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

Affiliations
  • 1Center for Translational Medicine, Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan.
  • 2Department of Bioindustry Technology, Da-Yeh University, Changhua, Taiwan.
  • 3Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan.
  • 4Instrumentation Center, Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan.
  • 5Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan. jawji@vghtc.gov.tw
  • 6Institute of Biomedical Sciences, National Chung-Hsing University, Taichung, Taiwan.
  • 7Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan.

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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Reference

1. Platts-Mills TA, Vervloet D, Thomas WR, Aalberse RC, Chapman MD. Indoor allergens and asthma: report of the Third International Workshop. J Allergy Clin Immunol. 1997; 100:S2–24.
2. Tsai JJ, Kao MH, Huang SL. Comparison of major aeroallergens in Taipei and Kin-Men. J Formos Med Assoc. 1997; 96:985–989.
3. Tsai JJ, Shen HD, Chua KY. Purification of group 2 Dermatophagoides pteronyssinus allergen and prevalence of its specific IgE in asthmatics. Int Arch Allergy Immunol. 2000; 121:205–210.
4. Trompette A, Divanovic S, Visintin A, Blanchard C, Hegde RS, Madan R, et al. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein. Nature. 2009; 457:585–588.
5. Chiou YL, Lin CY. Der p2 activates airway smooth muscle cells in a TLR2/MyD88-dependent manner to induce an inflammatory response. J Cell Physiol. 2009; 220:311–318.
6. Hsu SC, Chen CH, Tsai SH, Kawasaki H, Hung CH, Chu YT, et al. Functional interaction of common allergens and a C-type lectin receptor, dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN), on human dendritic cells. J Biol Chem. 2010; 285:7903–7910.
7. Jia HP, Kline JN, Penisten A, Apicella MA, Gioannini TL, Weiss J, et al. Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2. Am J Physiol Lung Cell Mol Physiol. 2004; 287:L428–L437.
8. Tsai JJ, Liu SH, Yin SC, Yang CN, Hsu HS, Chen WB, et al. Mite allergen Der-p2 triggers human B lymphocyte activation and Toll-like receptor-4 induction. PLoS One. 2011; 6:e23249.
9. Liu YH, Kao MC, Lai YL, Tsai JJ. Efficacy of local nasal immunotherapy for Dp2-induced airway inflammation in mice: using Dp2 peptide and fungal immunomodulatory peptide. J Allergy Clin Immunol. 2003; 112:301–310.
10. Liao EC, Tsai JJ. Clinical effectiveness of Tyrophagus putrescentiae allergy by local nasal immunotherapy using strips of Dermatophagoides pteronyssinus. J Asthma. 2011; 48:957–964.
11. Merkle D, Zheng D, Ohrt T, Crell K, Schwille P. Cellular dynamics of Ku: characterization and purification of Ku-eGFP. Chembiochem. 2008; 9:1251–1259.
12. Rostkowska-Nadolska B, Latocha M, Gawron W, Kutner A, Bochnia M. The influence of calcitriol and tacalcitol on proliferation of fibroblasts cultured from nasal polyps. Adv Clin Exp Med. 2007; 16:213–219.
13. Sato H, Ogino Y, Takagi H, Hata J, Asano S, Ohta T, et al. Pharmacological profiles of high-concentration (20 microg/g) tacalcitol ointment: effects on cutaneous inflammation, epidermal proliferation, and differentiation in mice. J Dermatol. 2003; 30:510–524.
14. Tukaj S, Trzonkowski P, Tukaj C. Regulatory effects of 1,25-dihydroxyvitamin D3 on vascular smooth muscle cells. Acta Biochim Pol. 2012; 59:395–400.
15. Clements D, Asprey SL, McCulloch TA, Morris TA, Watson SA, Johnson SR. Analysis of the oestrogen response in an angiomyolipoma derived xenograft model. Endocr Relat Cancer. 2009; 16:59–72.
16. Giaid A, Michel RP, Stewart DJ, Sheppard M, Corrin B, Hamid Q. Expression of endothelin-1 in lungs of patients with cryptogenic fibrosing alveolitis. Lancet. 1993; 341:1550–1554.
17. Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem. 1999; 274:10689–10692.
18. Lien E, Sellati TJ, Yoshimura A, Flo TH, Rawadi G, Finberg RW, et al. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem. 1999; 274:33419–33425.
19. Wu TT, Chen TL, Loon WS, Tai YT, Cherng YG, Chen RM. Lipopolysaccharide stimulates syntheses of toll-like receptor 2 and surfactant protein-A in human alveolar epithelial A549 cells through upregulating phosphorylation of MEK1 and ERK1/2 and sequential activation of NF-kappaB. Cytokine. 2011; 55:40–47.
20. Tanyaratsrisakul S, Jirapongsananuruk O, Thomas WR, Piboonpocanun S, Voelker DR. Der p2 stimulate inflammatory responses from lung epithelial cells and macrophages through the TLR2 and MAPK pathway. J Allergy Clin Immunol. 2012; 129:AB140.
21. Xue ML, Zhu H, Thakur A, Willcox M. 1 alpha,25-Dihydroxyvitamin D3 inhibits pro-inflammatory cytokine and chemokine expression in human corneal epithelial cells colonized with Pseudomonas aeruginosa. Immunol Cell Biol. 2002; 80:340–345.
22. Rostkowska-Nadolska B, Sliupkas-Dyrda E, Potyka J, Kusmierz D, Fraczek M, Krecicki T, et al. Vitamin D derivatives: calcitriol and tacalcitol inhibits interleukin-6 and interleukin-8 expression in human nasal polyp fibroblast cultures. Adv Med Sci. 2010; 55:86–92.
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