Multiplex cytometric bead assay Cell lysate of the apical lobe of the mouse lung was used for cytokine measurements. BD Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine kit (BD Biosciences) was used to measure IL-2, IL-4, IL-6, IFN-c, TNF, IL-17A and IL-10 in the lung tissue. Data were acquired on a FACS ARIA and samples were analysed using FCAP Array Software (BD Biosciences). Preparation of tissue for laser capture microdissection (LCM) The frozen lung tissue is sectioned at 7 mm in a cryostat (Leica Microsystems Nussloch GmbH, Germany) and a minimum of 10 sections per animal was prepared for LCM. The frozen sections were placed on Nuclease Free – MembraneSlides NF 1.0 PEN (Carl Zeiss MicroImaginG GmbH, M u¨nchen, Germany) and then stained with Cresyl Violet acetate (Sigma-AldrichH) according to the instruction ‘‘RNA extraction from frozen section’’ (PALM Laboratories, ZEISS) . The slides were allowed to air-dry com- pletely before stored desiccated at 280uC to prevent activation of endogenous RNase in the tissues. Laser capture microdissection (LCM) The Carl Zeiss Laser Microdissection and Pressure Catapulting (LMPC) technology (http://PALM-microlaser.com) was used, the specimen was microdissected by a focused laser beam, and then a defined laser pulse transports the cut piece of the specimen out of the object plane into a collection device, AdhesiveCap opaque (Carl Zeiss MicroImaginG GmbH, Mu¨ nchen, Germany). The lung section to be subjected to LCM was visualised with the PALM- MicroBeam microscope (Carl Zeiss, Germany) at a magnification of x 5. Typically, on each tissue section most of the peribronchial, perivascular and alveolar tissue were obtained and at least 10 sections were used for each mouse. The time required was about 3 h/per section. RNA isolation LCM cells were collected from three different microenvironments of three OVA sensitised and exposed (OVA/OVA) mice. Whole lung tissue section of 50 mm from OVA sensitized and PBS exposed (OVA/ PBS) mouse was used as control. The RNA was extracted using a QIAGEN RNeasyH Micro Kit (QIAGEN) according to manufactur- er’s protocol for the purification of total RNA from microdissected cryosections (QIAGEN). The RNA quality and quantity was determined using an Agilent Bioanalyzer with a RNA 6000 Nano Assay (Agilent Technologies, Deutschland GmbH, Waldbronn). Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) RNA from three OVA/OVA mice were pooled based on three microenvironments whereas RNA from one OVA/PBS mouse was used as a control. Each cDNA reaction was prepared using the SABiosciences’s RT2 First Strand Kit and 135 ng of total RNA according to the manufacturer’s protocol. Real-time RT-PCR was carried out using RT2 ProfilerTM PCR Array (PAMM-0011D) plates containing 84 mouse inflammatory cytokines, chemokines and receptor genes from Super array Bioscience Corporation, USA. SABiosciences’s RT2 SYBR Green qPCR Master Mix was used for detection and the array plates were run in a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad) according to manufacturer’s instructions (SABioscience). Results were moni- tored using different controls available on the plates. Gene expression levels were measured by the threshold cycle (Ct) and samples showing more than one peak in the melting curve were Lung Expansion of Th1, Th2, Th17 and Tregs excluded from the analysis performed using CFX Manager 2.0 software (Bio-Rad). Twenty-four genes were not considered as they did not pass our quality control, leaving 60 genes for further analysis. Data were normalized with five housekeeping genes [Gusb (Glucuronidase, beta), Hprt1 (Hypoxanthine guanine phosphoribosyl transferase 1), Hsp90ab1 (Heat shock protein 90kDA alpha (cytosolic), class B member 2), Gapdh (Glyceralde- hyde-phosphate dehydrogenase), Actb (Actin, beta cytoplasmic)] available in the plates. Finally, fold-changes compared to the control mice were calculated using the manufacturer’s software (SABioscience). A fold-change cutoff $3 was used to identify molecules whose expression was differentially regulated. Bioinformatic Analysis Genes of interest identified using the RT2 ProfilerTM PCR Array were further analysed by Ingenuity Pathways Analysis (IPA; version 9.0) (IngenuityH Systems, www.ingenuity.com), specifically in regards to their interactions. The fold change values were compared to the control. IPA utilises the Ingenuity Pathways Analysis Knowledge Base (IPA KB), a manually curated database of protein interactions from the literature, to analyse data. This KB was used to annotate the genes of interest and reveal their associations. Data analysis All data were expressed as mean 6 SEM (standard error of the mean). Statistical analyses were carried out using a non-parametric analysis of variance. The Mann-Whitney U test was used to determine the significant differences between the individual groups. A p value ,0.05 was considered statistically significant. Results Lung eosinophilia after allergen exposure All animals were sensitised to OVA and exposed to either OVA or PBS on five consecutive days, with the protocol presented in Figure 1A. The number of CD45+CCR3+SSChigh granulocytes, representing mature eosinophils, was significantly increased in the lung tissue after OVA exposure, compared to control PBS exposure (Figure 1B), confirming that a model of allergic airway inflammation had been established. Lung CD4+ T cell proliferation de novo and in situ after allergen exposure The total number of CD4+ T cells in lungs did not significantly differ between OVA and PBS exposed mice (Figure 2A). However, the total number of CD4+CD25+ cells in the lung significantly increased after repeated OVA exposure (Figure 2A). This finding concurs with other studies, demonstrating that regulatory T cells in the thymus are exclusively CD25+, while those in the periphery are both CD25+ and CD25- subsets of mature CD4+ T cells [12–14]. CD4+CD252Foxp3+ T cells have indeed been shown to be more effective than CD4+CD25+Foxp3+ T cells in mediating tolerance, emphasising their putative importance in vivo [15,16] and enforcing the importance of determining the presence of both the CD25+ and CD252 subsets. The expansion of the CD4+CD25+ subset of cells could be explained by local proliferation in the lung during repeated OVA exposure. An established FACS method was used to quantify the presence of newly produced cells by measuring the incorporation of BrdU and 7-AAD [17]. Indeed, both CD4+CD252 and CD4+CD25+ cells proliferated in the lung during allergen exposure (Figure 2C–D), confirming our hypothesis. As the relative number of CD4+CD252 cells is larger in the control group, more CD4+CD252 cells are newly produced (Figure 2D). Thus, cells PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e19889