Molecular Cloning And Immune Functional Characterization Of Pigeon Toll-like Receptor 7

The innate immune system acts as the front line of defense against invading microorganisms by recognizing pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), and activates the downstream signaling pathways to induce the expression of inflammatory cytokines and type I interferon. Toll-like receptor 7 (TLR7), located in the cellular endosome, is responsible for recognizing viral single-stranded RNA and antiviral imidazoquinoline compounds, and leads to the activation of the innate immune responses, playing a fundamental role in the defense against viral infection. In contrast to mammalian TLR7 orthologs, little is known about the sequence variation and biological function of avian TLR7. Most researches about avian TLR7 were focused on chicken, duck, goose or other waterfowl and terrestrial birds, while little was reported about aerial bird like pigeon. In this study, we cloned and analyzed the pigeon TLR7 sequence, identified the key ligand-recognition sites, and investigated its function in the innate immune response, aimed at accumulating theoretical basis for further research about the structure and biological function of pigeon TLR7 as well as the innate immunity of birds.1. Molecular cloning, sequence analysis and expression profile of pigeon TLR7In this study, we cloned the TLR7 gene from King pigeon for the first time. The entire coding region of the TLR7 gene was successfully amplified by PCR using conserved primers overlaying the coding region. Sequencing results show the pigeon TLR7 open reading frame is 3144 bp and encodes a 1047-amino acid protein, consisting of a canonical TLR composition with a signal peptide sequence,15 leucine-rich repeat (LRR) domains, a leucine-rich repeat C-terminal (LRR-CT) domain, and an intracellular Toll-interleukin-1 receptor (TIR) domain. Prediction for protein interaction and tertiary structure showed that pigeon TLR7 intramembranous domain could form a typical "m" shaped homodimer. Prediction for glycosylation and phosphorylation sites suggested that pigeon TLR7 contains 18 potential N-glycosylation sites and 38 phosphorylation sites. The amino acid sequence of pigeon TLR7 shared 65.0% identity with human,62.2% with mouse,81.5% with chicken and 83.8% with duck. The tissue distribution of pigeon TLR7 analyzed by RT-PCR suggested that immune-associated tissues, especially the spleen and liver, have high level of TLR7 expression.2. Identification of the immune function and potential ligand-recognition sites of pigeon TLR7The eukaryotic expression plasmid pCMV-PiTLR7 was constructed and transfected to HEK293T cells. The expression of TLR7 was verified by Western blotting. HEK293T cells transfected with pigeon TLR7 plasmid responded to the agonist R848, indicating a functional TLR7 homolog. Amino acid-inserting modifications were found at position 15 of LRR2, LRR11, LRR13, and LRR14 and position 10 of LRR10. Mutated pigeon TLR7 fragments, in which the insertions were deleted respectively, were amplified with an overlap-PCR method, and inserted into the expression vector pCMV. The immune functions of the TLR7 mutants were determined with an NF-κB luciferase assay of transfected cells. The deletion of the insertions absolutely abolished TLR7-NF-κB signaling. Thus, the irregular LRR insertions were crucial for pigeon TLR7 signaling, which could be key interaction sites for the ligand recognition. With quantitative real-time PCR and sandwich enzyme-linked immunosorbent assay, we observed that stimulation with R848 failed to induce the expression of interleukin 8 (IL-8) in any of the mutant-TLR7-transfected cells, consistent with their lack of NF-κB activity. However, the expression of interferon α (IFN-α) and tumor necrosis factor α (TNF-α) was significantly upregulated in the De110IN10 and De114IN15 groups. Remarkably, the levels of pigeon TLR7 expression were significantly increased in all the TLR7-mutated groups. Therefore, we speculate that another part of the deficient TLR7 mediates the induction of IFN-a and TNF-a by increasing the expression of TLR7 as compensation. However, the increased expression of TLR7 in the Del11IN15 group failed to induce the production of IFN-α, IL-8, or TNF-α, indicating that a false compensation occurred when the crucial LRR insertion was deleted.3. Pigeon TLR7-mediated innate immune responses stimulated by TLR7 agonist and Newcastle disease virusFollowing R848 stimulation of pigeon peripheral blood mononuclear cells, the levels of IFN-γ, IL-6, IL-8, CCL5, and IL-10 mRNA, assessed using quantitative real-time PCR, were significantly up-regulated at 12 h and 24 h following stimulation. However, the expression level of pigeon TLR7 did not change much. After Newcastle disease virus vaccine strain LaSota inoculation and agonist R848 injection, the level of TLR7 mRNA in the spleen of pigeons increased significantly in the R848-injected group, but decreased in the LaSota-inoculated group at 3 day post-infection (d.p.i.). The mRNA levels of inflammatory cytokines and chemokines were significantly upregulated in both LaSota-inoculated and R848-injected groups, suggesting that the pigeon TLR7-mediated immune response was not associated with the expression of TLR7. Triggering pigeon TLR7 could contribute to the NF-κB activation, and lead to robust up-regulation of inflammatory cytokines and chemokines, suggesting its important role in the innate immunity against viral infection.