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The NLRC4 inflammasome, which recognizes flagellin and components of the type III secretion system, plays an important role in the clearance of intracellular bacteria. Here, we examined the genomic sequences carrying two genes encoding key components of the NLRC4 inflammasome-NLR family, CARD-containing 4 (NLRC4), and NLR apoptosis inhibitory protein (NAIP)-in pigs. Pigs have a single locus encoding NLRC4 and NAIP. Comparison of the sequences thus obtained with the corresponding regions in humans revealed the deletion of intermediate exons in both pig genes. In addition, the genomic sequences of both pig genes lacked valid open reading frames encoding functional NLRC4 or NAIP protein. Additional pigs representing multiple breeds and wild boars also lacked the exons that we failed to find through genome sequencing. Furthermore, neither the NLRC4 nor the NAIP gene was expressed in pigs. These findings indicate that pigs lack the NLRC4 inflammasome, an important factor involved in monitoring bacterial proteins and contributing to the clearance of intracellular pathogens. These results also suggest that genetic polymorphisms affecting the molecular functions of TLR2, TLR4, TLR5, and other pattern recognition receptors associated with the recognition of bacteria have a more profound influence on disease resistance in pigs than in other species.

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.

Dectin-1, a C-type lectin receptor that recognizes fungal β-glucans, is involved in antifungal immunity and the regulation of intestinal immune homeostasis. Dectin-1 is involved in both synthesis and maturation of interleukin-1β, a key pro-inflammatory cytokine in immunity. Here, we assessed the genetic diversity in the gene encoding dectin-1 (CLEC7A) within various pig populations and examined the influence of these polymorphisms on the two different signaling pathways after ligand recognition. An amino-acid polymorphism located in the carbohydrate-recognition domain, leucine to serine at position 138 (L138S), which occurred exclusively in Japanese wild boars at low frequency, significantly increased NF-κB induction but not caspase-8 activity after stimulation with zymosan. In contrast, other amino-acid polymorphisms present at comparatively high frequency in commercial pig populations had little influence on ligand recognition. These results suggest that functionally neutral polymorphisms in dectin-1 are widespread in pig populations.

Pathogens localized extracellularly or incorporated into endosomes are recognized mainly by Toll-like receptors, whereas pathogens and pathogen-derived molecules that invade into the cytoplasm of host cells typically are recognized by intracellular pattern recognition receptors (PRRs), such as retinoic acid-inducible gene (RIG)-like helicases (RLHs) and nucleotide-binding oligmerization domain (NOD)-like receptors (NLRs). RIG-I and melanoma differentiation-associated gene 5 (MDA5), which belong to the RLH family, recognize viral genomic RNA, whereas NOD2, a member of the NLR family, responds to microbial peptidoglycans. These receptors may play an important role in pig opportunistic infectious diseases, such as pneumonia and diarrhea, which markedly impair livestock productivity, such that polymorphisms of these receptor genes are potential targets of pig breeding to increase disease resistance. Here, we report single nucleotide polymorphisms (SNPs) in porcine DDX58, IFIH1, and NOD2, which encode RIG-I, MDA5, and NOD2, respectively. Interestingly, compared with DDX58 and IFIH1, NOD2 abounded in nonsynonymous SNPs both throughout the coding sequence and in sequences encoding domains important for ligand recognition, such as helicase domains for RIG-I and MDA5 and leucine-rich repeats in NOD2. These differences in the distribution of SNPs in intracellular PRRs may parallel the diversity of their ligands, which include nucleic acids and peptidoglycans.