Exploring Determinants of Intrinsic DNA Load Towards Inflammaging

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PROJECT SUMMARY Sterile inflammation is exhibited in aging‐related conditions, including heart disease, cancer, diabetes, and cognitive decline. Such hyperinflammation typically displays an altered immune response comprising a type I interferon response and downstream senescence-associated secretory phenotype (SASP). The intrinsic factors that induce this chronic low-grade inflammation are not well understood. In studying the initiation of autoimmune polyarthritis in mice lacking the lysosomal endonuclease Dnase2a, we discovered a cell‐autonomous pathway through which damaged nuclear DNA is trafficked to the cytosol where it is recognized by the innate cytosolic DNA sensing cGAS-STING axis, triggering an inflammatory cascade. In healthy cells, nuclear DNA found in the cytosol is removed via autophagy for lysosomal degradation by DNASE2A, which degrades double-stranded DNA (dsDNA)—ligand for the DNA sensor cGAS. Consistent with this notion that damaged or irreparable nuclear DNA is a trigger of immunity when mis- localized and accumulated in the cytosol, elevated loads of extranuclear DNA in replicative senescent cells and cells from ataxia (AT) and progeria (HGPS) patients engage the same DNA sensing pathway leading to persistent inflammation. Controlling the intrinsic DNA load is thus critical to avoid sterile inflammation. While nucleases can clear DNA, unknown DNA export factors may facilitate DNA exit to the cytosol. By targeting curated sets of potential nucleases and DNA export factors, we propose to identify unknown intrinsic DNA controlling factors in a single-cell based CRISPR knockout optical screen by assaying dsDNA content using immunofluorescence. Hits will be validated for their DNA export or degradative function and ranked for their involvement in senescence and STING activation downstream of cGAS. Selected tops hits, along with DNASE2A, will be evaluated for their capacity in suppressing inflammation and SASP in senescent cells and AT and HGPS patient cells. In Dnase2a knockout mice, we will investigate immune cell phenotype in tissues to understand the mechanisms underlying the systemic senescent pathology. The identification and characterization of new determinants in inflammation and senescence will add new insights to the basic biology of DNA-mediated immunity and open up novel strategies in modulating inflammation to benefit aging-associated pathologies, including interferonopathy and laminopathy, autoimmunity and cancer.