The cGAS-STING pathway is a critical component of the innate immune system responsible for the detection of cytosolic DNA, which is key in identifying viral or bacterial infection. Activation results in the transcription of type I interferons (IFN), including IFN-β, and other proinflammatory cytokines that lead to the recruitment of immune cells to help control the infection. To explore this pathway, we use a system of conditionally immortalized macrophages (CIMs) derived from Cas9-transgenic i-TOMCAT mice. The Cas9 transgene allows us to make gene-specific knockout (KO) CIMs using CRISPR, while the i-TOMCAT allele, which drives expression of a fluorescent reporter (TdTomato) from the endogenous Ifnb1 locus, allows us to quantify type I interferon expression using flow cytometry. Using the STING agonist DMXAA, I found that CIMs in the progenitor state undergo rapid cell death upon STING activation, while terminally differentiated CIMs were resistant to cell death and produced high levels of IFN-β. Further experiments using the pan-caspase inhibitor Z-VAD-FMK and the necroptosis inhibitor necrostatin-1 showed that cell death in progenitor CIMs following STING activation is independent of apoptosis and necroptosis. This project aims to explore the signaling pathways downstream of STING that dictate either a cell death of IFN-β response. While considerable progress has been made in understanding how cGAS-STING signaling leads to IFN-β secretion, the cell death pathways associated with this cascade remain enigmatic. With recent studies showing that cGAS-STING activation can lead to tumor suppression by preventing cell division, promoting immunosurveillance, and triggering the release of tumor-specific antigens, research into this pathway may help guide the development of improved cancer immunotherapies.

The Type 2 inflammatory response occurs during intestinal parasitic helminth infection that affects over a billion people worldwide. Type 2 inflammation involves the activation of intestinal epithelial stem cells (ISCs) in the remodeling of the intestinal epithelium. ISCs differentiate into effector-like cell lineages such as goblet cells and tuft cells that lead to the production of mucus that flushes the helminth out of the intestines. However, the mechanisms that promote and regulate this epithelial remodeling and its long-term consequences remain unclear. For this project, I am analyzing the role of the bioactive lipid prostaglandin D₂ (PGD₂) in the regulation of goblet and tuft cell differentiation by creating enteroids that carry fluorescent reporters that mark ISCs. Enteroids are 3-D cell cultures formed from stem cells that model the intestinal epithelium without the presence of immune cells or other mediators; thus they allow me to hone in on the epithelial intrinsic factors that impact the immunological response in vivo. I hypothesize that PGD₂, and its receptor CRTH2, is necessary for the resolution of the Type 2 epithelial response during helminth infection. By treating enteroids with PGD₂, a PDG₂ antagonist, and inflammatory type 2 interleukins (IL) such as IL-4 and IL-13, I can analyze the role of these elements in the initiation and termination of the Type 2 epithelial response. This research is impactful because understanding the basic principles of intestinal epithelial regulation will be key to furthering the field of therapeutics and treatments for intestinal inflammatory diseases.

The adaptor protein MAVS plays an essential role in the immune system's antiviral defenses. Upon sensing viral RNA in the cytosol, RIG-I-like receptors trigger MAVS to form a signalosome with other proteins and induce interferon (IFN) expression. Recent datasets have identified three regions in the MAVS protein that might bind to RNA. However, the functions underlying these RNA-binding regions (RBRs 1, 2, 3) are still not understood. In this study, we aim to identify the differential functions of these RBRs on regulating MAVS interactions. I tested the ability of FLAG-tagged MAVS constructs with different RBR deletions to induce IFNB1 when overexpressed in MAVS knockout (KO) 293T cells. Different combinatorial deletions of these RBR regions lead to differential levels of IFN induction; deletion of RBR2 abolishes IFN expression, while constructs with additional deletions of RBRs 1 or 3 slightly restores IFN expression. We then investigated where the RBRs perform their regulatory role in the MAVS signaling pathway. We hypothesized that RBRs may be involved in MAVS-TRAF binding or MAVS ubiquitination, both of which would induce differing levels of IFN expression if disrupted. I used immunoprecipitation to find that the RBR2-deleted construct abrogated MAVS-TRAF binding, while any constructs with RBR1 deleted showed increased MAVS-TRAF binding, consistent with their restoration of IFNB1 expression. I will also use immunoprecipitation to measure the influence of RBRs on HA-tagged degradative K48 or activating ubiquitin K63. If the constructs interact with these ubiquitin mechanisms, we expect HA pulldowns from RBR1 deletions to show increased MAVS-K63 binding and/or the RBR2-deletion to increase MAVS-K48 binding. This study provides analysis of key regulatory regions that control the downstream IFN production and antiviral defense through MAVS, which could expose therapeutic targets for either treating viral infection or reducing the effects of abnormal IFN production such as in autoimmune disorders.

Retinoic acid-inducible gene (RIG-I) receptors (RLRs) are pathogen recognition receptors (PRRs) that play a major role in innate immune defense against RNA virus infection. RLR RIG-I and melanoma differentiation-associated protein 5 (MDA5) interact with viral RNA ligands in the cytoplasm via their N-terminal caspase recruitment domains (CARDS) to modulate antiviral immunity during an infection. Activated RLRs then signal through mitochondrial antiviral-signaling protein (MAVS) to stimulate the production of type 1 interferon (IFN). IFN upregulates transcription of interferon stimulated genes (ISGs) which encode proteins implicated in inducing an antiviral state to eliminate invading viruses.
Herein, we continue the evaluation of RLRs and their subsequent signaling partners in response to viral infection. To this aim, we utilize West Nile Virus (WNV), a neurotropic virus belonging to the Flaviviridae RNA virus family and commonly used to model positive sense RNA viruses and their role in RLR induced immunity. Two strains of WNV, WNV Madagascar (WNV MAD) which is attenuated in mice and WNV Texas (WNV TX) which is virulent in mice are used. The results demonstrated that the RLRs, specifically RIG-I, are significant contributors to the difference in virulence of pathogenic WNV TX and the non-pathogenic WNV MAD strains. Replication kinetics in knockout immunocompetent cell lines demonstrate that primarily RIG-I signaling through MAVS, not other RLRs (MDA5), restrict viral replication of WNV MAD but WNV TX in cell culture models. We also show the role of RIG-I and its restriction of WNV MAD intracellular viral protein accumulation. The specific mechanism of the restriction of WNV by RLR signaling is further elucidated, specifically the role of IFN in our cell culture model. Further studies will allow for the development of small molecule inhibitors or treatments that may aid in the prevention of severe disease caused by positive sense viral pathogens.