Measuring Activation of the Cytosolic DNA Sensing Pathway

In mammalian cells, DNA is normally sequestered within the confines of the nucleus or mitochondria. Entrance of DNA into the cytosol, whether foreign or self in origin, acts as a danger signal that triggers a host innate immune response. Cytosolically localized DNA is sensed by cyclic GMP-AMP synthase (cGAS), which synthesizes a novel second messenger known as cyclic GMP-AMP (2'3'-cGAMP). 2'3'-cGAMP, in turn, binds to and activates the ER resident adaptor Stimulator of Interferon Genes (STING), which triggers downstream signaling that culminates in the production of type-I interferons and other immune modulatory molecules. The pathway underlies the recognition of pathogenic DNA necessary to quell microbial infections, as well as the aberrant detection of self-DNA responsible for inducing certain autoimmune diseases. Such appreciation for the involvement of cGAS-cGAMP-STING signaling in numerous clinical phenotypes necessitates development of tools that can outline the extent of its contribution to various diseases. Additionally, numerous questions remain regarding the regulation of cGAS-cGAMP signaling. As 2'3'-cGAMP production is a hallmark of the pathway's activation, we sought to develop a robust method to monitor its formation in vivo, and quantify its levels in a wide variety of settings. Herein we present the development of an antibody of high sensitivity and specificity for this small molecule second messenger, capable of recognizing and quantifying 2'3'-cGAMP production in vivo. We show it can be adapted for use in a variety of techniques, to track and measure levels of 2'3'-cGAMP quantitatively, to visualize 2'3'-cGAMP produced in cells, and to quickly identify cGAMP-positive cell populations within live samples. We show this antibody to be an invaluable tool to elucidate outstanding questions in the field, and demonstrate its potential to detect patients with aberrant activation of the cGAS-STING pathway. We foresee a future in which the 2'3'-cGAMP antibody is used to quantify activation of the cGAS pathway in a variety of clinical and research settings.