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Research Article

volume 52 issue 6 (june 2018) : 851-857,   Doi: 10.18805/ijar.B-3290

Molecular characterization of duck (Anas platyrynhos) Toll-like receptors, mRNA expressions profile in day-old duckling’s tissues and cytokine response to in vitro TLR agonsists stimulation

T
T.R. Kannaki
P
P.C. Verma
M
M. R. Reddy
M
M. Shanmugam
1Directorate of Poultry Research, Rajendranagar, Hyderabad-500 030, Telangana, India.
Cite article:- Kannaki T.R., Verma P.C., Reddy R. M., Shanmugam M. (2017). Molecular characterization of duck (Anas platyrynhos) Toll-like receptors, mRNA expressions profile in day-old duckling’s tissues and cytokine response to in vitro TLR agonsists stimulation. Indian Journal of Animal Research. 52(6): 851-857. doi: 10.18805/ijar.B-3290.

ABSTRACT

TLR repertoire of duck, profiling of their mRNA expression in a range of duckling tissues and cytokine gene expressions upon TLR agonists stimulation in in vitro assay have been investigated. All ten TLR genes orthologous to chicken TLR repertoire were found in duck. Duck TLR genes showed 77-83% similarity at amino acid level to their chicken counterparts. All ten TLRs-TLR1LA, 1LB, 2A, 2B, 3, 4, 5, 7, 15 and 21 mRNA expressions were significantly higher in bursa than other tissues studied, whereas in muscle all TLRs mRNA expressions were significantly lower except for TLR15 (P<0.01). TLR7 gene expression was significantly higher in spleen, bursa and also in lung tissues (P<0.01). The cytokine gene expression levels in duck PBMCs upon LPS and poly I:C stimulation have been quantified. IL-1g gene expression level in LPS stimulated duck PBMC culture was significantly higher at both 12 h and 24 h time intervals (P<0.05). However, there were no significant changes in IFN-ã gene expression levels in poly I:C stimulated duck PBMC culture at both the intervals. TLR gene expression in young ducklings together with cytokine response upon LPS stimulation demonstrates the innate preparedness of younger birds to encounter pathogens and their functional ability to respond to their ligands.

REFERENCES

  1. Adams SC, Xing Z, Li J and Cardona CJ ( 2009) Immune-related gene expression in response to H11N9 low pathogenic avian influenza virus infection in chicken and Pekin duck peripheral blood mononuclear cells. Mol Immunol 46(8-9):1744-9
  2. Brownlie R, Zhu J, Allan B, Mutwiri GK, Babiuk LA, Potter A and Griebel P (2009) Chicken TLR21 acts as a functional homologue to mammalian TLR9 in the recognition of CpG oligodeoxynucleotides. Mol Immunol 46(15):3163–3170
  3. Cheng Y, Sun Y, Wang H, Shi S, Yan Y, Li J, Ding C and Sun J (2015) Cloning, expression and functional analysis of the duck Toll-like receptor 5 (TLR5) gene. Vet Sci 16 :37-46 
  4. Cormican P, Lloyd AT, Downing T, Connell SJ, Bradley D and O’Farrelly C (2009) The avian Toll-like receptor pathway– subtle differences amidst general conformity. Dev Comp Immunol 33: 967–973
  5. Ewald SE, Lee BL, Lau L, Wickliffe KE, Shi GP, Chapman HA and Barton GM (2008) The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature 456:658-662
  6. Gomis S, Babiuk L, Allan B, Willson P, Waters E, Ambrose N, Hecker R and Potter A (2004) Protection of neonatal chicks against a lethal challenge of Escherichia coli using DNA containing cytosine-phosphodiester-guanine motifs. Avian Dis 48: 813–822
  7. Gomis S, Babiuk L, Godson DL, Allan B, Thrush T, Townsend H, Willson P, Waters E, Hecker R and Potter A (2003) Protection of chickens against Escherichia coli infections by DNA containing CpG motifs. Infect Immun 71: 857–863
  8. Guan Y, Chen H, Li K, Riley S, Leung G, Webster R, Peiris J and Yuen K (2007) A model to control the epidemic of H5N1 influenza at the source. BMC Infect Dis 7: 132
  9. He H, Genovese KJ, Nisbet DJ and Kogut MH (2007) Synergy of CpG oligodeoxynucleotide and double-stranded RNA (poly I:C) on nitric oxide induction in chicken peripheral blood monocytes. Mol Immunol 44: 3234–3242
  10. He H, Genovese KJ, Nisbet DJ and Kogut MH (2006) Profile of Toll-like receptor expressions and induction of nitric oxide synthesis by Toll-like receptor agonists in chicken monocytes. Mol Immunol 43:783-9
  11. Higgs R, Cormican P, Cahalane S, Allan B, Lloyd AT, Meade K, James T, Lynn DJ, Babiuk LA and O’Farrelly C (2006) Induction of a novel chicken Toll-like receptor following Salmonella enteric serovar Typhimurium infection. Infect Immun 74: 1692–1698
  12. Huang Y, Yingrui Li, David W Burt, Hualan Chen and Yong Zhang et.al (2013) The duck genome and transcriptome provide insight into an avian influenza virus reservoir species Nature Genetics 45: 776–783 
  13. Iqbal M, Philbin VJ and Smith AL (2005) Expression patterns of chicken Toll-like receptor mRNA in tissues, immune cell subsets and cell lines. Vet Immunol Immunopathol 104: 117-127
  14. Jia H, Li G, Li J, Tian Y, Wang D, Shen J, Tao Z, Xu J and Lu L(2012) Cloning expression and bioinformatics analysis of the duck TLR 4 gene. Br Poult Sci 53:190-7 
  15. Kannaki TR, Reddy MR, Verma PC and Murugesan S (2012) Expression analysis of turkey (Meleagris gallopavo) toll-like receptors and molecular characterization of avian specific TLR15. Mol Biol Rep 39: 8539-49 
  16. Keestra AM, de Zoete MR, Bouwman LI and van Putten JP (2010) Chicken TLR21 is an innate CpG DNA receptor distinct from mammalian TLR9. J Immunol 185: 460-467 
  17. Kogut MH, Genovese KJ and He H (2007) Flagellin and lipopolysaccharide stimulate the MEK-ERK signaling pathway in chicken heterophils through differential activation of the small GTPases, Ras and Rap1. Mol Immunol 44:1729-36
  18. Kogut MH, Iqbal M, He H, Philbin V, Kaiser P and Smith A (2005) Expression and function of Toll-like receptors in chicken heterophils. Dev Comp Immunol 29: 791-807
  19. Kogut MH, Swaggerty C, He H, Pevzner I and Kaiser P (2006) Toll-like receptor agonists stimulate differential functional activation and cytokine and chemokine gene expression in heterophils isolated from chickens with differential innate responses. Microbes Infect 8:1866-74
  20. Kolluri G, Ramamurthy N, Churchil RR, Dhinakar Raj G and Kannaki TR (2014) Influence of age, sex and rearing systems on Toll-like receptor 7 (TLR7) expression pattern in gut, lung and lymphoid tissues of indigenous ducks. Br Poult Sci 55: 59-67
  21. Livak KJ and Schmittgen TD(2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408
  22. MacDonald MR, Xia J, Smith AL and Magor KE (2008) The duck toll like receptor 7: genomic organization, expression and function. Mol Immunol 45: 2055–2061
  23. MacKinnon KM, He H, Nerren JR, Swaggerty CL, Genovese KJ and Kogut MH (2009) Expression profile of toll-like receptors within the gastrointestinal tract of 2-day-old Salmonella enteriditis-infected broiler chickens. Vet Microbiol 137: 313-319
  24. Nerren JR, He H, Genovese K and Kogut MH (2010) Expression of the avian-specific toll-like receptor 15 in chicken heterophils is mediated by Gram-negative and Gram-positive bacteria, but not TLR agonists. Vet Immunol Immunopathol 136: 151–156
  25. Nerren JR, Swaggerty CL, MacKinnon KM, Genovese KJ, He H, Pevzne I and Kogut MH (2009) Differential mRNA expression of the avian-specific toll-like receptor 15 between heterophils from Salmonella-susceptible and -resistant chickens. Immunogenetics 61: 71–77
  26. Nishimura M and Naito S (2005) Tissue-specific mRNA expression profiles of human toll-like receptors and related genes. Biol Pharm Bull 28:886-92
  27. Peter ME, Kubarenko AV, Weber AN and Dalpke AH (2009) Identification of an N-terminal recognition site in TLR9 that contributes to CpG-DNA-mediated receptor activation. J Immunol 182: 7690-7697
  28. Philbin VJ, Iqbal M, Boyd Y, Goodchild MJ, Beal RK, Bumstead N, Young J and Smith AL (2005) Identification and characterization of a functional, alternatively spliced Toll-like receptor 7 (TLR7) and genomic disruption of TLR8 in chickens. Immunology 114: 507– 521
  29. Roach JC, Glusman G, Rowen L, Kaur A, Purcell MK, Smith KD, Hood LE and Aderem A (2005) The evolution of vertebrate Toll-like receptors. Proc Natl Acad Sci USA 102: 9577–9582
  30. Schultz J, Milpetz F, Bork P and Ponting CP (1998) SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci USA 95: 5857–5864
  31. Taghavi A, Allan B, Mutwiri G, Van Kessel A, Willson P, Babiuk L, Potter A and Gomis S (2008) Protection of neonatal broiler chicks against Salmonella Typhimurium septicemia by DNA containing CpG motifs. Avi Dis 52: 398–406
  32. Takeda K, Akira S (2005) Toll-like receptors in innate immunity. Int Immunol 17: 1-14 Takeda K, Kaisho T and Akira S (2003) Toll-    like receptors. Annu Rev Immunol 21: 335-376 
  33. Takeda K, Kaisho T and Akira S (2003) Toll-like receptors. Annu Rev Immunol 21: 335-376
  34. Temperley ND., Berlin, S., Paton, I.R., Griffin, D.K., Burt, D.W., 2008. Evolution of the chicken Toll-like receptor gene family: a story of gene gain and gene loss. BMC Genomics 9, 62.
  35. Vinkler M, Bryjová A, Albrecht T and Bryja J (2009) Identification of the first Toll-like receptor gene in passerine birds: TLR4 orthologue in zebra finch (Taeniopygia guttata). Tissue Antigens 74: 32-41
  36. Webster RG, Bean WJ, Gorman OT, Chambers TM and Kawaoka Y (1992) Evolution and ecology of influenza A viruses. Microbiol Rev 56:152–179 
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Indian Journal of Animal Research
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    Research Article

    volume 52 issue 6 (june 2018) : 851-857,   Doi: 10.18805/ijar.B-3290

    Molecular characterization of duck (Anas platyrynhos) Toll-like receptors, mRNA expressions profile in day-old duckling’s tissues and cytokine response to in vitro TLR agonsists stimulation

    T
    T.R. Kannaki
    P
    P.C. Verma
    M
    M. R. Reddy
    M
    M. Shanmugam
    1Directorate of Poultry Research, Rajendranagar, Hyderabad-500 030, Telangana, India.
    Cite article:- Kannaki T.R., Verma P.C., Reddy R. M., Shanmugam M. (2017). Molecular characterization of duck (Anas platyrynhos) Toll-like receptors, mRNA expressions profile in day-old duckling&rsquo;s tissues and cytokine response to in vitro TLR agonsists stimulation. Indian Journal of Animal Research. 52(6): 851-857. doi: 10.18805/ijar.B-3290.

    ABSTRACT

    TLR repertoire of duck, profiling of their mRNA expression in a range of duckling tissues and cytokine gene expressions upon TLR agonists stimulation in in vitro assay have been investigated. All ten TLR genes orthologous to chicken TLR repertoire were found in duck. Duck TLR genes showed 77-83% similarity at amino acid level to their chicken counterparts. All ten TLRs-TLR1LA, 1LB, 2A, 2B, 3, 4, 5, 7, 15 and 21 mRNA expressions were significantly higher in bursa than other tissues studied, whereas in muscle all TLRs mRNA expressions were significantly lower except for TLR15 (P<0.01). TLR7 gene expression was significantly higher in spleen, bursa and also in lung tissues (P<0.01). The cytokine gene expression levels in duck PBMCs upon LPS and poly I:C stimulation have been quantified. IL-1g gene expression level in LPS stimulated duck PBMC culture was significantly higher at both 12 h and 24 h time intervals (P<0.05). However, there were no significant changes in IFN-ã gene expression levels in poly I:C stimulated duck PBMC culture at both the intervals. TLR gene expression in young ducklings together with cytokine response upon LPS stimulation demonstrates the innate preparedness of younger birds to encounter pathogens and their functional ability to respond to their ligands.

    REFERENCES

    1. Adams SC, Xing Z, Li J and Cardona CJ ( 2009) Immune-related gene expression in response to H11N9 low pathogenic avian influenza virus infection in chicken and Pekin duck peripheral blood mononuclear cells. Mol Immunol 46(8-9):1744-9
    2. Brownlie R, Zhu J, Allan B, Mutwiri GK, Babiuk LA, Potter A and Griebel P (2009) Chicken TLR21 acts as a functional homologue to mammalian TLR9 in the recognition of CpG oligodeoxynucleotides. Mol Immunol 46(15):3163–3170
    3. Cheng Y, Sun Y, Wang H, Shi S, Yan Y, Li J, Ding C and Sun J (2015) Cloning, expression and functional analysis of the duck Toll-like receptor 5 (TLR5) gene. Vet Sci 16 :37-46 
    4. Cormican P, Lloyd AT, Downing T, Connell SJ, Bradley D and O’Farrelly C (2009) The avian Toll-like receptor pathway– subtle differences amidst general conformity. Dev Comp Immunol 33: 967–973
    5. Ewald SE, Lee BL, Lau L, Wickliffe KE, Shi GP, Chapman HA and Barton GM (2008) The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature 456:658-662
    6. Gomis S, Babiuk L, Allan B, Willson P, Waters E, Ambrose N, Hecker R and Potter A (2004) Protection of neonatal chicks against a lethal challenge of Escherichia coli using DNA containing cytosine-phosphodiester-guanine motifs. Avian Dis 48: 813–822
    7. Gomis S, Babiuk L, Godson DL, Allan B, Thrush T, Townsend H, Willson P, Waters E, Hecker R and Potter A (2003) Protection of chickens against Escherichia coli infections by DNA containing CpG motifs. Infect Immun 71: 857–863
    8. Guan Y, Chen H, Li K, Riley S, Leung G, Webster R, Peiris J and Yuen K (2007) A model to control the epidemic of H5N1 influenza at the source. BMC Infect Dis 7: 132
    9. He H, Genovese KJ, Nisbet DJ and Kogut MH (2007) Synergy of CpG oligodeoxynucleotide and double-stranded RNA (poly I:C) on nitric oxide induction in chicken peripheral blood monocytes. Mol Immunol 44: 3234–3242
    10. He H, Genovese KJ, Nisbet DJ and Kogut MH (2006) Profile of Toll-like receptor expressions and induction of nitric oxide synthesis by Toll-like receptor agonists in chicken monocytes. Mol Immunol 43:783-9
    11. Higgs R, Cormican P, Cahalane S, Allan B, Lloyd AT, Meade K, James T, Lynn DJ, Babiuk LA and O’Farrelly C (2006) Induction of a novel chicken Toll-like receptor following Salmonella enteric serovar Typhimurium infection. Infect Immun 74: 1692–1698
    12. Huang Y, Yingrui Li, David W Burt, Hualan Chen and Yong Zhang et.al (2013) The duck genome and transcriptome provide insight into an avian influenza virus reservoir species Nature Genetics 45: 776–783 
    13. Iqbal M, Philbin VJ and Smith AL (2005) Expression patterns of chicken Toll-like receptor mRNA in tissues, immune cell subsets and cell lines. Vet Immunol Immunopathol 104: 117-127
    14. Jia H, Li G, Li J, Tian Y, Wang D, Shen J, Tao Z, Xu J and Lu L(2012) Cloning expression and bioinformatics analysis of the duck TLR 4 gene. Br Poult Sci 53:190-7 
    15. Kannaki TR, Reddy MR, Verma PC and Murugesan S (2012) Expression analysis of turkey (Meleagris gallopavo) toll-like receptors and molecular characterization of avian specific TLR15. Mol Biol Rep 39: 8539-49 
    16. Keestra AM, de Zoete MR, Bouwman LI and van Putten JP (2010) Chicken TLR21 is an innate CpG DNA receptor distinct from mammalian TLR9. J Immunol 185: 460-467 
    17. Kogut MH, Genovese KJ and He H (2007) Flagellin and lipopolysaccharide stimulate the MEK-ERK signaling pathway in chicken heterophils through differential activation of the small GTPases, Ras and Rap1. Mol Immunol 44:1729-36
    18. Kogut MH, Iqbal M, He H, Philbin V, Kaiser P and Smith A (2005) Expression and function of Toll-like receptors in chicken heterophils. Dev Comp Immunol 29: 791-807
    19. Kogut MH, Swaggerty C, He H, Pevzner I and Kaiser P (2006) Toll-like receptor agonists stimulate differential functional activation and cytokine and chemokine gene expression in heterophils isolated from chickens with differential innate responses. Microbes Infect 8:1866-74
    20. Kolluri G, Ramamurthy N, Churchil RR, Dhinakar Raj G and Kannaki TR (2014) Influence of age, sex and rearing systems on Toll-like receptor 7 (TLR7) expression pattern in gut, lung and lymphoid tissues of indigenous ducks. Br Poult Sci 55: 59-67
    21. Livak KJ and Schmittgen TD(2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408
    22. MacDonald MR, Xia J, Smith AL and Magor KE (2008) The duck toll like receptor 7: genomic organization, expression and function. Mol Immunol 45: 2055–2061
    23. MacKinnon KM, He H, Nerren JR, Swaggerty CL, Genovese KJ and Kogut MH (2009) Expression profile of toll-like receptors within the gastrointestinal tract of 2-day-old Salmonella enteriditis-infected broiler chickens. Vet Microbiol 137: 313-319
    24. Nerren JR, He H, Genovese K and Kogut MH (2010) Expression of the avian-specific toll-like receptor 15 in chicken heterophils is mediated by Gram-negative and Gram-positive bacteria, but not TLR agonists. Vet Immunol Immunopathol 136: 151–156
    25. Nerren JR, Swaggerty CL, MacKinnon KM, Genovese KJ, He H, Pevzne I and Kogut MH (2009) Differential mRNA expression of the avian-specific toll-like receptor 15 between heterophils from Salmonella-susceptible and -resistant chickens. Immunogenetics 61: 71–77
    26. Nishimura M and Naito S (2005) Tissue-specific mRNA expression profiles of human toll-like receptors and related genes. Biol Pharm Bull 28:886-92
    27. Peter ME, Kubarenko AV, Weber AN and Dalpke AH (2009) Identification of an N-terminal recognition site in TLR9 that contributes to CpG-DNA-mediated receptor activation. J Immunol 182: 7690-7697
    28. Philbin VJ, Iqbal M, Boyd Y, Goodchild MJ, Beal RK, Bumstead N, Young J and Smith AL (2005) Identification and characterization of a functional, alternatively spliced Toll-like receptor 7 (TLR7) and genomic disruption of TLR8 in chickens. Immunology 114: 507– 521
    29. Roach JC, Glusman G, Rowen L, Kaur A, Purcell MK, Smith KD, Hood LE and Aderem A (2005) The evolution of vertebrate Toll-like receptors. Proc Natl Acad Sci USA 102: 9577–9582
    30. Schultz J, Milpetz F, Bork P and Ponting CP (1998) SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci USA 95: 5857–5864
    31. Taghavi A, Allan B, Mutwiri G, Van Kessel A, Willson P, Babiuk L, Potter A and Gomis S (2008) Protection of neonatal broiler chicks against Salmonella Typhimurium septicemia by DNA containing CpG motifs. Avi Dis 52: 398–406
    32. Takeda K, Akira S (2005) Toll-like receptors in innate immunity. Int Immunol 17: 1-14 Takeda K, Kaisho T and Akira S (2003) Toll-    like receptors. Annu Rev Immunol 21: 335-376 
    33. Takeda K, Kaisho T and Akira S (2003) Toll-like receptors. Annu Rev Immunol 21: 335-376
    34. Temperley ND., Berlin, S., Paton, I.R., Griffin, D.K., Burt, D.W., 2008. Evolution of the chicken Toll-like receptor gene family: a story of gene gain and gene loss. BMC Genomics 9, 62.
    35. Vinkler M, Bryjová A, Albrecht T and Bryja J (2009) Identification of the first Toll-like receptor gene in passerine birds: TLR4 orthologue in zebra finch (Taeniopygia guttata). Tissue Antigens 74: 32-41
    36. Webster RG, Bean WJ, Gorman OT, Chambers TM and Kawaoka Y (1992) Evolution and ecology of influenza A viruses. Microbiol Rev 56:152–179 
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