Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections / 生物医学与环境科学（英文）

OBJECTIVE@#Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.@*METHODS@#A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.@*RESULTS@#Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.@*CONCLUSION@#Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

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.

Effect of Retinoic Acid on the Differentiation and Lysozyme Expression in Human Airway Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Retinoic acid (RA)-deprived cultures of normal human tracheobronchial epithelial (NHTBE) cells became squamous metaplastic, failed to produce mucin and instead secreted or released large amounts of lysozyme (LZ). The purpose of this study was to elucidate the relationship between RA-deficiency induced squamous metaplasia and increased LZ as a function of time. MATERIALS AND METHOD:The change of lysozyme protein and lysozyme mRNA was investigated over time in cultures using passage-2 normal human tracheobronchial epithelial (NHTBE) cells and passage-2 normal human keratinocytes (NHK). The amount of lysozyme and mucin was measured with dot blot, message of lysozyme with RT-PCR, and cornifin mRNA with Northern blot. RESULTS: Lysozyme message levels were consistently higher in RA-sufficient than RA-deficient cultures. Intracellular and extracellular LZ increased to a peak on the day 16 and thereafter decreased in the RA-deficient cultures. LZ gene expression in the RA-deficient cultures was barely detectable on the day 7 but was clearly expressed between days 10 and 14, but thereafter message levels decreased markedly. On day 12, large numbers of cells began to exfoliate in the RA-deficient cultures. Extracellular LZ appeared simultaneously at the apical surface, presumably released from the exfoliated cells, which contained high concentrations of LZ. Intracellular LZ levels were more than 11 fold less in NHK cells compared to NHTBE cells. CONCLUSION: This study suggests that cellular accumulation of lysozyme protein is a unique feature of metaplastic squamous differentiation. Further studies are needed to find out what mechanisms are involved.

Regulation of Mucin and Non-Mucin Secretions and Gene Expression by Retinoic Acid in Human Airway Epithelium / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Airway hypersecretion is a frequent feature of several respiratory tract diseases including rhinitis, sinusitis, and otitis media. Efforts are being made in several laboratories to elucidate mechanisms involved in the regulation of secretion. There are several factors which modulate expression of the secretory phenotype, such as retinoic acid (RA), triiodothyronine, steroid, and extracellular matrix. We have been interested in elucidating the role of retinoids in regulating differentiation of mucin and non-mucin secretions. MATERIALS AND METHODS: Retinoic acid was removed from the culture media of normal human tracheobronchial epithelial cells grown in the air-liquid interface cultures. The effects on cell phenotype and mucin, lysozyme (LZ), and the secretory leukocyte protease inhibitor (SLPI) secretion and gene expression were examined. RESULTS: Removal of RA from the media induced squamous differentiation and caused a drastic decrease in mucin secretion and a decrease in expression of the mucin genes, MUC2 and MUC5AC. Lysozyme and SLPI secretions were increased in RA-depleted cultures. Paradoxically, LZ mRNA was decreased, while the SLPI mRNA levels were increased. A most intriguing finding was the paradoxical response of LZ to RA-depletion. The reason for this apparant incongruity between mRNA and protein levels is currently under investigation. CONCLUSION: Our studies show that RA is an important factor for mucous differentiation.

Roles of NF-?B in expression of hBD-2 mRNA in human primary epithelial cells induced by TNF-? / 第三军医大学学报

Objective To explore the roles of NF-?B in expression of human ?-defensin-2(hBD-2) mRNA induced by TNF-? in the human airway primary epithelial cells.Methods After the human bronchial primary epithelial cells were stimulated with TNF? or first with NF-?B inhibitor PDTC,then TNF-?,the expression of hBD-2 mRNA was detected by RT-PCR.The I?B-? protein level in the cytoplasm was detected by Western blotting and the nuclear factor-kappa B(NF-?B) binding activity was analyzed by electrophoretic mobility shift assays.Results The hBD-2 mRNA could be detected after 2.5 h of TNF-? stimulation and expressed in a dose-dependent manner.The NF-?B could be activated after 0.5 h of TNF-? stimulation.The supershifts assays indicated that the p65-p50 heterodimer formed complexes of NF-?B were involved in the activated of NF-?B.Conclusion TNF-? can induce the expression of hBD-2 mRNA in a dose-dependent manner.The p65-p50 heterodimer formed complexes of NF-?B play an important role in the regulation of hBD-2 gene expression in response to TNF-?.