, Volume 36, Issue 3, pp 582–591 | Cite as

Small Interfering RNA Targeting T-cell Ig Mucin-3 Decreases Allergic Airway Inflammation and Hyperresponsiveness

  • Xiao-Xia Lu
  • Karen S. McCoy
  • Jia-Li Xu
  • Wei-Kun Hu
  • He-Bin Chen


Since CD4+ T cells play a pivotal role in the development of airway inflammation and hyperresponsiveness, targeting activated CD4+ T cell subsets and increasing the cells with regulatory function would be a logical therapeutic approach. We showed that this outcome can be achieved by local therapy with Tim-3, which is a negative regulator of CD4+ T cells. Tim-3 expression was up-regulated by ovalbumin (OVA) induction. Attenuating Tim-3 expression by RNA interference suppressed allergen-induced immune responses. Intranasal application of Tim-3 shRNA diminished airway inflammation and hyperresponsiveness. Multiple mechanisms were involved in the inhibitory effects, including regulation the imbalance of Th1/Th17 and increasing Treg cell expression. Our results indicate that the Tim-3 pathway is highly involved in the regulation of asthma. Targeting Tim-3 by siRNA may hold therapeutic potential in preventing the development of allergic asthma.


asthma Tim-3 CD4+ T cell siRNA 



T-cell Ig and mucin domain-containing molecule-3


Airway hyperresponsiveness


Broncho-alveolar lavage fluid


Real-time polymerase chain reaction


Flow cytometer


Enzyme linked immunosorbent assay




Small interfering RNA



This work was supported by grant Wu Jieping Medical Fund 320.6750.1285, grant 2010CDB08804 from the Natural Science Foundation of Hubei Province, and grant WX11B10 from the Foundation of Wuhan Health Bureau.


  1. 1.
    Busse, W.W., and R.F. Lemanske Jr. 2001. Asthma. New England Journal of Medicine 344: 350–362.PubMedCrossRefGoogle Scholar
  2. 2.
    Wakashin, H., K. Hirose, Y. Maezawa, S. Kagami, A. Suto, N. Watanabe, Y. Saito, M. Hatano, T. Tokuhisa, Y. Iwakura, P. Puccetti, I. Iwamoto, and H. Nakajima. 2008. IL-23 and Th17 cells enhance Th2-cell-mediated eosinophilic airway inflammation in mice. American Journal of Respiratory and Critical Care Medicine 178: 1023–1032.PubMedCrossRefGoogle Scholar
  3. 3.
    Zhu, J., H. Yamane, and W.E. Paul. 2010. Differentiation of effector CD4 T cell populations. Annual Review Immunology 28: 445–89.CrossRefGoogle Scholar
  4. 4.
    Murphy, K.M., and S.L. Reiner. 2002. The lineage decisions of helper T cells. Nature Reviews Immunology 2: 933–944.PubMedCrossRefGoogle Scholar
  5. 5.
    Park, H., Z. Li, X.O. Yang, S.H. Chang, R. Nurieva, Y.H. Wang, Y. Wang, L. Hood, Z. Zhu, Q. Tian, and C. Dong. 2005. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nature Immunology 6: 1133–1141.PubMedCrossRefGoogle Scholar
  6. 6.
    Gruning, G., M. Warnock, A.E. Wakil, R. Venkayya, F. Brombacher, D.M. Rennick, D. Sheppard, M. Mohrs, D.D. Donaldson, R.M. Locksley, and D.B. Corry. 1998. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282: 2661–2663.Google Scholar
  7. 7.
    Robinson, D.S., Q. Hamid, S. Ying, A. Tsicopoulos, J. Barkans, A.M. Bentley, C. Corrigan, S.R. Durham, and A.B. Kay. 1992. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. New England Journal of Medicine 326: 298–304.PubMedCrossRefGoogle Scholar
  8. 8.
    Laan, M., L. Palmberg, K. Larsson, and A. Linden. 2002. Free, soluble interleukin-17 protein during severe inflammation in human airways. European Respiratory Journal 19: 534–537.PubMedCrossRefGoogle Scholar
  9. 9.
    Barczyk, A., W. Pierzchala, and E. Sozanska. 2003. Interleukin-17 in sputum correlates with airway hyperresponsiveness to methacholine. Respiratory Medicine 97: 726–733.PubMedCrossRefGoogle Scholar
  10. 10.
    Chakir, J., J. Shannon, S. Molet, M. Fukakusa, J. Elias, M. Laviolette, L.P. Boulet, and Q. Hamid. 2003. Airway remodeling-associated mediators in moderate to severe asthma: effect of steroids on TGF-β, IL-11, IL-17, and type I and type III collagen expression. Journal of Allergy and Clinical Immunology 111: 1293–1298.PubMedCrossRefGoogle Scholar
  11. 11.
    Schnyder-Candrian, S., D. Togbe, I. Couillin, I. Mercier, F. Brombacher, V. Quesniaux, F. Fossiez, B. Ryffel, and B. Schnyder. 2006. Interleukin-17 is a negative regulator of established allergic asthma. Journal of Experimental Medicine 203: 2715–2725.PubMedCrossRefGoogle Scholar
  12. 12.
    Harrington, L.E., R.D. Hatton, P.R. Mangan, H. Turner, T.L. Murphy, K.M. Murphy, and C.T. Weaver. 2005. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nature Immunology 6: 1123–1132.PubMedCrossRefGoogle Scholar
  13. 13.
    Kim, J.M., J.P. Rasmussen, and A.Y. Rudensky. 2007. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nature Immunology 8: 191–197.PubMedCrossRefGoogle Scholar
  14. 14.
    Lewkowich, I.P., N.S. Herman, K.W. Schleifer, M.P. Dance, B.L. Chen, K.M. Dienger, A.A. Sproles, J.S. Shah, J. Kohl, Y. Belkaid, and M. Wills-Karp. 2005. CD4+CD25+ T cells protect against experimentally induced asthma and alter pulmonary dendritic cell phenotype and function. Journal of Experimental Medicine 202: 1549–1561.PubMedCrossRefGoogle Scholar
  15. 15.
    Monney, L., C.A. Sabatos, J.L. Gaglia, A. Ryu, H. Waldner, T. Chernova, S. Manning, E.A. Greenfield, A.J. Coyle, R.A. Sobel, G.J. Freeman, and V.K. Kuchroo. 2002. Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature 415: 536–541.PubMedCrossRefGoogle Scholar
  16. 16.
    Sabatos, C.A., S. Chakravarti, E. Cha, A. Schubart, A. Sanchez-Fueyo, X.X. Zheng, A.J. Coyle, T.B. Strom, G.J. Freeman, and V.K. Kuchroo. 2003. Interaction of Tim-3 and Tim-3 ligand regulates T helper type 1 response and induction of peripheral tolerance. Nature Immunology 4: 1102–1110.PubMedCrossRefGoogle Scholar
  17. 17.
    Nakae, S., Y. Iwakura, H. Suto, and S.J. Galli. 2007. Phenotypic differences between Th1 and Th17 cells and negative regulation of Th1 cell differentiation by IL-17. Journal of Leukocyte Biology 81: 1258–68.PubMedCrossRefGoogle Scholar
  18. 18.
    Sánchez-Fueyo, A., J. Tian, D. Picarella, C. Domenig, X.X. Zheng, C.A. Sabatos, N. Manlong, O. Bender, T. Kamradt, V.K. Kuchroo, J.C. Gutierrez-Ramos, A.J. Coyle, and T.B. Strom. 2003. Tim-3 inhibits T helper type 1-mediated auto- and alloimmune responses and promotes immunological tolerance. Nature Immunology 4: 1093–1101.PubMedCrossRefGoogle Scholar
  19. 19.
    Seki, M., S. Oomizu, K.M. Sakata, A. Sakata, T. Arikawa, K. Watanabe, K. Ito, K. Takeshita, T. Niki, N. Saita, N. Nishi, A. Yamauchi, S. Katoh, A. Matsukawa, V. Kuchroo, and M. Hirashima. 2008. Galectin-9 suppresses the generation of Th17, promotes the induction of regulatory T cells, and regulates experimental autoimmune arthritis. Clinical Immunology 127: 78–88.PubMedCrossRefGoogle Scholar
  20. 20.
    Niwa, H., T. Satoh, Y. Matsushima, K. Hosoya, K. Saeki, T. Niki, M. Hirashima, and H. Yokozeki. 2009. Stable form of galectin-9, a Tim-3 ligand, inhibits contact hypersensitivity and psoriatic reactions: a potent therapeutic tool for Th1- and/or Th17-mediated skin inflammation. Clinical Immunology 132: 184–194.PubMedCrossRefGoogle Scholar
  21. 21.
    Frisancho-Kiss, S., J.F. Nyland, S.E. Davis, M.A. Barret, S.J. Gatewood, D.B. Njoku, D. Cihakova, E.K. Silbergeld, N.R. Rose, and D. Fairweather. 2006. Cutting edge: T cell Ig mucin-3 reduces inflammatory heart disease by increasing CTLA-4 during innate immunity. Journal of Immunology 176: 6411–6415.Google Scholar
  22. 22.
    Umetsu, D.T., S.E. Umetsu, G.J. Freeman, and R.H. DeKruyff. 2008. TIM gene family and their role in atopic diseases. Current Topics in Microbiology and Immunology 321: 201–15.PubMedCrossRefGoogle Scholar
  23. 23.
    Sehrawat, S., A. Suryawanshi, M. Hirashima, and B.T. Rouse. 2009. Role of Tim-3/Galectin-9 inhibitory interaction in viral-induced immunopathology: shifting the balance toward regulators. Journal of Immunology 182: 3191–3201.CrossRefGoogle Scholar
  24. 24.
    Yeung, M.Y., M. Mc Grath, and N. Najafian. 2011. The emerging role of the TIM molecules in transplantation. American Journal of Transplantation 11: 2012–2019.PubMedCrossRefGoogle Scholar
  25. 25.
    Stampfli, M.R., R.E. Wiley, G.S. Neigh, B.U. Gajewska, X.F. Lei, D.P. Snider, Z. Xing, and M. Jordana. 1998. GM-CSF transgene expression in the airway allows aerosolized ovalbumin to induce allergic sensitization in mice. Journal of Clinical Investigation 102: 1704–1714.PubMedCrossRefGoogle Scholar
  26. 26.
    Elbashir, S.M., J. Harhorth, K. Weber, and T. Tuschl. 2002. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26: 199–213.PubMedCrossRefGoogle Scholar
  27. 27.
    Chae, W.J., H.K. Lee, J.H. Han, S.W. Kim, A.L. Bothwell, T. Morio, and S.K. Lee. 2004. Qualitatively differential regulation of T cell activation and apoptosis by T cell receptor zeta chain ITAMs and their tyrosine residues. International Immunology 16: 1225–1236.PubMedCrossRefGoogle Scholar
  28. 28.
    Chuang, Y.H., C.L. Fu, Y.C. Lo, and B.L. Chiang. 2004. Adenovirus expressing Fas ligand gene decreases airway hyper-responsiveness and eosinophilia in a murine model of asthma. Gene Therapy 11: 1497–1505.PubMedCrossRefGoogle Scholar
  29. 29.
    Handerson Jr., W.R., L.O. Tang, S.J. Chu, S.M. Tsao, G.K. Chianq, F. Jones, M. Jonas, C. Pae, H. Wang, and E.Y. Chi. 2002. A role for cysteinyl leukotriense in airway remodeling in a mouse asthma model. American Journal of Respiratory and Critical Care Medicine 165: 108–116.CrossRefGoogle Scholar
  30. 30.
    Kearley, J., S.J. McMillan, and C.M. Lloyd. 2007. Th2-driven, allergen-induced airway inflammation is reduced after treatment with anti–Tim-3 antibody in vivo. Journal of Experimental Medicine 204: 1289–1294.PubMedCrossRefGoogle Scholar
  31. 31.
    Hu, W.K., X.X. Lu, S. Yang, G.P. Xu, F. Lan, S.X. Chen, W. Ni, W.N. Xiong, and S.D. Xiong. 2009. Expression of the Th1-specific cell-surface protein Tim-3 increases in a murine model of atopic asthma. Journal of Asthma 46: 872–877.PubMedCrossRefGoogle Scholar
  32. 32.
    Ray, A., A. Khare, N. Krishnamoorthy, Z. Qi, and P. Ray. 2010. Regulatory T cells in many favors control asthma. Mucosal Immunology 3: 216–229.PubMedCrossRefGoogle Scholar
  33. 33.
    Hammad, H., and B.N. Lambrecht. 2006. Recent progress in the biology of airway dendritic cells and implications for understanding the regulation of asthmatic inflammation. Journal of Allergy and Clinical Immunology 118: 331–336.PubMedCrossRefGoogle Scholar
  34. 34.
    McIntire, J.J., S.E. Umetsu, Q. Akbari, M. Potter, V.K. Kuchroo, G.S. Barsh, G.J. Freeman, D.T. Umetsu, and R.H. DeKruyff. 2001. Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family. Nature Immunology 2: 1109–1116.PubMedCrossRefGoogle Scholar
  35. 35.
    Graves, P.E., V. Siroux, S. Guerra, W.T. Klimecki, and F.D. Martinez. 2005. Association of atopy and eczema with polymorphisms in T-cell immunoglobulin domain and mucin domain–IL-2-inducible T-cell kinase gene cluster in chromosome 5q33. Journal of Allergy and Clinical Immunology 116: 650–656.PubMedCrossRefGoogle Scholar
  36. 36.
    Fukushima, A., T. Sumi, K. Fukuda, N. Kumagai, T. Nishida, H. Akiba, K. Okumura, H. Yagita, and H. Ueno. 2007. Antibodies to T-cell Ig and mucin domain-containing proteins (Tim)-1 and −3 suppress the induction and progression of murine allergic conjunctivitis. Biochemical and Biophysical Research Communications 353: 211–216.PubMedCrossRefGoogle Scholar
  37. 37.
    Freeman, G.J., J.M. Casasnovas, D.T. Umetsu, and R.H. DeKruyff. 2010. TIM genes: a family of cell surface phosphatidylserine receptors thatregulate innate and adaptive immunity. Immunological Reviews 235: 172–189.PubMedGoogle Scholar
  38. 38.
    Bettelli, E., Y. Carrier, W. Gao, T. Korn, T.B. Strom, M. Oukka, H.L. Weiner, and V.K. Kuchroo. 2006. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441: 235–238.PubMedCrossRefGoogle Scholar
  39. 39.
    Popescu, F.D. 2005. Antisense-and RNA interference-based therapeutic strategies in allergy. Journal of Cellular and Molecular Medicine 9: 840–853.PubMedCrossRefGoogle Scholar
  40. 40.
    Maneechotesuwan, K., Y. Xin, K. Ito, E. Jazrawi, K.Y. Lee, O.S. Usmani, P.J. Barnes, and I.M. Adcock. 2007. Regulation of Th2 cytokine genes by p38 MAPK-mediated phosphorylation of GATA-3. Journal of Immunology 178: 2491–2498.Google Scholar
  41. 41.
    Meinicke, H., Y. Darcan, and E. Hamelmann. 2009. Targeting allergic airway diseases by siRNA: an option for the future? Current Molecular Medicine 9: 483–494.PubMedCrossRefGoogle Scholar
  42. 42.
    Darcan-Nicolaisen, Y., H. Meinicke, G. Fels, O. Hegend, A. Haberland, A. Kuhl, C. Loddenkemper, M. Witzenrath, S. Kube, W. Henke, and E. Hamelmann. 2009. Small interfering RNA against transcription factor STAT6 inhibits allergic airway inflammation and hyperreactivity in mice. Journal of Immunology 182: 7501–7508.CrossRefGoogle Scholar
  43. 43.
    Weckmann, M., A. Collison, J.L. Simpson, M.V. Kopp, P.A. Wark, M.J. Smyth, H. Yagita, K.I. Matthaei, N. Hansbro, B. Whitehead, P.G. Gibson, P.S. Foster, and J. Mattes. 2007. Critical link between TRAIL and CCL20 for the activation of TH2 cells and the expression of allergic airway disease. Nature Medicine 13: 1308–1315.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Xiao-Xia Lu
    • 1
  • Karen S. McCoy
    • 2
  • Jia-Li Xu
    • 1
  • Wei-Kun Hu
    • 3
  • He-Bin Chen
    • 1
  1. 1.Department of pulmonary MedicineWuhan Women and Children Health Care CenterWuhanChina
  2. 2.Department of Pediatrics, Division of Pulmonary MedicineNationwide Children’s Hospital and Ohio State University School of Medicine and Public HealthColumbusUSA
  3. 3.Department of Ophthalmology, Tongi Hospital, Tongi Medical CollegeHuazhong University of Science and TechnologyWuhanChina

Personalised recommendations