Association of FCGR3A and FCGR3B haplotypes with rheumatoid arthritis and primary Sjögren's syndrome [POSTER PRESENTATION]

Background Rheumatoid arthritis (RA) is an autoimmune disease that is thought to arise from a complex interaction between multiple genetic factors and environmental triggers. We have previously demonstrated an association between a Fc gamma receptor (FcγR) haplotype and RA in a cross-sectional cohort of RA patients. We have sought to confirm this association in an inception cohort of RA patients and matched controls. We also extended our study to investigate a second autoanti-body associated rheumatic disease, primary Sjögren's syndrome (PSS). Methods The FCGR3A-158F/V and FCGR3B-NA1/NA2 functional polymorphisms were examined for association in an inception cohort of RA patients (n = 448), and a well-characterised PSS cohort (n = 83) from the United Kingdom. Pairwise disequilibrium coefficients (D') were calculated in 267 Blood Service healthy controls. The EHPlus program was used to estimate haplotype frequencies for patients and controls and to determine whether significant linkage disequilibrium was present. A likelihood ratio test is performed to test for differences between the haplotype frequencies in cases and controls. A permutation procedure implemented in this program enabled 1000 permutations to be performed on all haplotype associations to assess significance. Results There was significant linkage disequilibrium between FCGR3A and FCGR3B (D' = -0.445, P = 0.001). There was no significant difference in the FCGR3A or FCGR3B allele or genotype frequencies in the RA or PSS patients compared with controls. However, there was a significant difference in the FCGR3A-FCGR3B haplotype distributions with increased homozygosity for the FCGR3A-FCGR3B 158V-NA2 haplotype in both our inception RA cohort (odds ratio = 2.15, 95% confidence interval = 1.1–4.2 P = 0.027) and PSS (odds ratio = 2.83, 95% confidence interval = 1.0–8.2, P = 0.047) compared with controls. The reference group for these analyses comprised individuals who did not possess a copy of the FCGR3A-FCGR3B 158V-NA2 haplotype. Conclusions We have confirmed our original findings of association between the FCGR3A-FCGR3B 158V-NA2 haplotype and RA in a new inception cohort of RA patients. This suggests that there may be an RA-susceptibility gene at this locus. The significant increased frequency of an identical haplotype in PSS suggests the FcγR genetic locus may contribute to the pathogenesis of diverse autoantibody-mediated rheumatic diseases

Developments in the clinical understanding of lupus

Advances in genetics and new understanding of the molecular pathways that mediate innate and adaptive immune system activation, along with renewed focus on the role of the complement system as a mediator of inflammation, have stimulated elaboration of a scheme that might explain key mechanisms in the pathogenesis of systemic lupus erythematosus. Clinical observations identifying important comorbidities in patients with lupus have been a recent focus of research linking immune mechanisms with clinical manifestations of disease. While these advances have identified rational and promising targets for therapy, so far the therapeutic trials of new biologic agents have not met their potential. Nonetheless, progress in understanding the underlying immunopathogenesis of lupus and its impact on clinical disease has accelerated the pace of clinical research to improve the outcomes of patients with systemic lupus erythematosus

Recent advances in the understanding of renal inflammation and fibrosis in lupus nephritis

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THE ROLE OF THE MACROPHAGE IN ASBESTOS INDUCED AUTOANTIBODY PRODUCTION

Environmental exposure to silicate compounds such as silica and asbestos has been associated with increased autoimmune responses and the development of autoimmune disease in humans. Residents of Libby, MT have experienced significant asbestos exposure due to an asbestos contaminated vermiculite mine near the community over several decades. Residents have developed numerous asbestos-related diseases as well as increased autoimmune responses. However, the exact mechanism by which Libby amphibole asbestos generates autoimmune responses is unclear. To elucidate a possible mechanism for asbestos induced autoimmunity, the cellular effects of Libby amphibole asbestos were characterized in vitro using a phagocytic murine macrophage cell line, which are characteristic of alveolar macrophages. Our results indicate that Libby amphibole asbestos generates oxidative stress in murine macrophages similar to crocidolite asbestos. However, Libby asbestos induces distinct cellular effects compared to crocidolite asbestos. Therefore, the cellular effects of amphibole asbestos may be a combined consequence of its chemical composition as well as the activation of distinct cellular pathways during exposure. Libby amphibole asbestos also induces apoptosis in murine macrophages resulting in the translocation of SSA/Ro52 to cell surface blebs of apoptotic cells. These apoptotic cell surface blebs are recognized by autoantibodies from mice exposed to amphibole asbestos, suggesting that these cell surface structures may be antigenic when presented in a pro-inflammatory context. These results suggest that the induction of apoptosis may play a key role in environmentally induced autoimmunity. Interestingly, autoantibodies found in the sera of the Libby cohort also recognize the SSA/Ro52 autoantigen, indicating that humans and mice exposed to amphibole asbestos generate similar AA profiles and that the alterations of the immune response by amphibole asbestos may be comparable. We hypothesize that asbestos-induced autoimmunity is generated through a two hit mechanism. First, autoantigens become visible to the immune system during apoptosis, which results in the accumulation of autoantigens on the cell surface. The subsequent uptake and processing of apoptotic cells by antigen-presenting cells in a pro-inflammatory context will activate self-reactive T cells, inducing the loss of tolerance and generate the autoimmune responses

New developments on the pathogenesis of systemic lupus erythemstosus

Tese de doutoramento, Medicina (Pediatria), Universidade de Lisboa, Faculdade de Medicina, 2018Systemic Lupus Erythematosus (SLE) is a challenging autoimmune disease, with a complex etiopathogenesis and an unpredictable clinical course. In a large cohort of juvenile-onset SLE patients, we found that major infections were common, were associated with active disease and its treatment, and resulted in noteworthy morbidity. New biomarkers to guide the judicious use of immunosuppressive drugs and new treatment strategies with fewer side effects would, therefore, have an enormous impact in the management of these patients. In order to reach these goals we used the modern tools of molecular biology and focused on two of the most important complications of SLE: lupus nephritis and macrophage activation syndrome. Firstly, we identified the kidney lupus nephritis specific microRNA (miRNA) signature, which reflected mainly cell proliferation. MiRNAs are noncoding RNAs responsible for post-transcriptional gene silencing. These key regulatory molecules control the expression of multiple genes, so its dysregulation can contribute to sustained pathology. We showed that miR-26a and miR-30b were significantly decreased in the kidneys and urine of lupus nephritis patients. In vitro, the knockdown of miR-26a and miR-30b caused the proliferation of human mesangial cells and increased the expression of genes related to the cell cycle, including CCNE2, E2F8, MAD2L1, MYBL1 and POLQ. The Human Epidermal Growth Factor Receptor 2 (HER2) is a protein previously known to regulate miR-26a and miR-30b expression. Trastuzumab, a monoclonal antibody against HER2, used in breast cancer treatment, produces therapeutic actions precisely by up-regulating miR-26a and miR-30b. In human mesangial cells we also found that trastuzumab increases these two miRNAs. We hypothesized that HER2 also played a role in the pathogenesis of lupus nephritis and indeed we identified a dramatic overexpression of HER2 in the glomeruli and tubular compartments of the kidneys of lupus nephritis patients. The same pattern was not seen in the kidneys of healthy individuals or in other proliferative glomerulonephritides, including post-streptococcus glomerulonephritis, IgA nephropathy and granulomatosis with polyangiitis. Furthermore, in the lupus-prone NZM2410 mice we identified a highly increased expression of HER2, which correlated with disease activity. Finally, we showed that urinary HER2 was significantly increased in lupus nephritis and that its levels increased during flares, were higher in class III and class IV lupus nephritis and correlated with urinary proteincreatinine and monocyte chemoattractant protein 1 (MCP1) and vascular cell adhesion protein 1 (VCAM1) levels. We, therefore, established a strong rationale to use trastuzumab to block HER2 and decrease cell proliferation and damage in lupus nephritis. Regarding the macrophage activation syndrome, we were interested in the characterization of hemophagocytes. These are activated macrophages that have engulfed other hematopoietic cells. Traditionally they have been associated with the development of cytopenias in several life-threatening cytokine storm syndromes. New data have challenged this concept, since pancytopenia occurs in the absence of hemophagocytosis in mice and, in humans, over 40% of patients with macrophage activation syndrome do not have hemophagocytes in bone marrow aspirates. On the other hand, subclinical hemophagocytosis is detected in more than 50% of patients with systemic juvenile idiopathic arthritis, but only 10% develop a life-threatening macrophage activation syndrome. Thus the function and significance of hemophagocytes remained mysterious. Recent evidence demonstrated that environmental factors, particularly the cytokine milieu, determine the macrophage activation status in a continuum ranging from M1 to M2. M1 macrophages, driven by interferon γ, typically acquire proinflammatory properties and are associated with tissue damage, whereas M2 macrophages have more heterogeneous stimuli and functions, being associated with immunoregulation, tissue remodeling and fibrosis. Since interferon γ is the hallmark cytokine of hemophagocytic lymphohistiocytosis, one would expect to find that hemophagocytes express M1 surface receptors. We characterized the transcriptional phenotype of mouse Toll-like receptor 9 (TLR9) – induced hemophagocytes and described the surface phenotype of human bone marrow hemophagocytes. Interestingly, murine hemophagocytes had up-regulation of genes associated with the M2 and not the M1 phenotype. Immunohistochemical analyses in bone marrow samples from a uniquely diverse cohort of patients with hemophagocytic syndromes showed universal staining of hemophagocytes for the M2 marker CD163, but rarely for CD206 or CD64. Collectively, these data support the hypothesis that hemophagocytes have immunoregulatory functions and open new doors for the study of the pathogenesis of this syndrome. Finally, we were interested in characterizing the transcriptome of SLE, by RNAsequencing, in order to better understand the mechanisms that are responsible for chronicity. Monocytes from SLE patients exhibited a globally dysregulated gene expression. The transcriptome was not simply altered by the activation of a set of genes, but was also qualitatively different. Splicing patterns and polyadenylation were significantly altered and SLE monocytes expressed novel transcripts, an effect that was replicated by exposing control monocytes to lipopolysaccharide (LPS). We further identified increased circulating endotoxin in SLE patients, suggesting that chronic microbial translocation could contribute to the immunologic dysregulation in SLE, a new potential disease mechanism. In conclusion, with these different projects, which globally focused on the transcriptome and epigenome of SLE, we identified novel pathways and challenged the current paradigms. We described new mechanisms of disease, including the role of LPS in transforming qualitatively the SLE transcriptome and a putative immunoregulatory function for hemophagocytes. We demonstrated that miR-26a, miR-30b and HER2 control cell proliferation in lupus nephritis and that are promising biomarkers. Most importantly, our work rose the possibility of using anti-HER2 drugs for lupus nephritis management, opening the door to a new treatment strategy in this disease.Fundação Calouste Gulbenkian, Fundação Champalimau

New Horizons in the Genetic Etiology of Systemic Lupus Erythematosus and Lupus-Like Disease: Monogenic Lupus and Beyond

Systemic lupus erythematosus (SLE) is a clinically and genetically heterogeneous autoimmune disease. The etiology of lupus and the contribution of genetic, environmental, infectious and hormonal factors to this phenotype have yet to be elucidated. The most straightforward approach to unravel the molecular pathogenesis of lupus may rely on studies of patients who present with early-onset severe phenotypes. Typically, they have at least one of the following clinical features: childhood onset of severe disease (\u3c5 \u3eyears), parental consanguinity, and presence of family history for autoimmune diseases in a first-degree relative. These patients account for a small proportion of patients with lupus but they inform considerable knowledge about cellular pathways contributing to this inflammatory phenotype. In recent years with the aid of new sequencing technologies, novel or rare pathogenic variants have been reported in over 30 genes predisposing to SLE and SLE-like diseases. Future studies will likely discover many more genes with private variants associated to lupus-like phenotypes. In addition, genome-wide association studies (GWAS) have identified a number of common alleles (SNPs), which increase the risk of developing lupus in adult age. Discovery of a possible shared immune pathway in SLE patients, either with rare or common variants, can provide important clues to better understand this complex disorder, it\u27s prognosis and can help guide new therapeutic approaches. The aim of this review is to summarize the current knowledge of the clinical presentation, genetic diagnosis and mechanisms of disease in patents with lupus and lupus-related phenotypes