The Influence Of Neonatal TLR2 And TLR4 Immune Pathway To The Development Of Asthma And Other Allergic Diseases

In recent years, the morbidity of asthma and other allergic diseases increase constantly in population, especially in children. Allergic diseases are considered as"the diseases which need special investigation and prevention in the 21th century"by World Health Organization(WHO)。The allergic family medical history is high risk for the children to develop allergic diseases. For these children with high risk, it is very important to prevent in early life, and even in neonatal period. It is thought that asthma and other allergic diseases are induced by the imbalance of Th1 and Th2, and the up-regulation of Th2 cells. But now more and more studies show that the abnormality of regulatory T cells plays an important role in the pathogenesis of allergic diseases. Intervention of regulatory T cells maybe conduces to the therapy and prevention to allergic diseases. Recently, some studies show that TLRs play an important role in the modulation to the function and homeostasis of regulatory T cells. TLRs family belong to IL-1R/ TLR superfamily. TLRs distribute on immune cells, such as macrophage and dendritic cells. Different TLRs recognize different pathogen associated molecular patterns (PAMPs). The ligand of TLR2 is peptidoglycan (PPG), which is the component of cell wall of G+ bacteria. TLR4 recognizes lipopolysaccharide (LPS), which is the component of cell wall of G- bacteria, and lipids A(LPA), which is the bioactive component of LPS. Some studies show that there is correlation between the gene polymorphism of TLR2 and TLR4 and the development of asthma and other allergic diseases. Maternal allergic diseases history is relevant to the low expression of TLR2 and TLR4 mRNA in the neonatal cord blood. The expression level of TLR2 and TLR4 mRNA is relative to maternal and offspring's allergic response. Meanwhile, some studies show that the morbidity of allergic diseases in farming children is lower than that in city children, which is correlate to the higher content of microbiotic substrates in the living environment of farming children and farming pregnant mother. As the ligands of microbiotic substrates, TLR2 and TLR4 may prevent the allergic diseases through their expression content and gene polymorphism. So, we look TLR2 and TLR4 immune pathway as object, investigate their function on development of neonatal allergic diseases, and explore a potential prevention way to allergic diseases in the neonatal period and even in embryonic period.CBMC and PBMC were isolated from cord blood and adult peripheral blood, and then were cultured with stimuli of PHA(mitogen), LPA (TLR4 ligand), PPG (TLR2 ligand), Derp1 (extract from indoor dust and mite) or stable dust(extract from stable dust) separately. After culture for 72 hours, the following indexes were detected: lymphocyte proliferation with 3H-Thymidine incorporation; the secretion concentration of IFN-γ, IL-13,IL-10 and IL-17 with the Human Cytokine Multiplex Assay Kit; the percent of CD4+CD25+T cells in CBMC or PBMC with the flow cytometry; the expression of regulatory cells related genes: FOXP3,GITR,CTLA-4,LAG-3 and TGF-βwith RT-PCR; the suppress function of CD4+CD25+T cells to the division of effector cells with beads antibodies isolation, CFSE incorporation and flow cytometry.Firstly, we investigated systematically the characteristic of neonatal immune system on TLR2 and TLR4 immune pathway, in contrast to the adult immune system. Based on the study to 25 healthy adults and 31 healthy neonates, we found that on the TLR2 and TLR4 response pathway, lymphocyte proliferation of CBMC were lower than PBMC (P<0.0001). With the stimulation of PPG, the secretion concentration of IL-10 in CBMC had the tendency to be lower than PBMC (P=0.19). And the percent of CD4+CD25+T cells in CBMC was lower with the stimuli of LPA or PPG than that in PBMC (P=0.0017) (P=0.0006). Meanwhile, the suppress function of CD4+CD25+T cells in vitro to the division of effector cells in CBMC was weaker than in PBMC (P=0.01). In addition, we found that the suppress function of CD4+CD25+T cells in 2 adults with allergic diseases trend to be lower than healthy adult (P=0.12). A better understanding of the characteristics of the TLR2 and TLR4 pathway and regulatory T cells responses in neonatal immune system may help us to study further the differences among the neonatal immune systems with different backgrounds on the TLR2 and TLR4 response pathway.Secondly, based on the important influence of maternal allergic disease history to the development of allergic diseases in offspring, we investigated systematically the characteristic of neonate with allergic mother on TLR2 and TLR4 immune pathway, in contrast to the neonate with non-allergic mother. We found that CBMC from neonates with allergic mother secreted less IL-10 than neonate with non-allergic mother after the stimulation of PPG (P=0.05). But for the secretion of IFN-γand IL-13, there were the reverse tendency: allergic group were higher than non-allergic group (P=0.16,P=0.21). In neonates with allergic mother, the expression of LAG-3 were lower than the neonates with non-allergic mother with PHA,LPA or PPG ( P<0.05); the expression of GITR and TGF-βin allergic group were lower than non-allergic group with PHA and PPG stimuli( P<0.05), and were tendency to be lower than non-allergic group with LPA stimulant( P=0.09) ( P=0.09); the expression of FOXP3 and CTLA-4 in allergic group were tendency to be lower than non-allergic group with LPA stimulant( P=0.15) ( P=0.11). The suppress function of CD4+CD25+T cells in vitro to the division of effector cells in neonates with allergic mother were tendency to be lower than that in neonates with non- allergic mother. In short, on the TLR2 and TLR4 immune pathway, the function of regulatory T cells in neonates with allergic mother was weaker than that in neonates with non-allergic mother. The down-regulation of the function of regulatory T cells may weaken their suppression to the Th2 cells, accordingly increase the susceptibility to allergic diseases. So we introduced the hypothesis: we can prevent the development of allergic diseases in neonates with family allergic diseases history by interfering in the immune pathway of TLR2 and TLR4.To prove our hypothesis further, we investigated systematically the characteristic of neonates with farming mother on TLR2 and TLR4 immune pathway, in contrast to the neonates with non-farming mother. We found that on the response pathway of TLR2 and TLR4, especial TLR2, the percent of CD4+CD25+T cells in CBMC and the secretion concentration of IL-10 in neonates with farming mother were higher than those in neonates with non-farming mother( P=0.03) ( P=0.01). The suppress function of CD4+CD25+T cells in vitro to the division of effector cells in neonates with farming mother were tendency to be stronger than that in neonates with non-farming mother.These results documented that farming exposure could protect and prevent the development of allergic diseases through the immune pathway of TLR2 and TLR4, which up-regulated the amount and function of regulatory T cells and increased suppression to Th2 cells, accordingly decreased the susceptibility to allergic diseases. These results provided powerful evidence for our hypothesis.Furthermore, we first time investigate the characteristic of IL-17 on neonatal TLR2 and TLR4 immune pathway. We found that the secretion concentration of IL-17 in neonate trend to be lower than adult (P=0.17) (P=0.09). The CBMC from neonates with allergic mother secreted less IL-17 than that from neonates with non-allergic mother( P=0.13) ( P=0.09). There were positive correlation between IL-17 and IFN-γor IL-13, but the correlation between IL-17 and IL-10 trend to be negative, and this negative correlation was significant in the neonates with allergic mother. These results suggested that except for regulatory T cells, there was abnormality on the IL-17 secretion on the TLR2 and TLR4 immune pathway, which could influence the development of allergic diseases in neonate.Above all, our study first time investigated systematically the characteristic of neonatal TLR2 and TLR4 immune pathway; the influences of maternal allergic diseases and farming exposure to the neonatal immune system on the TLR2 and TLR4 pathway. We found that maternal allergic history could influence neonate through the TLR2 and TLR4 immune pathway, and down-regulate the function of regulatory T cells. Maternal farming exposure could influence neonate through the TLR2 and TLR4 immune pathway, and up-regulate the function of regulatory T cells, and then protect and prevent the development of allergic diseases, which provided reliable evidence for the prevention of allergic diseases on the neonates with high risk by TLR2 and TLR4 immune pathway. Meanwhile, in contrast to adult, neonatal immune system was immature on TLR2 and TLR4 response pathway, which provided adequate time point to interfere and prevent the development of allergic diseases. So, to the neonates with high allergic risk, interfering in the neonatal period even in the fetal period by TLR2 and TLR4 immune pathway may be a good potential prevention way.