Young, StephenAlghamdi, Abdullah2023-05-152023-05-152023https://hdl.handle.net/20.500.14154/68063Background: A number of genes including HLA-DRB1 and variants of PTPN22 (which codes for Lyp phosphatase) are associated with the development of rheumatoid arthritis (RA). Environmental factors, particularly cigarette smoking also promote RA and preliminary data suggested that cigarette smoke could impact Lyp phosphatase function in immune cells. Moreover, unpublished data suggested that Lyp may be involved in regulating metabolism in T cells. This project sought to discover then mechanisms by which PTPN22 and cigarette smoke may interact to alter immune cell metabolism and function. Methods: Isolated naïve and memory T cells from human blood cones were treated with cigarette smoke extract CSE or Lyp inhibitor for 24h and then stimulated with anti CD3/CD28 for 72h. Protein expression and phosphorylation were assessed using immunoblotting, changes in energy metabolism were assessed using the Seahorse instrument and cytokines expression was assessed using Luminex assay. All the experiments and the analysis were done after 24h of CSE or Lyp inhibitor treatment and 72h of stimulation. Results: CSE increased Lyp phosphatase activity in naïve, whereas Lyp inhibitor increased Lyp activity in memory only. Both treatments increased the phosphorylation of ZAP-70 in naïve and memory but not Lck. Lyp inhibitor increased Vav1 and Akt phosphorylation in naïve but not in memory, whereas CSE did not show any. CSE increased mTORC1 in naïve but not in memory, whereas CSE increased AMPK only in memory. Both treatments increased calcium (Ca2+) flux made in response to TCR stimulation. Moreover, CSE and Lyp inhibitor increased glycolysis and oxidative phosphorylation of naïve and memory T cells. Generally, CSE increased inflammatory cytokines such as IL6, IL-1β, IFNγ and TNFα, IL8, but not IL10. Conclusions: Overall, these data suggest that CSE may promote altered TCR function through interaction with Lyp. One consequence is an alteration in energy metabolism through changes in signalling pathway and cytokines production. This may explain part of the underlying mechanisms by which cigarette smoke promote RA development.262enRACSEAktLckThesis