Identification of new CpG oligodeoxynucleotide motifs that induce expression of interleukin-1β and nitric oxide in avian macrophages

Unmethylated CpG motifs are known to stimulate mammalian toll-like receptor-9 expressing cells such as macrophages. However, the magnitude of immune-stimulation by CpG-motif can be sequence- and host-specific, implying the importance of identifying new immune-stimulatory motifs. This study aimed to determine the frequency distribution of 256 unique hexamers CpG-motifs in the Salmonella genome and to characterize their immune-stimulatory activity in avian host. We synthesized 256 CpG oligodeoxynucleotides (CpG-ODNs) each containing triplicates of a unique hexamer CpG-motif and tested their ability to induce expression of pro-inflammatory cytokine IL-1β in avian macrophages using q-RT PCR in four rounds of screening assays. CpG-ODNs that induced significantly higher IL-1β expression were also subjected to Griess assay to determine their ability to induce nitric oxide (NO) production in avian macrophages. This analysis resulted in identification of 7 CpG-ODNs that consistently induced IL-1β expression and NO production in avian macrophages at a level similar to the expression achieved using commercially available PTO-CpG-ODN 2007 and LPS derived from Salmonella. To the best of our knowledge, this is the first report showing comprehensive screening of all possible unique CpG hexamer (n=256) motifs for their ability to induce IL-1β expression and NO production in avian macrophages. We also show that the newly identified CpG-motifs with high immune-stimulatory activity are widely distributed in Salmonella genome. The CpG-ODNs identified in this study may serve as promising immunoprophylactics to potentiate innate responses in chickens against Salmonella and other infectious agents.