Allergy Asthma Immunol Res.  2015 Jul;7(4):393-403. 10.4168/aair.2015.7.4.393.

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

• Yin SC
• Liao EC
• Chiu CL
• Chang CY
• Tsai JJ

• KMID: 2166687
• DOI: http://doi.org/10.4168/aair.2015.7.4.393

Abstract

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.

Keyword

Internalization; Der p2; human airway epithelium; Toll-like receptor 2; inflammation; calcitriol

MeSH Terms

Antibodies
Calcitriol
Dexamethasone
Enzyme-Linked Immunosorbent Assay
Epithelium*
Humans
Immunoblotting
Inflammation
Interleukin-6
Microscopy, Confocal
Nasal Polyps
Polymerase Chain Reaction
RNA
RNA, Messenger
Toll-Like Receptor 2
Toll-Like Receptors
Transfection
Antibodies
Calcitriol
Dexamethasone
Interleukin-6
RNA
RNA, Messenger
Toll-Like Receptor 2
Toll-Like Receptors

Figure

• 

  Fig. 1 Images of Der p2-EGFP and co-localized with ER-RFP in BEAS-2B cells by confocal laser scanning biological microscopy. Cells were cultured with 5 µg/mL of rDer p2-EGFP or rEGFP alone. No fluorescence could be detected in cells incubated with rEGFP alone (A). Fluorescence from Der p2-EGFP appeared in the cytoplasm after 4-hour (B) and 24-hour (C) incubation. Cells were also co-transduced with CellLight™ ER-RFP. Der p2-EGFP co-localized with ER-RFP after 24-hour incubation (C). A representative image and an actual figure representative of the 3 independent experiments with similar findings are shown. DIC, differential interference contrast; EGFP, enhanced green fluorescent protein; ER-RFP, endoplasmic reticulum-red fluorescent protein.

• 

  Fig. 2 Increased expression of MD2 mRNA and Der p2/MD2 protein in rDer p2-treated BEAS-2B cells and MD2 in nasal polyps. The expression of MD2 mRNA after 1 (1X) and 2 (2X) consecutive rounds of PCR amplification (4 hours) (A), Der p2 and MD2 protein by immunoblotting (24 hours) (B) in BEAS-2B cells incubated with 10 µg/mL of rDer p2. (C) Nasal polyps were cultured (4 hours) with medium only (left) or 10 µg/mL of rDer p2 (right). Tissue sections were immunostained with anti-MD2 antibody using DAB as a peroxidase substrate, and the mean intensity of staining was measured objectively (D). A representative image and an actual figure representative of the 3 independent experiments with similar findings are shown.

• 

  Fig. 3 Enhanced effects of LPS and reduced effects of anti-MD2 antibody on IL-6/IL-8 protein secretion by Der p2-activated BEAS-2B cells. Cells were cultured with rDer p2 in conjunction with LPS without (A) and with (B) additional anti-MD2 antibody for 24 hours. Fold changes in expression are shown in comparison to control treatment with medium. Data are presented as mean±SEM of the 3 separate experiments. A representative image and an actual figure representative of the 3 independent experiments with similar findings are shown. **P<0.01; ***P<0.001.

• 

  Fig. 4 MD2 overexpression increased IL-6 secretion by Der p2-activated BEAS-2B cells. MD2 overexpression was confirmed by immunoblotting with anti-MD2 antibody (A), the increasing fold of IL-6 secretion are shown in comparison to non-transfected control cells treated with medium (B). Data are presented as mean± SEM of the 3 separate experiments. A representative image and an actual figure representative of the 3 independent experiments with similar findings are shown.

• 

  Fig. 5 Effects of Der p2 with or without LPS on expression of mRNA encoding Toll-like receptors and cytokines in BEAS-2B cells cultured for 3, 6, and 16 hours. (A) Der p2 notably increased the expression of mRNA encoding IL-6 and IL-8, and this effect was further augmented in the presence of LPS. There was little or no change in the expression of mRNA encoding TLR4, whereas TLR2 and IL-1β were somewhat increased, especially in conjunction with LPS. Quantitative data on band densities were acquired by densitometric analysis and expressed relative to those of the β-actin loading control, which are shown under the bands. (B) Densitomeric analysis data are presented as the mean±SEM. A representative image and an actual figure representative of the 3 independent experiments with similar findings are shown.

• 

  Fig. 6 Effects of various inhibitory agents on mRNA expression in LPS + rDer p2-treated BEAS-2B cells and effects of TLR2 knockdown on the internalization of Der p2 by BEAS-2B cells. (A) Messenger RNA encoding TLR2, IL-6, and IL-8, and β-actin loading control in BEAS-2B cells cultured (6 hours) with rDer p2 in conjunction with LPS. Quantitative data were acquired by densitometric analysis and normalized to the expression of β-actin, which are shown under the bands. (B) Reduced fold induction of IL-6 and IL-8 mRNA induced by rDer p2 in conjunction with LPS. The most effective inhibitors on IL-6/IL-8 mRNA expression were dexamethasone, SP600125 (JNK inhibitor), calcitriol, SB203580 (p38 inhibitor), and BAY 11-7082 (IκB inhibitor). Reduction of fold induction (%) = [1- (LPS + rDer p2 stimulation with inhibitor/without inhibitor)] ×100%. Data are presented as the mean±SEM. (C) TLR2-knockdowned cells were incubated with rDer p2 for 24 hours, followed by rDer p2 protein analysis with immunoblotting. The concentration of Der p2 protein was notably reduced in the cellular lysates of shTLR2-knockdowned and rDer p2-treated cells. A representative image and an actual figure representative of the 3 independent experiments with similar findings are shown. *P<0.05; **P<0.01.

• 

  Fig. 7 Effects of various inhibitory agents on IL-6/IL-8 protein secretion in LPS + rDer p2-treated BEAS-2B cells. (A) Transcription-signalling inhibitors, (B) anti-inflammatory drugs and (C) neutralizing antibodies on IL-6/IL-8 protein secretion (24 hours) by LPS (50 ng/mL)- and rDer p2 (5 µg/mL)-stimulated BEAS-2B cells. (D) Reduction of fold induction by various agents on rDer p2 in conjunction with LPS-induced IL-6/IL-8 secretion. The most effective inhibitors on IL-6/IL-8 protein secretion were dexamethasone, SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), anti-TLR2 neutralizing antibody, and calcitriol. Reduction of fold induction (%) = [1 - (LPS + rDer p2 stimulation with inhibitor/without inhibitor)] ×100%. Data presented as mean±SEM of 3 independent experiments. **P<0.01; ***P<0.001.

• 

  Fig. 8 Schematic diagram of Der p2 internalization and induction of human airway epithelial cell activation. Der p2 was internalized by epithelial cells, and the up-regulated MD2 and TLR2 resulted in IL-6/IL-8 secretion, which were confirmed by anti-MD2 antibody, anti-TLR2 antibody, and shTLR2. The secretion of IL-6/IL-8 was also inhibited by MAPK/IκB inhibitors, dexamethasone, and calcitriol.

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