Defective infiltration of DCs, CD4⁺ T cells and CD8⁺ T cells in the lungs of PVM-infected P2Y₂-deficient mice.

<p><b>A.</b> The percentage of DCs (MHC II⁺ CD11c⁺ CD11b⁺), CD4⁺ T cells and CD8⁺ T cells were determined by flow cytometry analysis in BALFs of P2Y₂^+/+ and P2Y₂^−/− mice at day 8 and days 10 (d8 and d10) post-infection (N = 5). <b>B.</b> Analysis of viral titer in P2Y₂^+/+ and P2Y₂^−/− lungs after infection with PVM. PVM viral titer was quantified by quantitative PCR in lung homogenates of P2Y₂^+/+ and P2Y₂^−/− mice 8 or 10 days post-infection. Data were normalized to the viral titer obtained for P2Y₂^+/+ lungs at d8. <b>C.</b> IL-12, IFN-γ, TNF-α and IL-6 levels were determined by ELISA in the BALFs of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice (N = 11). <b>D.</b> The level of DC recruiters was determined by ELISA (MIP-3α, IP-10) or qPCR (BRAK) in the BALFs of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice (N = 5).</p

Cellular infiltration in the lungs of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice.

<p>50 µL of the viral suspension were instilled into the nostrils of the anesthetized mouse maintained in a vertical position as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050385#s2" target="_blank">Materials and Methods</a>. <b>A</b>, BALF was collected and the total number of cells was evaluated at days 8, 9, 10 and 12 (N = 7). <b>B.</b> Quantification of ATP level in the BALF of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice. ATP level was quantified in the BALF of P2Y₂^+/+ and P2Y₂^−/− mice using ATP detection assay system ATPlite at d8, d9 and d10 post-infection with PVM. <b>C</b>, Histological analysis of representative lungs of P2Y₂^+/+ and P2Y₂^−/− mice 10 days and 15 days after infection with PVM. Paraffin sections (7 µM) of lungs of P2Y₂^+/+ and P2Y₂^−/− mice infected by PVM were stained with haematoxylin-eosin (magnification: ×200).</p

Higher mortality rate in PVM-infected P2Y₂-deficient mice.

<p>Following intranasal inoculation of PVM (1000 PFUs), P2Y₂^+/+ and P2Y₂^−/− mice were monitored daily for survival (<b>A</b>) and weight loss (<b>B</b>). Weight curves (mean ± SEM) are relative to initial body weight. The displayed data result from the pooling of four independent experiments.</p

Quantification of neutrophils and macrophages, and their recruiters in the lungs of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice.

<p><b>A–D.</b> The level of the chemokines KC/CXCL-1 (A), MIP-2/CXCL-2 (B), MIP-1α/CCL3 (C) and MCP-1/CCL2 (D) was determined by ELISA in the BALFs of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice. (N = 9) <b>E.</b> Flow cytometry quantification of neutrophils and macrophages in the BALFs of PVM-infected P2Y₂^+/+ and P2Y₂^−/− mice at day 8 and 10 post-inoculation (N = 12). <b>F.</b> Cytospin preparations were made from BALFs of P2Y₂^+/+ and P2Y₂^−/− mice at day 8 post-infection using a Shandon III cytocentrifuge and were stained using Diff-Quick staining. Magnification: ×400.</p