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WO2015019381A1 - Procédé de diagnostic in vitro de fibrose pulmonaire idiopathique - Google Patents

Procédé de diagnostic in vitro de fibrose pulmonaire idiopathique Download PDF

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WO2015019381A1
WO2015019381A1 PCT/IT2014/000214 IT2014000214W WO2015019381A1 WO 2015019381 A1 WO2015019381 A1 WO 2015019381A1 IT 2014000214 W IT2014000214 W IT 2014000214W WO 2015019381 A1 WO2015019381 A1 WO 2015019381A1
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pulmonary fibrosis
iron
pulmonary
interstitial pneumonia
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Cesare Saltini
Francesca Mariani
Emanno PUXEDDU
Massimo Amicosante
Gabriella PEZZUTO
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Definitions

  • the present invention regards a method for the in vitro diagnosis of idiopathic pulmonary fibrosis. More precisely, the invention regards a method for the in vitro diagnosis of a form of pulmonary fibrosis characterized by altered regulation of intracellular iron and by the consequential exaggerated generation of chelatable iron-dependent oxygen radicals. This is characterized in vitro by the presence of allelic variants of HFE, the gene for Type 1 hereditary hemochromatosis, by the abnormal expression in alveolar macrophages of SLC11A1/NRAMP1 gene transcripts, non coding for the full length protein, and by the quantity of intracellular free (chelatable) iron in pulmonary alveolar macrophages as compared to healthy subjects.
  • pulmonary fibrosis is included in the group of idiopathic interstitial pneumonias, preferably Idiopathic pulmonary fibrosis "IPF", not excluding nonspecific interstitial pneumonia or "NSIP”, in its cellular (Cellular NSIP) or fibrosing (Fibrosing NSIP) forms, other fibrosing idiopathic interstitial pneumonias, even when associated with other diseases, in particular systemic illnesses.
  • the idiopathic interstitial pneumonias also include cryptogenic organizing pneumonia or "COP”, acute interstitial pneumonia or "AIP”, respiratory bronchiolitis-associated interstitial lung disease or "RB-ILD”, desquamative interstitial pneumonia or "DIP” and lymphocytic interstitial pneumonia or "LIP”.
  • IPF Idiopathic pulmonary fibrosis
  • UPF/UIP histopathologically characterized by the usual interstitial pneumonia (UIP) pattern
  • IPF/UIP differs from the other idiopathic interstitial pneumonias by its rapid progression, with an average survival of 2-4 years, in contrast to the higher mean survival of ten years in patients with NSIP or DIP.
  • Idiopathic pulmonary fibrosis is characterized by cell and tissue damage and interstitial inflammation and fibrosis affecting the pulmonary parenchyma: the pulmonary alveoli, the alveolar interstitium (the space between the pulmonary alveolar epithelium and the capillary endothelium), the peri-bronchial and the peri-vascular interstitium, leading to anatomic and functional damage.
  • the diagnosis, in the absence of a specific causal factor and specific biomarkers, is made on the basis of clinical criteria which include: the history, the physical exam, chest radiology, and, when the chest radiological findings are not sufficiently specific, the histopathology of lung tissue.
  • clinical criteria include: the history, the physical exam, chest radiology, and, when the chest radiological findings are not sufficiently specific, the histopathology of lung tissue.
  • IPF generally presents as a sporadic illness, but there are reported cases of familial aggregations which suggests an important component of genetic susceptibility.
  • Certain genes have been associated with susecpitibility in the familial form of pulmonary fibrosis: in particular the genes that code for surfactant protein C (SFTPC), surfactant protein A (SFTPA2) or telomerase reverse transcriptase (TERT).
  • SFTPC surfactant protein C
  • SFTPA2 surfactant protein A
  • TERT telomerase reverse transcriptase
  • iron plays an important role. Macrophagaes (i) phagocytize old and damaged red blood cells, thus recovering heme iron, (ii) return iron to the circulation, after binding it to transferrin, rendering it available for hemopoiesis and numerous vital cell processes, (iii) sequester iron in its inactive form in siderosomes, subcellular structures containing iron bound to ferritin, which reduces its toxic potential or (iv) use the iron for the production of oxygen radicals in order to destroy microorganisms.
  • the phagocytosis of mircroorganisms determines in the alveolar macrophages the activation of the production of superoxide anion (0 2 ' ) [Saltini C, Brugni N., Magnani C, Capitolo S., Capelli O., Velluti G., Bisetti A. Chemiluminescence measurement of phagocytic activity of pulmonary macrophages in sarcoid alveolitis. Respiration 1984; 45: 291-295], a reactive compound of oxygen that can generate hydrogen peroxide (H2O2) by disproportionation on the part of the superoxide dismutase enzyme (SOD).
  • H2O2O2 hydrogen peroxide
  • Hydrogen peroxide can activate the production of the hydroxyl radical ( ⁇ ), which is generated in the reaction between O 2 “ and H 2 O 2 , the Haber Weiss reaction, which produces ⁇ + OH " + O2, or in the Fenton reaction which, in the presence of free iron, leads to the reduction of ferric iron (Fe 3+ ) to ferrous iron (Fe 2+ ) by O 2 " , with the successive reduction of H 2 O 2 to ⁇ + OH " on the part of Fe 2+ interaction with H2O2 and the consequent regeneration of Fe 3+ .
  • ⁇ hydroxyl radical
  • the Haber Weiss reaction which produces ⁇ + OH " + O2
  • Fenton reaction which, in the presence of free iron, leads to the reduction of ferric iron (Fe 3+ ) to ferrous iron (Fe 2+ ) by O 2 " , with the successive reduction of H 2 O 2 to ⁇ + OH " on the part of Fe 2+ interaction with H2O2 and the consequent regeneration of Fe 3+ .
  • Hereditary hemochromatosis is a disease characterized by exaggerated absorption of alimentary iron and the consequent appearance of tissue damage, including hepatic cirrhosis, cardiomyopathy, diabetes mellitus, hypogonadism andpigmentation of the skin, without involving the respiratory system.
  • the disease is caused by allelic variants of four genes which code for proteins that regulate iron homeostasis and the level of iron absorption.
  • the HFE gene down-regulates transferrin receptor affinity for transferrin-bound iron, thus preventing the transport of iron, for example from plasma, into the cell, in order to maintain a stable level of intracellular iron.
  • the pathogenetic mutations reduce the capacity of the HFE protein to regulate the activity of the transferrin receptor thereby leading to an increase in intracellular iron.
  • HH also called non- HFE HH
  • HAMP hepcidin
  • TFR2 transferrin receptor 2 gene
  • SLC40A1 ferroportin
  • Macrophages and in particular the alveolar macrophages, are important in the homeostasis of pulmonary iron both through the increase in expression of the gene that codes for Ferritin H, the principal protein of the accumulation of intracellular iron, and through the reduction in the expression of the Ferroportin gene, coding for the protein that transports the iron outside of the cell, thus trapping the iron within the macrophage.
  • SLC11A1 solute carrier family 11 (proton-coupled divalent metal ion transporters), member 1] previously named NRAMP1 [Natural resistance associated macrophage protein 1] which codes for a transporter of cations capable of transporting ferrous iron from phagolysosomes to the cytoplasm, depriving the phagocytized microbes of iron and favoring the acidification of the lysosome by the activation of the lysosomal lytic enzymes and vice versa, transporting ferrous iron into the phagolysosome hence activating the production of intra phagolysosomal hydroxyl radicals.
  • Alterations in the function of the SLC11A1/NRAMP1 gene are associated with the susceptibility to infection by intracellular microorganisms in experimental animals.
  • polymorphisms in the sequence of the SLC11A1/NRAMP1 gene have been associated with the susceptibility to pulmonary tuberculosis (Jabado N, Jankowski A, Dougaparsad S, Picard V, Grinstein S, Gros P.
  • idiopathic interstitial pneumonia identified as idiopathic pulmonary fibrosis IPF/UIP
  • IPF/UIP idiopathic pulmonary fibrosis
  • HAMP Hepcidin
  • TFR2 Transferrina 2 receptor
  • SLC40A1 Ferroportin
  • idiopathic interstitial pneumonia identified as idiopathic pulmonary fibrosis IPF/UIP
  • the dysregulation of the SLC1 1A1/NRAMP1 gene characterized by: (i) the missing expression of the isoform of messenger RNA capable of being translated into a protein of 550 amino acids (aa) in length (SLC1 1A1 -001 ) in the absence of expression of other transcriptis of the gene SLC1 1A1/NRAMP1 ; or (ii) the expression of some of the diverse truncated isoforms of mRNA, produced by alternate splicing of the pre- mRNA in particular SLC11A1_201 and SLC11A1_003, in the absence of the SLC1 1A1 -001 isoform coding for the protein of 550 aa in length; or (iii) the expression of a coding isoform for a truncated protein of 483 amino acids (GeneBank: D3817
  • the SLC1 1A1/NRAMP1 gene expression abnormalities do not derive from a generalized disorder of the expression of mRNA of regulatory genes for cellular functions in macrophages, including homeostasis of iron and of oxidative stress, in the sense that the pulmonary alveolar phagocytes in patients with idiopathic pulmonary fibrosis express normal transcription levels for a number of genes coding for structural, iron-binding and anti-oxidant proteins.
  • IPF/UIP idiopathic interstitial pneumonia
  • ferritin labile iron or lalbile iron pool (LIP)
  • ⁇ hydroxyl radical
  • H2O2 hydrogen peroxide
  • allelic variants of the HFE gene is associated with, and represents, a factor of risk for the eccessive iron dependent generation of oxygen radicals, in particular hydroxyl radical, by pulmonary macrophages in these patients.
  • allelic variants of the HFE gene in patients with pulmonary interstitial disease different from IPF/UIP is also associated with a more elevated iron dependent production of oxygen radicals.
  • This condition in the context of the observations that non fibrotic interstitial pneumonias, at the onset of the illness, may evolve into usual interstitial pneumonia, thus appears not only to be a diagnostic indicator of IPF/UIP, but also an indicator of progression towards pulmonary fibrosis in patients who present with a radiological picture of "possibile" IPF/UIP or of NSIP.
  • the present invention thus defines for the first time a form of pulmonary fibrosis characterized by the alterated regulation of cellular iron and consequent exaggerated generation of iron chelatable-dependent oxygen radicals, included, preferably but not exclusively, within the category of idiopathic interstitial pneumonias (MPs), which includes in particular idiopathic pulmonary fibrosis "IPF7UIP, and also non specific interstitial pneumonia or "NSIP", in its cellular form (Cellular NSIP) or fibrosing form (Fibrosing NSIP), even when associated with other diseases.
  • MPs idiopathic interstitial pneumonias
  • IPF7UIP idiopathic pulmonary fibrosis
  • NSIP non specific interstitial pneumonia or NSIP
  • This definition is provided on the basis of specific genetic characteristics and on the consequent metabolic alterations plausibily causing the damage, as demonstrated by the altered expression of regulatory genes for the importation of iron into the cells (HFE) and the intralysosomal traffic of iron (SLC11A1/NRAMP1) in the macrophages, and the consequent elevated concentration of labile iron and ensuing elevated iron-dependent generation of oxygen radicals in these cells.
  • the present invention demonstrates for the first time that in the category of fibrosing interstitial pneumonias there is an identifiable pulmonary fibrosis/idiopathic interstitial pneumonia, currently identified, but not exclusively, as idiopathic pulmonary fibrosis IPF/UIP and characterized by the conditions i-iii listed below:
  • the excessive concentration of intracellular free iron can be revealed by measuring the spontaneous generation, non stimulated in culture, of oxygen radicals, in particular the OH radical, and by quantification of the inhibition with iron chelators, which is significantly higher than normal and is proportional to the concentration of free intracellular iron.
  • the above described conditions i-iii may all be present in the subject affected by the form of pulmonary fibrosis according to the present invention or in subjects at risk for developing the disease. Alternatively, only one of the above conditions may by present, either i or ii or iii. In other cases, the subject may present a combination of the above conditions, i.e. i and ii, ii and iii, i and iii.
  • This consists of an in vitro diagnostic assay for the diagnosis of pulmonary fibrosis associated with iron dysregulation in subjects with suspected interstitial pulmonary fibrosis and a clinical presentation compatible with "confident" or “possible” IPF/UIP, NSIP (cellular or fibrosing NSIP), other fibrosing idiopathic interstitial pneumonias and fibrosing interstitial pneumonias associated with other systemic and pulmonary illnesses, without the necessity of performing biopsies, endoscopy or surgery, based on the in vitro demonstration of abnormal regulation of iron homeostasis in pulmonary alveolar macrophages, and in circulating mononuclear phagocytes, associated with the condition of carrier of variant alleles and/or polymorphisms, and abnormal expression of regulatory genes of cellular iron homeostasis.
  • This assay may be carried out on samples of peripheral venous blood and on samples of pulmonary alveolar macrophages obtained ex vivo from bronchoscopy and bronchoalveolar lavage (BAL), with experiments employing moleculare and cellular biology as listed below.
  • the present invention concerns, thus, a method for the in vitro diagnosis of pulmonary fibrosis characterized by a greater quantity of free intracellular iron in pulmonary alveolar macrophages than that in healthy subjects, or for the identification of the risk of developing said pulmonary fibrosis, said pulmonary fibrosis being defined also as idiopathic pulmonary fibrosis, usual interstitial pneumonia, non specific interstitial pneumonia, preferably fibrosing, preferably idiopathic pulmonary fibrosis, said method comprising or consisting of the identification of one or more allelic variants and/or one or more polymorphisms of the HFE gene (OMIM 613609, localized onl chromosom 6p21.3) of DNA from a biological sample.
  • OMIM 613609 localized onl chromosom 6p21.3
  • the particular form of pulmonary fibrosis according to the present invention is characterized by a greater quantity of intracellular free iron in pulmonary alveolar macrophages as compared to a healthy subject.
  • the quantity of intracellular free iron can be determined using known methods. For example, it is possibile to determine said quantity of iron using an indirect method based on the measure of oxygen radical production, preferably hydroxyl radical, produced in a sample of alveolar macrophages with and without iron chelators and a fluorescent indicator of oxygen radicals.
  • the decrease in the quantity of oxygen radicals produced in the presence of iron chelators, as compared to the control sample without iron chelators represents the quantity of chelatable free intracelluar iron.
  • allelic variants are chosen from the group consisting of V53M, V59M, H63D, S65C, Q127H, E168Q, E168X, W169X, C282Y Q283P, P160 DEL C, preferably H63D, C282Y e/o S65C and the polymorphism is IVS2+4 T>C.
  • the method includes the revelation of H63D, C282Y, S65C e IVS2+4 T>C.
  • the method can be carried out on a biological sample chosen from the group consisting of blood, pulmonary alveolar cells or lung tissue.
  • the method uses the following couples of primers respectively for the amplification and, but not exclusively, the sequencing in two directions of the exons 1, 2A, 2B, 3, 4, 5 of the HFE gene.:
  • the present invention concerns, in addition, a method for the in vitro diagnosis of pulmonary fibrosis characterized by a greater quantity of intracellulare free iron in pulmonary alveolar macrophages as compared to that of a healthy subject, or for the identification of risk of developing said pulmonary fibrosis, said pulmonary fibrosis being defined also as idiopathic pulmonary fibrosis, usual interstitial pneumonia, non specific interstitial pneumonia, preferably fibrosing, preferably idiopathic pulmonary fibrosis, said method comprising or consisting of the evaluation of the expression of the SLC11A1/NRAMP1 gene in a biological sample which consists of cells from the alveolar bronchial tract obtained by bronchoscopy and BAL, in particular pulmonary alveolar macrophages, wherein said expression in a subject affected with said pulmonary disease is dysregulated with respect to a healthy subject and characterized by the absence of expression of SLC11A1/NRAMP1 gene mRNA isoform which codes for the mature protein of
  • the above mentioned method may further comprise the evaluation, on cDNA reverse transcribed from mRNA, for the expression of isoforms of cDNA reverse transcribed from mRNA of the SLC11A1/NRAMP1 gene, said isoforms coding for truncated proteins or non coding.
  • the method according to the invention can be carried out using Reverse Transcriptase-PCR, RT-PCR.
  • the following primers can be used for the amplification of cDNA reverse transcribed from mRNA from the SLC11A1/NRAMP1 gene for the identification of said isoforms:
  • the method can use the following couples of primers for the amplification of said isoforms of cDNA reverse transcribed from mRNA from the SLC11A1/NRAMP1 gene:
  • the invention further regards a method for the in vitro diagnosis of pulmonary fibrosis characterized by an increased quantity of intracellular free iron in the pulmonary alveolar macrophages as compared to healthy subjects, for the identification of the risk of developing pulmonary fibrosis, or for monitoring the biological response to the antifibrotic treatment with one or more iron chelators or antioxidant drugs, said pulmonary fibrosis being defined also as idiopathic pulmonary fibrosis, usual interstitial pneumonia, non specific interstitial pneumonia, preferably fibrosing, preferably idiopathic pulmonary fibrosis, said method comprising o consisting of the measure of the quantity of an oxygen radical, preferably the hydroxyl radical, produced by pulmonary alveolar macrophages and/or by the quantity of intracellular free iron not bound to ferritin in the pulmonary alveolar macrophages, wherein said quantity of hydroxyl radical and/or intracellular free iron not bound to ferritin in pulmonary alveolar macrophages in a subject affected with said pulmonary illness
  • the present invention also regards a method for the in vitro diagnosis of pulmonary fibrosis characterized by an increased quantity of intracellular free iron in the pulmonary alveolar macrophages as compared to healthy subjects, or for the identification of the risk of developing pulmonary fibrosis, said pulmonary fibrosis also being defined as idiopathic pulmonary fibrosis, usual interstitial pneumonia, non specific interstitial pneumonia, preferably fibrosing, preferably idiopathic pulmonary fibrosis, said method comprising or consisting of the combination of two or more methods as defined above.
  • kits for the in vitro diagnosis of pulmonary fibrosis characterized by an increased quantity of intracellulare free iron in the pulmonary alveolar macrophages as compared to healthy subjects, or for the identification of the risk of developing said pulmonary fibrosis, said pulmonary fibrosis also being defined as idiopathic pulmonary fibrosis, usual interstitial pneumonia, non specific interstitial pneumonia, preferably fibrosing, preferably idiopathic pulmonary fibrosis, said kit comprising or consisting of the following primers respectively for the amplification and, but not exclusively, the sequencing in two directions of the exons 1 , 2A, 2B, 3, 4, 5 of the HFE gene:
  • the present invention concerns a kit for the in vitro diagnosis of pulmonary fibrosis characterized by an increased quantity of intracellular free iron in the pulmonary alveolar macrophages as compared to healthy subjects, or for the identification of the risk of developing pulmonary fibrosis, said pulmonary fibrosis also being defined as idiopathic pulmonary fibrosis, usual interstitial pneumonia, non specific interstitial pneumonia, preferably fibrosing, preferably idiopathic pulmonary fibrosis, said kit comprising or consisting of the following primers for the amplification of cDNA of the SLC1 1A1/NRAMP1 gene coding for the mature protein of 550 amino acids:
  • kits can futher comprise the following primers for the amplification of the SLC1 1A1/NRAMP1 gene isoform of cDNA, said isoforms coding for truncated proteins or non coding, for the identification of the isoforms:
  • the present invention concerns a kit for the in vitro diagnosis of pulmonary fibrosis characterized by an increased quantity of intracellular free iron in the pulmonary alveolar macrophages as compared to healthy subjects, for the identification of the risk of developing pulmonary fibrosis, or for monitoring the biological response to the antifibrotic treatment with one or more iron chelators or antioxidant drugs, said pulmonary fibrosis also being defined as idiopathic pulmonary fibrosis, usual interstitial pneumonia, non specific interstitial pneumonia, preferably fibrosing, preferably idiopathic pulmonary fibrosis, said kit comprising or consisting of at least one iron chelator, at least one fluorescent indicator of intracellular reactive oxygen species.
  • Buffer A NaCL 137 mM, KCI 2.7 mM, Na2HP04 7.8 mM, KH2P04 1.5 mM, pH 7.2
  • Buffer B RPMI without pheol red or glutamine, with NaHC03 Reagent A: deferoxamine (DFO) 10 mM, or deferiprone 5 mM (DFP), in Buffer B REAGENT B: solution of 500 ⁇ M CM-H2DCFDA in dimethylformamide (DMF)
  • Reagent C 2% solution of auto-fluorescent spheres (FluoSpheres Sulfate Microspheres, 1 m yellow-green, Invitrogen, Molecular Probes, Carlsbad, CA, USA)
  • Chelatable Fluorescence Units (Sample A Fluorescence Units - Sample B Fluorescence Units) x (160/ Sample C Fluorescence Units). Pathological values > 31 .2 FU in the assay with deferiprone and >42 in the assay with deferoxamine.
  • the present invention concerns as well one or more iron chelators for the use in the prevention of the progression and in the treatment of pulmonary fibrosis characterized by a quantity of intracellular free iron in pulmonary alveolar macrophages as compared to healthy subjects, said pulmonary fibrosis being defined also as idiopathic pulmonary fibrosis, usual interstitial pneumonia, non specific interstitial pneumonia, preferably fibrosing, preferably idiopathic pulmonary fibrosis, wherein said one or more iron chelators are chosen from the group that consists of deferiprone, deferoxamine, deferasirox, ethylenediaminotetracetic acid (EDTA), dimercaptopropanol (dimercaprol), dimercaptosuccinic acid (DMSA), trientine hydrochloride, curcumin - a polyphenol derived from cucurma (Cucurma longa), baicalein - a flavone glucuronide extracted from Scutellaria - and its
  • Figure 1 Demonstrates the intracellular accumulation of free iron in patients with pulmonary fibrosis/idiopathic pulmonary fibrosis measured with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA), by the use of chelators capable of being accumulated in lysosomes, such as deferoxamine (100 mcM) and of permeating the cell membrane such as deferiprone (50 mcM).
  • the fluorometric assay permits the measurement of free iron by the reduction of the emission of fluorescence due to inihibiton by the chelators.
  • Figure 2A shows the quantity of deferoxamine chelatable fluorescence produced by the iron-dependent generation of oxygen radicals by cells recovered from BAL, not stimulated in vitro, from normal control subjects and patients with idiopathic pulmonary fibrosis, and demonstrates that the patients with idiopathic pulmonary fibrosis produce a greater quantity of free iron-dependent oxygen radicals compared to controls (p ⁇ 0.0001).
  • Figure 2B shows the same values, stratified for the presence of variant alleles of the HFE gene.
  • HFE- empty circles
  • HFE+ the controls and the subjects afflicted with IPF who express variant alleles for HFE are shown as closed circles.
  • HFE- the patients with idiopathic pulmonary fibrosis carrying allelic variants of the HFE genes produce a greater quantity of iron-dependent oxygen radicals as compared to patients homozygous for the wild type alleles (HFE-) of the gene (p ⁇ 0.05), who in turn produce a greater quantity of iron-dependent oxygen radicals as compared to healthy subjects (p ⁇ 0.005).
  • FIG. 3 Shows a schematic representation comparing the functional domains identified in the amino acid sequences of four isoforms of the SLC1 1A1/NRAMP1 protein by Smart Modular Architecture Research Tool (SMART) analysis.
  • SMART Smart Modular Architecture Research Tool
  • This isoform was found in the cell line U937 and the sequence deposited in GeneBank. It is interesting to note in the fourth isoform the presence of a leader sequence, or signal peptide, in the first 36 aa, that may insert the protein into the membrane of the lysosome.
  • FIG. 4 Demonstrates schematically the method for the execution of the Nested RT-PCR.
  • the nested RT-PCR which consists in a first reaction of the cDNA with the primers Pfor5'UTR-Prev (called external or “outer”, which produces a PCR product of 744 base pairs) and a second reaction (called internal or “inner”, which produces a PCR product of 570 base pairs) with the primers PforB-Prev3, using 0.5 microliters of the "outer” reaction.
  • the figure shows a schematic of the reaction of Nested PCR constructed on the messenger RNA of SLC1 1A1/Nramp1.
  • the primers for the external or outer RT-PCR are located in the region 5'UTR (Pfor5'UTR) and on Exon 6 (P rev), respectively.
  • the primers for the internal or inner RT-PCR reaction are located in the region 5'UTR (Pfor B) and on Exon 5 (Prev 3), respectively.
  • Figure 5 Demonstrates the quantity of deferoxamine-chelatable fluorescence produced by the iron-dependent generation of oxygen radicals by the cells obtained from BAL, not stimulated in vitro, in subjects with IPF/UIP.
  • the values of deferoxamine- chelatable fluorescence are stratifiied by the presence of allelic variants of the HFE gene and for the lack of expression of the SLC1 1A1/NRAMP1 gene transcript coding for the "full length" protein of 550 aa.
  • EXAMPLE 1 Study on the prevalence of allelic variants of the HFE gene, on the expression of the SCL11A1/NRAMP1 gene and on the dysregulation of iron in alveolar macrophages in patients with idiopathic pulmonary fibrosis/usual interstitial pneumonia and on the effect of iron chelators used for the diagnosis of pulmonary fibrosis.
  • bronchoalveolar lavage BAL
  • the control samples were obtained from healthy smoking subjects, with more than 10 pack years of smoking history, and from non smoking subjects, enrolled in an interventional study, approved by the Independent Ethics Committee of the University of Tor Vergata Medical Center (Rome, Italy), after informed consent was obtained.
  • the BAL was performed according to standard protocol, by instilling three aliquots of physiological saline solution, throught the operative channel of a fiberoptic bronchoscope, Olympus BF 1T 189, (Olympus, Hamburg, Germany), in the right middle lobe, the lingula and the antero-basal segment of the left lower lobe (Saltini C, Hance A.J., Ferrans V.J., Basset F., Bitterman P.B., Crystal R.G. Accurate quantification of cells recovered by bronchoalveolar lavage. Am. Rev. Respir. Dis. 1984; 130: 650-658.).
  • Bronchoavleolar lavage samples were first filtered through sterile gauze to eliminate the mucus and then transferred to 50 ml tubes. The cells were counted and the cell viability evaluated by Tryptan blue uptake. The differential cell count was carried out by staining cytopreps prepared using cytocentrifugation (Cytospin Shandon, Thermo Scientific, Waltham, MA, USA) with May Grumwald Giemsa Quick Stain (Bio-Optica, Milan, Italy). Sample contamination by epithelial cells was assessed and only samples containing ⁇ 5% epithelial cells were used thereafter.
  • the cell fraction of the BAL was separated from the liquid fraction by centrifugation for 5 minutes, at 170 g, in a refrigerated centrifuge (ALC 4237R, ALC International SRL, Cologno Monzese, Italy), and washed two times in buffered saline solution (PBS, Sigma-Aldrich, St Louis, MO, USA).
  • ALC 4237R ALC International SRL, Cologno Monzese, Italy
  • PBS buffered saline solution
  • ELF epithelial lining fluid
  • the volume of ELF recovered was calculated using the concentration of urea in the BAL liquid recovered (Urea QuantiChrom Assay Kit, BioAssay Systems, Hayward, CA, USA)as a marker of dilution as described by Rennard et al.
  • Fluorometric measure of the production of hydroxy! radical as a measure of free iron in alveolar macrophages Fluorometric measure of the production of hydroxy! radical as a measure of free iron in alveolar macrophages.
  • CM-H2DCFDA 5-(and-6)-chloromethyl-2' ,7' -dichlorodihydrofluorescein diacetate acetyl ester
  • CM-DCFDA non fluorescent deacetylated compound that rapidly generates a fluorescent product (CM-DCF) in the presence of hydroxyl radical ( ⁇ ).
  • CM-DCF fluorescent product
  • the intensity of fluorescence produced is proportional to the quantity of intracellular ROS, and in particular to hydroxyl radical ( ⁇ ).
  • CM-H2DCFDA (Invitrogen, Molecular Probes, Carlsbad, CA, USA) was stored at -20°C in the dark, to prevent photo oxidation, and reconstituted immediately before the experiment (0,5 mM CM-H2DCFDA in dimethylformamide [DMF]).
  • the fluorescence was measured by the fluorometer Luminescence Spectrometer LS50B (Perkin Elmer, Waltham, Massachusetts, USA) with an excitation length of 485 nm and an emission length of 535 nm, using a window of 2.5 nm.
  • ⁇ hydroxyl radical
  • cells were pretreated with deferiprone, (DFP) (Sigma Aldrich, St Louis, MO, USA), deferoxamine (DFO) (Sigma Aldrich, St Louis, MO, USA) or deferasirox (Swapnroop Drugs PVT Ltd Aurangabad-431004, India) at a final concentration of 50 ⁇ M, 100 ⁇ M e 500 ⁇ M, respectively.
  • DFP deferiprone,
  • DFO deferoxamine
  • deferasirox Swapnroop Drugs PVT Ltd Aurangabad-431004, India
  • the assay was conducted in the absence of pH indicators (phenol red) and serum in order to avoid the consequent alteration in the fluorochrome CM-DCF signal.
  • the cells were incubated for 1 hour at 37°C, 5% CO 2 , (NAPCO Model 5410), in the dark.
  • the CM- H2DCFDA was added at a final concentration of 5 ⁇ M.
  • the concentration of free, chelatable intracellular iron was estimated in fluorescent units of CM-H2DCFDA as the amount of fluorescence inhibited by deferoxamine (DFO) in comparison to the DFO untreated control and expressed as CM-H2DCFDA[DFO] FU/10 5 BAL cells.
  • the identification of the variant alleles and mutations was performed on genomic DNA, extracted and purified blood cells, lung cells and tissue samples (purification kit EZ1 DNA Blood 200 ⁇ [Qiagen, GmbH, Germany].
  • the coding regions and the intron/exon junction regions of the HFE gene were amplified in six amplification reactions using six specific copies of primers, followed by the sequence specific hybridization of the hybrid to identify the allelic variants of the HFE gene.
  • This experiemt was conducted using commercial reagents (kit AC066, Nuclear Laser Medicine, Milan, Italy) and by sequencing in both directions using six copies of primers for the identification of allelic variants and for coding and noncoding polymorphisms.
  • the six copies of primers are the following:
  • Reverse sequence 5'- CGTCCCAGGGTTCAAAGCTTTTCT (SEQ ID NO:23) Sequencing was carried out using a sequence kit (Applied Biosystem Big Dye Terminator v3.1 Cycle) for the identification of variant alleles and for coding and noncoding mutations.
  • the variants identified are V53M, V59M, H63D, S65C, Q127H, E168Q, E168X, W169X, C282Y Q283P, P160 DEL C, and the noncoding polymorphism IVS2+4 T>C.
  • allelic variants or mutations of the TFR2 gene was carried out by amplification of the sequences of interest, followed by the sequence-specific hybridization revealing 4 mutations of the TFR2 gene: Y250X, E60X, M172K, AVA Q 594-597 del, using a commercial kit for the diagnosis of hereditary hemochromatosis (Nuclear Laser Medicine, Milan, Italy).
  • allelic variants or mutations of the ferroportin gene (SLC40A1 , FPN) was carried out by amplification of the sequences of interest, followed by sequence-specific hybridization revealing 2 mutations: N144H, V162 del.
  • a commercial kit for the diagnosis of hereditary hemochromatosis was used (Nuclear Laser Medicine, Milan, Italy).
  • Table 2 shows the coding and noncoding isoforms, identified in data banks (GeneBank and GeneCards in addition to Ensembi Database).
  • the SPV_001 of the mRNA SLC11A1/Nramp1 gives rise to the complete protein of 550 aa
  • the SPV_201 (432 aa) gives rise to a truncated protein from a deletion at the N-terminal
  • the SPV_003 (163 aa) gives rise to a truncated protein at the C-terminal extremity that, supposedly, becomes degraded (nonsense-mediated decay) (see Figure 3).
  • the demonstration of the alterations of the expression of the SLC11A1/NRAMP1 gene is obtained by the identification of the isoforms of messenger RNA using amplification of cDNA with a panel of five couples of primers, described in Tables 3 and 4, that specifically identify the presence/absence of coding and non coding isoforms (see Table 5).
  • RNA/DNA/Protein Purification Kit kit RNA/DNA/Protein Purification Kit
  • Two micrograms of total RNA were reverse transcribed with Superscript III (Invitrogen, Milano, Italy) in a final volume of 50 microliters, at 50°C for 1 hour.
  • Superscript III Invitrogen, Milano, Italy
  • 2,5 microliters of cDNA were used, corresponding to 100 nanograms of total RNA.
  • Each sample of cDNA also underwent amplification with primers specific for the"housekeeping" gene, L34 ribosomal, in duplicate.
  • the Amplification Program used with the ABIPRISM SDS 7000 Thermal Cycler was the following : 95°C 45 secondi, 60°C 1 minute, 15 seconds, 40 cycles, with a Dissociation Curve at the end.
  • cDNA reverse transcribed from total mRNA obtained from alveolar macrophages removed from the lower respiratory tract of control subjects and from patients, was amplified using five couples of primer (primers NO:3, NO:4, NO:9, NO: 10, NO:11 , Table 3).
  • Usando i primer descritti nella tabella 3 sono pertanto state eseguite amplificazioni del cDNA retro-trascritto dello mRNA totale ottenuto da macrofagi alveolari prelevati dal tratto respiratorio inferiore dei soggetti di controllo e dei pazienti usando cinque coppie di primer ( descritti nella tabella 3 con le ID di sequenza: SEQ ID 3, 4, 9, 10, 11).
  • Table 5 shows, for the combination of primers listed in Table 4, the size of the expected amplification product for each isoform.
  • the electrophoretic bands specifically produced by each couple of primers are shown in Table 5. They allow to identify 15 different isoforms the of SLC11A1/NRAMP1 gene (14 of the 19 described in Ensembi and 1 described in GenBank) in the samples examined. The isoforms not identifiable with these primer combinations are shown in bold print (Table 5).
  • the Nested RT-PCR protocol consisted of an initial reaction of cDNA amplification using the Pfor5'UTR-Prev primers (defined as “outer”) and a second reaction (defined as “inner”) with the PforB- Prev3 primers, using 0.5 microliters from the outer reaction product (see Figure 3).
  • allelic variants and mutations associated with hereditary emochromatosis was conducted on a population of 89 patients with confident IPF/UIP as compared to 107 healthy controls and 54 patients with other pathology.
  • the frequency of the allelic variant of the HFE gene found in the patients with idiopathic pulmonary fibrosis was higher than that observed in normal subjects.
  • Table 6 shows the results of the analysis of the frequency of the variant alleles of the gene in the population of patients with IPF/UIP. None of the patients was a carrier for the mutation of the TFR2 gene or the SLC40A1 gene.
  • H63D H63D 0 0,0% 1 1 ,1%
  • HFE gene allelic variants associated with hereditary hemochromatosis, capable of conferring an elevated susceptibility for the accumulation of iron with an excess of free iron and generation of excessive oxygen radicals, in particular hydroxyl radical, capable of inducing cell/tissue damage and fibrosis.
  • oxygen radicals in particular hydroxyl radical
  • CM-H2DCFDA 5-(and-6)- chloromethyl-2' ,7' -dichlorodihydrofluorescein diacetate acetyl ester
  • OH hydroxyl radical
  • the fluorimetry with the fluorescent CM-H2DCFDA probe which reveals the production of hydroxyl radical in the Fenton reaction catalyzed by free iron, represents the indirect measure of cellular free iron.
  • the experiment allows the estimation of the size of the pool of iron inhibitable by chelators to be 14 and 16 fluorescent units, respectively, with deferoxamine and deferiprone.
  • the median spontaneous fluorescence was 35 units (25 ⁇ and 75 ⁇ percentiles: 27-56 units) which was reduced to 21 units of fluorescence (median, 25 ⁇ and 75 ⁇ percentiles: 17.8-31 ) after incubation with the iron chelator deferoxamine and 19 units of fluorescence (25 ⁇ and 75 ⁇ percentiles: 15-30) after incubation with the iron chelator deferiprone (50mcM).
  • the subjects afflicted with IPF/UIP spontaneously produced, without stimulation, a quantity of oxygen radicals, in particular, hydroxyl radical, signiticantly higher than that found in healthy controls [mean fluorescence 138.0 units (25 ⁇ e 75 ⁇ percentiles: 93-21 1 )].
  • the measure of the fluorescence induced by the production of free iron-dependent hydroxyl radical permitted the estimation of the pool of intracellular free iron in alveolar macrophages in patients with IPF/UIP to be 89 and 71 units of fluorescence, respectively with deferoxamine and deferiprone.
  • the chelator deferasirox in the same assay demonstrated an inhibition of 53 + 7 % of the spontaneous production of ( OH).
  • a panel of primers was used, capable of specifically identifying the 7 isoforms of the mRNA of the SLC11A1/NRAMP1 gene coding for protein and the 11 non coding isoforms of the same gene.
  • the table shows the percentage of samples that express the coding isoform for the complete SLC11A1/NRAMP1 protein of 550 amino acids (100% of healthy non smoking and smoking subjects versus 30% of subjects afflicted with IPF/UIP).
  • the table also shows the results of the analysis carried out for the presence of non "wild type” variant genotypes of the HFE gene and for the expression of non "full length” isoforms of the SLC11A1/NRAMP1 gene.
  • This analysis demonstrates that, including the non coding variants of HFE, a mere 10% of the population of patients with idiopathic pulmonary fibrosis normally express the most important genes in the accumulation and intracellular trsnsport of iron, in particular HFE and SLC1 1A1/NRAMP1 , versus 91 % of normal subjects tested.
  • results of 5 samples containing truncated isoforms are consistent with the presence of diverse sequences in the 5'UTR region and with varying relative quantities of each of them, so that they were difficult to amplify with a single reaction of RT-PCR.
  • the nested RT-PCR was able to reveal them, giving rise to PCR products compatibile with the mRNA coding for truncated proteins of 483, 432 and 401 aa (see Tables 2 and 5).
  • the experiments described define a new pathogenetic mechanism for pulmonary fibrosis/idiopathic interstitial fibrosis characterized by the non functioning allelic variants of the hereditary hemochromatosis HFE gene and the absent expression of the isoform of mRNA coding for the mature form of the 550 amino acid protein coded by the SCL11A1/NRAMP1 gene, divalent metal homeostasis regulator gene, in particular iron, selectively expressed by cells of the myeloid line.
  • the experimental data support the present invention demonstrating that, in alveolar macrophages in patients with idiopathic pulmonary fibrosis, the exaggerated production of hydroxyl radicals is mediated by intracellular free iron, is inhibitable by iron chelators and is associated with the expression of allelic variants of the HFE gene associated with hereditary emochromatosis.
  • the new pathogenetic model of disease permits a new diagnostic approach to be designed based on the demonstration, using specific molecular and cell biology assays, of the expression of allelic variants and mutations of the HFE gene for hereditary hemochromatosis, of the dysregulation of the SCL11A1/NRAMP1 gene with expression of splicing isoforms which do not code for the "full length" SCL11A1/NRAMP1 protein, or of the complete lack of expression of the SCL11A1/NRAMP1 gene isoforms of coding messenger RNA and of the exaggerated production of oxygen radicals, in particular OH, associated with the altered expression of the HFE and SLC11A1/NRAMP1 genes.
  • the new pathogenetic model of disease permits new protocols to be designed for the prevention of idiopathic pulmonary fibrosis/usual interstitial pneumonia based on the identification of genetic and functional alterations in individuals with, or at risk for, non fibrotic interstittal diseases.
  • This disease model allows for the rational application of protocols of therapy in humans, already experimented in animals, based on the use of iron chelators, which the data of the present invention demonstrate to be capable of reducing the production of hydroxyl radical to the point of interrupting the chain of pathological events that leads to the activation of fibrogenesis induced by the oxygen radical, OH in particular.

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Abstract

L'invention concerne un procédé pour le diagnostic in vitro d'une forme de fibrose pulmonaire caractérisée par la présence d'allèles ou de polymorphismes variants du gène pour l'hémochromatose héréditaire de type 1 (HFE), par l'expression anormale du transcrit non codant pour la protéine de longueur totale du gène SLC11A1/ NRAMP1 des macrophages alvéolaires pulmonaires, et par la quantité de fer intracellulaire libre dans les macrophages alvéolaires pulmonaires par comparaison à celle d'un sujet sain. De plus, l'invention concerne des chélateurs du fer en vue d'une utilisation dans le traitement de fibrose pulmonaire, caractérisée par une quantité supérieure de fer libre intracellulaire dans les macrophages alvéolaires.
PCT/IT2014/000214 2013-08-08 2014-08-08 Procédé de diagnostic in vitro de fibrose pulmonaire idiopathique Ceased WO2015019381A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017096013A1 (fr) * 2015-12-01 2017-06-08 Cornell University Utilisation de chélateurs du fer mitochondrial pour le traitement de la bronchopneumopathie chronique obstructive
US10905682B2 (en) 2015-12-01 2021-02-02 Cornell University Use of mitochondrial iron chelators for treatment of chronic obstructive pulmonary disease
CN112094861A (zh) * 2020-10-26 2020-12-18 南京林业大学 一种绿藻植物铁蛋白的表达纯化方法及其应用
RU2747653C1 (ru) * 2021-01-11 2021-05-11 Государственное бюджетное учреждение Санкт-Петербургский научно-исследовательский институт скорой помощи им. И.И. Джанелидзе Способ диагностики нарушения обмена железа при тяжелых формах covid-19

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