CD14 is a receptor involved in the recognition of lipopolysaccharide and other bacterial wall components that may be involved in the balance between infectious and allergic disease and the early polarization towards TH1. Our group has shown an association between polymorphisms in the 5′ flanking region of the CD14 gene and plasma soluble CD14 (sCD14) levels at 11 years of age. However, whether this association is present at birth and in infancy remains to be determined. In this study, we measured sCD14 levels in plasma from the umbilical cord (n=387) and at 3 months (n=357) and 1 year (n=312) of age in non-selected healthy infants to assess their relationship with CD14 genotypes at −4190, −2838, −1720 and −260 (relative to translation start site). There was no relation of CD14 genotypes with sCD14 at birth. However, there was a significant association between CD14 genotypes and sCD14 as early as 3 months. Longitudinal analysis suggests that CD14 polymorphisms modulate sCD14 levels up to 1 year of age. This association early in life may have an impact on TH1 polarization and subsequent protection against allergic disease.
In 1990, Wright et al.1 first described CD14 as a receptor involved in the recognition of lipopolysaccharide and other bacterial wall components. The CD14 gene is located on chromosome 5q31 and is predominantly expressed on the surface of monocytes, macrophages and neutrophils, and in a soluble form in the blood.1, 2, 3 It was not until 1999 that an association between CD14 and atopy was described. Baldini et al.4 found that the CD14/−260 polymorphism was associated with soluble CD14 (sCD14) levels and total immunoglobulin E (IgE) in 11-year-old subjects. Specifically, TT homozygotes had significantly higher sCD14 levels than did carriers of the CC and CT genotypes. TT homozygotes also had significantly lower levels of IgE than did carriers of the other two genotypes, but differences were significant only among children who were skin test positive. However, whether an association of genotype with sCD14 is present at birth and in infancy remains to be determined. Moreover, interferon-gamma and interleukin-4 responses were positively and negatively correlated, respectively, with sCD14 levels.4 Therefore, CD14 may be an important player in the balance between infectious and allergic disease, and the early polarization towards a TH1 response.5, 6 We tested the potential role of CD14 promoter polymorphisms, −260 as well as −4190, −2838 and −1720, on the development of sCD14 levels between birth and 1 year of age.
Results and discussion
The complete coding region of CD14, intronic sequence, approximately 6 kb of 5′ genomic and 2 kb of 3′ genomic were resequenced from DNA obtained from 23 European Americans as part of the Innate Immunity Program in Genomic Applications (PGA).7, 8 A total of 17 single nucleotide polymorphisms (SNPs) were identified; 15 of these had a minor allele frequency >10% (Figure 1). From the European-American-specific catalog of polymorphic sites, a tagging strategy based on bins of polymorphic sites that exceeded 10% minor allele frequency and a within-bin linkage disequilibrium (LD) exceeding 0.7 was employed using publicly available software as recently described.9 This algorithm resulted in four bins (Figure 1).
Using a high-throughput genotyping method based on 5′ exonuclease (TaqMan®) assay,10 we genotyped infants for CD14 polymorphisms at locations −4190 (major to minor allele: C to T), −2838 (C to T), −1720 (A to G) and −260 (T to C) relative to the translation start site using cord blood. These infants were recruited prenatally from healthy women between 32 and 35 weeks gestation participating in the Infant Immune Study, a prospective study of the development of immunological markers of asthma risk in infancy and early childhood.6 The minor allele frequencies for these loci were CD14/−4190=22.4% (n=375), CD14/−2838=27.5% (n=371), CD14/−1720=38.9% (n=369) and CD14/−260=47.1% (n=375) and all were found to be in Hardy–Weinberg equilibrium (data not shown). The strength of pairwise LD, as measured by r2, among the four SNPs is shown in Figure 2. LD was particularly strong between CD14/−1720 and CD14/–260 (r2=0.689). The genotype frequencies and LD values were comparable to those found in the European-American population used in the Innate Immunity PGA.7
Soluble CD14 was measured in plasma obtained at birth (n=387), 3 months (mean age 12.5 weeks ±5.4 s.d., n=357) and 1 year (mean age 56.0 weeks ±6.9 s.d., n=312). Plasma sCD14 increased significantly from birth to 3 months (geometric mean 0.60 vs 1.46 μg/ml; paired t-test P<0.0001) and from 3 months to 1 year (geometric mean 1.46 vs 1.88 μg/ml; paired t-test P<0.0001) (Figure 3). Cross-sectional analyses showed that there was no association between sCD14 levels at birth and CD14 genotypes at the −4190 (ANOVA P=0.4; n=327), −2838 (ANOVA P=0.7; n=322), −1720 (ANOVA P=0.9; n=321) or −260 (ANOVA P=0.8; n=326) loci (Figure 3).
The relationship between CD14 genotypes and post-natal sCD14 levels (at 3 months and 1 year) was investigated using random effects models (REMs). REMs are mixed models that offer several advantages in the analysis of longitudinal data:11 (1) they relax the assumption of independence between observations, (2) they control for the intra-subject serial correlation among repeated observations, (3) they are relatively insensitive to missing observations as long as they are missing at random and (4) they can adjust the associations of interest for potential confounders.
Using REMs and taking into account both 3-month and 1-year time points, we found that after adjusting for sex, age, ethnicity and parental asthma, there were notable differences for post-natal sCD14 levels among CD14 genotypes at loci −4190 (P=0.002), −1720 (P<0.0001) and −260 (P=0.0003) (Figure 3). Subjects who were homozygous for the T, A and C alleles at −4190, −1720 and −260, respectively, had decreased levels of sCD14 in their plasma compared to carriers of the other genotypes. Similar results have been observed in adolescent and adult subjects for CD14/−172012 and CD14/−260.4, 12, 13, 14 No association was found between CD14/−2838 and post-natal sCD14 levels. Haplotype analysis did not show any effect stronger than individual genotype data (data not shown).
To determine how early in life these genetic effects occur, we tested the associations (using REMs) between these genotypes and sCD14 levels at 3 months of age and found significant effects for the SNPs at −1720 and −260 (P=0.006 and 0.0007, respectively), whereas the effect of CD14/−4190 was borderline (P=0.07). No association was found between CD14/−2838 and 3-month sCD14 levels. Altogether, these results indicate that the CD14 polymorphisms at the −1720 and −260 loci modulate expression of sCD14 in humans as early as 3 months of age.
Our finding of an association between CD14 genotypes and sCD14 levels at 3 months and 1 year, but not at birth, suggests that genetic control of sCD14 may be affected by developmental factors and/or by the post-natal environment, such as exposure to microbial products.15 Alternatively, an effect of genetic variation on sCD14 levels at birth might have been masked by some other determinants of sCD14 levels that are active in utero or during the birthing process. Our results cannot be due to confounding by maternal CD14 genotype since there was no association between maternal genotype and sCD14 levels at 3 months of age (CD14/−4190, P=0.7; CD14/−2838, P=0.8; CD14/−1720, P=0.15; CD14/−260, P=0.12).
Functional studies have revealed a potential biologic mechanism for increased expression of sCD14 among carriers of the CD14/−260T allele. In separate in vitro studies, we found decreased affinity of DNA/protein interactions at the polymorphic Sp binding site with the T allele, which resulted in increased transcriptional activity of the CD14 promoter.16 This increased activity of the T allele was confirmed in this study as carriers of the CD14/−260T had higher levels of sCD14 at 3 months. Functionality of other CD14 polymorphic loci has not yet been demonstrated.
In summary, we identified four tag SNPs in the CD14 gene and assessed the extent of LD among them in the non-selected Infant Immune Study population. We evaluated the association between sCD14 levels (birth, 3 months, 1 year) and the four tag SNPs by using longitudinal analysis. This study provides support for the notion that genetic variation in the CD14 gene affects sCD14 levels as early as 3 months of age.
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This study was financially supported by Grants AI42268 and ES-00386 from the National Institutes of Health.
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LeVan, T., Guerra, S., Klimecki, W. et al. The impact of CD14 polymorphisms on the development of soluble CD14 levels during infancy. Genes Immun 7, 77–80 (2006). https://doi.org/10.1038/sj.gene.6364276
- innate immunity
- single nucleotide polymorphism
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