Allergy Asthma Immunol Res.  2017 Mar;9(2):133-141. 10.4168/aair.2017.9.2.133.

Neonatal Immune State Is Influenced by Maternal Allergic Rhinitis and Associated With Regulatory T cells

• Tan L
• Ou J
• Tao Z
• Kong Y
• Xu Y

• KMID: 2413387
• DOI: http://doi.org/10.4168/aair.2017.9.2.133

Abstract

PURPOSE
Maternal influences contribute to the origin of allergic diseases, but the mechanisms are not clear. The current literature prompted the role of epigenetics in the development of allergic diseases. We sought to investigate the roles of regulatory T (Treg) cells and Forkhead box p3 (Foxp3) DNA methylation in the process of maternal transmission of allergic rhinitis (AR) susceptibility.
METHODS
BALB/c female mice (AR mother) were sensitized by intraperitoneal injection of Dermatophagoides pteronyssinus (Der p) 1 on day 1 and 7. Then they mated with normal male mice on day 8. From day 21 to 28, the female mice were intranasal challenged with Der p 1 continuously. The normal controls were given with normal saline in the same way. On postnatal day 3, Female mice and their offspring were sacrificed to detect their histopathology in nasal mucosae, cytokines in sera of mother and spleen homogenates of offspring, Treg cells count, Foxp3 mRNA expressions, and Foxp3 DNA methylation levels in spleens.
RESULTS
Compared with the normal controls, neonatal offspring of Der p 1-stimulated female mice (AR offspring) showed the elevation of interleukin (IL)-4 (P<0.01) and IL-17 (P<0.01), the submission of IL-10 (P<0.01) in spleen homogenates. Further, Treg cells count in AR offspring decreased remarkably compared with the normal offspring (P<0.01). Though the difference of Foxp3 DNA methylation level between AR offspring and normal control offspring was not obvious, correlation analysis demonstrated that there was significantly positive correlation between Foxp3 DNA methylation level of mother and that of offspring (r=0.803, P<0.01).
CONCLUSIONS
Under the influence of Maternal AR, their neonatal offspring develop into T-helper type 2 (Th2) dominant immune state, which is closely associated with the recession of Treg cells. Foxp3 DNA methylation may be a mechanism responsible for that maternal effect but still need more studies to ensure.

Keyword

Allergic rhinitis; maternal; offspring; regulatory T cell; DNA methylation; forkhead box p3

MeSH Terms

Animals
Cytokines
Dermatophagoides pteronyssinus
DNA Methylation
Epigenomics
Female
Humans
Injections, Intraperitoneal
Interleukin-10
Interleukin-17
Interleukins
Male
Mice
Mothers
Nasal Mucosa
Rhinitis, Allergic*
RNA, Messenger
Spleen
T-Lymphocytes, Regulatory*
Cytokines
Interleukin-10
Interleukin-17
Interleukins
RNA, Messenger

Figure

• 

  Fig. 1 Experimental Protocol. After fed adaptively for 3 days, maternal mice were sensitized by initial intraperitoneal (i.p.) injections of 400 µL phosphate-buffered saline (PBS) containing 1 µg Der p1 (Indoor Biotechnologies, Charlottesville, Virginia) and aluminum hydroxide (4 mg) on day 1 and day 7. After the last maternal sensitization, the female mice were placed in cages to mate with normal male mice according to the female and male ratio 2:1 on day 8. From day 21 to day 28, the female mice were intranasal challenged with 20 µL PBS containing Der p 1 (2 µg) continuously. The normal mother group was sensitized and challenged with normal saline in the same way. The offspring were not stimulated with Der p 1, and were humanely killed for analysis 3 days after birth.

• 

  Fig. 2 HE and PAS staining of nasal mucosae (original magnification ×400). (A) The cytoplasm of eosinophils (black arrows) in the nasal lamina propria (LP) stains red by HE. (B) The cytoplasm of goblet cell in the nasal epithelial layer stains aubergine by PAS. Histopathology showed eosinophils accumulation and goblet cells hyperplasia in the nasal lamina propria in AR mother group and AR offspring group. HE, hematoxylin-eosin; PAS, periodic acid-schiff stain; AR, allergic rhinitis.

• 

  Fig. 3 Cytokines sections in sera of mother and spleen homogenates of offspring. The concentrations of IFN-γ, TGF-β, IL-10, IL-17, and IL-4 in sera of mother and spleen homogenates of offspring were measured by ELISA. (n=5 for each group; *P<0.05, **P<0.01). IFN, Interferon; TGF, transforming growth factor; IL, interleukin; ELISA, enzyme-linked immunosorbent assay.

• 

  Fig. 4 CD4+CD25+FOXP3+Tregs of total lymphocytes in spleens. Treg cells count in the spleen was measured by flow cytometry. (A) The analysis of CD4 vs FOXP3 expression was gated in CD4+CD25+ cells. (B) CD4+CD25+FOXP3+Tregs were calculated in each group (n=5 for each group; *P<0.01).

• 

  Fig. 5 Foxp3 mRNA Expressions. Foxp3 mRNA expression level in the spleen mononuclear cells was measured by RT-PCT. Relative Foxp3 mRNA levels were analyzed using the 2−ΔΔCT method (n=4–5 for each group; *P<0.01).

• 

  Fig. 6 Foxp3 DNA methylation level. Six CpG islands methylation level in Foxp3 promoter region measured by Bisulfite sequencing PCR. CpG islands methylation levels in the AR mother group had a tendency of elevation compared to normal controls. While CpG (−200), CpG (−70), CpG (−61), and CpG (−52) methylation levels in the AR offspring group were slightly increased than which in normal controls. But all the differences of CpG islands methylation levels were not distinguishable by statistical analysis (n=4–5 for each group; P>0.05).

• 

  Fig. 7 Correlation analysis between Foxp3 DNA methylation level of mother and that of offspring. Average DNA methylation level of CpG (−200), CpG (−70), CpG (−61) and CpG (−52) in all offspring was paired with that in their respective mothers. Based on linear regression model, correlation analysis demonstrated that there was significant positive correlation between Foxp3 DNA methylation level of mother and that of offspring (r=0.681, P<0.05).

• 

  Fig. 8 Correlation analysis between Foxp3 DNA methylation and mRNA expression. Average DNA methylation level of CpG (−200), CpG (−70), CpG (−61), and CpG (−52) was paired with Foxp3 mRNA expression in each individual mouse. Based on linear regression model, result demonstrated that there was significant negative correlation between average Foxp3 DNA methylation level and mRNA expression level in mother groups (r=0.924, P<0.01) and offspring groups (r=0.749, P<0.05).

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