Der p2 Internalization by Epithelium Synergistically Augments Toll-like Receptor-Mediated Proinflammatory Signaling

PURPOSE: House-dust-mite (HDM) major allergen Der p2 shares homology and function with Toll-like receptor (TLR) signaling protein myeloid differentiation-2 (MD2) and may lead to airway inflammation. Should Der p2 be internalized by human airway epithelium, it has the theoretical propensity to potentiate epithelium activation. This study aimed to demonstrate the internalization of Der p2 by airway epithelium and to investigate the effects of Der p2 on MD2 expression and epithelium activation. METHODS: Internalization of recombinant, enhanced green fluorescent protein-labelled Der p2 (rDer p2-EGFP) into human airway epithelium (BEAS-2B) was tracked by laser confocal microscopy and confirmed by immunoblotting. Reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemical staining were used to determine the effect of Der p2 on MD2 expression in vitro and ex vivo. Expression of messenger RNA (mRNA) encoding receptors/cytokines was measured by RT-PCR. Secretion of interleukin-6/interleukin-8 (IL-6/IL-8) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Internalization of Der p2 by BEAS-2B was confirmed by confocal microscopy and immunoblotting using rDer p2-EGFP and rDer p2, respectively. Expression of MD2 protein was increased in BEAS-2B and human nasal polyp airway epithelium cultured with rDer p2. Recombinant Der p2-cultured BEAS-2B caused little spontaneous IL-6/IL-8 secretion but significantly augmented by TLR ligand LPS. IL-6 secretion was up-regulated after MD2 transfection. Internalization of Der p2 was reduced by TLR2 RNA knockdown. Dexamethasone, calcitriol, anti-MD2/anti-TLR2 antibodies, and signalling inhibitors significantly reduced LPS+Der p2-induced IL-6/IL-8 secretion. CONCLUSIONS: Human airway epithelium may internalize Der p2, which potentiates the response to environmental proinflammatory stimuli through MD2 and TLRs. This study highlights a novel mechanism and alleviates IL-6/IL-8 secretion in mite-induced airway inflammation.

Association of Single Nucleotide Polymorphisms in the MD-2 Gene Promoter Region With Der p 2 Allergy

PURPOSE: Sensitization to house dust mite (Dermatophagoides pteronyssinus) is a considerable risk factor for the progression of allergic disease. The group 2 allergen from Dermatophagoides pteronyssinus, Der p 2, is considered a major one in patients with specific immunoglobulin E (IgE) to Der p 2. Der p 2 has structural homology with myeloid differentiation 2 (MD-2), which is involved in the lipopolysaccharide-binding component of the Toll-like receptor 4 signaling pathway and the development of inflammation. The aim of this study was to examine the genetic association of single nucleotide polymorphisms (SNPs) in the promoter region of MD-2 with Der p 2-sensitive allergy. METHODS: We investigated associations between cohort's characteristics, including 280 allergic and 80 healthy subjects by examining total IgE, eosinophils, D. pteronyssinus-specific IgE, Der p 2-specific IgE, the number of IgE-producing B cells induced by Der p 2, and the odds ratio of allergic symptoms. RESULTS: Based on the 1,000 genome project data, the minor allele frequencies of the rs1809441 and rs1809442 are 0.467 and 0.474, respectively. However, the correlation of linkage disequilibrium (LD) between these 2 SNPs is D'=1, the genotype frequencies of the 2 MD-2 (LY96) SNPs (rs1809441 and rs1809442) that are located nearby were significantly different between allergic and health subjects: the TT genotype of rs1809441 and the GG genotype of rs1809442 were more frequent in allergic subjects than in healthy subjects (16.1% vs 2.5% in both genotypes). The allergic patients with these genotypes exhibited significantly higher levels of D. pteronyssinus-specific IgE and Der p 2-specific Ig E, and a larger number of Der p 2-activated B cells. In addition, these 2 SNPs in the MD-2 promoter region were significantly associated with the prevalence of nasal, skin, and asthmatic allergic symptoms. CONCLUSIONS: Our results indicated that 2 SNPs in the MD-2 promoter region were significantly associated with Der p 2-specific Ig E, and thereby suggest that these SNPs may play a major role in susceptibility to Der p 2-triggered immune responses in a Taiwanese population.

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

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.

House dust mite allergy: environment evaluation and disease prevention

There are two groups of dust mites, house dust mites (HDMs) and storage mites (SMs), that have been identified in the household environment. Both could induce airway inflammation through activation of innate and adaptive immunity and lead to asthma. In order to monitor environmental dust mite infestation, different methods can be used to detect their presence, such as the use of floating methods, monoclonal antibodies, and nanostructured biosensor. SM could be identified in the storage room, mainly in contaminated food such as mushrooms and corn starch. In HDM-sensitive subjects and mice that were challenged with HDM or SM after sensitization, these mites could up-regulate IgE levels, T helper 2 associated cytokine production and airway hypersensitivity. Different age groups of subjects were sensitized by different species of mites. More subjects above 70 years were sensitized by SM and more subjects below the age of 40 years were sensitized to HDM. Different allergenic components of dust mite extracts, such as Der p 1, Der p 2, could activate innate immunity through activating pattern recognition receptor (PRR) and then lead to allergic inflammation. The best modality to treat HDM allergy is immunomodulation through Treg cells and IgA production. In the recent years, many studies indicated probiotics could increase IgA secretion and the number of Treg cells. However, some studies conducted in adults have contradictory effects in reducing allergic symptoms. Therefore, probiotics confer inconclusive benefits on the allergic symptoms.