Application of Next Generation Sequencing (RNA-seq and miRNA-seq) to study the molecular signature of Aluminium hydroxide adjuvant in ovine peripheral blood mononuclear cells (PBMCs) [RNA-Seq]

We report the application of RNA sequencing technology for high-throughput profiling of the aluminium hydroxide adjuvant mechanism of action in an in vivo experiment on sheep. We mapped about 52 million sequence reads per sample to the sheep genome (build Oar3.1) and identified 21,274 genes in the PBMCs of sheep treated with commercial vaccines or with the adjuvant alone. A total of 2473, 2980 and 429 differentialy expressed genes were identified in the vaccine tf VS vaccine t0, adjuvant tf VS adjuvant t0 and adjuvant tf VS vaccine tf comparisons, respectively. Previously reported genes related to alterations caused by aluminium adjuvant were found differentially expressed, namely: NLRP3, IL1B, IL8, TNF, NFKB2, RELA and RELB. In addition, most of the genes related to apoptosis were up-regulated in both group after treatment. In contrast, other genes related to immune response, inflammatory response and cell-cell signalling were up-regulated in Vac-injected sheep and whereas down-regulated in Adj-injected animals. Taken together, our analysis provides characterization of alterations in the transcriptome caused by the aluminium adjuvant and identifies increased expression of inflammatory cytokines, NF-KB family genes and apoptotic genes. Overall design: PBMCs Total RNA profiles of six age-matched Rasa sheep before and after inoculation (t0 and tf) of nine different commercial vaccines based of aluminium hydroxide (the vaccine group composed of 3 animals) or with the aluminium adjuvant alone in an equivalent dose (the adjuvant group composed of 3 animals) were generated by deep sequencing on an Illumina HiSeq2000 sequencer. In total, the study is composed of three biological replicates per group and thirteen libraries were sequenced (there was a tecnical replicate of a sample at the end of the experiment and two other samples where sequenced in two different sequencing runs).