WO2021063968A1 - Method and composition for diagnosing chronic obstructive pulmonary disease - Google Patents

Abstract

Chronic obstructive pulmonary disease (COPD), a cigarette smoking related disease, is one of the leading causes of death in the world, and represents a significant global healthcare burden. The inventors have previously demonstrated that PGE₂, a lipid mediator derived from the metabolism of phospholipase A2 (PLA2) and cyclooxygenase (COX), participate to the propagation of the senescence in COPD lung fibroblasts. They went further in the investigation of this signaling pathways by focusing on secreted PLA2 (sPLA2) and more particularly on its catalytically active isoforms XIIA and its receptors (PLA2R1 and heparan sulfate proteoglycans). Their results indicate an up-regulation of the sPLA2XIIA and its receptors in COPD fibroblast. Moreover, they found that a single exposure of sPLA2XIIA induces senescence (via heparan sulfate proteoglycans, ERK, p38, STAT3, ROS, AMPK/p53/p21) and that COPD fibroblasts are significantly more susceptible to these effects than controls cells. Taken altogether, these data define sPLA2XIIA as a circulating biomarker of COPD and establish a requirement for sPLA2 XIIA inhibition for the treatment or prevention of COPD.

Description

METHOD AND COMPOSITION FOR DIAGNOSING CHRONIC OBSTRUCTIVE

PULMONARY DISEASE

FIELD OF THE INVENTION:

The invention relates to the field of medicine, and more particularly to the detection and treatment of chronic obstructive pulmonary disease.

BACKGROUND OF THE INVENTION:

Chronic obstructive pulmonary disease (COPD), a cigarette smoking related disease, is one of the leading causes of death in the world, and represents a significant global healthcare burden (1). This pathologic state is characterized by airways remodeling and destruction of the alveolar wall, a condition called emphysema. Both processes lead to an irreversible airflow obstruction, which is the hallmark of the disease (2). However, the detailed pathogenesis of COPD remains uncertain.

Several studies show an increased cellular senescence in lung cells of COPD patients, including fibroblasts (3-6). Cell senescence is a state of irreversible growth arrest which can stem from shortening of telomeres during continuous cell replication (replicative senescence) or be triggered by other stressors, such as oxidative stress inducers (hydrogen peroxide, cigarette smoke, excessive ROS produced by a mitochondrial dysfunction) or inflammatory mediators (premature senescence) (7). In response to this signals, two main pathways: p53-p21 and pi 6-retinoblastoma protein (pRb), respectively were activated (8). In addition, senescent cells release several inflammatory mediators such as cytokines, chemokines, growth factors and MMPs referred as senescence-associated secretome or SASP (9). The SASP can have potent effects on neighboring cells and surrounding tissue and can act in both paracrine and autocrine manner. Indeed, it has been shown that vascular endothelial growth factor (VEGF), monocyte chemoattractant protein 1 (MCP-1) and CCL20 can induce senescence in heathy cells (Acosta et ah, 2013). Moreover, the SASP can reinforce both stress-induced, replicative and oncogene- induced senescence (OIS) growth arrest via IL-6 and IL-8 signaling, in a self-amplifying secretory network (10,11). Moreover, the clearance of senescent cells after a treatment with senolytics leads to an improvement of age-related diseases, suggesting that SASP suppression could explain the beneficial effects of senolysis. In the lung, cell senescence leads to a decreased regenerative capacity and an increased SASP by structural cells (epithelium, fibroblasts) (3,5,6). Thus, the SASP secreted by these cells could play a major role in lung inflammation and remodeling in COPD. However, the molecular mechanisms of senescence in COPD, and specifically how senescence is maintained and propagated from cell to cell, and how this propagation is related to inflammation remain unknown.

In a last study, the inventors demonstrated that PGE2_, a lipid mediator derived from the metabolism of phospholipids by phospholipase A2 (PLA2) and cyclooxygenase (COX), participate to the propagation of the senescence in COPD lung fibroblasts (6). They went further in the investigation of this signaling pathway by focusing on secreted PLA2 (sPLA2). This family contains 10 catalytically active isoforms (IB, IIA, IIC, IID, HE, IIF, III, V, X, and XIIA) and one inactive isoform (XIIB) in mammals (12). sPLA2s are Ca2+ -dependent low - molecular weight (14 - 18 kDa) enzymes that are released in plasma and biological fluids of patients with various inflammatory diseases. Their biological effects depends on the binding of sPLA2s to two different receptors: soluble and membrane-bound M-type sPLA2 receptor (PLA2R1) which drives or counter regulates the biological effects induced by sPLA2 and heparan sulfate proteoglycans such as syndecans and glypicans (13,14). Only two studies have showed a link between sPLA2 and senescence. Indeed, the sPLA2 (IB and IIA) and the over expression of PLA2R1 have induced senescence in dermal fibroblasts and in cancer cells respectively, suggesting that such an effect could be observed in COPD fibroblasts (15,16). However, if several studies have permitted to clarify the role of sPLA2 in the lung, the one of sPLA2 XIIA remains to elucidate. Therefore, the aim of the study was to investigate for the first time the potential role of sPLA2 XIIA in the senescence of lung COPD fibroblasts.

SUMMARY OF THE INVENTION:

In a first aspect, the invention relates to an in vitro method for diagnosing chronic obstructive pulmonary disease (COPD) in a subject, comprising the steps of (i) determining the expression level of the secreted PLA2XIIA in a sample obtained from said patient, and (ii) comparing the expression level determined at step i) with its predetermined reference value, and iii) concluding that the subject suffers from COPD when the expression level of the secreted sPLA2XIIA is higher than its predetermined reference value.

In a second aspect, the invention relates to an in vitro method for diagnosing chronic obstructive pulmonary disease (COPD) in a subject, comprising the steps of (i) determining the expression level of the receptors of sPLA2XIIA in a sample obtained from said patient, and (ii) comparing the expression level determined at step i) with its predetermined reference value, and iii) concluding that the subject suffers from COPD when the expression level of the receptors of sPLA2XIIA is higher than its predetermined reference value. In a third aspect, the invention relates to a method for preventing or treating COPD, comprising the steps of: (i) performing the method for diagnosing COPD of the invention, and (ii) administering to said patient a therapeutically effective amount of a sPLA2XIIA inhibitors.

DETAILED DESCRIPTION OF THE INVENTION:

Inventors previously demonstrated that PGE2_, a lipid mediator derived from the metabolism of phospholipase A2 (PLA2) and cyclooxygenase (COX), participate to the propagation of the senescence in COPD lung fibroblasts (6). They went further in the investigation of this signaling pathways by focusing on secreted PLA2 (sPLA2) and more particularly on its catalytically active isoforms XIIA and its receptors (PLA2R1 and heparan sulfate proteoglycans). Their results indicate an up-regulation of the sPLA2XQA and its receptors in COPD fibroblast. Moreover, they found that a single exposure of sPLA2XIIA induces senescence (via heparan sulfate proteoglycans, ERK, p38, STAT3, ROS, AMPK/p53/p21) and that COPD fibroblasts are significantly more susceptible to these effects than controls cells. Taken altogether, these data define sPLA2XIIA as a circulating biomarker of COPD and establish a requirement for sPLA2 XIIA inhibition for the treatment or prevention of COPD.

Accordingly, in a fist aspect, invention relates to an in vitro method for diagnosing chronic obstructive pulmonary disease (COPD) in a subject, comprising the steps of (i) determining the expression level of the secreted PLA2 XIIA (sPLA2 XIIA) in a biological sample obtained from said patient, and (ii) comparing the expression level determined at step i) with its predetermined reference value, and iii) concluding that the subject suffers from COPD when the expression level of the secreted sPLA2 XIIA is higher than its predetermined reference value.

In another aspect, the invention relates to an in vitro method for diagnosing chronic obstructive pulmonary disease (COPD) in a subject, comprising the steps of (i) determining the expression level of at least one sPLA2 XIIA receptor selected among PLA2R1, syndecan and glypican in a biological sample obtained from said subject, (ii) comparing the expression level determined at step i) with its predetermined reference value, and iii) concluding that the subject suffers from COPD when the expression level of the sPLA2 XIIA receptors is higher than its predetermined reference value.

In one embodiment, the sPLA2 XIIA receptor whose expression level is determined in step i) is PLA2R1. In one embodiment, the sPLA2 XIIA receptor whose expression level is determined in step i) is syndecan.

In one embodiment, the sPLA2 XIIA receptor whose expression level is determined in step i) is glypican.

In one embodiment, the sPLA2 XIIA receptors whose expression level are determined in step i) are PLA2R1 and syndecan.

In one embodiment, the sPLA2 XIIA receptors whose expression level are determined in step i) are PLA2R1 and glypican.

In one embodiment, the sPLA2 XIIA receptors whose expression level are determined in step i) are PLA2R1, glypican and syndecan.

In some embodiment, the sPLA2 XIIA receptors expression can be determined with sPLA2 XIIA expression to diagnose COPD.

Thus, in other word, the invention relates to an in vitro method for diagnosing chronic obstructive pulmonary disease (COPD) in a subject, comprising the steps of: (i) determining the expression level of sPLA2 XIIA and/or at least one sPLA2 XIIA receptors selected among PLA2R1, syndecan and glypican in a biological sample obtained from said subject, (ii) comparing the expression level determined at step i) with its predetermined reference value, and iii) concluding that the subject is at risk of having COPD when the expression level of the sPLA2 XIIA receptors is higher than its predetermined reference value.

In particular embodiment, the glypican whose expression level are determined according to the invention are glypican- 1, glypican-4 and/or glypican-6.

In particular embodiment, the syndecan whose expression level are determined in step i) are syndecan- 1 and/or syndecan-4.

In particular embodiment, the syndecan whose expression level are determined in step i) is syndecan-4.

As used herein, the term “diagnosing” refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.

As used herein, the term "chronic obstructive pulmonary disease" or "COPD" has its general meaning in the art and refers to a set of physiological symptoms including chronic cough, expectoration, exertional dyspnea and a significant, progressive reduction in airflow that may or may not be partly reversible. COPD is a disease characterized by a progressive airflow limitation caused by an abnormal inflammatory reaction to the chronic inhalation of particles. The Global Initiative for Chronic Obstructive Lung Disease (GOLD) has classified 4 different stages of COPD : stage I, where the forced expiratory volume in one second (FEV1) > 80 % normal, to stage IV where the FEV1<30% normal.

As used herein, the term “secreted phospholipase A2 group XIIA” or “sPLA2 XII A” refers to one of catalytically active isoforms of secreted phospholipase A2 (sPLA2) which catalyzes the calcium-dependent hydrolysis of the 2-acyl groups in 3-sn phosphoglycerides. The sequence of said protein can be found under the Uniprot accession number Q9BZM1. sPLA2s are released in plasma and biological fluids of patients with various inflammatory diseases. Indeed, sPLA2 has been shown to promote inflammation in mammals by catalyzing the first step of the arachidonic acid pathway by breaking down phospholipids, resulting in the formation of fatty acids including arachidonic acid. sPLA2s act through specific receptors: soluble and membrane-bound M-type sPLA2 receptor (PLA2R1) and heparan sulfate proteoglycans such as syndecans and glypicans. Inventors found also an up-regulation of these specific receptors in COPD versus control fibroblasts.

As used herein, the term “PLA2R1” has its general meaning in the art and refers to the secretory phospholipase A2 receptor. The sequence of said protein can be found under the Uniprot accession number QUO 18.

As used herein, the term “syndecan” has its general meaning in the art and refers to a single transmembrane domain proteins acting as coreceptors for G protein-coupled receptors. The syndecan protein family has four members: syndecan- 1, -2, -3 and -4 whose the amino acid length is 310, 201, 346 and 198 respectively. The subclassification of the family depending on the existence of GAG binding sites either at both ends of the ectodomain (syndecan- 1 and -3) or at the distal part only (syndecan-2 and -4) and a relatively long Thr-Ser-Pro-rich area in the middle of syndecan- 3’s ectodomain. The sequence of protein syndecan- 1, -2, -3, and -4 can be found under the Uniprot accession number P18827, P34741, 075056 and P31431, respectively.

As used herein, the term “glypican” has its general meaning in the art and refers to heparan sulfate proteoglycans. Glypicans are critically involved in developmental morphogenesis, and have been implicated as regulators in several cell signaling pathways. These include the Wnt and Hedgehog signaling pathways, as well as signaling of fibroblast growth factors and bone morphogenic proteins. The regulating processes performed by glypicans can either stimulate or inhibit specific cellular processes. The glypican protein family has six members: glypican- 1, -2, -3 -4, -5 and -6 whose the protein sequence can be found under the Uniprot accession number P35052, Q8N158, P51654, 075487, P78333 and Q9Y625 respectively. As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human. In a particular embodiment, the subject is a human who is susceptible to have COPD.

As used herein the term "biological sample" in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, bone marrow aspirate, urine, saliva, broncho-alveolar lavage (LBA) or any other bodily secretion or derivative thereof. As used herein "blood" includes whole blood, plasma, serum, circulating cells, constituents, or any derivative of blood. In a particular embodiment, the biological sample is a serum sample or a lung tissue sample.

As used herein, a “reference value” can be a “threshold value” or a “cut-off value”. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the sPLA2 XIIA, PLA2R1, syndecan or glypican expression level (obtained according to the method of the invention) with a defined threshold value.

In one embodiment of the present invention, the threshold value may also be derived from sPLA2 XIIA, PLA2R1, syndecan or glypican expression level (or ratio, or score) determined in a sample derived from one or more COPD patients. Furthermore, retrospective measurement of the sPLA2 XIIA, PLA2R1, syndecan or glypican level (or ratio, or scores) in properly banked historical patients samples may be used in establishing these threshold values. Reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of sSPLA2 XIIA, PLA2R1, syndecan or glypican expression level in samples previously collected from the patient under testing.

An additional object of the invention relates to an in vitro method for determining whether is at risk of having COPD in a subject, comprising the steps of (i) determining the expression level of sPLA2 XIIA and/or at least one sPLA2XIIA receptors selected among PLA2R1, syndecan and glypican in a biological sample obtained from said subject, (ii) comparing the expression levels determined at step (i) with their predetermined reference value, and iii) concluding that the subject is at risk of having COPD when the expression levels determined at step (i) is higher than their predetermined reference value.

As used herein, the term "risk" relates to the probability that an event will occur over a specific time period, as in the conversion to COPD, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no conversion.

An another object of the invention relates to an in vitro method for predicting the survival time of a subject suffering from COPD comprising the steps of (i) determining the expression level of sPLA2 XIIA and/or at least one sPLA2XIIA receptors selected among PLA2R1, syndecan and glypican in a biological sample obtained from said subject, (ii) comparing the expression levels determined at step (i) with their predetermined reference value, and iii) providing a good prognosis when the expression level determined at step (i) is lower than their predetermined reference value, or providing a bad prognosis when the expression level determined at step (i) is higher.

In another embodiment, methods according to the invention may be useful for predicting the overall survival (OS) of a patient suffering from COPD or for predicting the free survival (FS) of a patient suffering from COPD.

In a particular embodiment, the invention relates to an in vitro method for predicting the overall survival (OS) of a patient suffering from COPD comprising the steps of (i) determining the expression level of sPLA2 XIIA and/or at least one sPLA2XIIA receptors selected among PLA2R1, syndecan and glypican in a biological sample obtained from said subject, (ii) comparing the expression levels determined at step (i) with their predetermined reference value, and iii) providing a good prognosis when the expression level determined at step (i) is lower than their predetermined reference value, or providing a bad prognosis when the expression level determined at step (i) is higher than their predetermined reference value.

In a particular embodiment, the invention relates to an in vitro method for predicting the free survival (FS) of a patient suffering from COPD comprising the steps of (i) determining the expression level of sPLA2 XIIA and/or at least one sPLA2XIIA receptors selected among PLA2R1, syndecan and glypican in a biological sample obtained from said subject, (ii) comparing the expression levels determined at step (i) with their predetermined reference value, and iii) providing a good prognosis when the expression level determined at step (i) is lower than their predetermined reference value, or providing a bad prognosis when the expression level determined at step (i) is higher than their predetermined reference value.

As used herein, the term “Overall survival (OS)” denotes the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for a disease, such as AML (according to the invention). The overall survival rate is often stated as a five-year survival rate, which is the percentage of people in a study or treatment group who are alive five years after their diagnosis or the start of treatment.

As used herein, the term “Free Survival (FS)” (or Event-Free- Survival) denotes the length of time after primary treatment for a cancer ends that the patient remains free of certain complications or events that the treatment was intended to prevent or delay. These events may include the return of the cancer or the onset of certain symptoms, such as bone pain from cancer that has spread to the bone.

According to the invention, the expression level of the markers (sPLA2 XIIA and its receptors (PLA2R1, syndecan, glypican) listed above may also be measured by measuring the protein or mRNA expression level can be performed by a variety of techniques well known in the art.

Typically protein expression level may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS), flow cytometry, mass cytometry or ELISA performed on the sample.

In the present application, the “level of protein” or the “protein level expression” means the quantity or concentration of said protein. In particular embodiment, the protein is expressed at the cell surface for markers whose function is linked to their correct plasma membrane expression or total expression for markers whose function is not limited to membrane expression. In still another embodiment, the “level of protein” means the quantitative measurement of the proteins expression relative to a negative control. Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.

The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresis- mass spectroscopy technique (CE-MS).DOT BLOT etc. The reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein (sPLA2 XIIA, PLA2R1, syndecan or glypican) is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.

Particularly, a mass spectrometry-based quantification methods may be used. Mass spectrometry-based quantification methods may be performed using either labelled or unlabelled approaches [DeSouza and Siu, 2012] Mass spectrometry-based quantification methods may be performed using chemical labeling, metabolic labeling or proteolytic labeling. Mass spectrometry-based quantification methods may be performed using mass spectrometry label free quantification, a quantification based on extracted ion chromatogram (EIC) and then profile alignment to determine differential level of polypeptides. Particularly, a mass spectrometry-based quantification method particularly useful can be the use of targeted mass spectrometry methods as selected reaction monitoring (SRM), multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), data independent acquisition (DIA) and sequential window acquisition of all theoretical mass spectra (SWATH) [Moving target Zeliadt N 2014 The Scientist;Liebler Zimmerman Biochemistry 2013 targeted quantitation pf proteins by mass spectrometry; Gallien Domon 2015 Detection and quantification of proteins in clinical samples using high resolution mass spectrometry. Methods v81 pl5-23; Sajic, Liu, Aebersold, 2015 Using data-independent, high-resolution mass spectrometry in protein biomarker research: perspectives and clinical applications. Proteomics Clin Appl v9 p 307-21]

Particularly, the mass spectrometry-based quantification method can be the mass cytometry also known as cytometry by time of flight (CYTOF) (Bandura DR, Analytical chemistry, 2009).

Particularly, the mass spectrometry-based quantification is used to do peptide and/or protein profiling can be use with matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF), surface-enhanced laser desorption/ionization time of flight (SELDI-TOF; CLINPROT) and MALDI Biotyper apparatus [Solassol, Jacot, Lhermitte, Boulle, Maudelonde, Mange 2006 Clinical proteomics and mass spectrometry profiling for cancer detection. Journal: Expert Review of Proteomics V3, 13, p311-320 ; FDA K130831]

Methods of the invention may comprise a step consisting of comparing the proteins and fragments concentration in circulating cells with a control value. As used herein, "concentration of protein" refers to an amount or a concentration of a transcription product, for instance the proteins of the invention. Typically, a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a concentration. In a particular embodiment, "concentration of proteins" may refer to fragments of the proteins of the invention.

According to the invention, measuring the expression level of the mRNA selected from the group consisting of sPLA2 XIIA PLA2R1, syndecan and glypican in a sample obtained from the subject can be performed by a variety of techniques. For example the nucleic acid contained in the samples (peripheral-blood prepared from the subject) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. Conventional methods and reagents for isolating mRNA from a sample comprise High Pure miRNA Isolation Kit (Roche), Trizol (Invitrogen), Guanidinium thiocyanate-phenol- chloroform extraction, PureLink™ miRNA isolation kit (Invitrogen), PureLink Micro-to- Midi Total RNA Purification System (invitrogen), RNeasy kit (Qiagen), miRNeasy kit (Qiagen), Oligotex kit (Qiagen), phenol extraction, phenol-chloroform extraction, TCA/acetone precipitation, ethanol precipitation, Column purification, Silica gel membrane purification, Pure Yield™ RNA Midiprep (Promega), PolyATtract System 1000 (Promega), Maxwell® 16 System (Promega), SV Total RNA Isolation (Promega), geneMAG-RNA / DNA kit (Chemicell), TRI Reagent® (Ambion), RNAqueous Kit (Ambion), ToTALLY RNA™ Kit (Ambion), Poly(A)Purist™ Kit (Ambion) and any other methods, commercially available or not, known to the skilled person. The expression level of one or more mRNA in the sample may be determined by any suitable method. Any reliable method for measuring the level or amount of mRNA in a sample may be used. Generally, mRNA can be detected and quantified from a sample (including fractions thereof), such as samples of isolated RNA by various methods, including, for example, amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.), hybridization-based methods (e.g., hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.), and sequencing-based methods (e.g., next- generation sequencing methods, for example, using the Illumina or IonTorrent platforms). Other exemplary techniques include ribonuclease protection assay (RPA) and mass spectroscopy.

Many amplification-based methods exist for detecting the expression level of mRNA nucleic acid sequences, including, but not limited to, PCR, RT-PCR, qPCR, and rolling circle amplification. Other amplification-based techniques include, for example, ligase chain reaction (LCR), multiplex ligatable probe amplification, in vitro transcription (IVT), strand displacement amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art. A typical PCR reaction includes multiple steps, or cycles, that selectively amplify target nucleic acid species: a denaturing step, in which a target nucleic acid is denatured; an annealing step, in which a set of PCR primers (i.e., forward and reverse primers) anneal to complementary DNA strands, and an elongation step, in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target sequence. Typical PCR reactions include 20 or more cycles of denaturation, annealing, and elongation. In many cases, the annealing and elongation steps can be performed concurrently, in which case the cycle contains only two steps. A reverse transcription reaction (which produces a cDNA sequence having complementarity to a mRNA) may be performed prior to PCR amplification. Reverse transcription reactions include the use of, e.g., a RNA-based DNA polymerase (reverse transcriptase) and a primer. Kits for quantitative real time PCR of mRNA are known, and are commercially available. Examples of suitable kits include, but are not limited to, the High Capacity RNA-to-cDNA™ kit (Applied Biosystems) and the iScript™ Advanced cDNA synthesis kit (Bio-Rad). The mRNA can be ligated to a single stranded oligonucleotide containing universal primer sequences, a polyadenylated sequence, or adaptor sequence prior to reverse transcriptase and amplified using a primer complementary to the universal primer sequence, poly(T) primer, or primer comprising a sequence that is complementary to the adaptor sequence. In some embodiments, custom qRT-PCR assays can be developed for determination of mRNA levels. Custom qRT-PCR assays to measure mRNAs in a sample can be developed using, for example, methods that involve an extended reverse transcription primer and locked nucleic acid modified PCR. Custom mRNA assays can be tested by running the assay on a dilution series of chemically synthesized mRNA corresponding to the target sequence. This permits determination of the limit of detection and linear range of quantitation of each assay. Furthermore, when used as a standard curve, these data permit an estimate of the absolute abundance of mRNAs measured in the samples. Amplification curves may optionally be checked to verify that Ct values are assessed in the linear range of each amplification plot. Typically, the linear range spans several orders of magnitude. For each candidate mRNA assayed, a chemically synthesized version of the mRNA can be obtained and analyzed in a dilution series to determine the limit of sensitivity of the assay, and the linear range of quantitation. Relative expression levels may be determined, for example, according to the 2(- DD C(T)) Method, as described by Livak et ah, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-DD C(T)) Method. Methods (2001) Dec;25(4):402-8.

In some embodiments, two or more mRNAs are amplified in a single reaction volume. For example, multiplex q-PCR, such as qRT-PCR, enables simultaneous amplification and quantification of at least two mRNAs of interest in one reaction volume by using more than one pair of primers and/or more than one probe. The primer pairs comprise at least one amplification primer that specifically binds each mRNA, and the probes are labeled such that they are distinguishable from one another, thus allowing simultaneous quantification of multiple mRNAs.

Rolling circle amplification is a DNA-polymerase driven reaction that can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions (see, for example, Lizardi et al, Nat. Gen. (1998) 19(3):225-232; Gusev et al, Am. J. Pathol. (2001) 159(l):63-69; Nallur et al, Nucleic Acids Res. (2001) 29(23):E118). In the presence of two primers, one hybridizing to the (+) strand of DNA, and the other hybridizing to the (-) strand, a complex pattern of strand displacement results in the generation of over 109 copies of each DNA molecule in 90 minutes or less. Tandemly linked copies of a closed circle DNA molecule may be formed by using a single primer. The process can also be performed using a matrix- associated DNA. The template used for rolling circle amplification may be reverse transcribed. This method can be used as a highly sensitive indicator of mRNA sequence and expression level at very low mRNA concentrations (see, for example, Cheng et al., Angew Chem. Int. Ed. Engl. (2009) 48(18):3268-72; Neubacher et al, Chembiochem. (2009) 10(8): 1289-91). mRNA quantification may be performed by using stem-loop primers for reverse transcription (RT) followed by a real-time TaqMan® probe. Typically, said method comprises a first step wherein the stem-loop primers are annealed to mRNA targets and extended in the presence of reverse transcriptase. Then mRNA-specific forward primer, TaqMan® probe, and reverse primer are used for PCR reactions. Quantitation of mRNAs is estimated based on measured CT values. Many mRNA quantification assays are commercially available from Qiagen (S. A. Courtaboeuf, France), Exiqon (Vedbaek, Denmark) or Applied Biosystems (Foster City, USA).

Nucleic acids exhibiting sequence complementarity or homology to the mRNAs of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin). mRNA may be detected using hybridization-based methods, including but not limited to hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, and in situ hybridization.

Microarrays can be used to measure the expression levels of large numbers of mRNAs simultaneously. Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides, photolithography using pre- made masks, photolithography using dynamic micromirror devices, inkjet printing, or electrochemistry on microelectrode arrays. Also useful are microfluidic TaqMan Low-Density Arrays, which are based on an array of micro fluidic qRT-PCR reactions, as well as related microfluidic qRT-PCR based methods.

Microarrays can be used for the expression profiling of mRNAs. For example, RNA can be extracted from the sample and, optionally, the mRNAs are size- selected from total RNA. Oligonucleotide linkers can be attached to the 5' and 3' ends of the mRNAs and the resulting ligation products are used as templates for an RT-PCR reaction. The sense strand PCR primer can have a fluorophore attached to its 5' end, thereby labeling the sense strand of the PCR product. The PCR product is denatured and then hybridized to the microarray. A PCR product, referred to as the target nucleic acid that is complementary to the corresponding mRNA capture probe sequence on the array will hybridize, via base pairing, to the spot at which the capture probes are affixed. The spot will then fluoresce when excited using a microarray laser scanner. The fluorescence intensity of each spot is then evaluated in terms of the number of copies of a particular mRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular mRNA. Total RNA containing the mRNA extracted from the sample can also be used directly without size-selection of the mRNAs. For example, the RNA can be 3' end labeled using T4 RNA ligase and a fluorophore-labeled short RNA linker. Fluorophore-labeled mRNAs complementary to the corresponding mRNA capture probe sequences on the array hybridize, via base pairing, to the spot at which the capture probes are affixed. The fluorescence intensity of each spot is then evaluated in terms of the number of copies of a particular mRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular mRNA. Several types of microarrays can be employed including, but not limited to, spotted oligonucleotide microarrays, pre-fabricated oligonucleotide microarrays or spotted long oligonucleotide arrays.

In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).

Accordingly, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook- A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et ah, such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene-1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4- methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 - (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino] naphthalene- 1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), Diehl orotriazinylamino fluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2', 7'- difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6- carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the bandgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al, Science 281 :20132016, 1998; Chan et al., Science 281:2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can be produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlsbad, Calif.).

RT-PCR is typically carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of a specified wavelength and detect the fluorescence emitted by the excited fluorophore. The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and thermal polymerase. The majority of the thermocyclers on the market now offer similar characteristics. Typically, thermocyclers involve a format of glass capillaries, plastics tubes, 96-well plates or 384-well plates. The thermocylcer also involves software analysis. mRNAs can also be detected without amplification using the nCounter Analysis System (NanoString Technologies, Seattle, WA). This technology employs two nucleic acid-based probes that hybridize in solution (e.g., a reporter probe and a capture probe). After hybridization, excess probes are removed, and probe/target complexes are analyzed in accordance with the manufacturer's protocol. nCounter RNA assay kits are available from NanoString Technologies, which are capable of distinguishing between highly similar mRNAs with great specificity.

Mass spectroscopy can be used to quantify mRNA using RNase mapping. Isolated RNAs can be enzymatically digested with RNA endonucleases (RNases) having high specificity (e.g., RNase Tl, which cleaves at the 3'-side of all unmodified guanosine residues) prior to their analysis by MS or tandem MS (MS/MS) approaches. The first approach developed utilized the on-line chromatographic separation of endonuclease digests by reversed phase HPLC coupled directly to ESTMS. The presence of post-transcriptional modifications can be revealed by mass shifts from those expected based upon the RNA sequence. Ions of anomalous mass/charge values can then be isolated for tandem MS sequencing to locate the sequence placement of the post-transcriptionally modified nucleoside. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used as an analytical approach for obtaining information about post-transcriptionally modified nucleosides. MALDI- based approaches can be differentiated from EST-based approaches by the separation step. In MALDI-MS, the mass spectrometer is used to separate the mRNA.

Additional methods for mRNA detection and measurement include, for example, strand invasion assay (Third Wave Technologies, Inc.), surface plasmon resonance (SPR), cDNA, MTDNA (metallic DNA; Advance Technologies, Saskatoon, SK), and single-molecule methods such as the one developed by US Genomics. Multiple mRNAs can be detected in a microarray format using a novel approach that combines a surface enzyme reaction with nanoparticle-amplified SPR imaging (SPRI). The surface reaction of poly(A) polymerase creates poly(A) tails on miRNAs hybridized onto locked nucleic acid (LNA) microarrays. DNA-modified nanoparticles are then adsorbed onto the poly(A) tails and detected with SPRI. This ultrasensitive nanoparticle-amplified SPRI methodology can be used for mRNA profiling at attamole levels. mRNAs can also be detected using branched DNA (bDNA) signal amplification (see, for example, Urdea, Nature Biotechnology (1994), 12:926-928). mRNA assays based on bDNA signal amplification are commercially available. One such assay is the QuantiGene® Plex Gene Assay (Affymetrix, Santa Clara, CA). Northern Blot and in situ hybridization may also be used to detect mRNAs. Suitable methods for performing Northern Blot and in situ hybridization are known in the art. Advanced sequencing methods can likewise be used as available. For example, mRNAs can be detected using Illumina ® Next Generation Sequencing (e.g. Sequencing-By-Synthesis or TruSeq methods, using, for example, the HiSeq, HiScan, GenomeAnalyzer, or MiSeq systems (Illumina, Inc., San Diego, CA)). mRNAs can also be detected using Ion Torrent Sequencing (Ion Torrent Systems, Inc., Gulliford, CT), or other suitable methods of semiconductor sequencing.

In another embodiment, the expression level is determined by metabolic imaging (see for example Yamashita T et al., Hepatology 2014, 60:1674-1685 or Ueno A et al., Journal of hepatology 2014, 61 :1080-1087).

Expression level of a gene may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the response of antipsychotic treatment, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gen Q ACTB, ribosomal 18S gene, GUSB, PGK1, TFRC, GAPDH, TBP and A IJ . This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources. In a particular embodiment, expression levels are normalized by correcting the absolute expression level of mRNAs by comparing its expression to the expression of a reference mRNA.

Method for treatins chronic obstructive pulmonary disease (CO PD)

The subject as diagnosed with the method of the invention can be treated with an inhibitor of sPLA2 XIIA and/or with an inhibitor of its receptors (PLA2R1, syndecan and glypican).

Accordingly, in a second aspect, the invention relates to a method for treating COPD in a subject in need thereof comprising a step of administrating to said subject a therapeutically amount of an inhibitor of sPLA2 XIIA and/or an inhibitor of sPLA2XIIA receptor.

Thus, the invention relates to an inhibitor of sPLA2 XIIA and/or an inhibitor of sPLA2XIIA receptor for use in the treatment of COPD in a subject in need thereof.

In some embodiment, the inhibitor of sPLA2 XIIA and the inhibitor of sPLA2XIIA receptor are used as a combined preparation.

In other words, the invention relates to i) an inhibitor of sPLA2 XIIA and ii) an inhibitor of sPLA2XIIA receptor as combined preparation for simultaneous, separate or sequential use in the treatment of COPD in a subject in need thereof.

More particularly, the invention relates to a method of treating COPD comprising the steps of: i) determining whether a subject suffers from COPD according to the method as described above and ii) administrating to said subject a therapeutically amount of inhibitor of sPLA2 XII A and/or an inhibitor of sPLA2XIIA receptor when it is concluded at step i) that the subject suffers from COPD.

Thus, in another word, the invention relates to an inhibitor of sPLA2 XII A and/or an inhibitor of sPLA2XIIA receptor for use in the treatment of COPD comprising the steps of: i) determining whether a subject suffers from COPD according to the method of the invention and ii) administrating to the subject a therapeutically amount of said inhibitor of sPLA2 Xlla when it is concluded at step i) that the subject suffers from COPD.

In some embodiment, the inhibitor of sPLA2XIIA receptor is selected in the group consisting of inhibitor of PLA2R1, inhibitor of syndecan and inhibitor of glypican.

In some embodiment, the inhibitor of sPLA2XIIA receptor is inhibitor of syndecan-4.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

As used herein, the term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the immune checkpoint inhibitor of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the immune checkpoint inhibitor of the present invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the immune checkpoint inhibitor of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of the immune checkpoint inhibitor of the present invention employed in the pharmaceutical composition at levels lower than that required achieving the desired therapeutic effect and gradually increasing the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound, which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically, the ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors. A therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.

As used herein, the term “inhibitor of sPLA2 XIIA” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of sPLA2 XIIA. Such inhibitor inhibits the senescence of COPD cells that overexpress sPLA2 XIIA. Such inhibitor inhibits the senescence of COPD cells induced by sPLA2 XIIA.

In a particular embodiment, the inhibitor of sPLA2 XIIA is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.

In a particular embodiment, the inhibitor of sPLA2 XIIA is a peptide.

In a particular embodiment, the inhibitor of sPLA2 XIIA is a GAG-binding peptide.

As used herein, the term “GAG -binding peptide” refers to a peptide which is able to bind to the GAG-binding site of sPLA2 XIIA,

In a particular embodiment, the inhibitor of sPLA2 XIIA is a heparin-binding peptide

In other words, in a particular embodiment, the inhibitor of sPLA2 XIIA is a peptide which block GAG-sPLA2 XII interaction.

In a particular embodiment, the inhibitor of sPLA2 XIIA is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.In a particular embodiment, the inhibitor of sPLA2 XIIA is a small organic molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

In some embodiment, the inhibitor of sPLA2 XIIA is a glycosaminoglycan (GAG) or a glycosaminoglycan mimetic.

In some embodiment, the inhibitor of sPLA2 XIIA is a heparin or heparin mimetics such as 2,3-O-desulfated heparin (ODSH) and hybrid heparin-like compound isolated from Litopenaeus vannamei shrimp ^20-21 As used herein, the term “glycosaminoglycan” or “GAG” has its general meaning in the art and refers to a class of biomolecules expressed virtually on all mammalian cells and usually covalently attached to proteins, forming proteoglycans.

As used herein, the term “glycosaminoglycan mimetics” has its general meaning in the art and refers to compounds that block GAG-protein interaction. The glycosaminoglycan mimetic for use according to the invention can compete with heparan sulfate proteoglycans, i.e syndecan or glypican and block their interaction with sPLA2 XIIA. Examples of GAG mimetics includes heparin, heparin mimetics such as 2,3-O-desulfated heparin (ODSH) and hybrid heparin-like compound isolated from Litopenaeus vannamei shrimp ^{20 21}; fucoidane, chitine, chitosane modified polysaccharides, sulfated caffeic acid (CDS03), synthetically sulfated oligosaccharides, oligosaccharide-aglycone conjugates, non-carbohydrate-bases sulfated mimetics, muparfostat (PI-88); PG545; EP80061; SST0001 (roneparstat); fondaparinux (arixtra); Indraparinux; Idrabiotapariux; SR123781; AV5026 (semuloparin); M402

(necuparanib); GMI-1070 and regenerating agents (RGTAs) such as OTR4120 and OTR4131.

As used herein the term heparin has its general meaning in the art and refers to glycosaminoglycan of formula C12H19NO20S3 which is used as an anticoagulant (blood thinner). Its CAS Number is 9005-49-6.

In some embodiments, the inhibitor of sPLA2 XIIA is an antibody. As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMTP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Rabat et ak, 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al, 2006; Holliger & Hudson, 2005; Le Gall et al, 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0368 684, WO 06/030220 and WO 06/003388. In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B- cell hybridoma technique and the EBV-hybridoma technique.

In a particular, the inhibitor of sPLA2 XIIA is an intrabody having specificity for sPLA2 XIIA. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

In some embodiments, the inhibitor of sPLA2 XIIA is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of sPLA2 XIIA. In a particular embodiment, the inhibitor of sPLA2 XIIA expression is siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. In some embodiments, the inhibitor of sPLA2 XIIA expression is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 B1 and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffmi, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al, 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al, 2014, PLoS Negl. Trop. Dis., Vol. 8:e267T), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al, 2014 Cell Res. doi: 10.1038/cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e267L), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi:10.1534/genetics.113.160713), monkeys (Niu et al, 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.1 L), rats (Ma et al, 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

As used herein, the term “inhibitor of PLA2R1” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of PLA2R1. Such inhibitor can inhibit the senescence of COPD cells that overexpress sPLA2 XIIA.

In some embodiments, the inhibitor of PLA2R1 is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of PLA2R1. In a particular embodiment, the inhibitor of sPLA2 XIIA expression is shRNA.

As used herein, the term “inhibitor of glypican” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of glypican.

As used herein, the term “inhibitor of syndecan” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of syndecan.

In a particular embodiment, the inhibitor of glypican or syndecan is a synthetic xyloside such as b-D- xyloside; a heparan-sulfate lyase; a sulfotransferase inhibitor, a glycosyltransferase inhibitor; an antibody; an aptamers; siRNA or an antisense oligonucleotide.

In some embodiment, the inhibitor of syndecan is heparin.

In some embodiment, the inhibitor of sPLA2 XIIA and/or the inhibitor of sPLA2XIIA receptor is administered in combination with a classical treatment of COPD.

As used herein, the term “classical treatment” refers to any compound, natural or synthetic, used for the treatment of COPD.

In a particular embodiment, the classical treatment of COPD refers to p2-adrenergic agonists such as epinephrine ephedrine, albuterol, salbutamol, levalbuterol, pirbuterol, terbutaline, salmeterol, fomoterol, arfomoterol, bambuterol and indacaterol; anticholinergics such as ipratropium bromide, tiotropium, and aclidinoum; steroids such as fluticasone, budesonide, beclomethasone HFA, ciclesonide, mometasone; phosphodiesterase-4 inhibitors; theophylline; antibiotics and oxygen therapy. As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.

The medications used in the combined treatment according to the invention are administered to the subject simultaneously, separately or sequentially.

As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different

The subject as diagnosed according to the invention can also be treated with a classical treatment of COPD.

Accordingly, in a third aspect, the invention relates to a method of treating COPD in a subject in need thereof comprising a step of administrating to said subject a classical treatment of COPD.

In a particular embodiment, the invention relates to a method of treating COPD in a subject in need thereof comprising the steps of: i) determining whether a subject suffers from COPD according to the method as described above and ii) administrating to said subject a classical treatment of COPD when it is concluded at step i) that the subject suffers from COPD

A fourth aspect of the invention relates to a therapeutic composition comprising an inhibitor of sPLA2 XIIA according to the invention for use in the treatment of COPD in a subject as diagnosed according to the method as described above.

In another embodiment, the invention relates to a therapeutic composition comprising an inhibitor of sPLA2 XIIA according to the invention for use in the treatment of COPD in a subject with a bad prognosis as described above.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

Kit for yerformins the method according to the invention

In a fifth aspect, the invention relates to a kit for performing the methods of the present invention, wherein said kit comprises means for measuring the expression level of soluble sPLA2 XIIA in a biological sample. More particularly, the kit comprising:

(a) at least one reagent for measuring the expression of sPLA2 XIIA in a biological sample obtained from a subject, wherein when the expression level of soluble sPLA2 XIIA is higher than its predetermined reference value, it is indicative that the subject suffers from COPD;

(b) at least one control; and

(c) instructions for use.

The at least one reagent can be, for example, a sPLA2 XIIA-specific antibody, a pair of sPLA2 XIIA-specific primers, etc. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Up-regulation of sPLA2 XIIA and PLA2R1 in COPD fibroblasts at senescent stages. A. Gene expression level of sPLA2 XIIA in pulmonary fibroblasts derived from patients with chronic obstructive pulmonary disease (COPD, n=16) and non-smoker and smoker controls (NS-C, n=8; S-C, n=16 respectively). sPLA2 XIIA levels in culture media, n=8, 16 and 16 for NS-C, S-C, and COPD respectively. B. Correlation between sPLA2 XIIA at passage 7 and FEV1/VC and FEV1. C-E. Gene expression level of PLA2R1, Syndecan-1 (SDC-1) and Syndecan-4 (SDC-4) in pulmonary fibroblasts derived from patients with chronic obstructive pulmonary disease (COPD, n=19) and non-smoker and smoker controls (NS-C, n=9; S-C, n=16 respectively). * p<0.05 passage 7 vs. passage 3; J p<0.05, COPD vs. controls. £ p< 0.05 COPD vs. S-C. Data are presented as mean ± SEM.

Figure 2: Up-regulation of heparan sulfate proteoglycans in SC-C and COPD fibroblasts at non-senescent and senescent stages. Abbreviations are the same as those described in Figure 1. Gene expression level of glypicans 1, 4. * p< 0.05 passage 7 vs. passage 3; % p< 0.05, COPD vs. controls; # p< 0.05 COPD or S-C versus NS-C. Data are presented as mean ± SEM.

Figure 3: sPLA2 XIIA induces the accelerated senescence of non-senescent fibroblasts. Abbreviations are the same as those described in Figure 1. A. Percentage of S A b- Gal-positive cells. NS-C, S-C and COPD fibroblasts (n=8 15 and 15 respectively) were exposed to different concentrations of sPLA2 XIIA and stained after 24 hours exposure. Immunocytochemical expression and quantification p21 (green fluorescence) after exposure to sPLA2 XIIA. Nuclei were counterstained with DAPI. Scale-bar = 20 pm. Bar graphs represent the percentage of fibroblasts expressing P-p53 or p21 after treatment with sPLA2 XIIA. * p< 0.05 sPLA2 XIIA vs. vehicle (DMSO). J p< 0.05 COPD vs. controls, £ p< 0.05 COPD vs. S- C. B. Bar graph of cells stained for SA b-Gal activity after pre-treatment with either heparanase or antibody against heparan sulfate proteoglycans (anti-HS) or pifithrin a (Pifia, 25 pM), and exposed to sPLA2 XIIA at 2 ng.ml ¹. n=8 for NS-C, 15 for S-C and COPD. *p<0.05 sPLA2 XIIA vs. DMSO; $ p<0.05 sPLA2 XIIA vs. sPLA2 XIIA + PFTa or heparanase or anti-HS ; J p<0.05 COPD vs. controls, £ p<0.05 COPD vs. S-C. C. Bar graph of cells stained for p21 after the same treatments as the ones described in section B. Data are presented as mean ± SEM in the whole.

Figure 4: sPLA2 XIIA activates MAPK pathway. A. Bar graph of cells stained for SA b-Gal activity after pre-treatment with either an inhibitor of ERK (U0160, 10 mM) or inhibitor of p38 (SB 202190, 10 mM) or STAT3 inhibitor (S31201) and exposed to sPLA2 XIIA at 2 ng.ml ¹. n=8 for NS-C, 15 for S-C and COPD . <0.05 sPLA2 XIIA vs. DMSO; $ p< 0.05 sPLA2 XIIA vs. sPLA2 XIIA + PFTa or heparanase or anti-HS; t p< 0.05 COPD vs. controls, £ p< 0.05 COPD vs. S-C. B. Bar graph of cells stained for p21 after the same treatments as the ones described in section A. Data are presented as mean ± SEM in the whole.

Figure 5: sPLA2 XIIA induces ROS production. Abbreviations are the same as those described in Figure 1. A. The pretreatment of cells by either scavengers of total ROS (NAC) or mitochondrial ROS (mitoquinol), or anti-HS or inhibitors of MAPK pathways or STAT3 inhibitors decreased the ROS production measured by DCFH-DA fluorescence, * p< 0.05 PGE2 vs. DMSO; t p< 0.05 COPD vs. controls, £ p< 0.05 COPD vs. S-C; $ p<0.05 sPLA2 XIIA vs. sPLA2 XIIA + compounds. B. Bar graph of cells stained for SA b-Gal activity after pre-treatment with either NAC or mitoquinol and exposed to sPLA2 XIIA (2 ng/ml). C. Bar graph of cells stained for p21 after the same treatments as the ones described in section (C). * p< 0.05 PGE2 vs. DMSO; Ϊ p< 0.05 COPD vs. controls, £ p< 0.05 COPD vs. S-C; $ p< 0.05 cells exposed to sPLA2 XIIA + NAC or sPLA2 XIIA + mitoquinol vs. cells treated with sPLA2 XIIA alone. Data are presented as mean ± SEM in the whole Figure, n=8 for NS-C and 15 for S-C and COPD in the whole Figure.

Figure 6: sPLA2 XIIA activates the MIDAS. Expression of TFNa and IL-10 in S-C and COPD fibroblast after treatment with sPLA2XIIA (2ng/ml), alone or in combination with pifithrin a (Pifia, 25 mM). Data are presented as mean ± SEM

Figure 7: Expression of sPLA2 XIIA in the sera of patients. * p< 0.05 COPD patients versus non-smokers (NS-C) and smokers (S-C). Data are presented as mean ± SEM

Figure 8: sPLA2 XIIA induces ROS production. Abbreviations are the same as those described in Figure 1. (A) Production of ROS was measured by DCFH-DA fluorescence and by analysis of HO-1 protein expression by western blot. (B) The pretreatment of cells by either scavengers of total ROS (NAC) or mitochondrial ROS (mitoquinol), or anti-HS or syndecan-1 (SDC-1) or syndecan-4 (SDC-4) or inhibitors of MAPK pathways (SB202190, U 0160) or inhibitor of STAT3 pathways (S31201) decreased the ROS production measured by DCFH- DA fluorescence, * p< 0.05 PGE2 vs. DMSO; J p< 0.05 COPD vs. controls, £ p< 0.05 COPD vs. S-C; $ p<0.05 sPLA2 XIIA vs. sPLA2 XIIA + compounds.

EXAMPLE:

Material & Methods

Materials sPLA2 XIIA and inhibitors such as U0126, SB202190, pifithrin-a, N acethyl cysteine, Mitoquinol, S31201 were purchased from Abeam and Sigma (Saint Quentin Fallavier, France) respectively.

Used concentrations of pharmacological tools were chosen after performing dose- responses curves on cell viability (data not shown), and on the capacity to block the effects of sPLA2 on SA b-gal and p21 expression.

Patients

Primary lung fibroblasts from 16 subjects with COPD and 24 subjects without clinical, morphological or functional signs of COPD (control) were included (data not shown). Controls were divided into respectively 8 non-smokers and 16 smokers (NS-C and S-C groups respectively). Classification of COPD severity was based on the 2003 Global initiative for chronic obstructive lung disease (GOLD) criteria). All patients underwent surgery for lung tumor resection. None of the patients was treated with corticosteroids, bronchodilatator therapy, or had a history of cancer chemotherapy or radiotherapy.

Informed consent was obtained from all patients and the study was approved by the “Comite de Protection des Personnes lie de France IX”. The study protocol was consistent with national ethical and professional guidelines.

Isolation of pulmonary fibroblasts and culture

Lung fibroblasts were isolated from lung tissue from patients undergoing resective surgery for pulmonary carcinoma. Pleura- free parenchymal specimens were excised after careful macroscopic evaluation, from peripheral areas of the lobe as far away as possible from the tumor site. Lung fibroblasts were obtained by the explant method (17). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Cergy-Pontoise, France) containing 10% heat-inactivated fetal bovine serum (FBS) (PAA, Les Mureaux, France), 100 U/ml penicillin, 100 pg/ml streptomycin (Invitrogen, Cergy-Pontoise, France), at 37°C in a 5% CO2 atmosphere. Isolated cells were characterized as fibroblasts by morphological appearance and expression pattern of specific proteins by immunocytochemistry. Cells are stained positive with anti-vimentin, and stained always negative with anti-pancytokeratin and anti-CD31 (Dako Cytomation, Trappes, France) (data not shown). All cultures contained between 5 to 7% of a smooth muscle actin positive cells (Invitrogen, Cergy-Pontoise, France). To confirm a lack of cancer associated fibroblasts (CAF), parenchymal lung biopsies used for fibroblast isolation and positive control consisting in breast carcinoma (a gift from Dr Andujar, INSERM U955, Creteil) were stained with CAF markers (Desmin and PDGFP-R) according to the same protocol detailed in Immunohistochemistry section (data not shown).

Cell proliferation

Rate of cell proliferation was assessed by measuring cumulative population doubling levels (PDLs). Briefly, cells were seeded at 100.000 cells per 25 cm², grown for 1 week, harvested and seeded again until loss of cellular growth. PDLs calculated at each passage were summed to obtain a cumulative PDL.

Cell treatments

Fibroblasts isolated from non-smokers, smokers and COPD patients were used at non- senescent passage (passage 3). We checked that the concentration of all compounds used in this section didn’t alter the viability of cells by using MTT and LDH assays (data not shown).

Accelerated senescence.

Fibroblasts were exposed either to sPLA2 XIIA (0.5, 2 and 50 ng/ml) for 24 h or pre treated with different inhibitors before be treated with sPLA2 XIIA. In order to check that heparan sulfate proteoglycans were involved in the effects elicited by sPLA2 XIIA, fibroblasts were pre-exposed to either heparanase (I and III) or anti-heparan sulfate for 2h. To determine the role of ROS in the induction of senescence, cells were exposed to either NAC (5 mM) or MITOQ (25 nM) for 2 hours. Finally, in order to identify the cell signaling pathway activated, fibroblasts were pre-exposed to pifithrin-a (25 mM), a transcriptional inhibitor of phospho-p53 activity (ref) for 24 hours, or inhibitors of ERK (U0126, 10 pM) or P38 (SB202190,10 pM) and treated with sPLA2XIIA for one more day.

Staining for Senescence-associated b-Galactosidase (SA b-Gal).

Pulmonary fibroblasts were cultivated in an 8-well glass slide chamber. After exposure to either sPLA2 XIIA or sPLA2 XIIA and different inhibitors, staining for SA b-Gal activity (Ozyme, Saint-Quentin-en-Yvelines, France) was performed as described (18). Cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde for 10 minutes at room temperature. The slides were then rinsed with PBS and incubated with an SA b-Gal staining solution containing 40 mM sodium citrate (pH 6), 150 mM, NaCl, 5 mM potassium ferri cyanide, 5 mM potassium ferrocyanide, 2 mM MgCk and 1 mg/ml 5 bromo-4-chloro-3-indolyl^-D-galactoside. Positive cells stained with blue were counted under visible light by two independent observers.

Gene expression analysis Total RNA was extracted with an RNeasy kit (Qiagen, Courtaboeuf, France) according to manufacturer’s instructions. The gene expression level of different isoforms of sPLA2, PLA2R1, syndecans and glypicans, was analyzed by RT-qPCR using a lightcycler and expressed as the ratio to a house-keeping gene (S3FA1 and 36B4).

Preparation of cell homogenates for Western blot analysis

Lung fibroblasts samples were lysed on ice in a lysis buffer (10 mM Tris-HCl, pH 6.8, 150 mM NaCl, 10 mM Hepes, Saccharose 500 mM, Na2 EDTA 1 mM, 1.0% NP-40, 10% anti protease and 1% anti-phosphatase). The protein concentration of these cell extracts was quantified using Bradford protein assay (Bio-Rad Laboratories, Mame-La-Coquette, France). An equal amount of protein (30 pg/lane) from each cell extract was resolved on a 10% or 12% SDS-PAGE gel. Proteins were blotted to an Immuno-Blot polyvinylidenediflouride (PVDF) membrane (Bio-Rad Laboratories, Mame-La-Coquette, France) by electrophoresis. The membranes were blocked with TBS-T blocking buffer (10 % milk in 25 mM, Tris-HCl, pH 7.4, 3 mM; KC1, 140 mM; NaCl, and 0.05% Tween) and subsequently probed with following primary antibodies either overnight at 4°C: rabbit polyclonal pi 6, mouse monoclonal p21 (1:200, Santa Cruz Biotechnology, Heidelberg, Germany), or syndecan 1, 4 (Abeam, Cambridge, UK) or glypican 1, 4, P-ERK, ERK, P-p38, p38, p-AMPK, AMPK, SIRT3, P- NFKB, NKFB. After extensive washing with TBS-T, immunoblots were then incubated with an appropriate peroxidase-conjugated secondary antibody (GE Healthcare Europe, Orsay, France) for 1 h at room temperature. After three washes with TBS-T, immunoblots were detected using the ECL Western Blotting Detection Reagents (GE Healthcare Europe, Orsay, France) and recorded by exposure of the immunoblots to an X-ray film (Sigma, Saint Quentin Fallavier, France). The results were expressed as a ratio to b-actin expression. With this aim, immunoblots were incubated with b-actin primary antibody for 30 minutes at room temperature (Sigma, Saint Quentin Fallavier, France). After washes, immunoblots were incubated with alcalin phosphatase-conjugated secondary antibody (Bio-Rad Laboratories, Mame-La- Coquette, France) for 30 minutes at room temperature. Finally, immunoblots were revealed using the Immun-Star AP Substrate (Bio-Rad Laboratories, Mame-La-Coquette, France). X- ray films were quantified by using Image J software (NIH, USA).

Immunofluorescence

Fibroblasts were cultivated in 8-well glass slide chamber. After treatments, cells were fixed in either 4% paraformaldehyde or methanol for 15 min at 4°C. Cells were permeabilized with 0.05 % Triton x-100 (Sigma, Saint Quentin Fallavier, France) for 15 min, washed and blocked in 2% BSA at ambient temperature. Cells were incubated with following primary antibodies overnight at 4°C: rabbit polyclonal phospho (serine 15)-p53 (1:500, Abeam), mouse monoclonal p21 (1:50, Santa Cruz Biotechnology, Heidelberg, Germany). Fluorescence signal was detected by using a goat anti- mouse secondary antibody (1:500, conjugated with Alexa 594, Invitrogen, Cergy-Pontoise, France). The slides were mounted by using prolong DAPI (Invitrogen, Cergy-Pontoise, France). A fluorescence microscope coupled to a digital camera utilizing axiovision software was used to view and acquire images (Zeiss, Jena, Germany). Quantification was performed by using Image J software (NIH, MD, USA). sPLA2 XIIA assay

The concentration of sPLA2 XIIA was measured in conditioned medium collected at passage 3 and 7. sPLA2 XIIA was quantified using the quantitative sandwich enzyme immunoassay technique (Cusabio, foumisseur Clini sciences, France) according to the manufacturer's instructions (Bertin Pharma, Montigny-Le-Bretonneux, France). Concentration (pg/ml) was determined by generating a standard curve with known concentrations of sPLA2 XIIA and normalized with the number of cells used to generate the conditioned media.

Measurement of oxidative stress by DCFH2-DA assay and mitochondrial ROS production by MitoSOX Red assay

Endogenous ROS were quantified by oxidation of 2’, 7’ dichlorofluorescin diacetate (DCFH2-DA) into 2’, 7’ dichlorofluorescin (Thermofischer, France). Briefly, cells were cultivated in 6 wells culture plates and treated with either sPLA2 XIIA alone or sPLA2 XIIA with NAC or MitoQ. Cells were also treated with 250 mM H2O2 as a positive control (data not shown). Cells were incubated with 20 pM DCFH2-DA for 30 min at 37°C and fluorescence recorded for 45 minutes.

Statistical analysis

Data were analyzed with GraphPad Prism 5.0 (La Jolla, CA, USA). Comparisons between groups were performed with Kruskall-Wallis’ non-parametric analysis of variance test followed by two-by-two comparisons with Mann- Whitney’s U test when a significant difference was detected. p< 0.05 was considered statistically significant.

Results

Patient’s clinical and demographic features

The three groups of subjects were similar in age. Smokers and COPD patients had similar smoking history. Subjects with COPD displayed a mild to moderate degree of disease as revealed by GOLD stages I and II (4 and 11 subjects respectively) and an emphysema score of 42.6 ± 6.5 over 100. Up-regulation of the sPLA2XIIA and receptors (PLA2R1 and heparan sulfate proteoglycans) in COPD fibroblasts

We evaluated the expression of sPLA2, which are surrogated in the lung such as sPLA2 IB, IIA, IID, XIIA, V and X in pulmonary fibroblasts from non- smokers (NS-C), smokers (S- C) and COPD patients at non-senescent and senescent stages (data not shown). Among them, only the expression level of sPLA2 XIIA was altered during the acquisition of senescent profile. We analyzed the gene and protein expression level of sPLA2 XIIA and its receptors in pulmonary fibroblasts from non- smokers (NS-C), smokers (S-C) and COPD patients at non- senescent and senescent stages. sPLA2 XIIA mRNA expression was significantly higher in fibroblasts from COPD patients as compared with NS-C groups at passage 3 (Figure 1A). At passage 7, in COPD fibroblasts, secreted sPLA2 XIIA levels and its mRNA expression were significantly higher as compared with passage 3 and versus NS-C and S-C groups (Figure 1 A). The gene expression level of sPLA2 XIIA at passage 7 in all patients correlated positively with FEV1/VC (r= -0.28, P<0.05) (data not shown). Secreted sPLA2 XIIA levels at passage 7 in all patients correlated positively with FEV1/VC and FEV1 (r= -0.66, P< 0.01, and r= - 0.49, P< 0.05, respectively) (Figure IB). sPLA2s act through specific receptors, named PLA2R1 and heparan sulfate proteoglycan (syndecan and glypican). At passage 3, mRNA expression of PLA2R1 was significantly higher in S-C and COPD fibroblasts as compared to NS-C groups (Figure 1C), and this difference was maintained at passage 7. Moreover, its mRNA expression was significantly higher as compared with passage 3 in S-C and COPD fibroblasts. Concerning the heparan sulfate proteoglycans and interestingly, mRNA expression of syndecan 1, and glypican 4 were significantly higher in S-C and COPD fibroblasts as compared to NS-C groups at passage 3 whereas glypican 1 was significantly increased in COPD fibroblasts (Figure 2). At passage 3, syndecan 1 was higher expressed in S-C than in COPD fibroblasts whereas syndecan 4 was significantly increased in COPD fibroblasts than in SC group. At passage 7, mRNA expression of glypican 1 in COPD fibroblasts were significantly higher as compared with passage 3 and versus controls groups mRNA expression of syndecan 4 in COPD and S-C fibroblasts were significantly higher compared with NS-C.

Overall, these results indicate an up-regulation of the sPLA2 XIIA and receptors (PLA2R1, syndecan 1, glypican 1 and 4) in COPD versus control fibroblasts.

A single exposure to sPLA2 XIIA induces accelerated senescence of lung fibroblasts We further questioned whether a single exposure to sPLA2 XIIA at concentrations close to those found in the secretome of senescent fibroblasts could induce senescence of non- senescent fibroblasts, and whether COPD fibroblasts were more susceptible to this effect than controls cells.

Lung fibroblasts were exposed to three doses of sPLA2 XIIA: 0.5; 2 and 50 ng/ml. Twenty-four hours incubation of non-senescent fibroblasts with sPLA2 XIIA at the dose of 0.5 and 2 ng/ml induced a dose-response increase in the percentage of cells expressing SA b-gal, P-p53, p21 and pl6 in S-C and COPD fibroblasts (Figure 3A). These inductions were significantly higher in COPD compared with control fibroblasts whereas no significant effects were detected in NS-C fibroblasts at the dose of 0.5 ng/ml. Nevertheless, at the dose of 50 ng/ml, the percentage of senescent cells decreased to return to the one observed at 0.5 ng/ml whatever the kind of fibroblasts to be considered suggesting a negative feedback. An absence of cleaved caspase-3 and no detection of phosphatidyl serine residues were observed in fibroblasts of the three groups of patients (data were not shown).

The effects observed being maximum at 2 ng/ml, the following experiments were performed only at this dose.

A single exposure of sPLA2 XII activates MAPK and p21/p53 pathways

In order to determine the role of the different receptors in the induction of senescence, fibroblasts from S-C and COPD patients were treated with either shRNA PLA2R1 or heparanase I and III to cleave the heparan sulfate chains on proteoglycans. The exposure to heparanase significantly reduced sPLA2 XIIA-induced SA b-gal and p21 expression in COPD fibroblasts (Figure 3B-C). These effects were confirmed by an anti-heparan sulfate antibody (Figure 3B-C), reinforcing the involvement of heparan sulfate proteoglycans in sPLA2XIIA- induced senescence. To go further, we explored the heparin binding sites in sPLA2 XIIA with specific peptides. Hep d23 decreased sPLA2 XIIA-induced SA b-gal and p21 expression in COPD and S-C fibroblasts (figure 3D and E). Finally, we assessed the role of syndecans which have been shown to have a tissue specific function. The antibody directed against syndecan 1 lead to the decrease of b-gal and p21 expression in COPD fibroblasts and abolished the effects induced by sPLA2 in both controls whereas the antibody directed against syndecan 4 deceased the senescence induced by sPLA2XIIA only in COPD. Interestingly, this treatment takes back the level of b-gal and p21 expression at the same level than the one of S-C fibroblasts, confirming a preponderant role of syndecan 4 in these cells. Therefore, since p21 is a main downstream effector of p53, we investigated the role of p53 on sPLA2XQA2-induced SA-b gal, p21 expression. Pifithrin-a, an inhibitor of nuclear p53 translocation (19), completely prevented SA b-gal, p21 expression (Figure 3B-C) induced by sPLA2 XIIA in both control and COPD fibroblasts (Figure 3A).

Since, PLA2R1 is known to activate several signaling pathways such as MAPK, JAK/STAT (15, 16), we investigated the time-course (from 5 minutes to 2 hours) protein expression level of MAPK and JAK/STAT pathways after treatment with sPLA2 XIIA at 2 ng/ml. The protein expressions of P-ERK, P- p38 and P Tyr707 STAT3 increased after 5 minutes of treatment in S-C and COPD fibroblasts. However, these effects were transient in S- C fibroblasts whereas they were maintained in COPD fibroblasts (data not shown). Moreover, activated STAT3 can exert its transcriptional activity after a second phosphorylation (ref). We observed that P-ser 727- STAT3 is increased after 5 minutes of treatment and maintained during 1 hour whatever the kind of fibroblasts considered (data not shown). Moreover, the MAPK are known to phosphorylate STAT3 on the Serine 727. The inhibition of MAPK led to abolish this phosphorylation (data not shown) . Blockage of ERK, STAT3 and P38 phosphorylation with U0126 and SB202190 and S31201 respectively abolished sPLA2 XIIA-induced SA b-gal and p21 expression in all fibroblasts, highlighting the involvement of this MAPK and STAT3 pathway in sPLA2XIIA-induced senescence (Figure 4A and 4B).

A single exposure of sPLA2 XII induces ROS production

Since, ROS are involved in the induction of senescence (8), we focused on their involvement in the effects of sPLA2 XIIA. The concentration of intracellular ROS increased in a dose-dependent manner in pulmonary fibroblasts treated with sPLA2 XIIA (Figure 8A). This induction was significantly higher in COPD as compared to control fibroblasts (Figure 8A). Twenty-four hours pre-treatment of fibroblasts with thiol antioxidant N-acetyl-cysteine (NAC) and Mitoquinol (a scavenger of mitochondrial ROS) significantly reduced the increase of ROS (Figure 5 A and 8B), SA b-gal (Figure 5B) and p21 expression (Figure 5C) induced by sPLA2 XIIA. Interestingly, a same abolition of ROS production was observed with the anti-heparan sulfate antibody, syndecan 4 and the inhibitors of MAPK and STAT3 showing an involvement of these proteins in the increase of ROS production.

A single exposure of sPLA2 XII activates a specific secretome

We researched the proteins secreted by the cells after treatment with sPLA2 XIIA. We observed a low expression of cytokines (IL-6, IL-la), which are known to be usually present in senescent cells. This fact is associated to an absence of NFkb increase, confirming a lack of activation of this pathway (data not shown). However, we showed by using Mitoquinol that a part of the ROS production induced by sPLA2 XIIA was due to an increase of mitochondrial ROS, suggesting an involvement of dysfunctional mitochondria. Indeed, multiples types of mitochondrial dysfunction such as a downregulation of SIRT3 and 5, inhibition of the electron transport chain, and depletion of mitochondria DNA induce mitochondrial dysfunction- associated senescence (MIDAS). MIDAS is characterized by a lack of several cytokines including IL-6, IL-8 and IL-1 and by the activation of AMPK-p53 pathway and the expression of TNFa, CCL27 and IL-10. In order to investigate if the MIDAS linked to this dysfunction was activated, we researched the protein expression of AMPK, TNFa, CCL27 and IL-10, after treatment with sPLA2 XIIA at 2 ng/ml. In parallel, we followed the expression of P-AMPK and we observed an increase after 15 minutes of treatment which was maintained for two hours in S-C and COPD fibroblasts, (data not shown). This induction was significantly higher after 15 minutes of treatment in COPD compared with control fibroblasts. However, AMPK is also known to be involved in the activation of P-p53. Indeed, the blockage of AMPK with compound C lead to a decrease of the phosphorylation of p53 induced by sPLA2 XIIA, showing its role in the senescence (data not shown). IL-10 and TNFa were increased after an exposure to sPLA2 XIIA and this increase was higher in COPD as compared to control fibroblasts (Figure 6). In interesting way, the pifithrin a abolished the effects of sPLA2 XIIA.

Expression of sPLA2 XIIA in sera of patients

In order to confirm the role of sPLA2XIIA in the COPD, we measured the expression level of sPLA2XIIA in the sera of controls (NS-C and S-C) and smokers with COPD obtained from a cohort of COPD patients established by Hopital Henri Mondor (Figure 7). These patients are completed different of those used in this study. The three groups were similar in age. Smokers and COPD patients had similar smoking history. Subjects with COPD displayed a mild to moderate degree of disease as revealed by GOLD stages I and II.

The level of sPLA2 XIIA was significantly higher in the sera of COPD patients than the one of controls, suggesting that this protein could be a potential marker of this pathology and a potential target for therapeutics treatments.

Collectively, these results demonstrate that a single exposure to sPLA2 XIIA induces senescence via heparan sulfate proteoglycans, ERK, p38, STAT-3, ROS, AMPK/p53/p21 and leads to an activation of secretome associated to MIDAS (data not shown). Moreover, COPD fibroblasts are significantly more susceptible to these effects than controls cells.

REFERENCES:

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Claims

1. An in vitro method for diagnosing chronic obstructive pulmonary disease (COPD) in a subject, comprising the steps of (i) determining the expression level of the secreted PLA2XIIA (sPLA2XIIA) in a sample obtained from said patient, and (ii) comparing the expression level determined at step i) with its predetermined reference value, and iii) concluding that the subject suffers from COPD when the expression level of the secreted sPLA2XIIA is higher than its predetermined reference value.

2. An in vitro method for diagnosing chronic obstructive pulmonary disease (COPD) in a subject, comprising the steps of (i) determining the expression level of at least one sPLA2XIIA receptor selected among PLA2R1, syndecan and glypican in a sample obtained from said patient, and (ii) comparing the expression level determined at step i) with its predetermined reference value, and iii) concluding that the subject suffers from COPD when the expression level of the receptors of sPLA2XIIA is higher than its predetermined reference value.

3. An in vitro method for determining whether is at risk of having COPD in a subject, comprising the steps of (i) determining the expression level of sPLA2 XIIA and/or at least one sPLA2XIIA receptors selected among PLA2R1, syndecan and glypican in a biological sample obtained from said subject, (ii) comparing the expression levels determined at step (i) with their predetermined reference value, and iii) concluding that the subject is at risk of having COPD when the expression levels determined at step (i) is higher than their predetermined reference value.

4. An in vitro method for predicting the survival time of a subject suffering from COPD comprising the steps of (i) determining the expression level of sPLA2 XIIA and/or at least one sPLA2XIIA receptors selected among PLA2R1, syndecan and glypican in a biological sample obtained from said subject, (ii) comparing the expression levels determined at step (i) with their predetermined reference value, and iii) providing a good prognosis when the expression level determined at step (i) is lower than their predetermined reference value, or providing a bad prognosis when the expression level determined at step (i) is higher than their predetermined reference value.

5. A method of treating COPD comprising the steps of: i) determining whether a subject suffers from COPD according to the method of claim 1 or 2 and ii) administrating to said subject a therapeutically amount of inhibitor of sPLA2 XIIA and/or an inhibitor of sPLA2XIIA receptors, when it is concluded at step i) that the subject suffers from COPD.

6. The method according to claim 5, wherein the inhibitor of sPLA2 XIIA is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.

7. The method according to claim 5, wherein the inhibitor of sLPA2 XIIA is selected in the group consisting of inhibitor of PLA2R1, inhibitor of syndecan and inhibitor of glypican

8. The method according to claim 7, wherein the inhibitor of PLA2R1 is siRNA.

9. The method according to claim 7, wherein the inhibitor of syndecan is a synthetic xyloside; a heparan-sulfate lyase; a synthetic glycosaminoglycan mimetic; an antibody; an aptamers; siRNA or an antisense oligonucleotide.

10. The method according to claim 7, wherein the inhibitor of glypican is a synthetic xyloside; a heparan-sulfate lyase; a sulfotransferase inhibitor, a glycosyltransferase inhibitory synthetic glycosaminoglycan mimetic; an antibody; an aptamers; siRNA or an antisense oligonucleotide.

11. The method according to claim 5, wherein the inhibitor of sPLA2 XIIA and/or the inhibitor of sPLA2 XIIA receptor is administered in combination with a classical treatment of COPD.

12. A method for treating COPD in a subject in need thereof comprising a step of administrating to said subject a therapeutically amount of an inhibitor of sPLA2 XIIA and/or an inhibitor of sPLA2XIIA receptors selected in the group consisting of inhibitor of PLA2R1, inhibitor of syndecan and inhibitor of glypican.

13. A therapeutic composition comprising an inhibitor of sPLA2 XIIA and/or an inhibitor of sPLA2XIIA receptors selected in the group consisting of inhibitor of PLA2R1, inhibitor of syndecan and inhibitor of glypican for use in the treatment of COPD in a subject as diagnosed according to the method of claim 1 or 2.

14. A kit comprising: (a) at least one reagent for measuring the expression of sPLA2 XIIA in a biological sample obtained from a subject, wherein when the expression level of soluble sPLA2 XIIA is higher than its predetermined reference, it is indicative that the subject suffers from COPD; (b) at least one control; and (c) instructions for use.