Lung Expansion of Th1, Th2, Th17 and Tregs Figure 6. The distribution of T cell transcription factors differs in the lung microenvironments during allergic inflammation. A) Photographs of immunohistochemistry of MBP (red) in OVA or PBS exposed sensitised mice, T-bet, GATA-3, Foxp3 (brown) and RORct (red) in peribronchial, perivascular and alveolar tissue after exposure to OVA in sensitised mice. B) Quantification (cells/mm2) of T-bet, GATA-3, Foxp3 and RORct expressing cells in different compartments of the lungs, including peribronchial tissue, perivascular tissue and alveolar tissue, after exposure to PBS or OVA. Data shown as mean+SEM, *,0.05. C) Pie chart showing the relative distribution of T-bet, GATA-3, Foxp3 and RORct expressing cells in the different lung compartments after exposure of mice to OVA. doi:10.1371/journal.pone.0019889.g006 were quantified after PBS or OVA exposure. No differences were found in the expression of IL-2 (Figure 7A), whereas both IL-6 and TNF were significantly increased after allergen exposure, indicat- ing a general inflammatory status (Figure 7B-C). IFN-c, IL-4 and IL-17A were all significantly increased, with IL-4 the most prominently increased, indicating a Th2 activity in the tissue (Figure 7D). This supports the presence of a Th2-polarised inflammatory process in the lung environment. It should be noted that a number of cells, both structural as well as leukocytes have the capacity to release many of these cytokines. Evaluation of the gene expression in peribronchial, perivascular and alveolar lung tissue as a marker of local inflammatory response To further evaluate the local inflammatory microenvironment in the different lung tissue compartments, we used laser capture microdissected peribronchial, perivascular and alveolar tissue from OVA/OVA mice and control mice, followed by a real-time RT- PCR analysis and quantification of expression of 60 different cytokines, chemokines or respective receptor genes (Figure 8A). The effect of allergen exposure in all three tissue compartments was expressed as fold change compared to gene expression in OVA/PBS animals (OVA-OVA/OVA-PBS). Allergic inflamma- tion resulted in a wide variation of inflammatory response from down-regulation (i.e. Il8.8 times in peribronchial tissue) to up- regulation (i.e. more than 20 fold for Ccl8 in perivascular tissue) (Table 1). Only genes that were changed $3 times in at least one tissue compartment were considered having biological importance and were involved in a further analysis. Firstly, we evaluated genes affected by allergic inflammation (Figure 8B). We found that 22 genes were upregulated and 6 genes were downregulated after allergen exposure. Fourteen of these were changed in all three compartments. Of these, twelve genes were upregulated (Casp1, ccl12, ccl20.Ccl24, Ccl7, Ccl8, Cxcl1, Cxcl9, Il13, Il1r2 and Spp1) and two were downregulated (Il18 and Tnfrsf1a). Peribronchial and perivascular tissue had change in expression of three genes in common (Ccl19 and Cxcr3 were upregulated Il-8rb was downregulated), while peribronchial and alveolar tissue had changed expression in two gene (Il-6st and Pf4/ Cxcl4), both downregulated. Alveolar and perivascular micro- environments shared change in expression of four genes (Ccl17, Ccl9, Il1r1 and Cd40lg) that were all upregulated. Finally, there were genes that changed only in one compartment, which included upregulation Ccl11 and Ccr6 in the perivascular compartment and Ccl4 and Ccr1 in the alveolar compartment, and downregulation of Ccxl15 in the peribronchial compart- ment (Figure 8B). Next we focus on the differences between the three compart- ments. For analysis we grouped genes in three categories; genes Figure 7. Cytokine concentration in lung during allergic inflammation. Concentration of IL-2(A), IL-6(B), TNF (C), IFN-c, IL-4, IL-17A and IL-10 (D) was assessed using CBA; Results are expressed as pg/gram lung tissue, in mice exposed to PBS or OVA. Data are shown as mean 6 SEM, *p,0.05, **p,0.01. doi:10.1371/journal.pone.0019889.g007 PLoS ONE | www.plosone.org 10 May 2011 | Volume 6 | Issue 5 | e19889