PC25 COMPARISON OF TOLL LIKE RECEPTOR- AND TH1-/TH2-RELATED MRNA LEVELS IN SPANISH VERSUS GERMAN PREGNANT WOMEN AND NEONATES

Abstract

Introduction: Epidemiological studies indicate that the exposure to microbes in early life is associated with a reduced risk to develop allergic disease. Toll-like receptors bind microbial compounds thereby triggering innate immune responses. We asked whether the mRNA expression levels of toll like receptors (TLR) and Th1-/Th2-related markers differ in placental cord blood of Spanish and German neonates. Methods: 47 pregnant women from Granada, Spain (n=23, mean age 29 yrs; habituary high fish intake) and Munich, Germany (n=24, mean age 32 yrs; habituary low fish intake) were participants of the NUHEAL study and were selected for this study. Cord blood and maternal peripheral blood were obtained immediately after delivery. After RNA-extraction and cDNA synthesis, IL-4, IL-13, CRTH2, CCR4 (Th2), IFN-gamma, CXCR3, (Th1) and IL-1, TLR2 and TLR4 (TLR) were quantified by real-time RT PCR (Biorad, USA). Relative mRNA expressions were calculated as follows: 1/(Expression Gene-of-interest / Expression GAPDH). All reactions were run in duplicates and included untranscribed mRNA as negative controls. The quantitation of cDNAs in low copy numbers was verified with fluorescing hydrolysis probes. Amplification of the desired cDNAs was confirmed by sequencing. For comparisons between groups Mann-Whitney-U test was applied. Correlations r0.3 were considered positive (Spearman rho; SPSS Version 11.5). Results: Cord blood from Spanish neonates had significantly higher TLR2 (p<0.01) and TLR4 (p<0.001) mRNA-expression levels than cord blood from German neonates. In peripheral blood from Spanish mothers, TLR2 (p<0.01) and TLR4 mRNA (p<0.001) expression levels were also significantly higher as compared to German mothers. In contrast, Spanish women had lower IL-1 (p<0.05) and IL-4 mRNA (p<0.05) expression levels compared to German women. TLR2 and TLR4 mRNA expressions were highly correlated in mothers and theis neonates. mRNA expressions of the remaining markers did not differ between habitants of both countries. Conclusion: TLR2 and TLR4 mRNA expressions were clearly higher in Spanish women and neonates. We speculate that the differences in TLR expression are due to a different microbial exposure in both cities. We further speculate that environmental factors may influence the fetal immune system already in utero