Next generation sequencing (NGS) in peripheral blood and hematopoietic stem cells (HSC) in SLE: mechanisms of disease, novel therapeutic targets and biomarkers for disease activity and response to therapy

Several types of cells are involved in SLE, all of which originate from HSC. We have used RNA Sequencing to uncover genes and their products (RNA or proteins) that can be used as markers to predict patients who may be more susceptible to certain serious manifestations of lupus as well as to interrogate the cells in the bone marrow (stem cells) to identify targets for new therapies.

Human Peripheral Blood RNA-seq

RNA-seq resulted in a comprehensive characterization of the transcriptome in SLE finding a higher number of DEGs and eQTLs. We also used machine learning techniques in order to detect the smallest set of genes predicting SLE disease activity from the same dataset and found:

• Distinct transcriptome disturbances at inactive and active stages (“susceptibility and activity signature”)
• The oxidative phosphorylation (mitochondrial hyperpolarization) pathway is implicated for the first time in the disease activity and severity
• Active nephritis has distinct transcriptome changes that reflect granulocyte activation, humoral immunity and the proteasome (all potentially drug-able targets)
• Organ involvement was predicted with high accuracy (accuracy=0.89, sensitivity=0.89, specificity=0.88 in the validation data) using 25 genes based on the elastic net generalised linear model. Among the 25 best predictors were MPO, ITGA3 and CD38.
• SLEDAI-2K could not be predicted with high accuracy (accuracy 0.75, sensitivity=0.79, specificity=0.67) using 50 genes based on the neural network model. Performance was still the same even when 1648 genes (after first feature selection step) were used as predictors of SLEDAI-2K.

Human HSC RNA-seq

• Transcriptome analysis of hematopoietic progenitors in the bone marrow of lupus vs healthy patients displayed enhanced proliferation/activation and myeloid skewing
• Comparable transcriptional profiles for both  human and murine hematopoietic progenitors

Murine HSC RNA-seq

Bone marrow (BM) transcriptome analysis in lupus mice before and during the disease onset demonstrates:

• Hypercellular BM and HSCs
• Lupus bone marrow produces more myeloid progenitors
• Differentiation arrest in the myeloid level of hematopoietic tree by suppression of conventional regulators of granulopoiesis with alternative granulopoiesis pathway
• Transcriptome reprogramming reminiscent of “trained immunity”
• Aberrant myelopoiesis might contribute to persistent inflammation and flares

SLE is the prototypic autoimmune disease and efforts are underway to better understand its cause and find new therapeutic approaches. To this end, we conducted a study where samples of SLE patients were analyzed to provide further insights into molecular (genomic) markers that predict the disease course, the response to different therapies and the damage caused by the disease to different tissues. For the first task, we used cutting edge biological and informatic approaches. Our identified novel genes and pathways that contribute to disease flares, severity and specific manifestations such as nephritis, which might be further explored as potential therapies. Following these analyses, we mapped a list of 15 genes that can predict major organ involvement (kidney, brain, etc.) in a given SLE patient, based upon the pattern of gene expression of these genes. Our analysis also confirmed the critical role of the immune system (e.g. over-expression of the antiviral interferon-alpha) in the causation of the SLE. These results could potentially assist the early diagnosis of SLE. Of interest, combining genetic variation (i.e. inter-individual changes in the DNA) with gene expression, we showed that besides immune cells in the blood, other organs such as the liver and the brain are involved in causing the disease.

In other studies run in parallel, we discovered that mouse and human bone marrow (the organ that makes the cells of the blood) in SLE produce more cells that cause inflammation. Moreover, a specific type of blood cells (neutrophils) is produced in a totally different fashion in SLE compared to healthy individuals. The comparison of hematopoietic progenitors between mice and humans provides a more clear picture of the biology of the lupus hematopoietic stem cell and a better understanding how bone marrow is involved in lupus.

• Grigoriou M, Banos A, et al. Transcriptome Reprogramming and Myeloid Skewing in Hematopoietic Stem and Progenitor Cells in Systemic Lupus Erythematosus. Ann Rheum Dis 2020;79:242-253
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• Bertsias G et al. Combined genetic and transcriptome analysis of patients with SLE: Distinct, targetable signatures for susceptibility and severity. Ann Rheum Dis. 2019 Aug;78(8):1079-1089
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• Nikolaos I Panousis, et al. Genomic dissection of Systemic Lupus Erythematosus: Distinct Susceptibility, Activity and Severity Signatures. doi: https://doi.org/10.1101/255109.
  Bioarxiv.
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• Grigoriou M, Anastasiou M, Verginis P, Pavlidis P, Nikolaou C, Bertsias G, Boumpas D T, Banos A. Rna-seq profiling of hematopoietic stem cells in murine systemic lupus erythematosus (sle): validation and functional characterisation. Ann Rheum Dis 2017;76:A57-A58.
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• A Banos, M Grigoriou, P Verginis, P Pavlidis, G Bertsias, DT Boumpas. Transcriptome profiling by next generation sequencing of hematopoietic progenitors in murine systemic lupus erythematosus (SLE). Ann Rheum Dis 2016;75:A50.
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• Bertsias G, Panousis N, Gergiannaki I, Tektonidou M, Trachana M, Banos A, Fanouriakis A, Pamfil C, Dermitzakis E, Boumpas D . Molecular characterization of SLE by RNA-Seq; Identification of genes and expression – quantitative trait loci contributing to pathogenesis, severity and tissue susceptibility. Clin Exp Rheumatol. 2016; 34(4): Suppl.99: S-49.
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Abstracts

• Filia A. et al RNA sequencing and machine learning techniques predict major organ involvement in patients with systemic lupus erythematosus. EULAR Meeting, Madrid, Spain. June 2019. Oral presentation and Best Abstract Award
• Filia A. et al Biomarkers for the activity of Systemic Lupus Erythematosus using RNA sequencing and machine learning techniques. European Conference on Computational Biology, Athens, Greece. September 2018.

• Banos A.*, Grigoriou M., Filia A., Giannouli S., Nikolopoulos D., Pieta A., Karali V., Mitroulis I., Verginis P., Boumpas DT., Disorders of the Hematopoietic Stem Cells in the Bone Marrow and Periphery of SLE Patients, 26th Panhellenic Rheumatology Congress, 6-9 December 2018, Athens, Greece
• Grigoriou M.*, Banos A., Filia A., Pavlidis P., Mitroulis I., Verginis P., Boumpas DT., Gene expression analysis of Hematopoietic Stem and Progentiors Cells in an experimental model of SEL: Disorders of the Myeloid Lineage, 26th Panhellenic Rheumatology Congress, 6-9 December 2018, Athens, Greece
• Grigoriou M.*, Verginis P., Nikolaou C., Pavlidis P., Dermitzakis E., Bertsias G., Boumpas DT., Banos A., Next Generation Sequencing in Hematopoietic Progenitors of murine SLE model reveals aberrant regulation of Cebp/a expression, 11th European Lupus Meeting, 21-24 March 2018, Dusseldorf, Germany
• Bertsias G, Panousis N, Gergianaki I, Tektonidou M, Trachana M, Pamfil C, Fanouriakis A, Dermitzakis E, Boumpas D. The genomic architecture of Systemic Lupus Erythemathosus (SLE) by RNA-seq: Distinct disease susceptibility, activity and severity signatures and extensive genetic effects on whole blood gene expression. Abstract EULAR 2017, Madrid – accepted as Oral Presentation.
• M. Grigoriou*, M. Anastasiou, P. Verginis, P. Pavlidis, C. Nikolaou, G. Bertsias, D.T. Boumpas, A. Banos, RNA-seq profiling of Hematopoietic Stem Cells in Murine Systemic Lupus Erythematosus (SLE): Validation and Functional characterization, 37th European Workshop for Rheumatology Research, March 2 – 4, 2017, Athens, Greece
• Banos A.*, Grigoriou M., Verginis P., Nikolaou C., Pavlidis P., Dermitzakis E., Bertsias G., Boumpas DT., Transcriptome Profiling by Next Generation Sequencing of Hematopoietic Progenitors in Murine Systemic Lupus Erythematosus (SLE), 10th European Lupus Meeting, 5-8 October 2016, Venice, Italy
• Banos A.*, Grigoriou M., Verginis P., Pavlidis P., Bertsias G., Boumpas DT., Gene Expression Analysis of Hematopoietic Stem Cells (HSCs) in Murine Systemic Lupus Erythematosus (SLE), Functional Genomics Workshop, 10-12th February 2016, St Thomas’ Hospital Campus, King’s College London, London, UK
• Banos A.*, Grigoriou M., Verginis P., Pavlidis P., Bertsias G., Boumpas DT., Transcriptome Profiling by Next Generation Sequencing of Hematopoietic Progenitors in Murine Systemic Lupus Erythematosus (SLE), 36th European Workshop for Rheumatology Research, February 25 – 27, 2016, York, United Kingdom
• A Banos, M Grigoriou, P Verginis, P Pavlidis, G Bertsias, DT Boumpas. Transcriptome profiling by next generation sequencing of hematopoietic progenitors in murine systemic lupus erythematosus (SLE). Ann Rheum Dis 2016; 75:A50.
• Grigoriou M., Verginis P., Bertsias G., Boumpas DT., and Banos A., The Role Of Hematopoietic Stem Cells (HSC) In Systemic Autoimmunity, 35th European Workshop for Rheumatology Research, March 5 – 7, 2015, Budapest, Hungary