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.