Related Experiment Videos
Molecular cloning and functional characterization of chicken toll-like receptors. A single chicken toll covers
A Fukui1, N Inoue, M Matsumoto
1Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Nakamichi, Higashinari-ku, Osaka 537-8511, Japan.
Abstract:
Toll-like receptors (TLR) in the innate immune system have not been identified in non-mammalian vertebrates. Two types of TLR were cloned from a chicken bursa cDNA library using degenerate primers based on the consensus sequences of mouse and Drosophila Toll and designated as chicken TLR (chTLR) type 1 and type 2. Of the nine human TLRs reported to date, these chTLRs showed the highest homology to human TLR2. The extracellular regions of type 1 and type 2 contained a distinct approximately 200-amino acid stretch and were 45.3 and 46.3% homologous to that of human TLR2. The intracellular Toll/interleukin-1R homology domain of type 1 and type 2 was perfectly identical to each other and highly homologous (80.7%) to that of human TLR2. Both types were widely detected by reverse transcriptase-polymerase chain reaction and immunoblotting in various chicken organs, especially those rich in connective tissue. Both genes were mapped to chromosome 4q1.1, suggesting that they arose by gene duplication. By reporter gene assay, type 2 and to a lesser extent type 1, selectively signaled the presence of mycoplasma macrophage-activating lipopeptide-2/M161Ag in the human embryonic kidney 293 cell system. Cotransfection of type 2 and human CD14 or MD-2 into human embryonic kidney 293 cells allowed the response to Escherichia coli lipopolysaccharide (LPS), whereas type 1 did not signal LPS or any other microbial components tested. These results indicated that chTLR type 2 covers two major microbe patterns, lipoproteins and LPS, which are regulated by TLR2 and TLR4 in mammals. In oviparous animals, the duplicated TLRs in the pattern-recognition system may function for host-pathogen discrimination in a manner that is distinct from that in mammals.
Insights
Two chicken Toll-like receptors (TLR) were identified, showing homology to human TLR2. Chicken TLR type 2 recognizes lipoproteins and LPS, suggesting a distinct innate immune recognition system in birds.
Area of Science:
- Immunology
- Innate Immunity
- Comparative Genomics
Background:
- Toll-like receptors (TLR) are crucial for innate immunity but their presence in non-mammalian vertebrates is largely uncharacterized.
- Identifying TLRs in diverse species aids in understanding the evolution and conservation of immune responses.
Purpose of the Study:
- To identify and characterize Toll-like receptors (TLR) in chickens (Gallus gallus).
- To investigate the functional responses of identified chicken TLRs (chTLR) to microbial components.
- To compare the chTLR system with mammalian TLRs for insights into immune system evolution.
Main Methods:
- Cloning of chTLR type 1 and type 2 from chicken bursa cDNA using degenerate primers.
- Homology analysis comparing chTLRs to human TLRs.
- Detection of chTLR expression in various chicken organs via RT-PCR and immunoblotting.
- Reporter gene assays in human embryonic kidney 293 cells to assess functional responses to microbial ligands like lipoproteins and lipopolysaccharide (LPS).
Main Results:
- Two chTLR types were cloned, exhibiting significant homology to human TLR2, particularly in the intracellular Toll/interleukin-1R domain.
- chTLRs were widely expressed across chicken organs, with higher expression in connective tissues.
- Chicken TLR type 2, with co-receptors, responded to mycoplasma macrophage-activating lipopeptide-2 and Escherichia coli lipopolysaccharide (LPS).
- Chicken TLR type 1 showed a weaker response to lipopeptide but not LPS or other tested microbial components.
Conclusions:
- Chicken TLR type 2 recognizes both lipoproteins and LPS, analogous to mammalian TLR2 and TLR4, respectively.
- The duplicated TLR system in chickens may represent a distinct pattern-recognition mechanism for host-pathogen discrimination compared to mammals.
- These findings contribute to understanding the evolution of innate immunity in vertebrates.