BackgroundAllergic disease can be characterized as manifestations of an exaggerated inflammatory response to environmental allergens triggers. Mite allergen Der-p2 is one of the major allergens of the house dust mite, which contributes to TLR4 expression and function in B cells in allergic patients. However, the precise mechanisms of Der-p2 on B cells remain obscure.Methodology/Principal FindingsWe investigated the effects of Der-p2 on proinflammatory cytokines responses and Toll-like receptor-4 (TLR4)-related signaling in human B cells activation. We demonstrated that Der-p2 activates pro-inflammatory cytokines, TLR4 and its co-receptor MD2. ERK inhibitor PD98059 significantly enhanced TLR4/MD2 expression in Der-p2-treated B cells. Der-p2 markedly activated mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) and decreased p38 phosphorylation in B cells. MKP-1-siRNA downregulated TLR4/MD2 expression in Der-p2-treated B cells. In addition, Der-p2 significantly up-regulated expression of co-stimulatory molecules and increased B cell proliferation. Neutralizing Der-p2 antibody could effectively abrogate the Der-p2-induced B cell proliferation. Der-p2 could also markedly induce NF-κB activation in B cells, which could be counteracted by dexamethasone.Conclusions/SignificanceThese results strongly suggest that Der-p2 is capable of triggering B cell activation and MKP-1-activated p38/MAPK dephosphorylation-regulated TLR4 induction, which subsequently enhances host immune, defense responses and development of effective allergic disease therapeutics in B cells.

PurposeHouse-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.MethodsInternalization 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).ResultsInternalization 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.ConclusionsHuman 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.

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PurposeMyeloid 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.MethodsThe 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.ResultsPatients with the MD-2 promoter SNPs (rs1809441/rs1809442) had increased mRNA expressions of MD-2, ε heavy chain of IgE (Cε), 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-1β, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-α 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-γ and decreased secretion of IL-4 were noted after LPS stimulation.ConclusionsThe 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.