Alterations in immune response and PPAR/LXR regulation in cystic fibrosis macrophages  Charlotte Andersson, Munir M. Zaman, Amanda B. Jones, Steven D. Freedman  Journal of Cystic Fibrosis  Volume 7, Issue 1, Pages 68-78 (January 2008) DOI: 10.1016/j.jcf.2007.05.004 Copyright © 2007 Terms and Conditions

Fig. 1 Analyses of cytokine secretion in CF and WT macrophages. Peritoneal macrophages were plated, rested over night and the next day and incubated for 4.5 h with various concentrations of LPS. TNFα (A) and IL-6 (B) secretion into the media was assayed by ELISA. Some animals were pretreated for 10 days with oral DHA 40 mg/day. n=6. ⁎ p<0.05 statistical difference compared with WT. † p<0.05 statistical difference compared with cftr−/− mice. Journal of Cystic Fibrosis 2008 7, 68-78DOI: (10.1016/j.jcf.2007.05.004) Copyright © 2007 Terms and Conditions

Fig. 2 Analyses of NFκB activity in WT and CF macrophages. Peritoneal macrophages were plated, rested over night and then stimulated with 100 ng/ml LPS for 30 min. A. The cells were lysed and 9 mg/well protein were analyzed for NFκB activity B. p65 activity in plated macrophages after LPS stimulation with and without oral administration of DHA for 10 days (40 mg/day). ⁎ p≤0.05, ⁎⁎ p<0.01, ⁎⁎⁎ p<0.001 comparing to control without LPS stimulation unless otherwise indicated; n=6. Journal of Cystic Fibrosis 2008 7, 68-78DOI: (10.1016/j.jcf.2007.05.004) Copyright © 2007 Terms and Conditions

Fig. 3 Nuclear receptor mRNA expression by RT-PCR as a function of LPS stimulation and CFTR function. Macrophages were plated and stimulated with no LPS (time 0) or with 100 nM LPS for 8 h, 16 h, and 24 h. mRNA expression was assessed by RT-PCR and normalized to RB23. A. PPARα; B. PPARγ; C. PPARδ; D. RXRα; E. LXR; F. ABCA1. ⁎ p<0.05 comparing WT and CF. † p<0.05, †† p<0.01, ††† p<0.001 compared with WT control. ‡ p<0.05 compared with CF control. Figures are representative of three different experiments, n≥6. Journal of Cystic Fibrosis 2008 7, 68-78DOI: (10.1016/j.jcf.2007.05.004) Copyright © 2007 Terms and Conditions

Fig. 4 PPAR activity in WT and CF macrophages by EMSA with and without DHA treatment. A. EMSA was performed on nuclear fractions from WT and CF macrophage samples. 5 μg protein was loaded per well. PPRE binding was decreased in CF macrophages compared to WT. The results are representative of three separate experiments. B. Quantification of PPRE binding in WT and CF macrophages. The PPAR/RXR band from three separate experiments was quantitated by densitometric scanning. ⁎⁎⁎ p<0.001 C. Effect of oral administration of DHA on PPARα induction in CF macrophages. WT and cftr−/− mice were fed peptamen or peptamen containing DHA 40 mg/day for 10 days. Representative results are shown from two WT and three cftr−/− mice on DHA as well as one CF control mouse on no DHA. 5 μg of nuclear protein was loaded per well. PPARα antibody and DHA pretreatment was used as indicated in the figure. Journal of Cystic Fibrosis 2008 7, 68-78DOI: (10.1016/j.jcf.2007.05.004) Copyright © 2007 Terms and Conditions

Fig. 5 Effect of oral DHA administration in CF and WT mice on mRNA expression of nuclear receptors and their target genes. RNA was extracted from peritoneal macrophages from mice with or without pretreatment with oral DHA. WT and CF macrophages were analyzed for mRNA expression of: A. PPARα; B Acyl-CoA-Oxidase; C. PPARγ; D. CD36; E. PPARδ; F. RXRα; G. LXRα; H. ABCA1. All data were normalized to RB23 expression. ⁎ p<0.05, ⁎⁎ p<0.01. Results are representative of 2 separate experiments, n=6 for each group. Journal of Cystic Fibrosis 2008 7, 68-78DOI: (10.1016/j.jcf.2007.05.004) Copyright © 2007 Terms and Conditions

Fig. 6 Effect of DHA and PPAR agonists on cytokine secretion in vitro. Peritoneal macrophages were plated and incubated with media alone (CON) or containing DHA (5 μM), Rosiglitazone (ROSI; 10 μM ), or WY14643 (WY; 50 μM) for 16 h. Cells were then stimulated with 100 ng/ml LPS for 4 h and TNFα and IL-6 were assayed in the media by ELISA with control values normalized to 100%. ⁎ p<0.05, ⁎⁎ p<0.01, ⁎⁎⁎ p<0.001 statistical difference compared with control. Data shown are the means±SEM from 3 different experiments. Journal of Cystic Fibrosis 2008 7, 68-78DOI: (10.1016/j.jcf.2007.05.004) Copyright © 2007 Terms and Conditions

Fig. 7 Effect of the PPARα inhibitor MK-886 and other fatty acids on cytokine secretion in vitro. Peritoneal macrophages were plated and incubated with media alone or containing DHA (5 μM), DHA+MK-886 (10 μM), MK-886, eicosapentaenoic acid (EPA; 5 μM), 22:5 n-3 (5 μM), 24:6 n-3 (5 uM) or linoleic acid (LA; 5 μM). Results are representative of 2 separate experiments, n=6 for each group. ⁎ p<0.05, ⁎⁎ p<0.01 comparing to control, † p<0.05, †† p<0.01 comparing to DHA. Journal of Cystic Fibrosis 2008 7, 68-78DOI: (10.1016/j.jcf.2007.05.004) Copyright © 2007 Terms and Conditions