Allergy Asthma Immunol Res.  2015 Sep;7(5):497-506. 10.4168/aair.2015.7.5.497.

Enhanced Allergic Inflammation of Der p 2 Affected by Polymorphisms of MD-2 Promoter

• Liao EC
• Hsieh CW
• Chang CY
• Yu SJ
• Sheu ML
• Wu SM
• Tsai JJ

• KMID: 2147959
• DOI: http://doi.org/10.4168/aair.2015.7.5.497

Abstract

PURPOSE
Myeloid differentiation-2 (MD-2) has been associated with endotoxin and inflammatory disorders because it can recognize lipopolysaccharide (LPS) binding and attenuate Toll-like receptor 4 (TLR4)-mediated signaling. However, its role in allergic inflammation has yet to be clarified. We examined whether single nucleotide polymorphisms (SNPs) in MD-2 promoter can affect MD-2 expression and aimed to clarify the relationship between Der p 2 allergy and SNPs of MD-2 promoter.
METHODS
The function of SNPs of MD-2 promoter and the effects of cytokines and immunoglobulin on the secretion and mRNA expression were investigated in 73 allergic subjects with different MD-2 gene promoter variants. Peripheral blood mononuclear cells were cultured with or without LPS in the presence of Dermatophagoides pteronyssinus group 2 allergen (Der p 2), followed by mRNA extraction and cytokine expression analysis. The culture supernatants were collected for cytokine measurement.
RESULTS
Patients with the MD-2 promoter SNPs (rs1809441/rs1809442) had increased mRNA expressions of MD-2, epsilon heavy chain of IgE (Cepsilon), and interleukin (IL)-8; however, only MD-2 and IL-8 were further up-regulated after Der p 2 stimulation. Patients with SNPs of MD-2 promoter tended to have high levels of IL-1beta, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-alpha after Der p 2 and LPS stimulation. Increased secretions of IL-6, IL-8, and IL-10 were found to be up-regulated by Der p 2 stimulation, and an increased secretion of IFN-gamma and decreased secretion of IL-4 were noted after LPS stimulation.
CONCLUSIONS
The high levels of proinflammatory cytokines secreted by Der p 2 were predetermined by MD-2 promoter SNPs (rs1809441/rs1809442). Through cytokine secretion by Der p 2 and LPS, these SNPs may serve as an indicator of the pathological phenotype of Der p 2-induced allergic inflammation.

Keyword

Dermatophagoides pteronyssinus; Der p 2; myeloid differentiation-2; single nucleotide polymorphisms; allergy

MeSH Terms

Cytokines
Dermatophagoides pteronyssinus
Humans
Hypersensitivity
Immunoglobulin E
Immunoglobulins
Inflammation*
Interleukin-10
Interleukin-4
Interleukin-6
Interleukin-8
Interleukins
Phenotype
Polymorphism, Single Nucleotide
RNA, Messenger
Toll-Like Receptor 4
Tumor Necrosis Factor-alpha
Cytokines
Immunoglobulin E
Immunoglobulins
Interleukin-10
Interleukin-4
Interleukin-6
Interleukin-8
Interleukins
RNA, Messenger
Toll-Like Receptor 4
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Effects of MD-2 promoter SNPs on transcription activity. (A) The promoter activity was investigated using a reporter gene assay system. A total of 5 plasmid constructs were prepared by inserting different genotypes of the MD-2 gene promoter region into a pGL3-Basic vector, which contained a promoterless (pGL3-basic), or genotype of CC (rs1809442) GG (rs1809441), or a single mutant genotype of CG and GT, or a combined mutant genotype of GG and TT, or a positive control of SV-40 promoter. (B) Relative luciferase activity (RLA) was assayed in epithelium cells (BEAS-2B) transfected with different plasmid constructs without Der p 2 stimulation. Luciferase activity was normalized for transfection efficiency using a control plasmid (pGL3-Basic). Results are expressed as the fold increase in RLA of the MD-2 promoter construct vector as compared to pGL3-Basic. The values of luciferase activity are expressed as means±SD of the results from each group (n=5). *P<0.05 compared to the MD-2 promoter genotype of rs1809441-G/rs1809442-C constructs; **P<0.01 compared to the MD-2 promoter genotype of rs1809441-G/rs1809442-C constructs. (C) The scoring scheme was performed by simple routine searches highly correlated sequence fragments versus TFMATRIX transcription factor binding site profile database. Score=100.0* ('weight sum' - min)/( max-min); *, sequence position of SNP rs1809441; #, sequence position of SNP rs1809442.

• 

  Fig. 2 The mRNA expressions of ε heavy chain of IgE (Cε) and GATA-3 in PBMCs from subjects with the SNP rs1809441/42 mutant and wild genotypes were evaluated using reverse-transcriptase polymerase chain reaction (RT-PCR). (A) The 0, 24, 48, and 72 represent the time points (hours) after allergen rDer p 2 (10 µg/mL) challenge. The GAPDH expression acted as an internal-control. (B) The Cε expression was evaluated using RT-PCR. Data are expressed as mean of each group. Medium, detected without allergen challenge; Der p 2, detected after rDer p 2 (10 µg/mL) challenge. (C) The expression of the transcription factor GATA-3 was evaluated using RT-PCR; **P<0.05 compared between the MD-2 promoter SNPs (+) and SNPs (-) under medium only; ***P<0.01 compared between MD-2 promoter SNPs (+) and SNPs (-) under Der p 2 (10 µg/mL) challenge. ##P<0.05 compared between medium and rDer p 2 (10 µg/mL) challeges in the group of MD-2 promoter SNPs (+).

• 

  Fig. 3 The SNPs of MD-2 gene promoter affect the MD-2 mRNA expression in PBMC. The MD-2 mRNA expression was evaluated using RT-PCR. Data are expressed as mean of each group. Medium, detected without allergen challenge; Der p 2, detected after the rDer p 2 (10 µg/mL) challenge; *P<0.05 compared between the MD-2 promoter SNPs (+) and SNPs (-) without treatment; **P<0.05 compared between the MD-2 promoter SNPs (+) and SNPs (-) under rDer p 2 challenge; ##P<0.05 compared between medium and rDer p 2 (10 µg/mL) challenge in the group of MD-2 promoter SNPs (+).

• 

  Fig. 4 The SNPs of the MD-2 promoter affect Cε and Iγ1/2-Cµ mRNA expressions in PBMCs. The expressions of ε heavy chain of IgE (Cε) and Iγ1/2-Cµ mRNA were evaluated using RT-PCR. Data are expressed as mean of each group. Medium, detected without treatment; Der p 2, detected after the rDer p 2 (10 µg/mL) challenge; LPS, detected after LPS (100 ng/mL) treatment. ##P<0.05 compared between the MD-2 promoter SNPs (+) and SNPs (-) under different treatments.

• 

  Fig. 5 The SNPs of MD-2 promoter affect the mRNA expressions of IL-1β and IL-8 in PBMCs. The mRNA expressions of IL-1β and IL-8 were evaluated using RT-PCR. Data are expressed as mean of each group. Medium, detected without treatment; Der p 2, detected after the rDer p 2 (10 µg/mL) challenge; ##P<0.05 compared between medium and rDer p 2 challenge; **P<0.05 compared between the MD-2 promoter SNPs (+) and SNPs (-) under medium only; ***P<0.01 compared between MD-2 promoter SNPs (+) and SNPs (-) under Der p 2 (10 µg/mL) challenge.

• 

  Fig. 6 MD-2 promoter SNPs (rs1809441/rs1809442) affect the binding affinity of GATA-3 and TH2-cytokine expression. The binding activity of GATA-3 and TH2-cytokine expression were analyzed by electrophoretic mobility shift assay (EMSA) and flow cytometry, respectively. The human promyelocytic leukemia cell line (HL-60) and human monocyte derived from donor' PBMCs were employed for GATA-3 binding activity. The binding activity of GATA-3 to these MD-2 promoter SNPs was analyzed by EMSA using an oligonucleotide containing the tandem GATA motif (-136 to -161). (A) The binding activity of the transcription factor GATA-3 was induced by Der p 2 (1.5 µg/mL) at different time points (0-8 hours) with the human promyelocytic leukemia cell line HL-60. (B) Human monocytes derived from subjects with (+) or without (-) MD-2 promoter SNPs were collected to analyze the binding affinity of GATA-3. SNP (+), MD-2 promoter SNPs (rs1809441-T/rs1809442-G); SNP (-), MD-2 promoter SNPs (rs1809441-G /rs1809442-C). Quantification: hybridization signals were quantified by the image analysis program. (C) Cytokines in activated TH cells derived from donor's leukocytes underwent immunofluorescence staining and flow cytometry. Three-color staining methods were used to analyze the expressions of IL-4 and interferon (IFN)-γ in CD4+ cells. Mean fluorescence was measured using a flow cytometer and is presented in percentages. A total of 5,000 cells were analyzed in each sample.

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