Impact of <i>MIF</i> Gene Promoter Polymorphism on F508del Cystic Fibrosis Patients

<div><p>Macrophage migration Inhibitory Factor (MIF) is a pro-inflammatory cytokine sustaining the acute response to gram–negative bacteria and a regulatory role for MIF in Cystic Fibrosis has been suggested by the presence of a functional, polymorphic, four-nucleotide repeat in this gene's promoter at position −794, with the 5-repeat allele displaying lower promoter activity. We aimed at assessing the association of this polymorphism with disease severity in a group of Cystic Fibrosis patients homozygous for F508del <i>CFTR</i> gene mutation. Genotype frequencies were determined in 189 Cystic Fibrosis and 134 control subjects; key clinical features of patients were recorded and compared among homozygous 5-allele patients and the other <i>MIF</i> genotypes. Patients homozygous for the 5-repeat allele of <i>MIF</i> promoter displayed a slower rate of lung function decline (p = 0.027) at multivariate survival analysis. Multiple regression analysis on age-normalized respiratory volume showed no association of the homozygous 5-repeat genotype with lung function under stable conditions and no correlation with <i>P.aeruginosa</i> chronic colonization. Therefore, only the Homozygous 5-repeat genotype at <i>MIF</i> −794 is associated with milder disease in F508del Cystic Fibrosis patients.</p></div

Cox regression analysis for age at first acute episode with FEV1 <60% of predicted value on 185 Cystic Fibrosis patients homozygous for the F508del mutation.

<p>Number of patients with acute episode  = 119</p><p>Number of censored patients = 66</p><p>Overall significance p-value  = 0.0010</p><p>MIF-CATT genotype: <i>MIF</i> gene -CATT repeat genotype at position −794</p><p>95% CI  = 95% Confidence Interval</p><p>Cox regression analysis for age at first acute episode with FEV1 <60% of predicted value on 185 Cystic Fibrosis patients homozygous for the F508del mutation.</p

Clinical data of 187 Cystic Fibrosis patients homozygous for the F508del mutation according to the genotype for the <i>MIF</i> gene -CATT repeat at position −794.

<p>Data are presented as mean and 95% Confidence Interval (95% CI). FEV1: forced expiratory volume in one second; BMI: body mass index; cc by PA: chronic colonization by <i>P. aeruginosa</i>.</p><p>* CF specific percentile according to Kulich <i>et al</i>, <i>Am J Respir Crit Care Med</i>, 2005.</p><p>Clinical data of 187 Cystic Fibrosis patients homozygous for the F508del mutation according to the genotype for the <i>MIF</i> gene -CATT repeat at position −794.</p

Characteristics of 189 Cystic Fibrosis patients homozygous for the F508del mutation recruited from 2 different European centers.

<p>Continuous data are presented as mean ± SD unless otherwise stated; categorical data are presented as counts and proportions. FEV1: forced expiratory volume in one second; cc by PA: chronic colonization by <i>P. aeruginosa.</i> The most recent FEV1 was used for each patient. MIF-CATT: <i>MIF</i> gene -CATT repeat genotype at position -794</p><p>* CF specific percentile according to Kulich <i>et al</i>, <i>Am J Respir Crit Care Med</i>, 2005.</p><p>Characteristics of 189 Cystic Fibrosis patients homozygous for the F508del mutation recruited from 2 different European centers.</p

Multiple regression analysis of FEV1 (Kulich)^* data on 189 Cystic Fibrosis patients homozygous for the F508del mutation.

<p>Overall R² = 0.274; multiple correlation coefficient  = 0.524</p><p>Overall significance p-value<0.0001;</p><p>FEV1: forced expiratory volume in one second; cc by PA: chronic colonization by <i>P. aeruginosa</i>. MIF-CATT genotype: <i>MIF</i> gene -CATT repeat genotype at position -794</p><p>*CF specific percentile according to Kulich <i>et al</i>, <i>Am J Respir Crit Care Med</i>, 2005.</p><p>Multiple regression analysis of FEV1 (Kulich)^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114274#nt113" target="_blank">*</a>} data on 189 Cystic Fibrosis patients homozygous for the F508del mutation.</p

Lung function decline in 185 Cystic Fibrosis patients grouped according to <i>MIF</i> -794 CATT genotypes.

<p>Kaplan-Meier plots relative to age at first acute episode with FEV1 <60% of predicted value. (A) Comparison between patients with MIF 5-5 (homozygous 5-CATT repeats) vs. not 5-5 genotype; (B) comparison between patients with at least one 5-CATT allele vs. the others. Ticks indicate censored subjects follow-up times. “Number at risk” at the bottom indicates the number of patients without acute episodes at a given time interval and whose follow- up extends at least that far into the curve.</p

Lung histological responses to bleomycin are attenuated by vardenafil.

<p>Lung histological sections of wild-type (a,b,e,f,i,j) and CF mice homozygous for the F508del mutation (c,d,g,h,k,l) 10 days after treatment with saline (NaCl; a–d), bleomycin (Bleo; e–h) or bleomycin and vardenafil (Bleo+Vard; i–l) were stained with hematoxylin and eosin (a,c,e,g,i,k); impregnated with silver (b,d,f,h,j,l) or stained with Masson’s trichrome (inserts). Bars, 100 µm in panels a,c,e,g,i,k; 20 µm in panels b,d,f,h,j,l; and 40 µm in inserts. Representative micrographs from 5 mice per group.</p

Vardenafil prevents overresponsive proinflammatory status in CF fibroblasts.

<p>a,b–g) mRNA and c) protein expression of proinflammatory cytokines in response to 0.1 mg/ml LPS in lung (a,b,e–g) and skin (c,d) cultured fibroblasts at the second passage purified from CF mice homozygous for the F508del mutation and from wild-type (WT) mice. At the mRNA level, markers were assessed 3 h after LPS stimulation. At the protein level, CCL-2 (c) was assessed 24 h after LPS stimulation. Vardenafil (Vard; 0.1 µM) was used for TNF-α (e), IL-1β (f) and IL-6 (g) mRNA expression studies. 18S RNA used as a reference gene. Values are means ± SEM of 3 multi(96)well cultures per group from a representative experiment selected from at least 3 experiments with similar results. *: <i>P</i><0.05; **: <i>P</i><0.01; *** <i>P</i><0.001 for comparison of mean values.</p

Exaggerated proliferation and myofibroblast differentiation of CF fibroblasts.

<p>Cell proliferation and myofibroblast differentiation in cultured lung (A–D) and skin (E–H) fibroblasts at the second passage purified from CF mice homozygous for the F508del mutation and from wild-type (WT) mice. A,e) Uptake of³H-thymidine (1 µCi/well) assessed in cultured cells seeded at 30×10³ cells/well, in the absence of any added growth factor to culture media or in the presence of 1 to 100 ng/ml human rPDGF-BB for 1 h. After 48 h, adherent cells were trypsinized before ³H-thymidine counting. Data expressed as counts per minute (cpm). b,f) Cell growth analysis assessed by daily counting, in a Neubauer chamber, of trypsinized cells cultured in the absence of any added growth factor to culture media. c,g) α-SMA mRNA expression, using 18S RNA as a reference, assessed in the absence of any added growth factor to culture media or in the presence of 1 to 100 ng/ml human rTGF-β1 for 24 h. d) α-SMA mRNA expression assessed in the presence of 0.1 to 50 µM vardenafil (Vard) for 6 h. h) Micrographs of fibroblast cultures under stimulation with 10 ng/ml human rTGF-β1. Arrows identify formation of cellular aggregates. Bars: 100 µm. Values are means ± SEM of 3 multi(96)well cultures per group from a representative experiment selected from at least 3 experiments with similar results. *: <i>P</i><0.05; **: <i>P</i><0.01; *** <i>P</i><0.001 for comparison of mean values.</p

Exaggerated CF lung responses to bleomycin are attenuated by vardenafil.

<p>a) Soluble collagen content in homogenized unlavaged lungs; b) lymphocyte counts, c) CCL-2, d) IL-6, and e) TGF-β1 in bronchoalveolar lavage (BAL); and f) TIMP-1 in homogenized unlavaged lungs from CF mice homozygous for the F508del mutation and from wild-type (WT) mice 10 days after deposition into the lungs by pharyngeal aspiration of a single dose of 0.015 U bleomycin (Bleo). In case of simultaneous treatment with bleomycin and vardenafil, animals were given a first intraperitoneal injection of 0.14 mg/kg vardenafil (Vard) on the day before the bleomycin dose and every day thereafter until the day before sampling. Values are means ± SEM of 5 animals per group from a representative experiment selected from a series of 3 experiments with similar results. *: <i>P</i><0.05; **: <i>P</i><0.01; *** <i>P</i><0.001 for comparison of mean values.</p