Systemic Lupus Erythematosus (SLE) is a debilitating disease primarily in women involving complex T and B cell dysregulation. SLE presents with dysfunctional HDL and we have previously found that HDL-microRNAs (miRNA) are significantly altered in SLE; however, miRNAs are just one of many types of small non-coding RNAs (sRNA). As such, we hypothesized that HDL-sRNA cargo and cell-to-cell communication in SLE extend beyond miRNAs. Using high-throughput sRNA sequencing (sRNA-seq), we found that tRNA-derived sRNAs (tDRs) were highly abundant on HDL and were significantly altered in SLE subjects (n=9) compared to controls (n=8,
P
<0.05). In addition, circulating levels of angiogenin, an RNaseIII enzyme responsible for tDR cleavage from parent tRNAs, was also found to be significantly increased in plasma (
P
<0.05) from SLE subjects compared to controls. To determine if tDRs are altered in CD4+ T cells in SLE subjects, real-time PCR was used to quantify candidate tDRs, and we found that tDR-GlyGCC levels were significantly increased 4.2-fold in SLE (
P
<0.01) and readily exported to HDL. Strikingly, total RNAseq,
in silico
analysis, and mRNA sequencing suggested that ROCK2, a critical regulator of CD4+ T cell differentiation, is a direct tDR-GlyGCC target gene which was confirmed with gene reported (luciferase) assays. Moreover, activated human CD4+ T cells transfected with tDR-GlyGCC mimetics, demonstrated reduced ROCK2 protein levels and STAT3 phosphorylation, and consequently reduced inflammatory cytokine secretion (IL-17 and IL-21;
P
<0.05). To determine if T cell exported tDR-GlyGCC is transferred between cells by HDL,
ex vivo
studies were completed using Trans-PhotoActivatable-Ribonucleoside-CrossLinking-ImmunoPrecipitation high-throughput Sequencing (Trans-PAR-CLIPseq). Using this approach, we found a cassette of CD4+ T cell-originating sRNAs, including tDR-GlyGCC, that were transferred by HDL to recipient immune cells. Here, we demonstrate that HDL facilitates intercellular transfer of tDRs between immune cells and a critical role for tDR-GlyGCC in regulating T cell signalling.
Expression of genes involved in the T cell signalling pathway in circulating immune cells of cattle 24 months following oral challenge with Bovine Amyloidotic Spongiform Encephalopathy (BASE)