To better understand the gene expression response to the treatment, mice were treated with the aerosolized NTHi lysate, then euthanized after 2 h or 4 h for comparison to untreated mice. To reduce the lung leukocyte burden, the pulmonary vasculature was perfused and the airways lavaged with PBS. The lungs were mechanically homogenized, then total RNA was isolated from lung homogenates using the RNeasy system (Qiagen), and cRNA was synthesized and amplified from equal masses of total RNA using the Ilumina TotalPrep RNA amplification kit (Ambion). Amplified cRNA was hybridized and labeled on Sentrix Mouse-6 Expression BeadChips (Illumina), then scanned on a BeadStation 500 (Illumina). Primary microarray data were deposited at the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/ geo/) consistent with MIAME standards (GEO Accession GSE13740). Primary signal intensity was normalized between and within samples, and differentially expressed genes were identified based on signal change and inter-sample variation. Gene ontology analysis was performed using the NIAID Database for Annotation, Visualization and Integrated Discovery (DAVID) and the KEGG Database (GenomeNet). Differentially expressed genes were mapped to signaling pathways using Ingenuity Pathways Analysis 5.0 (Ingenuity Systems), and the pathway nodules were individually reviewed. C57BL/6 mice were used for the gene expression studies as they demonstrate similar NTHi lysate induced protection against all investigated pathogens, and because the genetically manipulated mice the authors plan to use to dissect the mechanisms of StIR are primarily on C57BL/6 backgrounds.