Background

As part of the innate immune system, both vertebrates and invertebrates possess pattern recognition receptors (PRRs), which respond to various kinds of molecules that characterize pathogens. Normally, PRR responses are quick, and their signals induce the production of proinflammatory cytokines and interferons, which are required for the appropriate exertion of other responses, including acquired immunity [1]. Vertebrates have fewer PRRs than invertebrates, but more than 30 genes encoding PRRs have been identified in each mammalian genome. In humans and mice, many studies have shown that some of the polymorphisms in PRR genes influence the immune response and disease susceptibility [2]. This suggests correlations between the immune response and PRR polymorphisms in pigs [3]. It is possible that these relationships could be used for breeding aimed at disease resistance. Here, we summarize the results of our searches for polymorphisms in PRRs and the influence of these polymorphisms on ligand recognition ability. The results of our preliminary study of the relationship between TLR2 polymorphism and the onset of pneumonia imply that PRR polymorphisms also affect disease resistance in pigs.

Methods

We used Landrace, Large White, Duroc, Berkshire, Hampshire, Middle Yorkshire, Clawn miniature, Potbelly, Meishan, and Jinhua breed pigs and Japanese Wild boars for polymorphisms detection [4–7]. Pig samples for exploration of polymorphisms were collected as ear notches, semen, peripheral blood, or muscle tissues, and genomic DNA was extracted by a standard method. cDNA was synthesized from RNA derived from peripheral blood. Polymorphisms were searched for by direct sequencing of PCR products covering the coding sequences generated with DNA and gene-specific primers. Targeted single nucleotide polymorphisms (SNPs) in additional samples were explored by mass spectrometry with a Sequenom MassARRAY Compact. To investigate the correlation between pneumonia and TLR2 genotype, we fed 626 Landrace pigs on a Mycoplasma hyopneumoniae (Mhp)-positive farm until they weighed 105 to 115 kg. The pigs were then sacrificed and inspected for hepatization of the lung lobes as an indicator of pneumonia. The experiment was conducted in accordance with the animal experiment regulations of Miyagi Livestock Experiment Station.

Results and discussion

Distribution of polymorphisms in pig PRRs

We searched for polymorphisms in the coding sequences (CDSs) of TLRs (TLR1, 2, 3, 4, 5, 6, 7, and 8), RIG-I-like helicases (DDX58 encoding RIG-I and IFIH1 encoding MDA5), and a NOD-like receptor (NOD2). We found many polymorphisms in the CDSs, particularly in the regions encoding domains required for ligand recognition [4–7]. Many SNPs changing the encoding of amino acids (nonsynonymous SNPs) were included in the ligand-recognizing regions. Notably, abundant nonsynonymous polymorphisms were observed in the ectodomains of TLRs expressed on cell surfaces (Table 1). In contrast, there were few nonsynonymous SNPs in the regions encoding domains related to signal transduction to the downstream pathways. (Table 1). The occurrence of polymorphisms in the domain in charge of ligand recognition may enable pig populations to cope with a broad variety of pathogens, whereas the occurrence of polymorphisms in the domain required for signal transduction has been strongly suppressed, possibly because of their harmful effects on the innate immunity of the hosts.

Table 1 Distribution of polymorphisms in CDSs of porcine PRRs
Full size table

Functional influence of polymorphisms in pig PRRs on molecular recognition

Polymorphisms affecting the encoded amino acids in PRRs may influence molecular function—particularly ligand specificity. However, it is possible that the mutations remaining in the pig population have no effects, and that we can observe only those polymorphisms that are not harmful to molecular function. Therefore, we conducted reporter assays using expression vectors carrying the observed polymorphisms. Human cells (HEK293) into which expression vectors carrying some of the PRR polymorphisms had been introduced showed varying responses to a ligand. For example, the response of pig NOD2 with a 1949T→C mutation to muramyldipeptide was completely abolished, whereas that of pig NOD2 with 2197A→C showed an increased response to the ligand [4]. Furthermore, we have so far detected several nonsynonymous SNPs in TLR2, TLR4, and TLR5 that affected their molecular function in ligand recognition (Shinkai et al., unpublished data).

Possible correlation between PRR polymorphisms and onset of infectious diseases

The influence of the polymorphisms on ligand recognition ability suggested a relationship between disease susceptibility and PRR genotype. We surveyed the incidence of pneumonia caused mainly by Mycoplasma hyopneumoniae in a Landrace population and investigated genotypes of TLR2, which is responsible for recognition of Gram-positive bacteria. Among 626 pigs, 37 had a G nucleotide instead of a C at position 406 in the CDS, causing an amino acid alteration from proline to alanine. Seventy-six percent (28 animals) of pigs with the G allele showed histopathological signs of pneumonia, whereas 57% (331 animals) of those without the G allele had pneumonia (Table 2). The former had a significantly greater incidence of pneumonia than the latter (odds ratio = 2.30; 95% interval: 1.07–4.97; P = 0.038). This suggests a relationship between PRR genotype and disease susceptibility, although we have to examine the susceptibility of animals with the homozygous G alleles to pneumonia. Further investigation with other pig populations may elucidate correlations between other SNPs in PRRs and disease susceptibility.

Table 2 Relationship between TLR2 genotype and occurrence of pneumonia
Full size table

Conclusions

Many polymorphisms occur in the genes encoding PRRs in pigs. Some of the polymorphisms have an influence on ligand recognition ability. Polymorphisms in PRRs may be correlated with disease resistance in pigs.