, Volume 41, Issue 4, pp 1266–1275 | Cite as

Cold Stimuli Facilitate Inflammatory Responses Through Transient Receptor Potential Melastatin 8 (TRPM8) in Primary Airway Epithelial Cells of Asthmatic Mice

  • Haipei Liu
  • Li Hua
  • Quanhua Liu
  • Jun Pan
  • Yixiao Bao


Bronchial asthma is a chronic inflammatory airway disease that can be aggravated by cold air. However, its mechanism remains largely unknown. As a thermo-sensing cation channel, transient receptor potential melastatin 8 (TRPM8) can be activated by cold stimuli (8–22 °C) and cooling agents. Whereas TRPM8 activation leads to enhanced expression of inflammatory cytokines and mucus hypersecretion in human bronchial epithelial cell lines, no previous study has examined its role in regulating the cold-induced inflammatory responses and its mechanism in asthmatic airway epithelium. Airway epithelial cells were isolated from asthma model mice and exposed to low temperature (18 °C). The TRPM8 overexpression plasmid and siRNA lentivirus were transfected to up- or downregulate the TRPM8 level. The expression of mRNAs of inflammatory cytokines was tested using real-time reverse transcription–polymerase chain reaction (RT-PCR). The activities of phosphorylated protein kinase C (PKC) and phosphorylated inhibitor of nuclear factor kappa B (IκB) were measured using the immunofluorescence assay. The expression of mRNAs of inflammatory cytokines [interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, IL-13, granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-α] increased significantly under cold conditions, which was boosted after TRPM8 overexpression and augmented further in the presence of PKC inhibitor, calphostin C. However, the downregulation of TRPM8 and nuclear factor kappa B (NF-κB) impaired the transcription of these cytokine genes. In addition, the phosphorylated PKC and phosphorylated IκB were activated by cold stimuli. Moreover, the expression of phosphorylated IκB protein improved in the presence of TRPM8, while disruption with the TRPM8 gene or TRPM8 antagonist prohibited the activation of IκB. Cold air could induce inflammatory responses through the TRPM8-mediated PKC/NF-κB signal pathway in primary airway epithelial cells of asthmatic mice.


airway inflammation asthma cold stimulus nuclear factor-κB transient receptor potential melastatin 8 protein kinase C 


Funding Information

This work was supported by the National Natural Science Foundation Youth Science Fund Project of China (No: 81300021).

Compliance with Ethical Standards

The study was approved by the Scientific and Ethics Committees at Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, China.

Conflict of Interest

The authors declare that they have no conflicts of interest.


  1. 1.
    Varricchi, G., G. Senna, S. Loffredo, D. Bagnasco, M. Ferrando, and G.W. Canonica. 2017. Reslizumab and eosinophilic asthma: one step closer to precision medicine? Frontiers in Immunology 8: 242. Scholar
  2. 2.
    Chanez, P., and M. Humbert. 2014. Asthma: still a promising future? European Respiratory Review 23: 405–407. Scholar
  3. 3.
    Chu, L.M., and P. Pahwa. 2017. Prevalence and associated factors for self-reported asthma in a Canadian population: the Canadian Community Health Survey, 2014. The Journal of Asthma 27: 1–9. Scholar
  4. 4.
    Sabnis, A.S., M. Shadid, G.S. Yost, and C.A. Reilly. 2008. Human lung epithelial cells express a functional cold-sensing TRPM8 variant. American Journal of Respiratory Cell and Molecular Biology 39: 466–474. Scholar
  5. 5.
    Behrendt, H.J., T. Germann, C. Gillen, H. Hatt, and R. Jostock. 2004. Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay. British Journal of Pharmacology 141: 737–745. Scholar
  6. 6.
    Li, M., Q. Li, G. Yang, V.P. Kolosov, J.M. Perelman, and X.D. Zhou. 2011. Cold temperature induces mucin hypersecretion from normal human bronchial epithelial cells in vitro through a transient receptor potential melastatin 8 (TRPM8)-mediated mechanism. The Journal of Allergy and Clinical Immunology 128 (626–634): e621–e625. Scholar
  7. 7.
    Cho, Y., Y. Jang, Y.D. Yang, C.H. Lee, Y. Lee, and U. Oh. 2010. TRPM8 mediates cold and menthol allergies associated with mast cell activation. Cell Calcium 48: 202–208. Scholar
  8. 8.
    Kim, J.H., Y.S. Jang, S.H. Jang, K.S. Jung, S.H. Kim, Y.M. Ye, and H.S. Park. 2017. Toluene diisocyanate exposure induces airway inflammation of bronchial epithelial cells via the activation of transient receptor potential melastatin 8. Experimental & Molecular Medicine 49: e299. Scholar
  9. 9.
    Xing, H., J.X. Ling, M. Chen, R.D. Johnson, M. Tominaga, C.Y. Wang, and J. Gu. 2008. TRPM8 mechanism of autonomic nerve response to cold in respiratory airway. Molecular Pain 4: 22. Scholar
  10. 10.
    Madouri, F., P. Chenuet, C. Beuraud, L. Fauconnier, T. Marchiol, N. Rouxel, A. Ledru, M. Gallerand, V. Lombardi, L. Mascarell, Q. Marquant, L. Apetoh, F. Erard, M. Le Bert, F. Trovero, V.F. Quesniaux, B. Ryffel, and D. Togbe. 2017. Protein kinase Ctheta controls type 2 innate lymphoid cell and TH2 responses to house dust mite allergen. The Journal of Allergy and Clinical Immunology 139: 1650–1666. Scholar
  11. 11.
    Green, T.D., A.L. Crews, J. Park, S. Fang, and K.B. Adler. 2011. Regulation of mucin secretion and inflammation in asthma: a role for MARCKS protein? Biochimica et Biophysica Acta 1810: 1110–1113. Scholar
  12. 12.
    Almeida, M., L. Han, E. Ambrogini, S.M. Bartell, and S.C. Manolagas. 2010. Oxidative stress stimulates apoptosis and activates NF-kappaB in osteoblastic cells via a PKCbeta/p66shc signaling cascade: counter regulation by estrogens or androgens. Molecular Endocrinology 24: 2030–2037. Scholar
  13. 13.
    Lutzny, G., T. Kocher, M. Schmidt-Supprian, M. Rudelius, L. Klein-Hitpass, A.J. Finch, J. Durig, M. Wagner, C. Haferlach, A. Kohlmann, S. Schnittger, M. Seifert, S. Wanninger, N. Zaborsky, R. Oostendorp, J. Ruland, M. Leitges, T. Kuhnt, Y. Schafer, B. Lampl, C. Peschel, A. Egle, and I. Ringshausen. 2013. Protein kinase c-beta-dependent activation of NF-kappaB in stromal cells is indispensable for the survival of chronic lymphocytic leukemia B cells in vivo. Cancer Cell 23: 77–92. Scholar
  14. 14.
    Moscat, J., M.T. Diaz-Meco, and P. Rennert. 2003. NF-kappaB activation by protein kinase C isoforms and B-cell function. EMBO Reports 4: 31–36. Scholar
  15. 15.
    Blackwell, T.S., and J.W. Christman. 1997. The role of nuclear factor-kappa B in cytokine gene regulation. American Journal of Respiratory Cell and Molecular Biology 17: 3–9. Scholar
  16. 16.
    Secor, E.R., W.F. Carson, A. Singh, M. Pensa, L.A. Guernsey, C.M. Schramm, and R.S. Thrall. 2008. Oral bromelain attenuates inflammation in an ovalbumin-induced murine model of asthma. Evidence-based Complementary and Alternative Medicine 5: 61–69. Scholar
  17. 17.
    Lam, H.C., A.M. Choi, and S.W. Ryter. 2011. Isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface. Journal of Visualized Experiments.
  18. 18.
    Ohara, Y., T.E. Peterson, B. Zheng, J.F. Kuo, and D.G. Harrison. 1994. Lysophosphatidylcholine increases vascular superoxide anion production via protein kinase C activation. Arteriosclerosis and Thrombosis 14: 1007–1013.CrossRefPubMedGoogle Scholar
  19. 19.
    Huang, Y., Y. Liu, L. Li, B. Su, L. Yang, W. Fan, Q. Yin, L. Chen, T. Cui, J. Zhang, Y. Lu, J. Cheng, P. Fu, and F. Liu. 2014. Involvement of inflammation-related miR-155 and miR-146a in diabetic nephropathy: implications for glomerular endothelial injury. BMC Nephrology 15: 142. Scholar
  20. 20.
    Pezzulo, A.A., T.D. Starner, T.E. Scheetz, G.L. Traver, A.E. Tilley, B.G. Harvey, R.G. Crystal, McCray PB Jr., and J. Zabner. 2011. The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. American Journal of Physiology. Lung Cellular and Molecular Physiology 300: L25–L31. Scholar
  21. 21.
    Sabnis, A.S., C.A. Reilly, J.M. Veranth, and G.S. Yost. 2008. Increased transcription of cytokine genes in human lung epithelial cells through activation of a TRPM8 variant by cold temperatures. American Journal of Physiology. Lung Cellular and Molecular Physiology 295: L194–L200. Scholar
  22. 22.
    Wang, X.P., X. Yu, X.J. Yan, F. Lei, Y.S. Chai, J.F. Jiang, Z.Y. Yuan, D.M. Xing, and L.J. Du. 2017. TRPM8 in the negative regulation of TNFalpha expression during cold stress. Scientific Reports 7: 45155. Scholar
  23. 23.
    Li, C.M., J.M. Perelman, V.P. Kolosov, and X.D. Zhou. 2011. Effects of transient receptor potential melastatin 8 cation channels on inflammatory reaction induced by cold temperatures in human airway epithelial cells. Chin J Tubero Respir Dis 34: 757–761. Scholar
  24. 24.
    Premkumar, L.S., M. Raisinghani, S.C. Pingle, C. Long, and F. Pimentel. 2005. Downregulation of transient receptor potential melastatin 8 by protein kinase C-mediated dephosphorylation. The Journal of Neuroscience 25: 11322–11329. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pediatric Respiratory MedicineXin Hua Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghaiChina
  2. 2.Institute for Pediatric ResearchXin Hua Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghaiChina
  3. 3.Department of Pediatrics, Shanghai EverBetter Pubin Children’s HospitalShanghaiChina

Personalised recommendations