AB0143 Immunomodulation followed by quantitative transcriptional profiling to characterize the functional role of the sjÖgren's-associated ncrna ac092580.4
Authors: JA Ice, I Adrianto, A Rasmussen, M Joachims, A Johnston, C Montgomery, K Sivils, CJ Lessard
Journal: Annals of the Rheumatic Diseases (2017)

<h3>Background</h3> Despite concerted efforts to characterize dysregulated transcriptional responses observed in Sjögren9s syndrome and related autoimmune disorders (both in whole blood and target tissues), the functional roles of non-coding RNAs (ncRNAs), many of which have been identified as critical players in transcriptional regulation of disease, remain poorly defined. <h3>Objectives</h3> In the present study, we describe ongoing efforts to functionally characterize the upregulated ncRNA identified by RNA-seq and <i>in silico</i> approaches, <i>AC092580.4</i> (FC=2.54), which we hypothesize plays a role in T and NK cell responses. <h3>Methods</h3> To study the immunomodulation of the ncRNA <i>AC092580.4</i>, we carried out a time-course experiment (0–36 hrs) using either PMA/I (500x dil) or universal Type I Interferon. Relative gene expression changes were determined using the Livak method by qPCR using optimized primers for <i>GZMA</i> and <i>AC092580.4</i> normalized to <i>GAPD</i>. Healthy PBMCs were subjected to stimulation by PHA (1mg/mL; 3 days), PMA/I (500x dil; 3 days), or anti-CD3/CD28 (50uL/1x10<sup>6</sup>cells; 1 day). An average 150-bp RNA-seq reads were generated for each sample; alignment was carried out using STAR (hg38) and comparisions of stimulated vs unstimulated cells were done using DEseq. Pearson9s correlation (<i>r</i>) was calculated for all 3,748 differentially expressed (DE) transcripts to identify transcripts co-expressed with <i>AC092580.4</i>. <h3>Results</h3> Of the transcripts showing DE in our SS RNA-seq study, we identified 8 as having significantly correlated expression with <i>AC092580.4</i> in the SS<sup>Ro-</sup> expression matrix (<i>r</i>&gt;0.70 or &lt;-0.65). To understand the possible effects of immunomodulation on relevant cells, we stimulated HSB-2 cells with PMA/I at various time points and assessed <i>AC092580.4</i> expression by. We observed downregulation of <i>AC092580.4</i> and the co-expressed transcript <i>GZMA</i> by PMA/I (trough: 12–16hrs; FC=0.09) followed by slow recovery at 36hrs (FC=0.59). To characterize these transcriptional changes further, we performed RNA-seq using healthy PBMCs exposed to various T cell stimulants. We observed marked upregulation of both <i>AC092580.4</i> and <i>GZMA</i> at 24/36hrs by all stimulants (FC=4.89–5.98). Other transcripts showed variable responses. <i>CAV2</i> is upregulated by PMA/I, but downregulated by CD3/CD28 and PHA. Stimulation by PHA leads to upregulation of <i>CD3D</i> (FC=1.56) and <i>SNRPD1</i> (FC=3.28) with little change in <i>RPL36A</i> (FC=1.09). Stimulation by CD3/CD28 similarly leads to upregulation of <i>CD3D</i> (FC=2.85) and <i>SNRPD1</i> (FC=10.04), but clear downregulation of <i>RPL36A (</i>FC=0.64<i>)</i>. We assessed <i>AC092580.4</i> expression in HSB-2 cells exposed to I IFN and observed initial upregulation (6hrs, FC=1.46) followed by gradual downregulation (36hrs, FC=0.18). <h3>Conclusions</h3> In the present study, we have initiated stimulation studies with to understand the immune relevance of <i>AC092580.4</i> and co-expressed targets. <i>AC092580.4</i> shows transcriptional induction by potent inducers of T cell responses (PMA/I, PHA, CD3/CD28) but is downregulated by type I IFN. Transcripts showing co-expressed with <i>AC092580.4</i> by whole-blood RNA-seq show divergent expression patterns according to the specific stimulus, suggesting a complex regulatory network governing dysregulated T and NK responses. <h3>Disclosure of Interest</h3> None declared