Mechanisms involved in the recognition of apoptotic cells by B lymphocytes

As many tumours are poorly immunogenic, one approach for cancer therapy aims to improve natural anti-tumour immunity. Recently it has been shown that anthracycline-induced apoptosis in tumour cells results in the relocation of the ER resident chaperone calreticulin to the cell surface. Interestingly, this relocation directly stimulates T cell-mediated anti-tumour immunity following tumour cell phagocytosis by dendritic cells. In addition, evidence from medullary carcinoma of the breast patients shows a correlation between tumour-infiltrating B cells and a more favourable prognosis. Surprisingly, many of these infiltrating B cells recognise proteins relocated from their normal intracellular distribution to the tumour cell surface, also as a result of apoptosis. As B cells have recently been shown to be an important population of antigen presenting cells capable of extracting membrane antigens from target cells, these findings raise the possibility that tumour infiltrating B cells may be capable of presenting tumour antigens promoting the generation of anti-tumour immunity. In particularly, this thesis envisages that following apoptosis induction in tumour cells, antigen specific B cells will be able to acquire relocated tumour antigens, therefore directly promoting antigen-specific T cells. To provide a proof of principle I have characterized the processes involved in the extraction of membrane-tethered antigens by B cells from target cells using flow cytometry and a novel proliferation bio-assay. Unexpectedly, surface antigen acquisition by B cells has been demonstrated to be rapid, proceeding in times as short as 1 min. Furthermore, I have also characterised the processes involved in the relocation of antigens to the cell surface of dying target cells from various intracellular locations using anthracyclines and UVC as immunogenic apoptotic agents. This has allowed me to demonstrate that antigens from several intracellular compartments following apoptosis induction are acquired by antigen-specific B cells. Importantly, this antigen acquisition was sufficient to induce CD4+ T cell activation as confirmed by proliferation assay. These in vitro findings demonstrate that B cells are capable of activating CD4+ T cells following interaction with apoptotic target cells. Therefore, further investigations concerning the role of B cells in natural anti-tumour immunity may improve current anti-cancer therapies.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Effectiveness of soluble CTLA-4-Fc in the inhibition of bone marrow T-cell activation in context of indoleamine 2.3-dioxygenase (IDO) and CD4+Foxp3+ Treg induction

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease with systemic inflammation finally resulting in damaged joints. One of the RA development models suggests bone marrow (BM) as a place of inflammation development further leading to disease progression. We aimed to investigate the potential of CTLA-4-Fc molecule in inducing tolerogenic milieu in BM measured as indoleamine 2,3-dioxygenase (IDO) expression, CD4 METHODS: Bone marrow mononuclear cells (BMMC) were stimulated by pro-inflammatory cytokines and CTLA-4-Fc. Next IDO expression, CD4 RESULTS: We found that CTLA-4-Fc and IFN-γ stimulation results in IDO production by BMMC. CTLA-4-Fc induced tryptophan catabolism can inhibit mitogen-induced CD4 CONCLUSION: This study sheds light on some immunosuppression aspects present or induced in BM. The potential of IDO-mediated pathways were confirmed in the periphery, what may represent the promising candidates for therapeutic strategies in RA

Epigenetic Modulation as a Therapeutic Prospect for Treatment of Autoimmune Rheumatic Diseases

Systemic inflammatory rheumatic diseases are considered as autoimmune diseases, meaning that the balance between recognition of pathogens and avoidance of self-attack is impaired and the immune system attacks and destroys its own healthy tissue. Treatment with conventional Disease Modifying Antirheumatic Drugs (DMARDs) and/or Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) is often associated with various adverse reactions due to unspecific and toxic properties of those drugs. Although biologic drugs have largely improved the outcome in many patients, such drugs still pose significant problems and fail to provide a solution to all patients. Therefore, development of more effective treatments and improvements in early diagnosis of rheumatic diseases are badly needed in order to increase patient’s functioning and quality of life. The reversible nature of epigenetic mechanisms offers a new class of drugs that modulate the immune system and inflammation. In fact, epigenetic drugs are already in use in some types of cancer or cardiovascular diseases. Therefore, epigenetic-based therapeutics that control autoimmunity and chronic inflammatory process have broad implications for the pathogenesis, diagnosis, and management of rheumatic diseases. This review summarises the latest information about potential therapeutic application of epigenetic modification in targeting immune abnormalities and inflammation of rheumatic diseases

Tuning Monocytes and Macrophages for Personalized Therapy and Diagnostic Challenge in Rheumatoid Arthritis

Monocytes/macrophages play a central role in chronic inflammatory disorders, including rheumatoid arthritis (RA). Activation of these cells results in the production of various mediators responsible for inflammation and RA pathogenesis. On the other hand, the depletion of macrophages using specific antibodies or chemical agents can prevent their synovial tissue infiltration and subsequently attenuates inflammation. Their plasticity is a major feature that helps the switch from a pro-inflammatory phenotype (M1) to an anti-inflammatory state (M2). Therefore, understanding the precise strategy targeting pro-inflammatory monocytes/macrophages should be a powerful way of inhibiting chronic inflammation and bone erosion. In this review, we demonstrate potential consequences of different epigenetic regulations on inflammatory cytokines production by monocytes. In addition, we present unique profiles of monocytes/macrophages contributing to identification of new biomarkers of disease activity or predicting treatment response in RA. We also outline novel approaches of tuning monocytes/macrophages by biologic drugs, small molecules or by other therapeutic modalities to reduce arthritis. Finally, the importance of cellular heterogeneity of monocytes/macrophages is highlighted by single-cell technologies, which leads to the design of cell-specific therapeutic protocols for personalized medicine in RA in the future

DNA Methylation as a Future Therapeutic and Diagnostic Target in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a long-term autoimmune disease of unknown etiology that leads to progressive joint destruction and ultimately to disability. RA affects as much as 1% of the population worldwide. To date, RA is not a curable disease, and the mechanisms responsible for RA development have not yet been well understood. The development of more effective treatments and improvements in the early diagnosis of RA is direly needed to increase patients’ functional capacity and their quality of life. As opposed to genetic mutation, epigenetic changes, such as DNA methylation, are reversible, making them good therapeutic candidates, modulating the immune response or aggressive synovial fibroblasts (FLS—fibroblast-like synoviocytes) activity when it is necessary. It has been suggested that DNA methylation might contribute to RA development, however, with insufficient and conflicting results. Besides, recent studies have shown that circulating cell-free methylated DNA (ccfDNA) in blood offers a very convenient, non-invasive, and repeatable “liquid biopsy”, thus providing a reliable template for assessing molecular markers of various diseases, including RA. Thus, epigenetic therapies controlling autoimmunity and systemic inflammation may find wider implications for the diagnosis and management of RA. In this review, we highlight current challenges associated with the treatment of RA and other autoimmune diseases and discuss how targeting DNA methylation may improve diagnostic, prognostic, and therapeutic approaches

Small Molecule Inhibitors in the Treatment of Rheumatoid Arthritis and Beyond: Latest Updates and Potential Strategy for Fighting COVID-19

The development of biological disease-modifying antirheumatic drugs (bDMARDs) and target synthetic DMARDs (tsDMARDs), also known as small molecule inhibitors, represent a breakthrough in rheumatoid arthritis (RA) treatment. The tsDMARDs are a large family of small molecules targeting mostly the several types of kinases, which are essential in downstream signaling of pro-inflammatory molecules. This review highlights current challenges associated with the treatment of RA using small molecule inhibitors targeting intracellular JAKs/MAPKs/NF-κB/SYK-BTK signaling pathways. Indeed, we have provided the latest update on development of small molecule inhibitors, their clinical efficacy and safety as a strategy for RA treatment. On the other hand, we have highlighted the risk and adverse effects of tsDMARDs administration including, among others, infections and thromboembolism. Therefore, performance of blood tests or viral infection screening should be recommended before the tsDMARDs administration. Interestingly, recent events of SARS-CoV-2 outbreak have demonstrated the potential use of small molecule inhibitors not only in RA treatment, but also in fighting COVID-19 via blocking the viral entry, preventing of hyperimmune activation and reducing cytokine storm. Thus, small molecule inhibitors, targeting wide range of pro-inflammatory singling pathways, may find wider implications not only for the management of RA but also in the controlling of COVID-19

S100A8 and S100A12 Proteins as Biomarkers of High Disease Activity in Patients with Rheumatoid Arthritis That Can Be Regulated by Epigenetic Drugs

Rheumatoid arthritis (RA) is an autoimmune chronic inflammatory disease that is still not well understood in terms of its pathogenesis and presents diagnostic and therapeutic challenges. Monocytes are key players in initiating and maintaining inflammation through the production of pro-inflammatory cytokines and S100 proteins in RA. This study aimed to test a specific DNA methylation inhibitor (RG108) and activator (budesonide) in the regulation of pro-inflammatory mediators—especially the S100 proteins. We also searched for new biomarkers of high disease activity in RA patients. RNA sequencing analysis of healthy controls (HCs) and RA monocytes was performed. Genes such as the S100 family, TNF, and IL-8 were validated by qRT-PCR following DNA-methylation-targeted drug treatment in a monocytic THP-1 cell line. The concentrations of the S100A8, S100A11, and S100A12 proteins in the sera and synovial fluids of RA patients were tested and correlated with clinical parameters. We demonstrated that RA monocytes had significantly increased levels of S100A8, S100A9, S100A11, S100A12, MYD88, JAK3, and IQGAP1 and decreased levels of IL10RA and TGIF1 transcripts. In addition, stimulation of THP-1 cells with budesonide statistically reduced the expression of the S100 family, IL-8, and TNF genes. In contrast, THP-1 cells treated with RG108 had increased levels of the S100 family and TNF genes. We also revealed a significant upregulation of S100A8, S100A11, and S100A12 in RA patients, especially in early RA compared to HC sera. In addition, protein levels of S100A8, S100A11, and S100A12 in RA synovial fluids compared to HC sera were significantly increased. Overall, our data suggest that the S100A8 and S100A12 proteins are strongly elevated during ongoing inflammation, so they could be used as a better biomarker of disease activity than CRP. Interestingly, epigenetic drugs can regulate these S100 proteins, suggesting their potential use in targeting RA inflammation

Impact of Adipose-Derived Mesenchymal Stem Cells (ASCs) of Rheumatic Disease Patients on T Helper Cell Differentiation

Complex pathogenesis of systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) is associated with an imbalance of various Th-cell subpopulations. Mesenchymal stem cells (MSCs) have the ability to restore this balance. However, bone marrow-derived MSCs of SLE and SSc patients exhibit many abnormalities, whereas the properties of adipose derived mesenchymal stem cells (ASCS) are much less known. Therefore, we examined the effect of ASCs obtained from SLE (SLE/ASCs) and SSc (SSc/ASCs) patients on Th subset differentiation, using cells from healthy donors (HD/ASCs) as controls. ASCs were co-cultured with activated CD4+ T cells or peripheral blood mononuclear cells. Expression of transcription factors defining Th1, Th2, Th17, and regulatory T cell (Tregs) subsets, i.e., T-bet, GATA3, RORc, and FoxP3, were analysed by quantitative RT-PCR, the concentrations of subset-specific cytokines were measured by ELISA, and Tregs formation by flow cytometry. Compared with HD/ASCs, SLE/ASCs and especially SSc/ASCs triggered Th differentiation which was disturbed at the transcription levels of genes encoding Th1- and Tregs-related transcription factors. However, we failed to find functional consequences of this abnormality, because all tested ASCs similarly switched differentiation from Th1 to Th2 direction with accompanying IFNγ/IL-4 ratio decrease, up-regulated Th17 formation and IL-17 secretion, and up-regulated classical Tregs generation