Background and purpose All blood cell lineages that have implicated to the pathogenesis of SLE originate from the Hematopoietic Stem Cells (HSCs). Studying HSCs may help to dissect fundamental immune aberrations in SLE and elucidate the HSC contribution to the pathogenesis of the disease.
Materials and methods HSCs were isolated from either healthy C57/BL6 or NZBxNZW/F1 lupus-prone mice bone marrow. The selection markers used are Lin-Sca-1+c-Kit+for LSK compartment that encompasses both long- and short-term HSCs as well as multipotent progenitors (MPP). Flow cytometry cell sorting of LSK was used for enumeration, RNA extraction, qPCR and cell cultures. Paired-end RNA-sequencing analysis was performed by the Illumina HiSeq 2000 platform.
Results We found significantly increased numbers of LSK in the BM of lupus NZBxNZW/F1 mice with established disease as compared to pre-diseased NZBxNZW/F1 mice in combination with evidence of them exiting the latent state and progression of cell cycle and aberrant differentiation with skewing towards the myeloid lineage. Transcriptome analysis revealed 800 differentially expressed genes (DEGs) (FC>1.5, q<0.05) in diseased lupus mice compared to pre-diseased with enrichment in transcription factors involved in hematopoiesis, regulation of immune responses and HSC function/homeostasis. We selected Cebpα (FC −0,88) -a master regulator of myeloid differentiation, self-renewal and resistance to stress-induced apoptosis of HSCs- for further investigation. qPCR analysis showed decreased Cebpα expression during the progression of the disease in SLE but increased Cebpα in aged healthy C57/BL6 mice. In vitro stimulation with IFNα decreased Cebpα expression in lupus -but not in healthy LSK. Serum from pre-diseased NZBxNZW/F1 decreased Cebpα expression only in pre-diseased LSK. Experiments to reverse Cebpα downregulation (using lenti-virus and modifiers of the metabolomics) are in progress.
Conclusions HSC RNA-seq analysis suggests both intrinsic and extrinsic influences resulting in downregulation of Cebpα in murine lupus. SLE HSCs have pronounced expansion, enhanced proliferation and aberrant differentiation -in part due to the effects of IFNα. Together these results suggest a decreased capacity of lupus HSCs to respond to stressors which may account for the cytopenias and the infections in SLE.
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