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BACKGROUND: Rheumatoid Arthritis (RA) is an autoimmune disease with autoantibodies leading to a localized inflammation reaction in the joints. An important type of antibody involved in RA include antibodies against citrullinated peptides/proteins. Citrullination is a normal cellular process, however, RA is marked by a state of pathologic hypercitrullination. Specific risk factors, such as genetic predisposition, gender, and smoking, have been identified as contributing to the development of RA, but the mechanism behind how autoantibodies lead to RA pathology, what leads to pathological hypercitrullination and the generation of anti-citrullinated protein antibodies (ACPA) is insufficiently understood. It has been established that RA patients have antibodies to human endogenous retroviruses (HERVs), and more specifically, to the HERV-K family of such viruses. HERV-Ks are ancient retroviruses that have been integrated into the human genome and rendered inactive due to the accumulation of mutations. However, the presence of HERV-K antibodies suggests that there may be the capability to restore viral transcription in certain cases. If HERV-K was to be expressed, it would elicit the same immune reaction that exogenous viruses do, however, since it is a part of the human genome, this would effectively be an autoimmune reaction. OBJECTIVE: This thesis aims to explore the RA immune response to HERV-K, with a focus on neutrophils. Further, the relationship between HERV-K immunity and pathological hypercitrullination will also be explored. METHODS: Our studies utilized protein/antibody detection techniques to quantitate specific proteins/antibodies in sera and leukocytes isolated from RA patients. These techniques included enzyme-linked immunosorbent assay (ELISA), flow cytometry, and western blot analysis. Antibodies against HERV-K envelope (Env), an important viral protein, and citrullinated-Env (citEnv) were affinity-purified from patient sera to be used in these experiments. Results: Our experiments first replicated a result that has been previously established in the literature: RA patients produce antibodies against HERV-K. More specifically, we found that RA patient’s Env antibodies almost exclusively recognize the surface portion of the protein. Our data also presents the finding that patient antibodies are more reactive to citEnv than they are to unmodified Env, and that citEnv is expressed on patient neutrophils. Lastly, we found that some patient neutrophils are associated with the membrane attack complex of complement. CONCLUSION: The fact that RA patient’s neutrophils express citEnv suggests a link between HERV-K and the pathological hypercitrullination that’s a hallmark of RA. Additionally, the association of the membrane attack complex (MAC) with patient neutrophils suggests a potential mechanism of how this may occur: neutrophils express Env (following the re-initiation of HERV-K transcription), which then undergo citrullination and get recognized by ACPA, stimulating an immune response. This leads to complement activation and eventually MAC-induced neutrophil lysis. In doing so, neutrophils, which are enriched in citrullinating enzymes, can spill such enzymes into the extracellular space, leading to further citrullination and ACPA development. Further studies need to be done to confirm these data, as well as to fill gaps in knowledge regarding the novel hypothesis that HERV-K expression in RA patients may play a role in hypercitrullination and the disease’s pathogenesis. |
