Investigating the role of endothelial and fibroblastic reticular cells in lung inflammation caused by STING gain-of-function autoinflammation

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a cytosolic double- stranded DNA (dsDNA) sensing pathway critical for regulating immune homeostasis. A series of gain-of-function (GOF) mutations result in constitutive activation of STING, causing an autoinflammatory disease called STING- Associated Vasculopathy with Onset in Infancy (SAVI). SAVI patients succumb to treatment resistant inflammatory lung disease and respiratory failure. There is little known about the mechanisms by which inflammation occurs. To address the urgent need to develop safe and effective therapies, we have developed a murine model for the most common STING gain-of-function mutation, STINGV154M/WT (VM). These mice recapitulate the lung inflammation exhibited by human SAVI patients. To identify the specific cell types involved in causing lung inflammation, we developed a novel VM conditional knock-in (CKI), allowing specific targeting of the VM mutation to different cell types. We demonstrated that endothelial cell (EC) STING GOF is sufficient in driving bronchus-associated lymphoid tissue (BALT) formation. However, the mechanism of action remains to be elucidated. Moreover, we have previously described SAVI lung disease as independent of type I interferon (IFN) and IRF3, signaling proteins downstream of STING activation. STING activation leads to downstream signaling of other pathways including NF-κB and autophagy. The signaling mechanism causing lung inflammation is also unknown. Additionally, STING GOF in ECs was insufficient to cause the extent of lung inflammation seen in VM mice, suggesting STING GOF in cells other than ECs is required for lung disease. Upon ubiquitous VM expression, we find evidence of fibroblast activation in the lung tissue. Fibroblastic reticular cells (FRCs) are a subset of fibroblasts that define the function and structure of lymphoid organs such as BALT. In addition to ECs, STING is highly expressed in FRCs, yet the role of STING in FRCs and contributions to lung disease is unknown. Thus, we hypothesize that coordinated interactions between ECs and FRCs exacerbate SAVI lung autoinflammation, which is dependent on NF-κB activation. In this proposal, Aim 1 will investigate how STING GOF mutation in ECs initiates immune cell recruitment. Aim 2 will determine the synergistic effects of STING GOF mutation in ECs and FRCs on lung autoinflammation. We propose to utilize in vivo, ex vivo, and in vitro techniques to test our hypothesis. The studies proposed in this application will provide critical insights that will enable us to design the best therapies. Furthermore, these studies will provide an opportunity to study the impact of STING activation on stromal cell types, an area of research that requires further exploration. Our findings will discern the role of ECs and FRCs in VM lung autoinflammation and will broadly provide insight into stromal cell-driven mechanisms of other lung disorders.