Intrinsic Role of CFTR in Skeletal Muscle Figure 5. Hyperinflammatory phenotype of Cftr2/2 skeletal muscle cells in vitro. (A) Upper panels: Cytokine mRNA expression levels in Cftr+/+ versus Cftr2/2 diaphragmatic myotubes under control conditions (CTL) and after 4 hours of stimulation (TNF-a 1 ng/ml, IL-1a 5 U/ml, IFN-c 20 U/m, LPS 1 ng/ml). N = 5 per group; * p,0.05 for control versus stimulated groups, { p,0.05 for Cftr+/+ versus Cftr2/2. Lower panels: Representative RNase protection assays from diaphragm (left panel) and limb muscle (right panel). (B) NFkB p65 levels in nuclear extracts (normalized for protein) obtained from diaphragmatic myotubes in Cftr+/+ versus Cftr2/2 groups stimulated for the indicated time periods (0–60 min). N = 4–5 per group; * p,0.05 for Cftr+/+ versus Cftr2/2. (C) Western analysis of phosphorylated (pERK1/2) and total ERK1/2 MAPK in cytoplasmic lysates from Cftr+/+ and Cftr2/2 diaphragmatic myotubes following stimulation with cytokines+LPS. Results are representative of 2 experiments. doi:10.1371/journal.pgen.1000586.g005 burden and number of inflammatory cells in bronchoalveolar lavage fluid were greater in infected Cftr2/2 mice (data not shown). At 2 days after the initiation of pulmonary infection, there was an upregulation of pro-inflammatory gene expression within the diaphragms of both Cftr2/2 and Cftr+/+ mice. However, as was the case in vitro, the diaphragmatic mRNA levels of chemokines (MIP-2, MIP-1a, RANTES) and other cytokines (TNF-a, IL-1b) were significantly higher in infected Cftr2/2 mice (Figure 6A). Therefore, the greater vulnerability of Cftr2/2 diaphragms to developing a hyperinflamma- tory phenotype in vitro was also observed in vivo. PLoS Genetics | www.plosgenetics.org 7 July 2009 | Volume 5 | Issue 7 | e1000586