Delineating the interplay between nucleic-acid-dependent phase separation and immune responses against cGAS in SLE

Project Summary:
This work is proposed in response to NOT-AR-23-006 as a mechanistic study of innate and
adaptive immune responses in systemic lupus erythematosus (SLE). SLE is a systemic rheumatic
disease that disproportionately impacts females and racially and ethnically disenfranchised
groups. Despite advances in treatment, the molecular mechanisms of SLE pathogenesis remain
poorly understood. Cyclic G/AMP (cGAMP) synthetase (cGAS) is a central driver of the cell-
intrinsic interferon (IFN) response which has been linked to SLE pathogenesis. When activated
by cytosolic double-stranded DNA (dsDNA), cGAS generates cGAMP, a key second messenger
for downstream IFN signaling. Resting cGAS is a monomer, and dsDNA-dependent activation
entails dimerization and subsequent higher-order oligomer formation, resulting in the generation
of phase-separated cGAS. In preliminary studies, we have identified the presence of
autoantibodies targeting cGAS in SLE patient sera, which are associated with SLE disease
activity. In addition, we have observed that cGAS can bind to and forms condensates on various
nucleic acids, but dsDNA is the only ligand that permits the dimerization required for enzymatic
activity and formation of hyrdo-gel-like condensates. Building upon these novel observations, we
propose to test the hypothesis that biophysical changes triggered by cGAS activation shape the
adaptive immune response that leads to the targeting of cGAS as an autoantigen in a subset of
SLE patients with active disease. In Aim 1, we will test the theory that dsDNA-dependent gel-like
cGAS condensates are preferentially targeted by SLE autoantibodies. To do this, we will develop
assays incorporating purified cGAS (monomer vs dimer) complexed with various nucleic acid
ligands (dsDNA, dsRNA, ssDNA, ssRNA, DNA-RNA hybrid). In Aim 2, we will determine if phase
separation and/or ligand binding alter cGAS uptake and presentation by antigen presenting cells
(APCs). To accomplish this, we will utilize monocyte-derived dendritic cells (moDCs) exposed to
the same panel of cGAS-ligand complexes used in Aim 1, and will analyze cGAS uptake and
subsequent moDC activation using confocal microscopy and flow cytometry. Then, we will
examine the effect of nucleic acid binding on cGAS peptide presentation in the context of MHC II
using a natural antigen processing assay (NAPA). We anticipate that this work will reveal interplay
between innate and adaptive immune components that drives aberrant immune phenomena
observed in SLE, providing a conceptual and experimental framework that could be readily
applied to the numerous nucleic acid-binding autoantigens featured in SLE.

Grant Number: 1R21AR085266-01
NIH Institute/Center: NIH
Principal Investigator: Brendan Antiochos