New developments on the pathogenesis of systemic lupus erythemstosus

Abstract

Systemic 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.