Lung Expansion of Th1, Th2, Th17 and Tregs Figure 8. Expression of inflammatory cytokine, chemokine and receptor genes in the lung after allergen exposure compared to control. A) The different lung tissue compartments (alveolar, perivascular and peribronchial) were isolated from lung tissue from OVA or PBS exposed mice using the PALM microBeam microscopy. Lung compartment samples were taken from three different animals and then pooled. RNA was extracted and the inflammatory response in each microenvironment was assessed using real-time RT-PCR array of 84 inflammatory cytokines, chemokines and receptors. The expression of these genes in each compartment is visualized as heat map. Red colour indicates up-regulated genes, green colour indicates down-regulated genes, and the dark colour indicates great fold change. B) Venn diagram of genes with fold change $3 times after allergen exposure in at least one tissue compartment compared to control. Up-regulated genes are shown in red font and down regulated genes are shown in green font. doi:10.1371/journal.pone.0019889.g008 involved in general inflammation, genes involved in allergic inflammation and genes from chemokines that can affect T effect cells migration, as reported in the literature, followed by analysis of their relationship using Ingenuity Pathways Analysis. To determine the role in allergic inflammation, 13 genes related to allergy were included (Figure 9A), revealing differences between the three compartments. Among them, eotaxin-2 (Ccl24) was the highest amplified gene and was expressed mainly in perivascular tissue. Eotaxin-1(Ccl11) was amplified in the same manner, but to a smaller degree. The main Th2 cytokine, Il13, was also expressed in all three compartments with emphasis in alveolar tissue. This was also seen for Spp1 (osteopontin), which showed a greater increase compare to Il13. Il18 was downregulated in all compartments, while Il8rb (Cxcr2) was downregulated mainly in the peribronchial and perivascular areas. Direct and indirect relationships between some of these allergy-associated genes was documented (Figure 9B). Our final goal was to describe the expression of chemokine genes that may have a role in the homing of Th1, Th2, Th17 and Treg cells to different compartments. The Th1 related chemokines Ccl7, Ccl2 and Cxcl9 were increased in all three compartments after allergen exposure, with two of them increased in alveolar tissue (Ccl7, Ccl2). Interestingly Pf4 (Cxcl4) was down-regulated. Among the Th2 related chemokines, Ccl8 showed the higher fold increase mainly in perivascular and then in peribronchial tissue. For Th17, Ccl20 was increased mainly in perivascular tissue. The Ccl4 a chemokine (Il17-related) was increased mainly in alveolar tissue. Finally Ccl19, a Treg cell related chemokine was increased mainly in perivascular tissue supporting immunohistochemistry data (Figure 10A). Analysis with ingenuity showed also a complex relation between the majority of above chemokines, supporting the concept of a sophisticated network of high complexity and high complexity and interaction (Figure 10B). PLoS ONE | www.plosone.org 11 May 2011 | Volume 6 | Issue 5 | e19889