9-/- mouse B cells produced more class-switched antibodies, and the autoantibody repertoire was skewed toward RNA-containing antigens. GN in these mice was associated with DC infiltration, and purified Sle1TLR-9-/- mouse renal DCs were more efficient at TLR-7-dependent antigen presentation and expressed higher levels of TLR-7 protein. Importantly, this increase in TLR-7 expression occurred prior to disease development, indicating a role in the initiation stages of tissue destruction. Conclusion The increase in TLR-7-reactive immune complexes, and the concomitant enhanced expression of their receptor, promotes inflammation and disease in Sle1TLR9-/- mice.

The presence of damaged and microbial DNA can pose a threat to the survival of organisms. Cells express various sensors that recognize specific aspects of such potentially dangerous DNA. Recognition of damaged or microbial DNA by sensors induces cellular processes that are important for DNA repair and inflammation. Here, we review recent evidence that the cellular response to DNA damage and microbial DNA are tightly intertwined. We also discuss insights into the parameters that enable DNA sensors to distinguish damaged and microbial DNA from DNA present in healthy cells.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that catalyses the synthesis of cGAMP, which serves as a ligand for stimulator of interferon (IFN) genes (STING). Activation of STING results in production of type I IFNs through phosphorylation of TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3). Type I IFNs are critical participants in the innate and adaptive immune recognition of cancer cells. Deficiencies in the cGAS-STING signalling pathway have been reported in many tumors. This mitigates expression of type I IFNs and may thus contribute to non-inflamed tumor microenvironment. In particular, a non-T-cell-inflamed tumor microenvironment correlates with poor patient survival. STING agonists may contribute to antitumor activity by upregulating proinflammatory cytokines and type I IFNs and various STING agonists are now being tested in clinical trials. The role of STING in immune cells is relatively well understood; however, its role in tumor cells has not yet been described in detail. Here we show that cGAS is able to bind to DNA present in the cytosol of tumor cells and subsequently induces STING signaling leading to expression of type I IFNs. Knockout of STING in mouse prostate TRAMP-C2 tumor cells resulted in higher tumor burden and reduced infiltration of immune cells such as dendritic and CD8+ T-cells into the tumor microenvironment. Consistently, treatment with the STING agonist cGAMP elevated type I IFNs levels in TRAMP-C2 cells and led to a reduced tumor volume compared to untreated control. This suggests a pivotal role of tumoral STING in antitumor immunity. However, despite intact STING expression, most tested human cancer cell lines were not responsive to various STING agonists and consequently failed to upregulate expression of type I IFNs. In contrast, all tested tumor cell lines responded to Poly(I:C)-induced TLR3 signaling, suggesting that failure to respond to cGAMP was due to a defect upstream of TBK1. Downregulation of cGAS did not render cells responsive to cGAMP, indicating that inability to respond to cGAMP is due to deficiencies of STING to activate TBK1. In the human and mouse prostate cancer cell lines DU145 and TRAMP-C2, respectively, autocrine IL-6 rendered cells unresponsive to STING agonists. While treatment with anti-IL-6 antibodies restored cGAMP responsiveness in DU145 cells, addition of recombinant IL-6 suppressed cGAMP-mediated upregulation of type I IFNs. In summary, our data suggest that cytosolic DNA activates the cGAS-STING signaling pathway. A functional STING is pivotal for eliciting an effective anticancer immune response. In most human cancer cell lines, however, STING signalling is inhibited. Since STING agonists are being evaluated in clinical trials, it is crucial to understand mechanisms that mediate STING unresponsiveness. We show that in tested prostate cancer cells, IL-6 signals contribute to unresponsiveness of STING and blocking of IL-6 can restore responsiveness towards STING agonists. Citation Format: Manuel Adrian Suter, Wendy Ya Ling Zhang, Muznah Bte Nazar Khan Khatoo, Nikki Ya Ling Tan, Chien Tei Too, Shubhita Tripathi, Paul A. MacAry, Veronique Angeli, Stephan Gasser. Tumoral STING is required for effective anticancer immunity [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B189.

Self-DNA is present in the cytosol of many cancer cells and can promote effective immune rejection of tumor cells, but the mechanisms leading to the presence of cytosolic DNA are unknown. Here, we report that the cleavage of genomic DNA by DNA structure-specific endonuclease MUS81 and PARP-dependent DNA repair pathways leads to the accumulation of cytosolic DNA in prostate cancer cells. The number of nuclear MUS81 foci and the amount of cytosolic dsDNA increased in tandem from hyperplasia to clinical stage II prostate cancers and decreased at stage III. Cytosolic DNA generated by MUS81 stimulated DNA sensor STING-dependent type I interferon (IFN) expression and promoted phagocytic and T cell responses, resulting in type I and II IFN-mediated rejection of prostate tumor cells via mechanisms that partly depended on macrophages. Our results demonstrate that the tumor suppressor MUS81 alerts the immune system to the presence of transformed host cells.