Specific primers and probes

Lungs were homogenized using a TissueLyser LT (Qiagen) and total RNA was extracted from the lung tissue supernatant using RNeasy Mini kit (Qiagen). RNA extraction from sorted lung cells was performed using Trizol (Invitrogen). Following the chloroform step, the aqueous phase containing RNA was further processed using the RNeasy Mini or Micro Kit according to manufacturer’s instructions (Qiagen). RNA concentration was determined by NanoDrop (Thermo Scientific). In all, 2 μg or 9 μl (sorted cells) of RNA was converted to cDNA using High Capacity RNA-to-cDNA kit (Applied Biosystems). RT-qPCR was performed with Quantitect Probe PCR Master Mix (Qiagen). For mRNA analysis of Gapdh, Cxcl1, Cxcl2, Il6, Csf2, and Ifna5 gene specific primers and probes were used (all Applied Biosystems). For absolute quantification of RSV L gene, TNF-α and IFN-γ mRNA, the exact number of copies of the gene of interest was calculated using a plasmid DNA standard curve for each gene and normalized to Gapdh.^{21 (link)} RT-qPCR was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems). To quantify relative mRNA expression the mean ΔCT was calculated for each target gene relative to Gapdh (encoding glyceraldehyde-3-phosphate dehydrogenase) and expressed as 2^−ΔCT. Analysis was performed using 7500 Fast System SDS Software (Applied Biosystems).