Abstract
TLR9 plays pivotal role in innate immune responses through upregulation of costimulatory molecules and induction of proinflammatory cytokines like type I interferons including interferon alpha (IFNA). The present study characterized IFNA cDNA and predicted protein sequences in goat and black buck. Response of the PBM cells to TLR9 agonist CpG ODN C and Phorbol Myristate Acetate (PMA) was evaluated by realtime PCR. IFNA coding sequences were amplified from leukocyte cDNA and cloned in pGEMT-easy vector for nucleotide sequencing. Sequence analysis revealed 570 bp, IFNA ORF encoding 189 amino acids in goat and black buck. Black buck and goat IFNA has 92.1% to 94.7% and 93% to 95.6% similarity at nucleotide level, 86.3% to 89.5% and 70.9% to 91.6% identity at amino acid level with other ruminants, respectively. Nonsynonymous substitutions exceeding synonymous substitutions indicated IFNA evolved through positive selection among ruminants. In spite of lower total leukocyte count, the innate immune cells like monocytes and neutrophils were more in black buck compared to goat. In addition, CpG ODN C-stimulated PBM cells revealed raised IFNA transcript in black buck than goat. These findings indicate sturdy genetically governed immune system in wild antelope black buck compared to domestic ruminant goat.
Highlights
TLR9 emerged as a robust pathogen recognition receptor of innate immune system in the present decade and is known to be present in the endosomal membrane of the Plasmacytoid dendritic cells [pDCs], macrophages, and Bcells where it functions as a known sensor for bacterial, viral, fungal, and parasitic foreign DNA [1]. pDCs have been proved as powerful natural Interferon alpha (IFNA) secreting cells in mouse, human, and porcine in response to bacterial/viral CpG motifs via TLR9 signaling [2,3,4]
Before going for the actual analysis of blood, total leukocyte count (TLC) and differential leukocyte count (DLC) ware performed in order to assess cellular status of their immune system
The recombinant plasmids were characterized by restriction enzyme digestion and nested PCR (140 bp), the nucleotide sequences of IFNA gene fragments were aligned to generate full-length ORF sequences (570 bp) similar to bovine IFNA, which were submitted to NCBI GenBank
Summary
TLR9 emerged as a robust pathogen recognition receptor of innate immune system in the present decade and is known to be present in the endosomal membrane of the Plasmacytoid dendritic cells [pDCs], macrophages, and Bcells where it functions as a known sensor for bacterial, viral, fungal, and parasitic foreign DNA [1]. pDCs have been proved as powerful natural Interferon alpha (IFNA) secreting cells in mouse, human, and porcine in response to bacterial/viral (unmethylated cytosine-guanine) CpG motifs via TLR9 signaling [2,3,4].Unmethylated CpG motifs or pathogen derived DNA rich in CpG motifs are known to be the cognate agonists for TLR9. TLR9 emerged as a robust pathogen recognition receptor of innate immune system in the present decade and is known to be present in the endosomal membrane of the Plasmacytoid dendritic cells [pDCs], macrophages, and Bcells where it functions as a known sensor for bacterial, viral, fungal, and parasitic foreign DNA [1]. PDCs have been proved as powerful natural Interferon alpha (IFNA) secreting cells in mouse, human, and porcine in response to bacterial/viral (unmethylated cytosine-guanine) CpG motifs via TLR9 signaling [2,3,4]. In response to TLR9 signaling, several downstream costimulatory molecules and cytokines like Type I interferons (IFN), IFN-γ, Interleukins IL1, IL6, IL10, IL12, and IL18, involved in evoking the adaptive immune system optimally against the invading pathogen, are produced. CpG (oligodeoxynucleotide) ODN is used as immunotherapeutic agents in clinical trials for cancer [7] and infectious diseases like influenza reviewed by [8] and parasitic diseases like Toxoplasmosis [9]
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