CFTR is a negative regulator of NFkappaB mediated innate immune response

Abstract

Background: Dysfunctional CFTR in the airways is associated with elevated levels of NFkappaB mediated IL-8 signaling leading to neutrophil chemotaxis and chronic lung inflammation in cystic fibrosis. The mechanism(s) by which CFTR mediates inflammatory signaling is under debate.

Methodology/principal findings: We tested the hypothesis that wt-CFTR down-regulates NFkappaB mediated IL-8 secretion. We transiently co-expressed wt-CFTR and IL-8 or NFkappaB promoters driving luciferase expression in HEK293 cells. Wt-CFTR expression in HEK293 cells suppresses both basal and IL1beta induced IL-8, and NFkappaB promoter activities as compared to the control cells transfected with empty vector (p<0.05). We also confirmed these results using CFBE41o- cells and observed that cells stably transduced with wt-CFTR secrete significantly lower amounts of IL-8 chemokine as compared to non-transfected control cells. To test the hypothesis that CFTR must be localized to cell surface lipid rafts in polarized airway epithelial cells in order to mediate the inflammatory response, we treated CFBE41o- cells that had been stably transduced with wt-CFTR with methyl-beta-cyclodextrin (CD). At baseline, CD significantly (p<0.05) induced IL-8 and NFkappaB reporter activities as compared to control cells suggesting a negative regulation of NFkappaB mediated IL-8 signaling by CFTR in cholesterol-rich lipid rafts. Untreated cells exposed to the CFTR channel blocker CFTR-172 inhibitor developed a similar increase in IL-8 and NFkappaB reporter activities suggesting that not only must CFTR be present on the cell surface but it must be functional. We verified these results in vivo by comparing survival, body weight and pro-inflammatory cytokine response to P. aeruginosa LPS in CFTR knock out (CFKO) mice as compared to wild type controls. There was a significant (p<0.05) decrease in survival and body weight, an elevation in IL-1beta in whole lung extract (p<0.01), as well as a significant increase in phosphorylated IkappaB, an inducer of NFkappaB mediated signaling in the CFKO mice.

Conclusions/significance: Our data suggest that CFTR is a negative regulator of NFkappaB mediated innate immune response and its localization to lipid rafts is involved in control of inflammation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. CFTR downregulates IL-8 and NFκB reporter activities.

HEK293 cells were transiently transfected with wt-CFTR and IL-8 (A) or NFκB (B) reporter constructs together with a renila luciferase internal control plasmid (n = 3). The cells were induced with 1 ng/ml IL1-β overnight. Data are normalized to the renila luciferase internal control and expressed as mean±SD. A. IL-8 promoter driven luciferase expression. IL1-β stimulates IL-8 promoter activity and wt-CFTR blunts both control and IL1-1β driven IL-8 (*p

Figure 2. CFTR downregulates IL-8 chemokine secretion.

CFBE41o- control and cells stably transduced with wt-CFTR were induced with 1 ng/ml IL1-β and IL-8 chemokine secretion in media was calculated after overnight treatment. Data are shown as mean±SD of three experiments. IL1-β stimulates IL-8 chemokine secretion while wt-CFTR blunts both control and IL1-1β driven IL-8 chemokine secretion (*p

Figure 3. Inhibition of CFTR regulated NFκB signaling under conditions that disrupt lipid rafts.

CFBE41o- cells stably transduced with wt-CFTR were transiently transfected with IL-8 or NFκB reporter constructs and a renila luciferase internal control plasmid (n = 3). The cells were induced with 1 ng/ml IL1-β and/or treated with 5 mM methyl-β-cyclodextrin (CD) for 6 hrs (A), and/or 10 µM CFTR-172 inhibitor overnight (B). The data are shown as mean±SD of IL-8 or NFκB promoter activities normalized to renila luciferase internal control. A. IL-1β induced IL-8 and NFκB promoter driven luciferase expression, and wt-CFTR dampened baseline and cytokine induced reporter activity (Figs 1&2). Pre-treatment with CD under conditions known to disrupt lipid rafts eliminated the effect of wt-CFTR, *p

Figure 4. The CFTR knock out mice show significant decline in body weight after LPS treatment.

The FABP-CFTR gut corrected CFTR knock-outs (CFKO) and wt- mice (n = 12) were injected i.p. with 15 mg/kg body weight of P. aeruginosa LPS, and body weights were recorded daily for 6 days. The CFKO mice show significant decline in body weight as compared to wild type mice (p<0.001; day 3–6). Wild type mice (blue) recover from weight loss by day 6 while CFKO mice (red) don't show significant recovery. The 25% of CFKO mice died during the course of experiment due to LPS induced chronic inflammation (green). The data suggest that CFTR is critical for LPS induced immune response and recovery from LPS induced inflammation. The data are shown as body weight in grams of each mouse in wt- and CFKO- group from day 0 to 6. The non-linear logarithmic regression was used to calculate the trend lines indicating the changes in body weight in wild type (blue) and CFKO (red) mice after LPS exposure.

Figure 5. The CFTR knock out mice show significant decline in recovery from inflammation after LPS treatment.

The FABP-CFTR gut corrected CFTR knock-outs (CFKO) and wt- mice (n = 12) were injected i.p. with 15 mg/kg body weight of P. aeruginosa LPS, and survival was recorded daily for 7 days. The data are shown as percentage of mice that survived at each time point. The CFKO mice (red) show a 25% decline in survival as compared to wild type mice (blue) by day 4 (*p<0.05). Wild type mice show 100% survival and recovery from inflammation at the given LPS dose while 25% of CFKO mice fail to recover and don't survive.

Figure 6. The CFTR knock out mice have elevated IκB-NFκB mediated inflammatory signaling.

The FABP-CFTR gut corrected CFTR knock-outs (CFKO) and wt- mice (n = 3) were injected i.p. with 15 mg/kg body weight of P. aeruginosa LPS or live bacteria (100 µl, 2×108), and IκB-p levels (24 hrs) were quantified by immunoblotting while serum IL1-β levels (6 or 24 hrs) were determined by ELISA. A. The representative immunoblot indicates the IκB-p protein levels in each group. The lung protein extracts of CFKO mice show inherent increase in IκB-p levels indicative of elevated NFκB signaling and activation. The densitometry analysis of IκB-p protein levels in CFKO lungs as compared to wild type mice show a 4.5 fold increase (n = 3, p<0.05) at baseline. B. The serum concentration of IL1-β (pg/ml) shown as mean±SD. The CFKO mice (red) have significantly higher pro-inflammatory cytokine, IL1-β, levels (***p<0.001 at 6 hrs while *p<0.05 at 24 hrs) on LPS induction as compared to wild type mice (green; **p<0.01).

Figure 7. Hypothetical model of CFTR mediated NFκB signaling and innate immune response.

The lack of functional CFTR on the cell surface or inhibition of lipid raft localization by methyl-β-cyclodextrin (CD) de-regulates pro-inflammatory signaling via TNFα-IL1β-TLR pathways resulting in an overactive innate immune response, chronic NFκB mediated inflammation, and lung destruction. Our data suggest that localization of functional CFTR at the cell surface in cholesterol rich lipid rafts serves as a negative regulator of NFκB signaling. We propose that restoration of an optimal amount of functional CFTR on the cell surface can control chronic inflammatory pathophysiology of CF lung disease by not only restoring the chloride efflux function but by also regulating the chronic NFκB mediated inflammatory signaling.