Toll-Like Receptor 21 of Chicken and Duck Recognize a Broad Array of Immunostimulatory CpG-oligodeoxynucleotide Sequences

Abstract

CpG-oligodeoxynucleotides (CpG-ODNs) mimicking the function of microbial CpG-dideoxynucleotides containing DNA (CpG-DNA) are potent immune stimuli. The immunostimulatory activity and the species-specific activities of a CpG-ODN depend on its nucleotide sequence properties, including CpG-hexamer motif types, spacing between motifs, nucleotide sequence, and length. Toll-like receptor (TLR) 9 is the cellular receptor for CpG-ODNs in mammalian species, while TLR21 is the receptor in avian species. Mammalian cells lack TLR21, and avian cells lack TLR9; however, both TLRs are expressed in fish cells. While nucleotide sequence properties required for a CpG-ODN to strongly activate mammalian TLR9 and its species-specific activities to different mammalian TLR9s are better studied, CpG-ODN activation of TLR21 is not yet well investigated. Here we characterized chicken and duck TLR21s and investigated their activation by CpG-ODNs. Chicken and duck TLR21s contain 972 and 976 amino acid residues, respectively, and differ from TLR9s as they do not have an undefined region in their ectodomain. Cell-based TLR21 activation assays were established to investigate TLR21 activation by different CpG-ODNs. Unlike grouper TLR21, which was preferentially activated by CpG-ODN with a GTCGTT hexamer motif, chicken and duck TLR21s do not distinguish among different CpG-hexamer motifs. Additionally, these two poultry TLR21s were activated by CpG-ODNs with lengths ranging from 15 to 31 nucleotides and with different spacing between CpG-hexamer motifs. These suggested that compared to mammalian TLR9 and grouper TLR21, chicken and duck TLR21s have a broad CpG-ODN sequence recognition profile. Thus, they could also recognize a wide array of DNA-associated molecular patterns from microbes. Moreover, CpG-ODNs are being investigated as antimicrobial agents and as vaccine adjuvants for different species. This study revealed that there are more optimized CpG-ODNs that can be used in poultry farming as anti-infection agents compared to CpG-ODN choices available for other species.

The Evasion of Antiviral Innate Immunity by Chicken DNA Viruses

“…chTLR21 is mainly expressed in immune-relevant tissues. The bursa of the Fabricius and spleen express the highest level of chTLR21, while the lung and thymus have a modest expression (Chuang et al, 2020). chTLR21 expression could be induced by stimulation with CpG-ODN.…”

“…It has been predicted that chTLR21 has three domains: a cytoplasmic (TIR) domain, a single transmembrane region, and an extracellular domain containing 27 LRRs with cysteine-rich capping structures at both ends (Keestra et al, 2010). Compared to TLR21 of other species, chTLR21 has high similarities with TLR21 of Xenopus tropicalis (61%) and Takifugu rubripes (57%) and appears more distantly to murine TLR13 (47%; Chuang et al, 2020). As for hTLR9, there is only 38% similarity in aa between chTLR21 and hTLR9, while two aa residues, which are essential for the interaction between CpG DNA and TLR9 (Asp534 and Tyr536), appear to be conserved in chTLR21 (Rutz et al, 2004;Wei et al, 2009).…”

“…As for hTLR9, there is only 38% similarity in aa between chTLR21 and hTLR9, while two aa residues, which are essential for the interaction between CpG DNA and TLR9 (Asp534 and Tyr536), appear to be conserved in chTLR21 (Rutz et al, 2004;Wei et al, 2009). Cellular distribution analysis using confocal laser microscopy on transfected HEK293 cells has suggested that the co-expressed chTLR21-FLAG and hTLR9 have similar localization profiles inside the cells (Chuang et al, 2020). chTLR21 is mainly expressed in immune-relevant tissues.…”

“…Although chTLR21 acts as a functional homolog of mammalian TLR9, there are differences between chTLR21 and hTLR9 in ligand specificities. For example, hTLR9 prefers the CpG-DNA hexameric motifs GTCGTT, while various CpG-ODNs hexamer motifs can activate chTLR21 (Keestra et al, 2010;Chuang et al, 2020). A recent study further revealed that CpG-ODNs with lengths between 15 and 31 nucleotides and different spaces between CpG-hexamer motifs could be sensed by chTLR21 (Chuang et al, 2020).…”

“…Stimulation of HEK293 cells in which chTLR21 was transfected with CpG-ODNs resulted in NF-κB activation and subsequently IL-1β and IL-6 mRNA expressions. Using RNA interference (RNAi) technology has confirmed that MyD88 is dependent on TLR21 in response to CpG-ODN in HD11 macrophages (Keestra et al, 2010;Chuang et al, 2020). However, the exact molecular mechanism of chTLR21 activation and signal transduction remains to be explored further.…”