ABSTRACT Background: We previously showed that treatment with Mycobacterium bovis BCG killed by extended freeze-drying (EFD BCG) modulates inflammation through regulatory T cells (Tregs) in an acute asthma model. In this study, we investigated the kinetics of Treg induction as well as their long-term homing in spleen and lungs correlating with reduced airway hyperresponsiveness (AHR) in a murine model of acute allergic asthma. We then evaluated the therapeutic implication of EFD BCG in a chronic asthma model.Methods: Tregs expressing Foxp3 were analyzed in various organs shortly and long-term after EFD BCG, live- and Heat Killed-(HK-) BCG treatments in an acute model of asthma. We further studied EFD BCG treatment on airway inflammation using a chronic model of asthma in mice. Results: Foxp3 expression peaked in the inguinal draining lymph-nodes (iDLNs) 2-4 days after EFD BCG treatment whereas it was long-term observed in spleen (days 7 to 90). This increase in Foxp3 expression was also found in lungs upon intranasal ovalbumin (OVA) challenge in OVA-sensitized mice. The loss of protection 4 months after EFD BCG treatment was correlated with the end of this phenomenon. Moreover, major lung inflammation hallmarks of severe asthma after multiple allergen challenges promoting chronic airway inflammation in OVA sensitized mice were reduced by EFD BCG treatment: AHR, eosinophils and neutrophils in bronchoalveolar lavage (BAL), mucus metaplasia, Th2 as well as Th17 cytokine levels in BAL and sera. EFD BCG treatment also enhances PPAR-γexpression and regulates NF-κBp65 translocation in lung extracts in this model of chronic asthma. Conclusions: EFD BCG treatment induced long-term protective effect associated to Foxp3 Tregs in the spleen and lungs in an acute model of asthma and inhibits AHR in a chronic model of asthma. EFD BCG could be a new and promising immuno-modulatory alternative treatment to corticoids in severe human asthma.
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M. Lagranderie, J. Vanoirbeek, B. Vargaftig, P. Guyonvarc’h, G. Marchal and X. Roux, "Therapeutic Administration of Mycobacterium bovis BCG Killed by Extended Freeze-Drying Modulates Airway Inflammation in a Chronic Murine Model of Asthma," Open Journal of Respiratory Diseases, Vol. 3 No. 2, 2013, pp. 79-88. doi: 10.4236/ojrd.2013.32013.
 W. W. Busse, S. Banks-Schlegel and S. E. Wenzel, “Pathophysiology of Severe Asthma,” Journal of Allergy and Clinical Immunology, Vol. 106, No. 6, 2000, pp. 1033-1042. doi:10.1067/mai.2000.111307
 P. Pala, S. D. Message, S. L. Johnston and P. J. Openshaw, “Increased Aeroallergen-Specific Interleukin-4-Producing T Cells in Asthmatic Adults,” Clinical and Experimental Allergy, Vol. 32, No. 12, 2002, pp. 1739-1744.
 N. Krug, T. Jung, U. Napp, K. Wagner, G. Schultze-Werninghaus, C. Heusser, C. H. Rieger, U. Schauer and H. Fabel, “Frequencies of T Cells Expressing Interleukin-4 and Interleukin-5 in Atopic Asthmatic Children. Comparison with Atopic Asthmatic Adults,” American Journal of Respiratory and Critical Care Medicine, Vol. 158, No. 3, 1998, pp. 754-759.
 T. Kiwamoto, Y. Ishii, Y. Morishima, K. Yoh, A. Maeda, K. Ishizaki, T. Iizuka, A. E. Hegab, Y. Matsuno, S. Homma, A. Nomura, T. Sakamoto, S. Takahashi and K. Sekizawa, “Transcription Factors T-Bet and GATA-3 Regulate Development of Airway Remodeling,” American Journal of Respiratory and Critical Care Medicine, Vol. 174, No. 2, 2006, pp. 142-151.
 C. Osterlund, H. Gronlund, N. Polovic, S. Sundstrom, G. Gafvelin and A. Bucht, “The Non-Proteolytic House Dust Mite Allergen Der p 2 Induce NF-kappaB and MAPK Dependent Activation of Bronchial Epithelial Cells,” Clinical and Experimental Allergy, Vol. 39, No. 8, 2009, pp. 1199-1208. doi:10.1111/j.1365-2222.2009.03284.x
 P. J. Barnes and I. M. Adcock, “Glucocorticoid Resistance in Inflammatory Diseases,” Lancet, Vol. 373, No. 9678, 2009, pp. 1905-1917.
 M. Spears, I. Donnelly, L. Jolly, M. Brannigan, K. Ito, C. McSharry, J. Lafferty, R. Chaudhuri, G. Braganza, P. Bareille, L. Sweeney, I. M. Adcock, P. J. Barnes, S. Wood and N. C. Thomson, “Bronchodilatory Effect of the PPAR-Gamma Agonist Rosiglitazone in Smokers with Asthma,” Clinical Pharmacology & Therapeutics, Vol. 86, No. 1, 2009, pp. 49-53. doi:10.1038/clpt.2009.41
 C. M. Hawrylowicz, “Regulatory T Cells and IL-10 in Allergic Inflammation,” Journal of Experimental Medicine, Vol. 202, No. 11, 2005, pp. 1459-1463.
 M. Lagranderie, M. Abolhassani, J. A. Vanoirbeek, C. Lima, A. M. Balazuc, B. B. Vargaftig and G. Marchal, “Mycobacterium bovis Bacillus Calmette-Guerin Killed by Extended Freeze-Drying Targets Plasmacytoid Dendritic Cells to Regulate Lung Inflammation,” Journal of Immunology, Vol. 184, No. 2, 2010, pp. 1062-1070.
 M. Gheorghiu, P. H. Lagrange, M. Lagranderie and A. M. Balazuc, “The Effects of Dispersed or Surface Grown Cultures, Manufacture and Control Methods on BCG Standardization,” Development in Biological Standardization, Vol. 58, No. Pt A, 1986, pp. 191-205.
 M. Lagranderie, M. Abolhassani, J. Vanoirbeek, J. Lefort, M. A. Nahori, E. S. J. R. Lapa, M. Huerre, B. Vargaftig and G. Marchal, “Mycobacterium bovis BCG Killed by Extended Freeze-Drying Reduces Airway Hyperresponsiveness in 2 Animal Models,” Journal of Allergy and Clinical Immunology, Vol. 121, No. 2, 2008, pp. 471-478. doi:10.1016/j.jaci.2007.09.033
 J. A. Vanoirbeek, M. Rinaldi, V. De Vooght, S. Haenen, S. Bobic, G. Gayan-Ramirez, P. H. Hoet, E. Verbeken, M. Decramer, B. Nemery and W. Janssens, “Noninvasive and Invasive Pulmonary Function in Mouse Models of Obstructive and Restrictive Respiratory Diseases,” American Journal of Respiratory Cell and Molecular Biology, Vol. 42, No. 1, 2010, pp. 96-104.
 M. Tarkowski, J. A. Vanoirbeek, H. M. Vanhooren, V. De Vooght, C. M. Mercier, J. Ceuppens, B. Nemery and P. H. Hoet, “Immunological Determinants of Ventilatory Changes Induced in Mice by Dermal Sensitization and Respiratory Challenge with Toluene Diisocyanate,” American Journal of Physiology—Lung Cellular and Molecular Physiologyl, Vol. 292, No. 1, 2007, pp. L207-214.
 D. Agnello, C. S. Lankford, J. Bream, A. Morinobu, M. Gadina, J. J. O’Shea and D. M. Frucht, “Cytokines and Transcription Factors that Regulate T Helper Cell Differentiation: New Players and New Insights,” Journal of Clinical Immunology, Vol. 23, No. 3, 2003, pp. 147-161.
 M. Lagranderie, C. Kluge, H. Kiefer-Biasizzo, M. Abolhassani, M. A. Nahori, C. Fitting, M. Huerre, A. Bandeira, H. Bercovier and G. Marchal, “Mycobacterium bovis Bacillus Calmette-Guerin Killed by Extended Freeze-Drying Reduces Colitis in Mice,” Gastroenterology, Vol. 141, No. 2, 2011, pp. 642-652, 652, e641-644.
 D. J. Evans, P. Cullinan and D. M. Geddes, “Cyclosporin as an Oral Corticosteroid Sparing Agent in Stable Asthma,” Cochrane Database of Systematic Reviews, No. 2, 2001, Article ID: CD002993.
 E. Xystrakis, S. Kusumakar, S. Boswell, E. Peek, Z. Urry, D. F. Richards, T. Adikibi, C. Pridgeon, M. Dallman, T. K. Loke, D. S. Robinson, F. J. Barrat, A. O’Garra, P. Lavender, T. H. Lee, C. Corrigan and C. M. Hawrylowicz, “Reversing the Defective Induction of IL-10-Secreting Regulatory T Cells in Glucocorticoid-Resistant Asthma Patients,” Journal of Clinical Investigation, Vol. 116, No. 1, 2006, pp. 146-155. doi:10.1172/JCI21759
 K. Asadullah, W. Sterry and H. D. Volk, “Interleukin-10 therapy—Review of a New Approach,” Pharmacological Reviews, Vol. 55, No. 2, 2003, pp. 241-269.
 K. J. Erb, J. W. Holloway, A. Sobeck, H. Moll and G. Le Gros, “Infection of Mice with Mycobacterium bovis-Bacillus Calmette-Guerin (BCG) Suppresses Allergen-Induced Airway Eosinophilia,” Journal of Experimental Medicine, Vol. 187, No. 4, 1998, pp. 561-569.
 M. A. Nahori, M. Lagranderie, J. Lefort, F. Thouron, D. Joseph, N. Winter, B. Gicquel, J. R. Lapa e Silva and B. B. Vargaftig, “Effects of Mycobacterium bovis BCG on the Development of Allergic Inflammation and Bronchial Hyperresponsiveness in Hyper-IgE BP2 Mice Vaccinated as Newborns,” Vaccine, Vol. 19, No. 11-12, 2001, pp. 1484-1495. doi:10.1016/S0264-410X(00)00345-5
 T. Major, G. Wohlleben, B. Reibetanz and K. J. Erb, “Application of Heat Killed Mycobacterium bovis-BCG into the Lung Inhibits the Development of Allergen-Induced Th2 Responses,” Vaccine, Vol. 20, No. 11-12, 2002, pp. 1532-1540.
 P. Stock, O. Akbari, G. Berry, G. J. Freeman, R. H. Dekruyff and D. T. Umetsu, “Induction of T Helper Type 1-Like Regulatory Cells that Express Foxp3 and Protect against Airway Hyper-Reactivity,” Nature Immunology, Vol. 5, No. 11, 2004, pp. 1149-1156. doi:10.1038/ni1122
 S. J. Szabo, S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman and L. H. Glimcher, “A Novel Transcription Factor, T-Bet, Directs Th1 Lineage Commitment,” Cell, Vol. 100, No. 6, 2000, pp. 655-669.
 H. Hammad, H. J. de Heer, T. Soullie, V. Angeli, F. Trottein, H. C. Hoogsteden and B. N. Lambrecht, “Activation of Peroxisome Proliferator-Activated Receptor-Gamma in Dendritic Cells Inhibits the Development of Eosinophilic Airway Inflammation in a Mouse Model of Asthma,” American Journal of Pathology, Vol. 164, No. 1, 2004, pp. 263-271.
 L. Klotz, I. Dani, F. Edenhofer, L. Nolden, B. Evert, B. Paul, W. Kolanus, T. Klockgether, P. Knolle and L. Diehl, “Peroxisome Proliferator-Activated Receptor Gamma Control of Dendritic Cell Function Contributes to Development of CD4 + T Cell Anergy,” Journal of Immunology, Vol. 178, No. 4, 2007, pp. 2122-2131.
 D. Hartl, B. Koller, A. T. Mehlhorn, D. Reinhardt, T. Nicolai, D. J. Schendel, M. Griese and S. Krauss-Etschmann, “Quantitative and Functional Impairment of Pulmonary CD4 + CD25hi Regulatory T Cells in Pediatric Asthma,” Journal of Allergy and Clinical Immunology, Vol. 119, No. 5, 2007, pp. 1258-1266.