A role for non-coding RNAs and alternative splicing in the regulation of neutrophil activation and gene expression in systemic lupus erythematosus

Abstract Excess neutrophil apoptosis and the release of neutrophil extracellular traps (NETs) in systemic lupus erythematosus (SLE) leads to accumulation of cell debris and production of auto-antibodies targeting nuclear proteins and DNA. SLE neutrophil activation is regulated by changes in gene expression, notably expression of type-I interferon-response genes and genes coding for granule proteins. This observational study measured both mRNA and small non-coding RNAs in SLE (n = 11) and healthy control (HC, n = 10) ultra-pure blood neutrophils to identify changes in expression that are involved in regulating neutrophil phenotype. Using RNAseq, we identified significant differential expression (DE) of 69 microRNAs, 63 other small non-coding RNAs, 236 piwiRNAs and 83 tRNA fragments in SLE neutrophils compared to HC (false discovery rate (FDR) adj. p < 0.05). We also identified 78 significant alternative splicing events across 64 genes (FDR adj. p < 0.05, Δpercent spliced in (PSI) > 0.1 or < -0.1). Bioinformatic analysis of miRNA:mRNA DE genes predicted significant activation of autophagy, neutrophil degranulation, interferon alpha/beta signalling, and apoptosis pathways in SLE neutrophils. Translation and mRNA processing were predicted to be down-regulated. microRNAs implicated in NETs production were miR-155-5p, miR-146a-5p and miR-let-7b-5p (FDR adj. p < 0.05). SNORD89 was identified as a potential promoter of apoptosis in SLE neutrophils, along with alternative splicing of apoptosis genes myeloid cell leukaemia-1 (MCL1), caspase-8 (CASP8) and death-associated protein kinase-2 (DAPK2) (FDR adj. p < 0.05). Our study describes for the first time, dysregulated expression of small non-coding RNAs in SLE neutrophils and proposes non-coding RNA and alternative gene splicing as regulators of neutrophil-driven disease pathology in SLE.