Background Hematopoietic Stem Cells (HSCs) give rise to all blood cell lineages, which have been implicated in the pathogenesis of Systemic Lupus Erythematosus (SLE). We reasoned that the fundamental immune aberrations in SLE –genetic or epigenetic- may be traced back to the HSC population. We are validating and functionally characterising the molecular targets of RNA-seq transcriptome profiling in the HSC compartment of lupus mice.
Materials and methods HSCs were isolated from either control C57/BL6 or NZBxNZW/F1 lupus prone mice bone marrow (n=15±5). The selection markers used are Lin-Sca-1+c-Kit+ for LSK compartment and intermediate progenitors as common lymphoid and myeloid progenitors were characterised. Flow cytometry cell sorting was utilised for enumeration, RNA extraction, cell cycle analysis and CFU assays. Lentiviral vectors of either overexpressing or silencing gene targets were constructed.
Results We identified increased frequencies (~3% pre-diseased vs ~5% diseased, p<0.05) as well as absolute numbers (80–100×103 pre-diseased vs 100–150×103 diseased, p<0.05) of HSCs in the BM of lupus NZBxNZW/F1 mice with established disease as compared to pre-diseased NZBxNZW/F1 or control C57/BL6 mice. Bone marrow populations such as hematopoietic stem progenitors cells (HSPCs), lymphoid and myeloid lineages differed in homogeneity depending upon either age or disease, suggesting alterations in HSC potential under inflammatory conditions. The frequency of common myeloid progenitors in pre-diseased mice is higher compared to control mice. Accordingly, serum from F1 pre-diseased mice promoted healthy HSCs to proliferation and skewed their differentiation towards to myeloid lineage. Transcriptome analysis by RNA-seq of HSCs from lupus mice revealed 294 differentially expressed genes (DEGs) (FC >1.5, q<0.05) in diseased lupus mice compared to pre-diseased. DEGs show enrichment in transcription factors (such as Fbxw7, Cebpα, Blimp1, Hif1α) involved in hematopoiesis regulation of immune responses during inflammation and autoimmune diseases and HSC function and homeostasis. We have constructed lentiviral vectors to silence Cebpα gene expression for further functional analysis of its impact in HSCs under inflammatory conditions.
Conclusions Transcription factors involved in hematopoiesis such as Cebpα are enriched in lupus HSCs and may drive the increased proliferation and myeloid differentiation observed within the inflammatory milieu of the disease.