Volume 5 Supplement 4
In silico analysis of candidate genes associated with humoral innate immune response in chicken
© Slawinska et al; licensee BioMed Central Ltd. 2011
Published: 3 June 2011
Production and function of natural antibodies (NAbs) constitutes an important mechanism of the humoral innate immunity in vertebrates. The level of NAbs in chicken is heritable and the genetic background has been partly investigated. However, to date the genetic determination of humoral innate immune response in avian species has not been fully described. The goal of this study was to propose a new set of candidate genes with a potential effect on the NAb phenotype for further SNP association study.
In silico analysis of positional and functional candidate genes covered 14 QTL regions associated with LPS, LTA & KLH NAbs and located on six chromosomes: GGA5, GGA6, GGA9, GGA14, GGA18 and GGAZ. The function of the genes was subsequently determined based on the NCBI, KEGG, Gene Ontology and InnateDB databases.
As a result, the core panel of 38 genes participating in metabolic pathways of innate immune response was proposed. Most of them were assigned to chromosomes: GGA14, GGA5, GGA6 and GGAZ (13, 9, 8 and 5 genes, respectively). These candidate genes encode proteins predicted to play a role in (i) proliferation, differentiation and function of B lymphocytes; (ii) TLR signalling pathway, and (iii) MAP signalling cascade.
Proposed set of candidate genes is recommended to be included in the follow-up studies to model genetic networks of innate humoral immune response in chicken.
Humoral innate immunity in vertebrates that establishes the first barrier against pathogens consists of two basic mechanisms – natural antibodies (NAbs) and complement system. Expanding the knowledge on this field of avian immunology might be of help to overcome the difficulties in poultry industry, struggling constantly with diseases outbreaks eg. Avian Influenza . In chicken, the level of NAbs proved to be heritable . However, the genetic determination of NAbs is not fully described as it lacks information on which genes can be considered as the regulators in the complicated network of NAbs creation and function. This study contributes to the discovery of genetic determination of humoral innate immunity as it lists the proposed positional and functional candidate genes that have the putative impact on the NAb phenotype.
Chromosomal regions for in silico candidate gene analysis were initially selected based on the location of the QTL associated with the NAb titres directed against LPS (lipopolysaccharide), LTA (lipoteichoic acid) and KLH (keyhole limpet hemocyanine) antigens in chicken. This step was performed based on results from two independent studies, i.e.
● Study 1 – LPS and LTA NAb QTL detection study ;
● Study 2 – LPS and LTA NAb QTL validation study; KLH NAb detection study (data not published).
Study 2 was carried out within a new chicken reference population, set-up as a F2 cross between commercially selected breed (WL, White Leghorn) and a Polish, unselected native chicken breed (GP, Green-legged Partridgelike). For a candidate gene analysis reported here, the chromosomal regions of interest included QTL associated with LPS and LTA NAb titres that had been detected in study 1 and consecutively validated in study 2 as well as QTL associated with KLH NAb titres that had been detected in study 2. These QTL were located in the following chicken chromosomes: GGA5, GGA6, GGA9, GGA14, GGA18 and GGAZ. The regions of interest were designated based on the physical location of the microsatellite markers flanking the QTLs. The list of candidate genes within the QTL regions was prepared based on NCBI database , and gene function was assessed with KEGG , InnateDB  and Gene Ontology . The genes meeting both the criteria, i.e. location within the QTL regions & function in innate immunity (including signalling pathways and B cell function) were listed in a panel of the candidate genes associated with humoral innate immune response.
Positional and functional candidate genes associated with innate humoral immune response
B cell linker
caspase recruitment domain family, member 11
BCR, TCR, NFκB
caspase 7, apoptosis-related cysteine peptidase
Regulation of NFκB activity
CD59 molecule, complement regulatory protein
T cell activation, complement system inhibition
T-cell antigen CD7 precursor
T cell activation, T and B cell interaction, component of mature T cells
Binding of proteins in cell membrane
class II, major histcompability complex, transactivator
LRR binding, MHCII transcription activation
chemokine (C-X-C motif) ligand 12
Leukocyte activation, T cell proliferation, chemotaxis
FAS (TNFRSF6)-associated via death domain
Apoptosis, NFκB cascade activation, early development of T cells
TNF receptor superfamily, member 6
TNFα, Fas, B and T cells
Ig production, immune response with (B cells) Homeostasis between B I T cells
fibroblast growth factor 10
TLR activation, inflammatory cytokine secretion (with APC)
fibroblast growth factor 8
MAPK cascade activation
v-fos FBJ murine osteosarcoma viral oncogene homolog
TLR, BCR, TCR, MAPK, JNK, IL
Synthesis of AP-1 transcription factor
immunoglobulin superfamily, mem. 6
B and T cells
Membrane receptor of T and B cells
interleukin 20 receptor beta
T and B cells proliferation and differentiation
interleukin 21 receptor
T and B cells proliferation and differentiation
interleukin 31 receptor A
MAPK, Jak-STAT, IL
MAPKKK cascade, cytokine and chemokine signal transduction, monocyte and macrophage differentiation
interleukin 4 receptor
T cells, IL
Th2 lymhocyte differentation, cytokine receptor
interleukin 6 signal transducer
Fragment of cytokine receptor complex
interleukin 9 receptor
Jak and STAT activation, cytokine receptor
Janus kinase 2
lipopolysaccharide induced TNF factor
Mitogen activated protein kinase kinase 3
MAPK, TLR, JNK, Fc, p38, TNFα, Jak-STAT, TRAIL
Mitogen activated protein kinase kinase 4
MAPK, TLR, Fas, JNK, Fc, TCR, Jak-STAT, TRAIL
MAP kinase activation, in response to different stimuli, survival signal for T cells
mitogen activated protein kinase kinase kinase 1
MAPK, TLR, Fas, JNK, Fc, p38, NFκB, TCR, BCR, INFγ, TRAIL, TNFα
Integration of enzyme fosforylation in response to different factors
mitogen-activated protein kinase kinase kinase 13
Activation of different MAP kinases
mitogen-activated protein kinase 8 interacting protein 3
MAPK and JNK integration
nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha
TLR, BCR, TCR, NFκB
programmed cell death 4 (neoplastic transformation inhibitor)
Negative JNK regulation, expression of the gene under control of T cells
recombination activating gene 2
B and T cells
B and T cells differentiation, gene conversion in Ig
retinol binding protein 4, plasma
Activation of Ig secretion
supressor of cytokine sygnalling 1
Inhibition of cytokine secretion & Jak-STAT cascade
Transcription factor 7-like 2
transforming growth factor, beta 3
MAPK, TGFβ, GPCR
MAPK activation, growth factor activity
TNF receptor superfamily, member 13B
AP-1, NFκB, TNF
Key role in humoral immune response
TNF receptor-associated factor 6
TNF, TLR, IL, NFκB, TCR
Signal transduction in many pathways, Th1 immune response, T cell activation
TNF receptor-associated factor 7
MAPKKK cascade activation
It can be summarized that these candidate genes encode proteins predicted to play a role in:
(i) Proliferation, differentiation and function of B lymphocytes, e.g. CXCL12, BLNK, IL21R, RBP4, CD59, TNFRSF13B;
(ii) TLR signalling pathway, e.g. TRAF6, FADD, NFκBIA, CARD11, FAS, FGF8, TGFB, IL31RA;
(iii) MAP signalling cascade, e.g. MAP2K3, MAP2K4, MAP3K1, MAP3K13, MAPK8IP3.
Immune response is a complicated process; encoded by multiple genes organized within the frames of functional networks rather than pathways and regulated by many interactions. However, prior to modelling the most probable genetic network, the information is needed on the genes that can be taken into account and their physiological function.
As mentioned above, the function of the proposed set of candidate genes was associated with three groups of cellular and physiological processes that can hypothetically affect innate humoral immune response in chicken. Briefly, production of antibodies, including NAbs takes place in B cells, stimulated by Th2 cytokines. Therefore, both B and T cells function is a crucial element in antibody release. CXCL12 gene is responsible for B cells proliferation . CXCL12-/- knockout mice produced drastically reduced number of B cells and died during the perinatal period . In turn, BLNK gene affects B cell development, which was completely inhibited in BLNK-/- knockout mouse . Finally, IL21R and RBP4 genes are responsible for maintenance of mature B cells function. Knocked out mice (both IL21R-/- and RBP4-/-) expressed impaired production of antibodies [11, 12].
TLR signalling pathway is triggered when molecular patterns (such as LPS or LTA) are recognized. Some of the proposed candidate genes are involved in TLR pathway, just to mention TRAF6 and FADD, as well as genes affecting NFκB expression and function, such as NFκBIA, CARD11, TNFRSF13B and FAS[13–15]. Furthermore, the analysis in silico pointed out a number of genes that activate MAPK cascade, a key signalling pathway initiated by TLR, for example FGF8, TGFB3 and IL31RA. Additionally, the candidate gene set includes such genes as MAP2K3, MAPK8IP3, MAP3K13, MAP2K4 and MAP3K1, which are the members of MAPK signal transduction pathway .
Chicken immune response is one of the major areas recently studied in life science research related to livestock. So far, different approaches have been applied to dissect the genetic bases of avian health traits. Rapid development of technology supporting high-throughput genomic studies provided an excellent tool for fast and efficient genotyping. Still, the accurate gene selection can pose a problem. Therefore, the additional criteria, like validated QTL regions may be of assistance to list the proper genes that can be further on evaluated and contribute to genetic network modelling of humoral immune response in chicken. For that reason we proposed a panel of candidate genes related to the level of LPS, LTA & KLH NAbs in chicken.
The study supported by the State Committee for Scientific Research (grant no. P 06D 012 30) and by the Integrated Regional Development Programme (grant no. SPS.IV-3040-UE/S05/2009)
This article has been published as part of BMC Proceedings Volume 5 Supplement 4, 2011: Proceedings of the International Symposium on Animal Genomics for Animal Health (AGAH 2010). The full contents of the supplement are available online at http://www.biomedcentral.com/1753-6561/5?issue=S4.
- Gauthier-Clerc M, Lebarbenchon C, Thomas F: Recent expansion of highly pathogenic avian influenza H5N1: a critical review. Int. J. Avian Sci. 2007, 149: 201-214.Google Scholar
- Parmentier HK, Lammers A, Hoekman JJ, de V Reilingh G, Zaanen ITA, Savelkoul HFJ: Different levels of natural antibodies in chickens divergently selected for specific antibody responses. Dev. Comp. Immunol. 2004, 28: 39-49. 10.1016/S0145-305X(03)00087-9.View ArticlePubMedGoogle Scholar
- Siwek M, Buitenhuis B, Cornelissen S, Nieuwland M, Knol EF, Crooijmans R, Groenen M, Parmentier H, van der Poel J: Detection of QTL for innate: non-specific antibody levels binding LPS and LTA in two independent populations of laying hens. Dev. Comp. Immunol. 2006, 30: 659-66. 10.1016/j.dci.2005.09.004.View ArticlePubMedGoogle Scholar
- National Center for Biotechnology Information: [http://www.ncbi.nlm.nih.gov]
- Kanehisa M, Goto S: KEGG: kyoto encyklopedia of genes and genomes. Nucleic Acids Res. 2000, 28: 27-30. 10.1093/nar/28.1.27.PubMed CentralView ArticlePubMedGoogle Scholar
- Lynn DJ, Winsor GL, Chan C, Richard N, Laird MR, Barsky A, Gardy JL, Roche FM, Chan THW, Shah N, Lo R, Naseer M, Que J, Yau M, Acab M, Tulpan D, Whiteside MD, Chikatamarla A, Mah B, Munzner T, Hokamp K, Hancock REW, Brinkman FSL: InnateDB: facilitating systems-level analyses of the mammalian innate immune response. Mol. Syst. Biol. 2008, 4: 218-10.1038/msb.2008.55.PubMed CentralView ArticlePubMedGoogle Scholar
- Gene Ontology Consortium: Creating the gene ontology resource: design and implementation. Genome Res. 2001, 11: 1425-33. 10.1101/gr.180801.View ArticleGoogle Scholar
- Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto T, Bronson RT, Springer TA: Impaired B-lyphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. PNAS. 1998, 95: 9448-53. 10.1073/pnas.95.16.9448.PubMed CentralView ArticlePubMedGoogle Scholar
- Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, Kitamura Y, Yoshida N, Kikutani H, Kishimoto T: Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature. 1996, 382: 635-8. 10.1038/382635a0.View ArticlePubMedGoogle Scholar
- Pappu R, Cheng AM, Li B, Gong Q, Chiu C, Griffin N, White M, Sleckman BP, Chan AC: Requirement for B Cell Linker Protein (BLNK) in B Cell Development. Science. 1999, 286: 1949-54. 10.1126/science.286.5446.1949.View ArticlePubMedGoogle Scholar
- Quadro L, Gamble MV, Vogel S, Lima AAM, Piantedosi R, Moore SR, Colantuoni V, Gottesman ME, Guerrant RL, Blaner WS: Retinol and Retinol-Binding Protein. Gut Integrity and Circulating Immunoglobulins. J Infect Dis. 2000, 182 Suppl 1: S97-S102. 10.1086/315920.View ArticlePubMedGoogle Scholar
- Ozaki K, Spolski R, Feng CG, Qi CF, Cheng J, Sher A, Morse HC, Liu C, Schwartzberg PL, Leonard WJ: A critical role for IL-21 in regulating immunoglobulin production. Science. 2002, 298: 1630-4. 10.1126/science.1077002.View ArticlePubMedGoogle Scholar
- Li X, Stark GR: NFκB-dependent signaling pathway. Exp. Hematol. 2002, 30: 285-96. 10.1016/S0301-472X(02)00777-4.View ArticlePubMedGoogle Scholar
- Cormican P, Lloyd AT, Downing T, Connell SJ, Bradley D, O'Farrelly C: The avian Toll-Like receptor pathway--subtle differences amidst general conformity. Dev Comp Immunol. 2009, 33: 967-73. 10.1016/j.dci.2009.04.001.View ArticlePubMedGoogle Scholar
- Lynn DJ, Lloyd AT, O'Farrelly C: In silico identification of components of the Toll-like receptor (TLR) signaling pathway in clustered chicken expressed sequence tags (ESTs). Vet Immunol Immunopathol. 2003, 93: 177-84. 10.1016/S0165-2427(03)00058-8.View ArticlePubMedGoogle Scholar
- Massagué J: Integration of Smad and MAPK pathways: a link and a linker revisited. Genes Dev. 2003, 17: 2993-7. 10.1101/gad.1167003.View ArticlePubMedGoogle Scholar
- Liu Y, Shepherd EG, Nelin LD: MAPK phosphatases-regulating the immune response. Nat. Rev. Immunol. 2007, 7: 202-12. 10.1038/nri2035.View ArticlePubMedGoogle Scholar
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