Periodic Reporting for period 3 - LUPUSCARE (PRECISION CARE IN SYSTEMIC AUTOIMMUNITY: AN INTEGRATED MULTI-TISSUE/LEVEL APPROACH FOR SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)) Reporting period: 2020-09-01 to 2022-02-28 Summary of the context and overall objectives of the project Systemic lupus erythematosus (SLE), is an autoimmune, potential life-threatening disease that affects predominantly women of the reproductive age. SLE is a complex and a heterogeneous disease whereby the interplay of environmental, genetic and epigenetic factors leads to perturbation of complex biological networks resulting into diverse clinical manifestations of varying severity. In SLE the immune system turns again itself producing cells and molecules that harm various organs including joints, skin, kidney, brain, lung, heart and blood. In this project, we have introduced novel concepts and targets for the pathogenesis, monitoring and management of human systemic lupus erythematosus (SLE) and have developed novel methods and technologies to be used for studying the disease. More specifically, we are employing powerful high-throughput laboratory methods to lay the foundations for a molecular-based taxonomy to be used for personalized therapy. Accordingly, we are defining for the first time the genomic architecture of human SLE and provide clues to the understanding of the systemic multiorgan nature of the disease and its marked clinical and molecular heterogeneity. Finally, we are discovering novel targets of therapy and biomarkers for diagnosis/monitoring. Increased sensitivity to sun and other environmental stressors is a key feature of SLE. In a systematic multi-tissue approach, we are defining the response of the cells to DNA damage providing evidence that the DNA damage response has a key role in promoting autoreactivity and increasing the risk for lymphoma. In order to explain the staggering female predominance in SLE, we are also delineating for the first time the transcriptomic basis of the female predominance in SLE defining specific genes involved in this process. Finally, we have introduced the concept that immune abnormalities in SLE can be traced back to the early progenitor cells in the bone marrow which produce all the cells of the blood that cause damage in SLE. By the use of novel methods and technologies such as single-cell RNA-seq, xenotransplantation and bone-marrow-on-a-chip- we are defining potential novel targets for lupus therapy by inhibiting the flow of cells from the bone marrow to the peripheral organs where they cause inflammation and damage. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far OverviewApproximately 5% of the population of Western Countries is affected by one of over 100 autoimmune diseases. Systemic lupus erythematosus (SLE), is the prototypic systemic (ie affecting multiple organs) autoimmune disease and affects approximately 80-100 people per 100,000 population. In this project, we have introduced novel concepts and targets for the pathogenesis, monitoring and management of human systemic lupus erythematosus (SLE) and have developed novel methods and technologies to be used in the investigation of this disease. The Genomic architecture of SLE and its implications We have defined for the first time the genomic architecture of human SLE providing important clues to the understanding of the systemic nature of the disease, its marked heterogeneity and novel targets of therapy and biomarkers for diagnosis/monitoring (Panousis et al Ann Rheum Dis 2019). Validation of these findings in several replication/validation cohorts during the second part of this project will increase their impact. Identifying modules of co-expressed transcripts for individual patients and linking it to the corresponding clinical phenotypes (i.e. nephritis, neuropsychiatric disease, antiphospholipid syndrome) and response to therapy is likely to facilitate patients’ stratification to distinct disease endotypes, facilitate personalized therapy, and improve clinical trial design by selecting patients more likely to respond to a particular agent.DNA damage and response in SLE and its pathologic correlatesIncreased DNA damage and defective DNA repair are key features of SLE in a process linking genetic with environmental factors such as ultraviolet irradiation. Genomic DNA damage may stimulate inflammatory responses and increase the risk for lymphoma. We have defined the DNA damage response in the immune cells of SLE and provided evidence that has a key role in autoreactivity and in contributing to the increased risk for lymphoma. We are also, introducing for the first time in autoimmunity the notion of Total Mutation Burden (TMB) and explore its impact to the severity of the disease.SLE a disease that epitomizes sex dimorphismSex dimorphism of various diseases - in females vs males- regarding susceptibility and severity of the disease and response to treatment is receiving increased attention. We have delineated for the first time the transcriptomic basis of the female predominance in SLE defining specific genes involved in this process. Analysis of RNA-seq data led to identification of 39 genes differentially expressed upon comparison of male and female patients, six of which did not demonstrate differential expression upon comparison of the control cohorts for both sexes: SMC1A, APOE, OPLAH, ARSD, MTCOP12, FRG1BP (Panousis et al Ann Rheum Dis 2019). Preliminary classification of expression data in all (total number 24) possible patterns suggests that the two most overrepresented, consisting of 3,650 genes demonstrate high relevance to the sexual dimorphism The emerging role of bone marrow and the myeloid lineage in SLE Most cells participating in the pathogenesis of SLE originate from bone marrow (BM) hematopoietic stem progenitor cells (HSPCs). HSPCs actively respond to inflammatory stimuli by myeloid skewing, but this may lead to exhaustion, decreased function, increased risk for inflammation, decreased adaptive immunity and increased cardiovascular mortality. We have introduced the concept that immune abnormalities in SLE can be traced back to the progenitor cells in the bone marrow and have produced data -by the use of novel methods and technologies such as single-cell RNA-seq, xenotransplantation and bone-marrow-on-a-chip- supporting this. In SLE, there is evidence of deregulation of hematopoiesis with skewing towards the myeloid lineage at the expense of lymphopoiesis and priming of HSPCs that exhibit a ‘trained immunity’ signature; this may contribute to inflammation and risk of flare (Grigoriou et al, Ann Rheum Dis 2020). Together these data indicate that abnormalities of immune cells in SLE can be traced back in the BM HSPCs, a disease where stem cell therapy has been considered for refractory cases Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The Genomic architecture of SLE and its implications This is the first comprehensive combined genetic and transcriptomic characterization of the ‘genomic architecture’ of SLE and provides important clues to the understanding of the systemic nature of the disease, its marked heterogeneity and novel targets of therapy and biomarkers for diagnosis/monitoring.More specifically, the discovery of three distinct gene expression signatures in SLE namely susceptibility and activity and severity is novel and could be capitalized for testing people at risk for SLE (first degree relatives of patients and patients with early/incomplete SLE) to diagnose early and inform treatment decisions based on the likelihood to develop severe disease with involvement of major organs such as the kidneys. Importantly, in the second round of experiments with the new cohorts we are exploring the value of transcriptomic analysis in predicting response to therapy and the differential response of various molecular signatures to various treatments used in SLE (immunosuppressive or biologic). Blood transcriptome discriminates SLE versus healthy individuals with high accuracy and can distinguish active versus inactive/low disease activity states. Several of the pathways discovered in this project (oxidative phosphorylation, DNA damage, interferon, plasmablast) are druggable either through testing novel compounds or repositioning of existing drugs. Combined eQTL analysis from the Genotype Tissue Expression (GTEx) project and SLE-associated genetic polymorphisms demonstrates that susceptibility variants may regulate gene expression in the blood but also in other tissues. A novel finding in this analysis is the involvement of the liver and brain as causal tissues in SLE -above and beyond the known suspects such as bone marrow and blood. DNA polymorphisms that confer susceptibility to SLE regulate gene expression not only in the blood but also in multiple other tissues, which may explain the multiorgan involvement in SLE. Finally, another novel finding is the fact that patients with SLE exhibit perturbed mRNA splicing in genes enriched in immune system and interferon signaling pathways leading to immune hyperreactivity both in innate and adaptive immunity in SLE. Perturbed mRNA spicing in autoimmunity is a novel finding. DNA damage and response in SLE and its pathologic correlatesIncreased DNA damage and defective DNA repair are key features of SLE in a process linking genetic with environmental factors such as ultraviolet irradiation. DNA damage occurs on exposure to genotoxic agents and during physiological DNA transactions. Genomic DNA damage may stimulate inflammatory responses. In SLE, we have demonstrated IFNα-mediated deregulation of mitochondrial metabolism and impairment of autophagic degradation, leading to cytosolic accumulation of mtDNA that is sensed via stimulator of interferon genes (STING) to promote induction of autoinflammatory DCs. Identification of mtDNA as a cell-autonomous enhancer of IFNα signaling underlines the significance of efficient mitochondrial recycling in the maintenance of peripheral tolerance. Antioxidant treatment and metabolic rescue of autolysosomal degradation emerge as drug targets in SLE and other IFNα-related pathologies. Our findings also identify key cells in the pathogenesis of SLE such as regulatory T cells, B cells and monocytes with increased DNA damage and defective DNA damage response elucidating the pathways involved as well as their functional implications and contribution to disease. Our data link oxidative stress in SLE with DDR with SLE pathology. In reference to Tregs, cells known to be cardinal feature of autoimmune pathologies, we found mitochondrial oxidative stress, deregulated autophagy and a robust DNA damage response leading to cell death. Of interest, preliminary data from this work suggest that defective DNA damage and response in T regs may be a generalized mechanism in autoimmunity (Alissafi et al, in preparation). Importantly scavenging of mtROS either chemically or through targeted expression of catalase to mitochondria of Foxp3+ Treg cells reverses their DNA damage response and ameliorated the autoimmune phenotype through contraction of Th1 and Th17 autoimmune responses. In reference to B cells, specific molecules for DNA damage response will be used for ex vivo inhibition of antibody production, cytokine secretion, and differentiation to autoantibody producing B cells. These will be tested on primary B cells from SLE patients and experimental animal models.SLE a disease that epitomizes sex dimorphismThe sex dimorphism of various diseases is receiving increased attention in terms of susceptibility and severity of the disease and response to treatment. Analysis of RNA-seq data led to identification of 39 genes differentially expressed upon comparison of male and female patients, 6 of which did not demonstrate differential expression upon comparison of the control cohorts for both sexes: SMC1A, APOE, OPLAH, ARSD, MTCOP12, FRG1BP. Preliminary classification of expression data in all (total number 24) possible patterns suggests that the 2 most overrepresented, consisting of 3,650 genes demonstrate high relevance to the sexual dimorphism. A large proportion of these genes belong the group of 6,730 differentially expressed genes upon comparison of healthy and disease states. Functional annotation bioinformatic (DAVID) analysis of genes belonging to these two overrepresented patterns of expression revealed enrichment of pathways relevant to SLE pathogenesis such as NOD-/TOLL-like receptor signaling, some of which (e.g. TLR signaling) already implicated in sexual dimorphism. Our data provide the first combined genetic and transcriptomic basis for this dimorphism pinpointing among others to genes located to the X chromosome and escaping X-chromosome inactivation such as SMC1A and ARSD. Using SMC1A as a model gene we are exploring the interaction of genetic, environmental and epigenetic factors in sexual dimorphisms. The emerging role of bone marrow and the myeloid lineage in SLE Most cells participating in the pathogenesis of SLE originate from bone marrow (BM) hematopoietic stem progenitor cells (HSPCs). HSPCs actively respond to inflammatory stimuli by myeloid skewing, but this may lead to exhaustion, decreased function, increased risk for inflammation, decreased adaptive immunity and increased cardiovascular mortality. In SLE, there is evidence of deregulation of hematopoiesis with skewing towards the myeloid lineage at the expense of lymphopoiesis and priming of HSPCs that exhibit a ‘trained immunity’ signature; this may contribute to inflammation and risk of flare (Grigoriou et al, Ann Rheum Dis 2020). These data indicate that abnormalities of immune cells in SLE can be traced back in the BM HSPCs, a disease where stem cell therapy has been considered for refractory cases. During systemic inflammation there is activation of HSPCs in the bone marrow, resulting in their proliferation and differentiation towards the myeloid lineage. These HSPCs are uniquely primed to respond to acute inflammation but may have a poor response to infectious challenges (Frangou, et al, Autoimmun Rev 2019). Re-establishment of the appropriate myeloid versus lymphoid balance and alleviation of cell exhaustion may improve transplantability of HSPCs and may restore immune function. This could also decrease risk of infection and atherosclerosis and attenuate inflammation, decreasing the risk of flare. Further analysis in single-cell RNA-seq data in the BM HSPCs to identify novel developmental transcription factor that promote SLE is in progress. We also plan to use siRNA experiments on human and mice hematopoietic progenitors in order to assess their kinetics in vitro. We also plan to perform siRNA experiments on human and mice hematopoietic progenitors in order to assess their kinetics in vitro, initially on SIRT7 silencing in circulating CD34+ progenitors in order to assess a change in engraftment potential and extramedullary colonization. In this project, we have created a device which represents a pure in vitro and scaffold-free BMoC device ( bone-marrow-on-a-chip), intended for both the generation and sustainment of the hematopoietic niche which could be as a study platform for studies of the pathogenesis and treatment of SLE (Kefallinou et al, submitted). Figure 8 Figure 3 Figure 11 Figure 5 Figure 12 Figure 7 Figure 2 Objectives&Work-packages Figure 1 Figure 6 Figure 9 Figure 4 Figure 10
