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Identification of the Q969R gain-of-function polymorphism in the gene encoding porcine NLRP3 and its distribution in pigs of Asian and European origin

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Abstract

The nucleotide-binding domain, leucine-rich-containing family, pyrin-domain containing-3 (NLRP3) inflammasome comprises the major components caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and NLRP3. NLRP3 plays important roles in maintaining immune homeostasis mediated by intestinal microorganisms and in the immunostimulatory properties of vaccine adjuvants used to induce an immune response. In the present study, we first cloned a complementary DNA (cDNA) encoding porcine ASC because its genomic sequence was not completely determined. The availability of the ASC cDNA enabled us to reconstitute porcine NLRP3 inflammasomes using an in vitro system that led to the identification of the immune functions of porcine NLRP3 and ASC based on the production of interleukin-1β (IL-1β). Further, we identified six synonymous and six nonsynonymous single-nucleotide polymorphisms (SNPs) in the coding sequence of NLRP3 of six breeds of pigs, including major commercial breeds. Among the nonsynonymous SNPs, the Q969R polymorphism is associated with an increased release of IL-1β compared with other porcine NLRP3 variants, indicating that this polymorphism represents a gain-of-function mutation. This allele was detected in 100 % of the analyzed Chinese Jinhua and Japanese wild boars, suggesting that the allele is maintained in the major commercial native European breeds Landrace, Large White, and Berkshire. These findings represent an important contribution to our knowledge of the diversity of NLRP3 nucleotide sequences among various pig populations. Moreover, efforts to exploit the gain of function induced by the Q969R polymorphism promise to improve pig breeding and husbandry by conferring enhanced resistance to pathogens as well as contributing to vaccine efficacy.

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Abbreviations

NLRP3:

Nucleotide-binding domain, leucine-rich-containing family, pyrin-domain containing-3

ASC:

Apoptosis-associated speck-like protein containing a caspase recruitment domain

PAMPs:

Pathogen-associated molecular patterns

SNPs:

Single-nucleotide polymorphisms

CDS:

Coding sequence

IL-1β:

Interleukin-1β

HEK:

Human embryonic kidney

PCR:

Polymerase chain reaction

ELISA:

Enzyme-linked immunosorbent assay

CARD:

Caspase recruitment domain

LRRs:

Leucine-rich repeats

NACHT:

Domain present in NAIP, CIITA, HET-E, and TP-1

References

  • Abderrazak A, Syrovets T, Couchie D, El Hadri K, Friguet B, Simmet T, Rouis M (2015) NLRP3 inflammasome: from a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases. Redox Biol 4:296–307

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Albin DM, Wubben JE, Rowlett JM, Tappenden KA, Nowak RA (2007) Changes in small intestinal nutrient transport and barrier function after lipopolysaccharide exposure in two pig breeds. J Anim Sci 85:2517–23

    Article  CAS  PubMed  Google Scholar 

  • Allen IC, Scull MA, Moore CB, Holl EK, McElvania-TeKippe E, Taxman DJ, Guthrie EH, Pickles RJ, Ting JP (2009) The NLRP3 inflammasome mediates in vivo innate immunity to influenza A virus through recognition of viral RNA. Immunity 30:556–65

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bell JK, Mullen GE, Leifer CA, Mazzoni A, Davies DR, Segal DM (2003) Leucine-rich repeats and pathogen recognition in Toll-like receptors. Trends Immunol 24:528–33

    Article  CAS  PubMed  Google Scholar 

  • Benko S, Philpott DJ, Girardin SE (2008) The microbial and danger signals that activate Nod-like receptors. Cytokine 43:368–373

    Article  CAS  PubMed  Google Scholar 

  • Conforti-Andreoni C, Ricciardi-Castagnoli P, Mortellaro A (2011) The inflammasomes in health and disease: from genetics to molecular mechanisms of autoinflammation and beyond. Cell Mol Immunol 8:135–45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Darwin C (1868) The variation of animals and plants under domestication. John Murray, London

    Google Scholar 

  • Di Virgilio F (2013) The therapeutic potential of modifying inflammasomes and NOD-like receptors. Pharmacol Rev 65:872–905

    Article  PubMed  Google Scholar 

  • Eisenbarth SC, Colegio OR, O’Connor W, Sutterwala FS, Flavell RA (2008) Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 453:1122–6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ewing B, Hillier L, Wendl MC, Green P (1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8:175–185

    Article  CAS  PubMed  Google Scholar 

  • Excoffier L, Laval G, Schneider S (2005) Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol Bioinform Online 1:47–50

    CAS  Google Scholar 

  • Excoffier L, Slatkin M (1995) Maximum-likelihood estimation of molecular haplotype frequencies in a diploid population. Mol Biol Evol 12:921–7

    CAS  PubMed  Google Scholar 

  • Gao Y, Han F, Huang X, Rong Y, Yi H, Wang Y (2013) Changes in gut microbial populations, intestinal morphology, expression of tight junction proteins, and cytokine production between two pig breeds after challenge with Escherichia coli K88: a comparative study. J Anim Sci 91:5614–5625

    Article  CAS  PubMed  Google Scholar 

  • Giuffra E, Kijas JM, Amarger V, Carlborg O, Jeon JT, Andersson L (2000) The origin of the domestic pig: independent domestication and subsequent introgression. Genetics 154:1785–91

    CAS  PubMed  PubMed Central  Google Scholar 

  • Groenen MA, Archibald AL, Uenishi H, Tuggle CK, Takeuchi Y, Rothschild MF, Rogel-Gaillard C, Park C, Milan D, Megens HJ, Li S, Larkin DM, Kim H, Frantz LA, Caccamo M, Ahn H, Aken BL, Anselmo A, Anthon C, Auvil L, Badaoui B, Beattie CW, Bendixen C, Berman D, Blecha F, Blomberg J, Bolund L, Bosse M, Botti S, Bujie Z, Bystrom M, Capitanu B, Carvalho-Silva D, Chardon P, Chen C, Cheng R, Choi SH, Chow W, Clark RC, Clee C, Crooijmans RP, Dawson HD, Dehais P, De Sapio F, Dibbits B, Drou N, Du ZQ, Eversole K, Fadista J, Fairley S, Faraut T, Faulkner GJ, Fowler KE, Fredholm M, Fritz E, Gilbert JG, Giuffra E, Gorodkin J, Griffin DK, Harrow JL, Hayward A, Howe K, Hu ZL, Humphray SJ, Hunt T, Hornshoj H, Jeon JT, Jern P, Jones M, Jurka J, Kanamori H, Kapetanovic R, Kim J, Kim JH, Kim KW, Kim TH, Larson G, Lee K, Lee KT, Leggett R, Lewin HA, Li Y, Liu W, Loveland JE, Lu Y, Lunney JK, Ma J, Madsen O, Mann K, Matthews L, McLaren S, Morozumi T, Murtaugh MP, Narayan J, Nguyen DT, Ni P, Oh SJ, Onteru S, Panitz F, Park EW et al (2012) Analyses of pig genomes provide insight into porcine demography and evolution. Nature 491:393–8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Janeway CA Jr, Medzhitov R (2002) Innate immune recognition. Annu Rev Immunol 20:197–216

    Article  CAS  PubMed  Google Scholar 

  • Jo EK, Kim JK, Shin DM, Sasakawa C (2016) Molecular mechanisms regulating NLRP3 inflammasome activation. Cell Mol Immunol 13:148–159

  • Jozaki K, Shinkai H, Tanaka-Matsuda M, Morozumi T, Matsumoto T, Toki D, Okumura N, Eguchi-Ogawa T, Kojima-Shibata C, Kadowaki H, Suzuki E, Wada Y, Uenishi H (2009) Influence of polymorphisms in porcine NOD2 on ligand recognition. Mol Immunol 47:247–252

    Article  CAS  PubMed  Google Scholar 

  • Kim J, Ahn H, Woo HM, Lee E, Lee GS (2014) Characterization of porcine NLRP3 inflammasome activation and its upstream mechanism. Vet Res Commun 38:193–200

    Article  PubMed  Google Scholar 

  • Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120

    Article  CAS  PubMed  Google Scholar 

  • Kumar S, Tsai CJ, Nussinov R (2000) Factors enhancing protein thermostability. Protein Eng 13:179–91

    Article  CAS  PubMed  Google Scholar 

  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–8

    Article  CAS  PubMed  Google Scholar 

  • Leemans JC, Cassel SL, Sutterwala FS (2011) Sensing damage by the NLRP3 inflammasome. Immunol Rev 243:152–62

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li H, Willingham SB, Ting JP, Re F (2008) Cutting edge: inflammasome activation by alum and alum’s adjuvant effect are mediated by NLRP3. J Immunol 181:17–21

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mariathasan S, Weiss DS, Newton K, McBride J, O’Rourke K, Roose-Girma M, Lee WP, Weinrauch Y, Monack DM, Dixit VM (2006) Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440:228–32

    Article  CAS  PubMed  Google Scholar 

  • Masumoto J, Taniguchi S, Ayukawa K, Sarvotham H, Kishino T, Niikawa N, Hidaka E, Katsuyama T, Higuchi T, Sagara J (1999) ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells. J Biol Chem 274:33835–8

    Article  CAS  PubMed  Google Scholar 

  • Masumoto J, Taniguchi S, Nakayama K, Ayukawa K, Sagara J (2001) Murine ortholog of ASC, a CARD-containing protein, self-associates and exhibits restricted distribution in developing mouse embryos. Exp Cell Res 262:128–33

    Article  CAS  PubMed  Google Scholar 

  • Matzinger P (2002) The danger model: a renewed sense of self. Science 296:301–305

  • Nickerson DA, Tobe VO, Taylor SL (1997) PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res 25:2745–2751

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rathinam VA, Vanaja SK, Waggoner L, Sokolovska A, Becker C, Stuart LM, Leong JM, Fitzgerald KA (2012) TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by gram-negative bacteria. Cell 150:606–19

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–25

    CAS  PubMed  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

  • Schultz J, Milpetz F, Bork P, Ponting CP (1998) SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci U S A 95:5857–64

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shigeoka AA, Mueller JL, Kambo A, Mathison JC, King AJ, Hall WF, Correia Jda S, Ulevitch RJ, Hoffman HM, McKay DB (2010) An inflammasome-independent role for epithelial-expressed Nlrp3 in renal ischemia-reperfusion injury. J Immunol 185:6277–85

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shimada K, Crother TR, Karlin J, Dagvadorj J, Chiba N, Chen S, Ramanujan VK, Wolf AJ, Vergnes L, Ojcius DM, Rentsendorj A, Vargas M, Guerrero C, Wang Y, Fitzgerald KA, Underhill DM, Town T, Arditi M (2012) Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity 36:401–14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shinkai H, Arakawa A, Tanaka-Matsuda M, Ide-Okumura H, Terada K, Chikyu M, Kawarasaki T, Ando A, Uenishi H (2012a) Genetic variability in swine leukocyte antigen class II and Toll-like receptors affects immune responses to vaccination for bacterial infections in pigs. Comp Immunol Microbiol Infect Dis 35:523–532

    Article  CAS  PubMed  Google Scholar 

  • Shinkai H, Matsumoto T, Toki D, Okumura N, Terada K, Uenishi H (2015) Porcine NOD1 polymorphisms with impaired ligand recognition and their distribution in pig populations. Mol Immunol 63:305–311

    Article  CAS  PubMed  Google Scholar 

  • Shinkai H, Okumura N, Suzuki R, Muneta Y, Uenishi H (2012b) Toll-like receptor 4 polymorphism impairing lipopolysaccharide signaling in Sus scrofa, and its restricted distribution among Japanese wild boar populations. DNA Cell Biol 31:575–581

    Article  CAS  PubMed  Google Scholar 

  • Sokalingam S, Raghunathan G, Soundrarajan N, Lee SG (2012) A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein. PLoS One 7:e40410

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Song-Zhao GX, Srinivasan N, Pott J, Baban D, Frankel G, Maloy KJ (2014) Nlrp3 activation in the intestinal epithelium protects against a mucosal pathogen. Mucosal Immunol 7:763–74

    CAS  PubMed  Google Scholar 

  • Tohno M, Shimosato T, Aso H, Kitazawa H (2011) Immunobiotic Lactobacillus strains augment NLRP3 expression in newborn and adult porcine gut-associated lymphoid tissues. Vet Immunol Immunopathol 144:410–6

    Article  CAS  PubMed  Google Scholar 

  • Tschopp J, Schroder K (2010) NLRP3 inflammasome activation: the convergence of multiple signalling pathways on ROS production? Nat Rev Immunol 10:210–5

    Article  CAS  PubMed  Google Scholar 

  • Verma D, Sarndahl E, Andersson H, Eriksson P, Fredrikson M, Jonsson JI, Lerm M, Soderkvist P (2012) The Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to excessive interleukin-1beta and IL-18 production. PLoS One 7:e34977

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vladimer GI, Marty-Roix R, Ghosh S, Weng D, Lien E (2013) Inflammasomes and host defenses against bacterial infections. Curr Opin Microbiol 16:23–31

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yen H, Sugimoto N, Tobe T (2015) Enteropathogenic Escherichia coli uses NleA to inhibit NLRP3 inflammasome activation. PLoS Pathog 11:e1005121

    Article  PubMed  PubMed Central  Google Scholar 

  • Yokota K, Satou K, Ohki S (2006) Comparative analysis of protein thermo stability: differences in amino acid content and substitution at the surfaces and in the core regions of thermophilic and mesophilic proteins. Sci Technol Adv Mater 7:255–262

    Article  CAS  Google Scholar 

  • Zimmerman JJ, Karriker LA, Ramirez A, Schwartz KJ, Stevenson GW (2012) Diseases of swine. 10th ed. Wiley-Blackwell, Ams

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Acknowledgments

We thank M. Ezure for technical support and helpful discussions. This study was supported by the Project of the Ministry of Agriculture, Forestry and Fisheries of Japan (Animal Genomics for Innovative Breeding Technology; AGB1002).

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Authors and Affiliations

  1. Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, 768 Senbonmatsu, Nasushiobara, Tochigi, 329-2793, Japan

    Masanori Tohno

  2. Animal Bioregulation Unit, Division of Animal Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Owashi, Tsukuba, Ibaraki, 305-8634, Japan

    Hiroki Shinkai & Hirohide Uenishi

  3. Animal Research Division, Institute of Japan Association for Techno-innovation in Agriculture, Forestry and Fisheries, 446-1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki, 305-0854, Japan

    Daisuke Toki & Naohiko Okumura

  4. Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, 2 Ikenodai, Tsukuba, Ibaraki, 305-0901, Japan

    Kiyoshi Tajima

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  1. Masanori Tohno
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  2. Hiroki Shinkai
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Correspondence to Masanori Tohno.

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Tohno, M., Shinkai, H., Toki, D. et al. Identification of the Q969R gain-of-function polymorphism in the gene encoding porcine NLRP3 and its distribution in pigs of Asian and European origin. Immunogenetics 68, 693–701 (2016). https://doi.org/10.1007/s00251-016-0917-y

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