Peroxiredoxin 6 Fails to Limit Phospholipid Peroxidation in Lung from Cftr-Knockout Mice Subjected to Oxidative Challenge

Oxidative stress plays a prominent role in the pathophysiology of cystic fibrosis (CF). Despite the presence of oxidative stress markers and a decreased antioxidant capacity in CF airway lining fluid, few studies have focused on the oxidant/antioxidant balance in CF cells. The aim of the current study was to investigate the cellular levels of reactive oxygen species (ROS), oxidative damage and enzymatic antioxidant defenses in the lung of Cftr-knockout mice in basal conditions and as a response to oxidative insult

Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis

Iron Homeostasis and Inflammatory Status in Mice Deficient for the Cystic Fibrosis Transmembrane Regulator.

Cystic Fibrosis (CF) is a frequent and lethal autosomal recessive disease caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Patients with CF suffer from chronic infections and severe inflammation, which lead to progressive pulmonary and gut diseases. Recently, an expanding body of evidence has suggested that iron homeostasis was abnormal in CF with, in particular, systemic iron deficiency and iron sequestration in the epithelium airway. The molecular mechanisms responsible for iron dysregulation and the relationship with inflammation in CF are unknown.We assessed the impact of CFTR deficiency on systemic and tissue iron homeostasis as well as inflammation in wildtype and CFTR knockout (KO) mice. First, in contrast to the systemic and intestinal inflammation we observed in the CFTR KO mice, we reported the absence of lung phenotype with regards to both inflammation and iron status. Second, we showed a significant decrease of plasma ferritin levels in the KO mice, as in CF patients, likely caused by a decrease in spleen ferritin levels. However, we measured unchanged plasma iron levels in the KO mice that may be explained by increased intestinal iron absorption.These results indicate that in CF, the lung do not predominantly contributes to the systemic ferritin deficiency and we propose the spleen as the major organ responsible for hypoferritinemia in the KO mouse. These results should provide a better understanding of iron dysregulation in CF patients where treating or not iron deficiency remains a challenging question

Analysis of Human Histone H2AZ Deposition In Vivo Argues against Its Direct Role in Epigenetic Templating Mechanisms

Chromatin is considered to be a principal carrier of epigenetic information due to the ability of alternative chromatin states to persist through generations of cell divisions and to spread on DNA. Replacement histone variants are novel candidates for epigenetic marking of chromatin. We developed a novel approach to analyze the chromatin environment of nucleosomes containing a particular replacement histone. We applied it to human H2AZ, one of the most studied alternative histones. We find that neither H2AZ itself nor other features of the H2AZ-containing nucleosome spread to the neighboring nucleosomes in vivo, arguing against a role for H2AZ as a self-perpetuating epigenetic mark

Erythrophagocytosis in presence of CFTR(inh)-172.

<p>Two independent experiments (Exp1, 2) of erythrophagocytosis (EP) using normal or aged red blood cells were performed with bone-marrow derived macrophages incubated or not with 10^-5M CFTR(inh)-172.</p

Duodenal and spleen iron-related proteins in CFTR KO mice.

<p>Duodenum (A) and spleen (B, C) were collected from WT and CFTR KO mice. Duodenum DcytB, DMT1 and ferroportin were analyzed using proteins from membrane enriched-fractions and L-ferritin from cytosolic fractions. The loading control, ß-actin, is shown (A). Perls’ blue staining of spleen section from WT and KO mice (B). Spleen ferroportin was analyzed using proteins from membrane enriched-fractions and L-ferritin from cytosolic fractions. The loading control, ß-actin, is shown. The right panel represents the quantification of the blots (arbitrary units). Data are presented as mean ± SEM. * P<0.05.</p

Red cells, iron indices, and iron-related genes in CFTR KO mice.

<p>Hematological and iron parameters were analyzed in WT and CFTR KO mice: RBC (red blood cells), Hb (hemoglobin), HC (hematocrit) and MCV (mean corpuscular volume) (A); plasma iron, TfS, (transferrin saturation) and ferritin (B). Liver hepcidin1 and BMP6 mRNA levels relative to cyclophilin-A were analyzed by real-time PCR (C). Data are presented as mean ± SEM. * P<0.05.</p

Lung iron phenotype in CFTR KO mice.

<p>Representative images of BAL (bronchoalveolar lavages) cytospin slides obtained from WT and CFTR KO mice and stained with Perl's Prussian blue. Iron loaded macrophages are indicated by arrows. Magnification X40 (A). Perls’ blue staining of lung section from WT and KO mice (B). Lung iron content (C) and L-ferritin analysis from cytosolic fractions; the right panel represents the quantification of the blots (arbitrary units) (D). Data are presented as mean ± SEM.</p

Inflammatory status of the CFTR KO mice.

<p>WBC (white blood cells), lymphocytes, monocytes and neutrophils were measured in the blood of WT and CFTR KO mice (A). Cytokines levels were measured by the V-PLEX Proinflammatory Panel1 (mouse) Kit in the plasma of WT and CFTR KO mice according to the manufacturer’s instruction (B). Duodenal mRNA levels relative to cyclophilin-A expression were assessed by real-time PCR for the indicated genes (C). Data are presented as mean ± SEM. * P<0.05.</p