Purpose Next-generation RNA-sequencing was applied to investigate SLE pathogenesis through a comparative transcriptomic analysis of a peripheral lymphoid organ (spleen) and end-organ tissues (kidneys, brain) of lupus-prone and healthy mice.
Methods NZB/W-F1 lupus-prone mice were sacrificed at the pre-puberty, pre-autoimmunity and nephritic stage of the disease. Age-matched C57/BL6 mice were used as controls. Spleen, kidneys and brain were removed and total RNA was extracted. Paired-end RNA-sequencing was performed with Illumina HiSeq 2000 platform. Relative expression levels of transcripts and differentially expressed genes (DEG) (FC >1.5, p<0.05) were calculated. Functional enrichment analysis was performed with IPA, RNEA and gprofiler.
Results To investigate SLE developmental biology, a comparative analysis between the same organs of the same model at different stages of the disease was performed. In the spleen, brain and kidneys of NZB/W-F1 mice, 277, 6 and 8 DEG at the pre-puberty vs pre-autoimmunity stage; 212, 6 and 8 DEG at the nephritic vs pre-puberty stage; and 15, 6 and 2 DEG at the nephritic vs pre-autoimmunity stage were identified, respectively. In the brain, kidneys and spleen, hierarchical clustering revealed 178, 1012 and 2105 genes respectively that were deregulated in at least 1 of 3 stages. Clusters were subjected to functional enrichment analysis. In the brain, genes were mainly downregulated and enriched for metabolic pathways (glycolysis/TCA cycle/Pentose-Phosphate pathway), whereas overexpressed genes were enriched for Jak/Stat signalling pathway. In kidneys, one of the biggest cluster points towards metabolic pathways, particularly to lipid metabolism. In the spleen, DEG were mainly overexpressed. Of note, early genes were particularly enriched in cell-cycle processes; intermediate genes in membrane-related and extracellular matrix functions; and late genes in inflammatory and immune response pathways. To investigate SLE genetic susceptibility, a comparative analysis of the same organ of lupus-prone vs healthy mice at different stages of the disease was performed. In addition to immune response pathways (interferon signalling/antigen-presentation), DEG were involved in canonical pathways such as the phagosome, platelet activation, epithelial adherence junction signalling and the extrinsic prothrombin activation pathway.
Conclusions We identified novel stage-specific tissue-dependent pathways involved in immune response, and tissue injury and response in SLE. Validation is in progress.
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