Apostolos Bossios* Abstract + + - Expansion of CD4 CD25 and CD25 T-Bet, GATA-3, Foxp3 and RORct Cells in Allergic Inflammation, Local Lung Distribution and Chemokine Gene Expression You Lu, Carina Malmha¨ ll, Margareta Sjo¨ strand, Madeleine Ra˚ dinger, Serena E. O’Neil, Jan Lo¨ tvall, Department of Internal Medicine, Krefting Research Centre, Institute of Medicine, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Allergic asthma is associated with airway eosinophilia, which is regulated by different T-effector cells. T cells express transcription factors T-bet, GATA-3, RORct and Foxp3, representing Th1, Th2, Th17 and Treg cells respectively. No study has directly determined the relative presence of each of these T cell subsets concomitantly in a model of allergic airway inflammation. In this study we determined the degree of expansion of these T cell subsets, in the lungs of allergen challenged mice. Cell proliferation was determined by incorporation of 5-bromo-29-deoxyuridine (BrdU) together with 7- aminoactnomycin (7-AAD). The immunohistochemical localisation of T cells in the lung microenvironments was also quantified. Local expression of cytokines, chemokines and receptor genes was measured using real-time RT-PCR array analysis in tissue sections isolated by laser microdissection and pressure catapulting technology. Allergen exposure increased the numbers of T-bet+, GATA-3+, RORct+ and Foxp3+ cells in CD4+CD25+ and CD4+CD25- T cells, with the greatest expansion of GATA-3+ cells. The majority of CD4+CD25+ T-bet+, GATA-3+, RORct+ and Foxp3+ cells had incorporated BrdU and underwent proliferation during allergen exposure. Allergen exposure led to the accumulation of T-bet+, GATA-3+ and Foxp3+ cells in peribronchial and alveolar tissue, GATA-3+ and Foxp3+ cells in perivascular tissue, and RORct+ cells in alveolar tissue. A total of 28 cytokines, chemokines and receptor genes were altered more than 3 fold upon allergen exposure, with expression of half of the genes claimed in all three microenvironments. Our study shows that allergen exposure affects all T effector cells in lung, with a dominant of Th2 cells, but with different local cell distribution, probably due to a distinguished local inflammatory milieu. Citation: Lu Y, Malmha¨ll C, Sjo¨ strand M, Ra˚dinger M, O’Neil SE, et al. (2011) Expansion of CD4+CD25+ and CD25- T-Bet, GATA-3, Foxp3 and RORct Cells in Allergic Inflammation, Local Lung Distribution and Chemokine Gene Expression. PLoS ONE 6(5): e19889. doi:10.1371/journal.pone.0019889 Editor: Carol Feghali-Bostwick, University of Pittsburgh, United States of America Received September 27, 2010; Accepted April 19, 2011; Published May 19, 2011 Copyright: 2011 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by a grant from the Swedish Heart-Lung Foundation (project no. 20070575) and Sahlgrenska University Hospital Foundation, Gothenburg, Sweden. Prof. Jan Lo¨ tvall is funded by the Herman Krefting Foundation Against Asthma/Allergy. The sponsors played no role in the study design, conduct, collection, analysis, interpretation, review, or approval of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: apostolos.bossios@gu.se Introduction Asthma is one of the most common chronic diseases world- wide, with an estimated total prevalence of 5% [1]. Eosinophilic inflammation is a common characteristic linked closely to allergic asthma. Beyond eosinophils, different T cells are important regulators of the inflammatory process in asthma. Indeed, the lung has an extensive network of antigen-presenting cells that provide antigen presentation to T cells in lung-associated lymphoid tissue [2]. CD4+ T cells are essential regulators of the immune response and inflammatory diseases. After encountering specific antigen, they become activated, expand their populations and differentiate into various effector T cell subsets, such as T helper type 1 (Th1), Th2, interleukin 17 (IL-17)-producing T helper (Th17) and regulatory T cells (Treg cells). The development of these cells is dictated by their specific transcription factors T-bet, GATA-3, RORct and Foxp3, respectively [3–6]. These cells have been suggested to regulate different aspects of allergic inflammation. For example, the development of allergy and the presence of eosinophilic inflammation are suggested to be driven by a Th2 over-activation, in relation to a reduced Th1 activity [7]. Beyond these two cell types, Th17 and Treg cells have been proposed to interact with other T cells, and interrelate with each other, to determine which type of inflammation is induced [8,9]. However, no study has directly determined the relative presence, tissue distribution or surrounding inflammatory milieu of each of these T cell subsets concomitantly in a model of allergic airway inflammation. The aim of the current study was to determine the degree of expansion of different T cell subsets expressing transcription factors for Th1, Th2, Th17 and Treg cells, in the lungs of sensitised mice exposed to allergen. To establish in which microenvironment these cells reside, immunohistochemistry for each transcription factor was performed. General lung inflamma- tion was estimated by measuring the concentration of cytokines released in the lung. The local inflammatory response in the microenvironments where different cells were located was estimated by measuring the expression of 60 inflammatory cytokine, chemokine and receptor genes using real-time RT- PCR array in tissue isolated by laser microdissection and pressure catapulting technology. PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e19889