| Abstract |
The leukocyte recruitment cascade is a well-orchestrated series of events leading to inflammatory cells crossing the vascular wall and reaching peripheral tissues. In this context, understanding how surface molecules on leukocytes react to signaling queues by the vascular endothelium is of paramount importance. In the present thesis work, I examined the roles of the transmembrane molecule CD43 on T-cells and the intracellular molecule STimulator of INterferon Genes (STING) on endothelial cells in the leukocyte recruitment and inflammatory processes.
Sialomucin CD43 is a transmembrane glycoprotein functioning as an E-Selectin ligand for Th17 cells in vitro that is required for rolling on the vascular endothelium and Th17 Cell recruitment during inflammation in vivo. In this thesis, I investigated how CD43 regulates the Th17 cell steps required for leukocyte recruitment. I focused on how CD43 acts in relation to intercellular adhesion molecule 1 (ICAM-1) due to its critical role to the leukocyte recruitment cascade across the endothelium. ICAM-1 engagement by leukocyte integrins activates several signaling pathways within both leukocytes and endothelium, thus priming the former for transmigration. To better understand the role of CD43 in Th17 cell adhesion to ICAM‐1, I used a combination of flow chamber studies, immunocytochemistry and flow cytometry techniques to assess Th17 interaction with ICAM-1 and expression levels of proteins involved. I found that CD43 promotes Th17 cell ICAM-1-dependent adhesion, apical migration and transendothelial migration. Specifically, I showed that CD43-/- Th17 cells adhesion to ICAM-1 was significantly impaired as compared to WT Th17 cells.
Next, I investigated the roles of STING, a cytosolic double stranded DNA sensor that functions as an adaptor molecule for type I interferon (IFN-I) signaling by activating IFN-I stimulated genes (ISG), in T cell recruitment in response to acute peritonitis, cardiac pressure-overload induced inflammatory responses as well as in the context of vascular injury. I found that T cell infiltration into the peritoneum in response to TNF-α is highly dependent on STING expression on endothelial cells, using global and inducible EC-specific STING-/- mice, a tool I generated during my thesis. T cell transendothelial migration (TEM) across mouse and human EC deficient in STING was strikingly reduced compared to control EC, whereas T cell adhesion was not impaired. I additionally found that T cell STING does not contribute to T cell TEM. This coupled with my results that STING did not affect IFNγ expression is why I focused on the IFN-I-IFNAR axis on EC. Mechanistically, I identified the EC IFN-I-IFNAR axis as a critical signaling cascade in response to TNF-α—induced T cell recruitment and pinpointed CXCL10, an ISG and a chemoattractant for T cells, as a key player in the process. Because Th1 recruitment is a critical step for cardiac inflammation and maladaptive cardiac remodeling in response to pressure overload induced heart failure, I investigated whether STING contributed to this process in heart failure. I showed that STING-/- mice exhibited reduced cardiac fibrosis and left ventricular hypertrophy and that correlated with reduced hallmarks of Heart failure (HF) such as the development of interstitial and perivascular fibrosis and left ventricular hypertrophy. My findings point to a role of STING in cardiac inflammation and open an avenue to further investigate its role in this model.
I also hypothesized that EC STING modulates other aspects of EC responses to stress, such as re-endothelialization post vascular injury, an important process for restoring tissue homeostasis and avoiding persistent inflammation and maladaptive remodeling. I subjected WT, STING-/-, and EC-STING-/- mice to wire induced carotid injury, an experimental model of vascular injury that mimics the EC damage induced by Percutaneous Coronary Interventions (PCI). I found that STING-/- mice exhibited delayed re-endothelialization and that EC-STING-/- mice recapitulated the phenotype of the global STING-/- mice, supporting a role for EC STING in reendothelialization post injury. Mechanistically, I performed scratch assays on WT and STING-/- mouse heart EC monolayers, as well as on human HUVEC monolayers cells lacking STING after CRISPR knock down. Real time imaging indicated that in EC lacking STING, longer healing times were needed post scratch, indicating roles of STING signaling in wound healing.
Altogether, this body of work contributes insights into mechanisms that involve T cell CD43 and EC-STING, which may be modulated in T cells and the vascular endothelium, respectively, to harness T cell-mediated acute and chronic inflammation.
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Investigating mechanisms of T Cell recruitment to sites of inflammation and injury : The novel roles of endothelial cell STING and T Cell CD43
