Tese de Doutoramento Envelhecimento e Doenças Crónicas 2023 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

Abstract Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE) are autoimmune diseases characterized by chronic inflammation and tissue damage resulting in severe functional limitations, and disability and therefore result in a burden on the health care system. Current therapies can delay disease progression and improve the patient's quality of life. However, some patients remain non responsive to these treatments, which also can lose efficiency over time and display off-target effects. Thus, understanding the pathogenic mechanisms underlying these diseases is crucial to pave the way for new effective therapies. The role of T cells in autoimmunity has been a spotlight in immunology research. Precisely, CD4+ T cells are central to RA and SLE pathogenesis through both antibody-dependent and independent mechanisms. Still, understanding how the microenvironment imprints pathogenic functions on CD4+ T cells remains elusive. The role of toll-like receptors (TLRs) in T cells has been overlooked and mainly described to function as a co-stimulatory signal that promotes proliferation and survival. Here, we advance that TLRs expressing T cells are present in peripheral blood and expanded in the inflamed sites, in patients with RA and SLE. Moreover, we demonstrated that TLR's role in T cells goes beyond acting only as a co-stimulatory signal and that they can elicit distinct pathogenic programs. In RA, we identified a TLR4+ Tfh-like subset that was more activated and proliferative and displayed chemokine receptors that allow them to home to inflamed sites. These cells could respond directly to TCR engagement and elicit production of IL-21 which favors antibody production. Also, they were able to sense synovial fluid components through the TLR4 which leads them to switch their pathogenic program to produce IL-17 pro-inflammatory cytokine that elicits osteoclastogenesis. Regarding SLE, we were able to identify discrete CD4+ T cell populations expressing different combinations of TLR4, TLR7, and TLR9. TLR4+ T cells in SLE displayed a similar phenotype as the subset identified in RA. Regarding TLR7+ and TLR9+ T cells we observed that they did not display a Tfh-like phenotype. Interestingly, we verified that TLR7+ T frequency correlated with disease activity and TLR9+ T cells' higher frequencies were present in the patients within the early stages of renal involvement. Accordingly, TLR9+ T cells upregulated CCR7 and IL-17R reinforcing that this subset may be of interest in patients that display lupus nephritis. In terms of cytokine production, TLR4 engagement induced an increase in TNF-α production. On another hand, TLR7 and TLR9 engagement alone could not elicit an increase in cytokine production. Instead, they acted as co-stimulatory signals and slightly increased IL-21 production. Altogether, our results support the idea that distinct TLRs may function differently depending on the disease stage and organ involved as they may be shaped by local components. Deciphering TLR's role in T cells is crucial to understand T cell response in different organs and diseases and their engaging in distinct pathogenic functions. Understanding their role could provide a great advance in the way the patients are evaluated as it could provide a better way to stratify the patients that could better benefit some therapies and also identify the patients that may develop more tissue destruction before occurring (e.g. bone damage and renal injury in RA and SLE, respectively). Moreover, manipulating the TLR intrinsic signaling could be a way to target T cell pathogenic function in autoimmune diseases.