Stewart, JamesALAidarous, Sondus2024-10-072024-07https://hdl.handle.net/20.500.14154/73168ThesisCD95L can induce a pathological nonapoptotic pathway and promote Th17 migration when cleaved from the cell surface by proteases. The targeted inhibition of this nonapoptotic pathway by blocking the calcium-inducing domain to impede IL-17 migration could be of therapeutic benefit in inflammation, as seen in a murine lupus model. We aimed to investigate the CD95L nonapoptotic pathway and factors involved in the cleavage of CD95L in the context of SARS-CoV-2 infection and pulmonary fibrosis. Three approaches were used: in vitro studies, in vivo murine models and human clinical sample-based studies. In vitro, the Jurkat cell line was stimulated with PHA, LPS, recombinant MMP9 and mouse sera to optimise conditions for the expression and cleavage of CD95L. MMP9 was investigated as a putative cleavage factor of CD95L and was found at higher levels in naïve lung supernatant than in serum. In a SARS-CoV-2 murine model, the effect of inhibitory peptide treatment on mice intranasally infected with 104 PFU SARS-CoV-2 was assessed. IL-17 serum levels in the peptide-treated group were significantly lower than in the control group, yet this difference did not translate to disease progression, measured by viral load, weight loss, histopathology and survival. IL-17 and sCD95L levels were tested in SARS-CoV-2 patient samples from a biobank cohort and healthy control samples. There was no difference between the groups, and sCD95L was undetectable in the SARS-CoV-2 patient group. Our hypothesis on pulmonary fibrosis was tested through a model of bleomycin-induced fibrosis in BALB/c mice across 36 days. Animals were challenged with 4x105 of murine gammaherpesvirus (MHV) intranasally to aid fibrosis development on day 0. Bleomycin was administered twice at 40 mg/kg on day 8 and day 17. Inhibitory peptide treatment, control peptide treatment or mock treatment was given the day after bleomycin administration. Post-mortem, cardiac bleeds and lung and liver tissue were collected. IL-17, MMP9 and sCD95L were measured by ELISA. Right lungs were processed using Trizol for qPCR, and left lungs, along with livers, were prepared for histopathology. No difference was found between groups in terms of IL-17, MMP9 or sCD95L serum levels, gene expression of il-17 or ccr6, fibrosis scores based on Masson’s trichrome staining, and weight loss or survival rate. However, a significant correlation was seen between weight loss and the fibrosis score. In human pulmonary fibrosis serum samples, IL-17 levels were barely detectable and did not differ from those in healthy controls. Interestingly, sCD95L levels were significantly higher and MMP9 levels were significantly lower than in healthy controls. In summary, blocking the calcium-inducing domain could reduce IL-17 levels but did not affect disease progression in a SARS-CoV-2 model. Moreover, these findings could not be translated to other respiratory diseases, like pulmonary fibrosis.178enRespiratoryCritical careImmunologymurine modelApoptosisPathwaysSARS-CoV-2COVIDPulPulmonary FibrosisThesis