Background
With increasing urbanization, the incidence of allergic diseases such as asthma has increased worldwide, which has in turn sharply affected quality of life [
1,
2]. China, in particular, is confronted with this severe problem, especially in some major cities such as Shanghai [
3]. In early life, long-time over-loading of microorganisms in the environment, which includes contacting with livestock, or drinking fresh milk that containing multiple bacteria, endotoxins and fungi, has an important role in preventing allergic diseases such as childhood asthma [
4,
5]. Exposure to microorganisms during infancy, or even at the fetal stage (via maternal exposure), decreases the morbidity due to allergic diseases in children [
6‐
9]. However, research on the effects of maternal exposure to microbes in rural areas on the neonatal immune system and allergic diseases in infants is still lacking in China.
Toll-like receptors (TLRs) of the immune system are involved in recognizing microorganisms; their expression and gene polymorphisms are involved in the process of neonatal immune maturation [
10]. The major ligands of TLR2 and TLR4 are peptidoglycans (PPG) and lipopolysaccharide (LPS), respectively. TLR2 gene polymorphisms have been associated with asthma, and high TLR2 expression has been shown in farming children in Europe [
9,
11]. Genetic variation in TLR2 and TLR4 is a major determinant of susceptibility to asthma and allergy in rural children [
12‐
14]. Further, the maternal TLR signaling pathway is required for protection of offspring against asthma [
15]. Data from the PARSIFAL team showed that maternal exposure to microbes improves the expression of TLR genes, and affects differentiation of fetal T cells by regulating the intrauterine microbial environment, consequently playing a protective role in offspring against allergic diseases [
5]. Previous studies have revealed four genetic polymorphisms in human TLR2, TLR4, and CD14 [
12,
13,
16,
17], such as TLR2 rs1898830, TLR2 rs4696480, TLR4 rs4986790 and CD14 rs2569190, which play a role in the pathogenic mechanism of allergic diseases and the protection mechanism of maternal exposure to farming.
Recently, the role of Tregs in pathogenesis of asthma has been attracting increasing attention. Several studies have demonstrated decreased quantity and function of Tregs in asthma [
15]. In our previous work, we showed that decreased quantity and function of Tregs in the cord blood of newborns, and it might present even prior to the Th1/Th2 imbalance in infants who had family history of allergy and were at high risk [
18]. Some research has shown that common microorganisms in rural areas induce up-regulation of Tregs through the TLR pathway, subsequently showing protective role against asthma and other allergic diseases [
19‐
21]. However, the underlying mechanisms of this protective effect remain to be determined.
Similar to the Th1/Th2 imbalance, the Treg/Th17 imbalance is also vital to the pathogenesis of asthma [
22]. Moreover, the role of Th9 cells, which are characterized by IL-9 secretion and specific expression of PU.1, in development of asthma has been increasingly addressed [
23,
24]; however, the association of Th9 cells with Tregs remains to be elucidated.
Since the Jilin province is a large agricultural province in China, in this study, we studied the main exposure factors in the rural environment of this area. We also attempted to investigate the effect of continual maternal exposure to the farming environment on development of the neonatal immune system, especially on the TLR2/4-Treg-Th (Th1, Th2, Th9, and Th17) axis, in the Jilin province. The results of our study will lay the theoretical foundation for allergic disease prevention in early life.
Methods
Study populations, maternal recruitment, and general information
One hundred and twenty-six pregnant women were recruited from the Jilin province from January 2014 to January 2016, and divided into non-farming (84 cases) and farming (42 cases) groups, with approval from the ethics committee of the Second Hospital of Jilin University. The farming group was defined as mothers living in rural areas over the last 5 years and during pregnancy, and performing routine agricultural work. The non-farming group was defined as mothers working and living away from the rural environment, having little chance of performing agricultural work. The exclusion criteria included maternal autoimmune disease history, history of medication that could affect the immune system (like systemic corticoid and immunosuppressor), infection history, and/or antibiotic usage during pregnancy, abnormal delivery process, and unhealthy newborn. Informed consent and questionnaires (items listed below) completed by the mothers before delivery were obtained.
Analysis of endotoxin and glucans in dust from the living rooms of the pregnant mothers
Dust from the living rooms of the pregnant mothers (1 month before delivery) was collected on pre-weighed glass fiber filters, using vacuum cleaners with sampling nozzles (ALK, Horsholm, Denmark) according to a standardized protocol with photo and video instructions. The dust-containing filters were weighed and extracted in a volume of 5–40 ml, and the net dust weight determined (< 0.5 g, 5 ml; 0.5–1.0 g, 10 ml; 1.0–2.0 g, 20 ml; > 2.0 g, 40 ml). Endotoxins were measured by the kinetic chromogenic Limulus Amebocyte Lysate (LAL) test, and glucans were assayed by an inhibition enzyme immunoassay (EIA) [
25].
Monitoring the health statuses of the mothers
Maternal allergy was defined as a history of doctor’s diagnosis of asthma and/or allergic rhinitis, and/or allergic dermatitis. Total IgE measurements were performed by the RAST method using peripheral blood serum of the mothers.
Umbilical cord blood collection and general neonatal status
Umbilical cord blood (20–30 ml) treated with anticoagulant was collected at childbirth, and used for cell isolation; the cells were cultured within 24 h of blood collection. Information on maternal gestational age and delivery type, and neonatal gender, height, body weight, and Apgar scores were obtained from medical records.
Cytological test of umbilical cord blood
Isolation of cord blood mononuclear cells (CBMCs), cell culture, and analysis of lymphocyte proliferation (LP)
Cord blood samples were collected from the umbilical vein after delivery and processed within 24 h. CBMCs were isolated by density-gradient centrifugation with Ficoll-Hypaque Plus (GE Healthcare, Piscataway, NJ, USA) after dilution in phosphate-buffered saline (PBS; Gibco, Carlsbad, CA, USA). The cells were washed in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) and diluted in 10% human serum (Sigma-Aldrich) to a concentration of 5 × 106 cells/ml. For the LP assay, 0.5 × 106 cells/well were seeded and stimulated with peptidoglycan (PPG; 10 µg/ml, Sigma-Aldrich), lipopolysaccharide (LPS; 0.1 µg/ml, Sigma-Aldrich), or phytohemagglutinin (PHA; 5 µg/ml, Sigma-Aldrich) for 3 days. After incubation at 37 °C in a humidified 5% CO2 incubation chamber, the samples were pulsed with 1 µCi 3H-thymidine for additional 6–10 h. The cells were harvested with a ComBI cell harvester (Skatron Instrument) onto filter plates, which were read using a β-counter.
Measurement of CD4+CD25+FOXP3+T cell percentage
CBMCs were cultured for 3 days with a concentration of 1 × 10
6 cells/ml. And then anti-human CD4–fluorescein isothiocyanate (FITC) and CD25–RPE-Cy5 (BD Biosciences) were used for surface staining, following the manufacturer’s instructions. After cell permeabilization was performed, Foxp
3-PE/corresponding isotype control antibodies (BD Biosciences,San Jose, CA, USA) were added, and then the cells were analyzed by flow cytometry (BD FACSAria II). The gating strategy was set up as done previously [
26] and is described in the Supporting Information.
Measurement of specific gene expression in Tregs (FOXP3, LAG-3, GITR, CTLA-4 and TGF-β) and TLR2/4
Total RNA from CBMCs after 3 days of culture was isolated using TriPure Isolation Reagent (Roch, Mannheim, Germany). Reverse transcription was performed (Invitrogen, Karlsruhe, Germany) for cDNA synthesis. Specific primer pairs were designed using the Primer Express software (Vector NTI advance10). The iCycler iQ-multicolor Real-Time PCR Detection System was used to detect the PCR product obtained by binding of SYBR Green (Applied Biosystems, Darmstadt) to dsDNA. CT indicates the number of PCR cycles required for the fluorescence signal to exceed the detection threshold value. The difference in CT values for two genes was used to calculate delta CT [ΔCT = CT (target gene) − CT (internal reference gene 18S)]. A higher ΔCT indicates lower gene expression. Here, we have presented the gene expression of FOXP3, LAG-3, GITR, CTLA-4, TGF-β, and TLR2/4 with un-stimulation and upon PHA, PPG, and LPS stimulation as ΔCT (Additional file
1: ST6). We also calculated the fold difference [fd = 2^(− ΔΔCT)]. ΔΔCT = ΔCT (a target sample-PHA/PPG/LPA stimulated) − ΔCT (a reference sample-unstimulated), and have presented the relative gene expression of the above genes (Fig.
3).
The designed primers were as follows:
-
18S rRNA: 5ʹ-AGTCCCTGCCCTTTGTACACA-3ʹ, 5ʹ-GATCCGAGGGCCTCACTAAAC-3ʹ
-
Foxp3: 5ʹ-ACCTTCCCAAATCCCAGTGC-3ʹ, 5ʹ-GAAGATGGTCCGCCTGGC-3ʹ
-
GITR: 5ʹ-GCACCACCCTTGTCCCCC-3ʹ, 5ʹ-CAGCGTTGTGGGTCTTGTTCC-3ʹ
-
CTLA4: 5ʹ-TGGCCCTGCACTCTCCTGT-3ʹ, 5ʹ-GGACCTCAGTGGCTTTGCCT-3ʹ
-
LAG-3: 5ʹ-CAATGGCGACTTTACCCTTC-3ʹ, 5ʹ-CCTCTGGGATGGGGTGTC-3ʹ
-
TGF-β: 5ʹ-ACAAGTTCAAGCAGAGTACACACAGC-3ʹ, 5ʹ-CACTGCCGCACAACTCCG-3ʹ
-
TLR2: 5ʹ-TTGGGGGTCATCATCAGCC-3ʹ, 5ʹ-GAGCCTGGAGGTTCACACACC-3ʹ
-
TLR4: 5ʹ-ACAAAATCCCCGACAACCTCC-3ʹ, 5ʹ-AGGGCTAAACTCTGGATGGGG-3ʹ
Measurement of specific Th1, Th2, Th9, Th17 and Treg cytokines
The supernatant from CBMCs after 3 days of culture was collected, incubated with mixed Capture Beads (IL-9, IL-10, IL-13, IL-17, IFN-γ) (BD Cytometric Bead Array (CBA) Human Soluble Protein Master Buffer Kit),and labled with PE detection reagents. Cytokines were measured using a flow cytometer under CBA Flex Sets (BD FACSAria II).
Analysis of the suppressive action of Tregs
CD4 + CD25- and CD4 + CD25 + T cells were isolated from the CBMCs by positive selection of CD4 + CD25 + T cells (Miltenyi Biotec, Köln, Germany). To show that the isolated CD4 + CD25 + T cells represent Tregs, we stained isolated them with either anti-human CD127-PE or FOXP3-PE, which are both important markers of Tregs. CD3- T cells were isolated (CD3-cell isolation kit, Miltenyi Biotec) and irradiated as antigen-presenting cells. The purity of the isolated CD3-, CD4 + CD25-, and CD4 + CD25 + T cells and the percentage of the CD127dim/- and FOXP3 + Tcells within the CD4 + CD25 + T cells were analyzed by flow cytometry (one sample shown in Fig.
4). The CD4 + CD25- T cells were labeled with CFSE [Cell Trace™ CFSE Cell Proliferation Kit (Invitrogen)] before culture. Then the CD4 + CD25- T cells were incubated for 3 days with irradiated CD3- cells in co-culture with/without CD4 + CD25 + T cells with PHA stimulation. Cell division of CD4 + CD25- T cells was assessed by flow cytometry by testing the CFSE content in the newly divided cells (one sample shown in Fig.
5). The cytokine concentrations in the supernatants (IFN-r, IL-13, IL-9, and IL-17) were measured using the BD Cytometric Bead Array (CBA) Human Soluble Protein Master Buffer Kit.
Detection of gene polymorphisms of TLR2/4 and CD14
Blood Genomic DNA Midi Kit was used for DNA extraction from umbilical cord blood. Nucleotide sequences of SNPs of TLR2-15607 (rs1898830, GG, SNP) and TLR2-16934 (rs4696480, AA, SNP), TLR4 rs4986790, and CD14 rs2569190 were obtained from the NCBI gene sequence database, and Primer5.0 was used for primer design as follows:
-
TLR2 rs1898830; 5ʹ-TTTAATGACTTATGAAAAAAATTACATATAAAA-3ʹ, 5ʹ-TTGTCTTGCCAGAGGTTCAT-3ʹ
-
TLR2 rs4696480: 5ʹ-AGAAAAGAGAGACAATAGAACATAAAACAA-3ʹ, 5ʹ-TCACCAAGGGAGCAGTTTATT-3ʹ
-
TLR4 rs4986790: 5ʹ-TTTAAATGTAATGAAAACTTGTATTCAAGG-3ʹ,
-
5ʹ-TTAACTAATTCTAAATGTTGCCATCC-3ʹ
-
CD14 rs2569190: 5ʹ-CCGGCTTCCAGGCTTC-3ʹ, 5ʹ-CCAGAGAAGGCTTAGGCTCC-3ʹ
Genotyping was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom Inc., San Diego, CA, USA) [
27]. PCR assays and associated extension reactions were designed using the Spectrodesigner software (Sequenom Inc.). The PCR amplification products were sequenced using the ABI3730 sequencing system (ABI). All amplification and extension reaction conditions were as previously described [
28]. Deviations from the Hardy–Weinberg equilibrium (HWE) were assessed for quality control of the genotyping procedures.
Statistical analysis
Data were analyzed using the SigmaStat1.0 software. Metric data were analyzed by either t-test [shown as means (SEM)] or Mann–Whitney rank test (shown as medians [25%/75% interquartile ranges (IQRs)]), depending on whether they were in normal distribution. One-way ANOVA and Tukey–Kramer test were performed for multiple groups. Attributes data were analyzed by either X2 test or Fisher’s exact test. Statistical significance was defined by P < 0.05.
Discussion
In our studies, we found that corn cultivation, collection, and drying; and livestock, poultry, and dog feeding, but not fresh-milk ingestion were the major exposure factors in rural areas in northeast China, which was remarkable different from Loss G [
4]. Endotoxin content in the living environment was significantly higher in the farming group. Farming mothers reduced their workload (by about 50%) during pregnancy, but did not change their living environment. This indicated that the increased endotoxin content (also the high microbial load) in the living environment may be the major rural exposure factor. Loss G, Carnes and Stein groups [
4,
29,
30] reported that the increased endotoxin content in the environment is a protective factor against asthma and other allergic diseases. In our study, we also conducted a telephonic follow-up and found a tendency of decreased mortality due to allergic disease in young children with farming mothers. However, more information needs to be collected in future studies.
After contact with microorganisms, the immune system of newborns with farming mothers shows overall weakened responses because of the consecutive high-load microbial stimulation during pregnancy, which may be the reason why CBMCs in the farming group presented weaker proliferation responses after LPS and PPG stimulation, when compared with CBMCs in the non-farming group. We found distinctly increased IFN-γ/IL-13 ratio after LPS stimulation in neonates with farming mothers, which indicated increased Th1/Th2 ratio. Our results are consistent with the data of Siwiec [
31]. Although it has been reported in previous studies that TH17 and Treg cell markers were positively correlated and influenced by maternal farm exposure [
32], we did not find any notable different secretion of IL-17 and positive correlation of TH17/Tregs in the infant with farming mother. Th9 cell number has been shown to be increased in allergic diseases such as asthma [
33]. We found that IL-9 secretion dropped after LPS stimulation in CBMCs from neonates in the farming group, which might be associated with their non-susceptibility to allergic diseases. Although IL-10-producing Tregs (Tr1) could secrete IL-10 [
34], we did not find any change in IL-10 production in neonates with farming mothers. The reason for the above results might be that Tr1 is not the main type of Tregs in the neonatal immune system, or that IL-10 is also secreted by Th2 cells, whose down-regulation offset the enhanced IL-10 production from Tregs.
In accord with the data of Lau et al. [
35], our research found that the quantity of the CD4 + CD25 + FOXP3 + Tregs and expression of FOXP3, GITR, and CTLA4 increased in newborns with farming mothers after LPS or PPG challenge. Recently, the function of neonatal Tregs had also been studied. Since in isolated CD4 + CD25 + T cells, the percentage of CD127dim/-T cells and FOXP3 + T cells reached more than 95%, isolated CD4 + CD25 + T cells were considered as Tregs in vitro to study the suppression of Thl, Th2, Th9, and Th17 cell differentiation. In accord with a previous hypothesis [
18,
19], our results revealed that Tregs showed stronger suppression of Th2-specific cytokine production in the farming group, while the suppressive effect on Th1 showed no difference from that in the non-farming group. It is known that reversing the innate Th2 advantage is a difficult point in preventing allergic diseases in infants and young children, because the immune state of innate Th2 advantage date back to fetal period and lasts probably for many years, then it gradually develops to Th1 polarity so as to adapt to the surrounding environment [
36,
37]. If this status, which is called “continuous fetal status of the immune system,” is not improved, the abnormality state of allergic response to foreign substances would not be eliminated [
38]. The delayed maturity of the Th1 immune system is a high-risk factor for allergic diseases after birth [
39]. Our study found that neonatal Th2 advantage status could also be influenced by parents and the environment. Increased exposure of pregnant women to microorganisms might reduce the risk to allergic diseases in offspring, which attributed partly to revising neonatal Th2 advantage status early by enhancing the suppressive action of Tregs on Th2 cell differentiation. Otherwise, the suppressive action of Tregs on Th9 cells tended to enhance in newborns with farming mothers (Fig.
6), which might be the reason for the declining IL-9 production. Given the important role of Th9 cells in maturation of the neonatal immune system [
40], it could be a new target for early prevention and immunotherapy of allergic diseases.
Some studies [
20,
41,
42] indicate that high-load of microorganisms alleviate airway inflammation, which is induced by dust mites extracts, in asthma mice. Common microorganisms in farming environment protect against asthma and other allergic diseases in animal models through modulating Tregs [
19,
21]. In some clinical researches, probiotics and prebiotics have been applied during maternal pregnancy or infancy to regulate the microbiome in infants and young children, which could prevent childhood allergy [
42].
TLR2/4 may bridge microbial exposure and Tregs and Th cells [
35]. Based on previous studies [
17], we studied four polymorphism sites on TLR2, TLR4, and CD14, and found that SNPs of TLR2-15607 and TLR2-16934 affected both neonatal Tregs and Th cells in the farming group to some extent. SNPs of TLR2-15607 and TLR2-16934 did not affect neonatal TLR2 gene expression dependent on maternal farming factor, probably because TLR2-15607 and TLR2-16934, which are locates at the TLR2 promoter regions, affected mainly TLR2 function instead of its expression [
43]. The difference in the TLR2-Treg-Th axis in infants between the farming and non-farming groups might partly be due to the functional change in TLR2. More work on the detailed mechanism and follow-up on farming infants needs to be performed, aiming to delineate the influence of neonatal immune status on the susceptibility to allergic diseases during childhood.
In conclusion, endotoxin content was higher in the farming area than in the non-farming area in the Jilin province, China. Maternal living in farming environment for more than 5 years affected the innate immune system of offspring, which might partly be due to neonatal TLR2 gene polymorphism and expression. In the farming group, through the TLR2/4 pathway, maternal microbial over-load affected the quantity and function of neonatal Tregs, which regulated the early differentiation of Th1/Th2, even Th9 cells, and then reduced the susceptibility to allergic diseases in offspring. Our findings provide a new target and probable method for protecting children against asthma and other allergic diseases at the early stage. But limited to the quantities of volunteer, parts of results showed only tendency but no statistic differences. So in the future we will recruit more pregnant women as well as finish the following-up to observe the allergic status of offspring.
Authors’ contributions
JL conceived and designed the experiments and final approval of the version to be published; JY performed the experiments and wrote the manuscript; XL and YL analyzed the data; SM, FW and BY contributed reagents/materials/analysis tools; YX, TM, JY contributed to the conception of the study. All authors read and approved the final manuscript.