Patent Application
20180355427
Inflammation of the myocardium or myocarditis is a disease with heterogeneous etiology, oftenly caused by an infectious pathogen (bacteria, virus, fungi or protozoa) (Andreoletti et al., 2009) or by an autoimmune disorder (Frustaci et al., 2009), which can result in dilated cardiomyopathy (Felker et al., 2000). T CD4+ cells are necessary and sufficient for the development of myocarditis (Smith and Allen, 1991; Smith and Allen, 1993). Specifically, TH17 lymphocytes (Sonderegger et al., 2006) and IL-23 cytokine are critical for the development of the pathology, being TH17 cells more immunopathogenic than TH1 cells when they are exogenously transferred (Rangachari et al., 2006). Furthermore, suppression of TH17 cell differentiation through the blocking of the IL-6 signaling pathway prevents the development of EAM (Experimental Autoimmune Myocarditis) (Sonderegger et al., 2008; Yamashita et al., 2011). With respect to IL-17, it has been observed that this cytokine does not participate in the initial development of myocarditis but it plays a crucial role in fibrosis and the remodeling that the heart experiences in more advanced stages of the disease (Baldeviano et al., 2010). In humans, the clinical relevance of TH17 cells is close to being verified, and in fact, in patients with viral myocarditis IL-17A and RORγt expression in the heart has already been detected and it has been observed that TH17 cells help B cells produce antibodies against cardiac proteins (Yuan et al., 2010). However, little research has been conducted concerning TH17 cells circulating in the blood of patients with myocarditis. Only one study is known in which the proportion of TH17 lymphocytes in the blood of patients with dilated cardiomyopathy was compared to that of healthy subjects, there being a higher percentage of TH17 cells and a larger number of IL-17, IL-6, IL-23 and RORγt cells in the blood of sick subjects with respect to the blood of healthy individuals (Yi et al., 2009).
Regulatory T cells are another population of T CD4+ cells that are crucial in the development of myocarditis. FoxP3 expression has been detected in endocardium biopsies of patients with dilated cardiomyopathy (Noutsias et al., 2011). Furthermore, the depletion of Treg cells allows the spontaneous development of myocarditis (Ono et al., 2006), whereas the exogenous transfer of Treg cells limits cardiac inflammation and attenuates viral replication in response to infection with CVB3 or Trypanosoma cruzi (Mariano et al., 2008; Shi et al., 2010). Treg cells are also responsible for inducing tolerance by nasal route against autoantigens, which prevents CVB3 infection from progressing to chronic myocarditis (Fousteri et al., 2011). Similarly, IL-10 cytokine limits autoimmune inflammation of the heart by means of tolerance induced by nasal route (Kaya et al., 2002). However, there is a substantial discrepancy with respect to the predictive value of the proportion of Treg lymphocytes circulating in blood in patients with acute and chronic heart pathologies (Ammirati et al., 2010; Carvalheiro et al., 2012; Sardella et al., 2007).
Recent studies estimate the prevalence of myocarditis in about. 22 of 100.000 patients annually (International Classification of Diseases, WHO), and the American Heart Association and American College of Cardiology Rank myocarditis as the third leading cause of sudden cardiac death in professional athletes. Moreover, between 1-5% of patients with acute viral infection develop myocarditis. The diagnosis of myocarditis made based on clinical, laboratory, ECG, and Echo findings is not always easy. Moreover, a safe and sensitive non-invasive diagnostic test to confirm the diagnosis of acute myocarditis, compared to other pathologies with myocardial injury like acute coronary disease, is not available nowadays. Endomyocardial biopsy (EMB) is still considered the gold standard diagnosis of myocarditis. However, EMB is not commonly performed due to safety reasons; 6% of wide range complications and 0.4 incidence of death due to perforation.
Acute myocarditis resolves in about 50% of cases in the first weeks, however 25% develop persistent heart failure that can progress to end-stage dilated cardiomyopathy and heart transplantation. Therefore, early diagnosis of the disease is vital for the specific treatment of the patient (antiviral, immunosuppressive, heart failure therapies, etc) to avoid the subsequent development of the disease. However, most acute myocarditis patients have common symptoms with Acute Myocardial Infartion patients (AMI). It is thus necessary to provide tools in clinical practice for the differential diagnosis of acute myocarditis and AMI since acute myocarditis often simulates a myocardial infarction and differential diagnosis is many times difficult as intramyocardial biopsy is not a routine diagnostic technique. This often makes these patients undergo coronary angiography and MRI with contrast to rule out ischemia, with consequent damage to the patient, delay in the election of immunosuppressive treatment of choice and high medical expenses associated with coronary angiography and MRI as the only reliable methods for distinguishing these Myocarditis from AMI. Myocarditis is therefore a diagnostic challenge that does not have a solution in clinical practice.
The present invention solves this problem by providing an assay capable of assuring an acceptable degree of specificity and sensitivity for diagnosing myocarditis, during an acute episode, by using blood samples, particularly plasma or serum samples.
The present invention provides an in vitro method for diagnosing a cardiomyopathy in a human subject, wherein the method comprises using, as an indicator, the expression levels, obtained from a biological sample isolated from the human subject, preferably a blood, plasma or serum sample, of one or more of the following miRNAs: miR-721 and/or miR155, and obtaining a result of the method by comparing the expression levels of said one or more miRNAs with a reference value or with the expression levels of a control, indicator of the absence of cardiomyopathy.
More particularly, the present invention provides an in vitro method for diagnosing Myocarditis during an acute episode. Myocarditis is a Th17-mediated disease characterized by the presence of immune infiltrates that triggers cardiomyocyte necrosis and fibrosis of the heart and, therefore, progressive heart dysfunction. It shares common symptoms with acute myocardial infarction so it is important to find biomarkers for the specific differential diagnosis of myocarditis, preferably during the acute episode, from acute myocardial infarction. MiR-721, found overexpressed in exacerbated Experimental Autoimmune Myocarditis (EAM) mouse model and Th17 cells, is shown herein as an excellent target for such differential diagnosis. In this sense, we found miR-721 overexpressed in the serum, draining lymph nodes and isolated Th17 cells from EAM mice. Furthermore, we were able to detect miR-721 in the serum of EAM but not in basal conditions. Since we found Th17 and miR-721 upregulated in myocarditis patients and no Th17 response in acute myocardial infarction patients, we estimate miR-721 to be a powerful diagnostic tool to distinguish between both pathologies.
In the present invention, based on the determination that mice with myocarditis present miR-721 and miR-155 expression levels in blood serum much higher than those of control mice, the expression of these miRNAs in the plasma of patients with different cardiomyopathies was analyzed. The result was an extraordinary increase in miR-721 and miR-155 expression in the plasma of patients with acute myocarditis compared to healthy subjects. Patients in the chronic phase of the disease also presented expression levels of these miRNAs that were much higher than those of healthy volunteers, but not as high as during the acute phase. Furthermore, with respect to Takotsubo cardiomyopathy, the spectacular increase in miR-721 and miR-155 expression in the blood of patients with this syndrome compared to control subjects is striking. These results point out the possible role of these miRNAs in the pathophysiology of myocarditis, preferably during the acute episode, and dilated cardiomyopathy. In this sense and based on these results, the present invention provides a novel strategy for the diagnosis of myocarditis, in particular during the acute episode, based on the determination of miR-721 and/or miR-155 in blood, serum or plasma samples.
In addition, it is noted that when a patient reaches the hospital with a pain in the chest it is often difficult to distinguish between acute myocardial infarction (AMI) and myocarditis because these two pathologies share most symptoms such as arrhythmias, elevation of the ST segment, circulating troponin levels or C-reactive protein (Fung et al, 2016) etc. . . . . Therefore, clinicians routinely proceed with invasive techniques to treat AMI since this disease is more harmful. However, the autoimmune phenotype characterized by a high CD4+ T cells and Treg/Th17 levels in myocarditis patients is not observed in AMI patients. In addition, recent results from our laboratory showed that miR-721 is not expressed in the serum of mouse models for myocardial infarction right after the episode, but only around two weeks later, matching the Th17 response peak developed post-infarction to solve the injury. Since miR-721 is highly upregulated in acute and chronic myocarditis patients when they come to the hospital, it is a powerful biomarker for myocarditis. Therefore, the non-invasive strategy proposed in the present invention is a fast and efficient approach to clarify the diagnosis between these two cardiovascular events.
Furthermore, the extraordinary increase in miR-721 expression in the plasma of patients with acute myocarditis compared to healthy subjects is due to the fact that miR-721 is significantly present in the exosomes of Th17 cells. To our knowledge, this is the first time that it is described that MiR-721 is upregulated in the exosomes from Th17 in diseases characterized by an exacerbated Th17 response (see examples 5.1 or 5.2) such as myocarditis, in particular during the acute episode. Such extraordinary increase due to its increased localization in the exosomes facilitates the detection of such biomarker (miR-721) in non invasive samples such as blood, serum or plasma biological samples and thus the diagnosis of diseases such as acute myocarditis in human subjects.
Hence, a first aspect of the invention refers to an in vitro method for obtaining data useful for diagnosing a cardiomyopathy in a human subject, preferably for the detection or diagnosis of a cardiomyopathy in a human subject, wherein the method comprises using, as an indicator, the expression levels, obtained from a biological sample isolated from the human subject, of one or more of the following miRNAs: miR-721 and/or miR155, and obtaining a result of the method by comparing the expression levels of said one or more miRNAs with a reference value or with the expression levels of a control, indicator of the absence of such cardiomyopathy.
In the present invention, SEQ ID NO 1 is understood as CAGUGCAAUUAAAAGGGGGAA (murine miR-721 sequence). Variations of said sequence associated with the different miR-721 sequences in different species are included in this definition, such as the miR-721 sequence in humans which is UCUUGCAAUUAAAAGGGGGAA illustrated herein as SEQ ID NO 4. Hsa-miR-721 is located within the region: NCBI Reference Sequence: NC_000008.11 (REGION: 95353371 . . . 95353588).
In the article Wheeler G. et al., FEBS Letters 2006, where the mouse miR-721 was identified, it is stated that no homologous sequences were found in human genome. However, a 21 nucleotides region of the mouse precursor is conserved in the monkey and human genome (see
In the present invention, SEQ ID NO 2 is understood as UUAAUGCUAAUUGUGAUAGGGGU (miR-155 murine) and SEQ ID NO 3 is understood as UUAAUGCUAAUCGUGAUAGGGGU (miR-155 human).
In a preferred embodiment of the first aspect of the invention, illustrative non-limiting examples of said biological samples include different types of tissue samples, as well as samples of biological fluids, such as blood, serum, plasma, cerebrospinal fluid, peritoneal fluid, and feces. Preferably, said samples are blood, serum or plasma biological samples.
In another preferred embodiment of the first aspect of the invention, the cardiomyopathy is myocarditis, Takotsubo syndrome, dilated cardiopathy, peripheral ischemia or Chagas. More preferably, the cardiomyopathy is acute myocarditis.
Another preferred embodiment of the first aspect of the invention or any of its preferred embodiments, uses, as an indicator, the expression levels of one or more miRNAs such as those defined in SEQ ID NOs: 3 or 4 above, and wherein the result is an indicator of the existence of a myocarditis, preferably an acute myocarditis, or of Takotsubo syndrome if:
In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the biological sample is selected from the group consisting of blood, serum or plasma and such biological sample is treated with a lysis agent such as a detergent composition or chaotropic agents and/or physical lysis (e.g. Ultrasounds, vigorous shaking), capable of disrupting the membrane of the exosomes so that such exosomes, in particular exosomes from Th17 cells, liberate their content to the exterior and thus liberate miRNAs such as miR-721.
In this sense it is noted that collection and handling procedures of blood, serum or plasma needs to be greatly improved for miRNA analysis in order to reliably detect differences between healthy and disease patients. Furthermore, ribonucleases present in blood can degrade free RNA upon collection rendering extracellular miRNA at risk of degradation. These factors have consequently decreased sensitivity and specificity of miRNA biomarker assays. miRNAs secreted from blood cells into exosomes are protected from degradation and have increased potential as biomarkers. To improve the use of miRNAs as biomarkers, exosomes have to be either isolated from blood, serum or plasma by different methods and then miRNAs need to be released from the inside with a lysis agent to disrupt the membrane of the exosomes or simply disrupted without previously isolating them so that they liberate their content to the exterior and thus liberate miRNAs such as miR-721 in the sample (examples of lysis agents useful in the present invention are shown in table 1 below). In any case, the fact that this is the first time that it is described that miR-721 is upregulated in the exosomes from Th17 cells in the blood, serum or plasma samples of patients suffering from acute myocarditis, unveils the use of miR-721 in these samples as a most value tool to reliably detect differences between healthy and disease patients.
In the context of the present invention, the term “detergent composition” is understood as a composition comprising amphipathic molecules (containing both polar hydrophilic heads and non-polar hydrophobic tails) that enables disruption and formation of hydrophobic-hydrophilic interactions among molecules. In biological research, detergents such as oxyethylene, tert-octylphenol or ethyleneglycoether polymers are used to solubilize cell-derived membranes such as plasmatic membranes, organelles membranes or extracellular exosomes in order to allow the release of their content. To not interfere with soluble proteins n-Octylglucoside or other mild non-denaturing detergent for the solubilization of proteins can be used.
In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the biological sample is selected from the group consisting of blood, serum or plasma and such biological sample is treated so that the exosomal fraction of the Th17 cells is obtained and the determination of the expression levels of miR-721 is performed in such exosomal fraction by using a lysis agent, such as a detergent composition or chaotropic agents and/or physical lysis (e.g. Ultrasounds, vigorous shaking), capable of disrupting the membrane of the exosomes so that such exosomes, in particular exosomes from Th17 cells, so that such exosomes liberate their content to the exterior and thus liberate miRNAs such as miR-721.
Exosomes are extracellular cell-derived vesicles with a diameter ranging from 30 to 300 nm present in biological fluids and cultured media of cell cultures. They contain proteins, metabolites and nucleic acids such as mRNA and non-coding RNAs coated in a lipid bilayer membrane. The exosomal fraction corresponds to all the exosomes of a particular biological sample or cell culture medium.
In the context of the present invention, the exosomal fraction from Th17 cells can be obtained from any known technique to the skilled person. For example, cell suspensions from lymph nodes 6 days after EAM induction were cultured (2×107 cells/ml) during 48 h in exosome-free TexMACS media (Miltenyl Biotech) with MyHC peptide (10 ug/ml) and IL-23 (10 ng/ml). Afterwards, sorting for IL-17eGFP using the BD FACs Aria II cell sorter/iCyt Synergy 4L cell sorter. Sorted cells were cultured again in TexMACS media with MyHC peptide and IL-23 for IL-17+ cells (Th17) expansion. Supernatants from post-sorted Th17 were collected after 48 h. Briefly, rest of cells were pelleted (2000 rpm for 10 min). For exosomes isolation culture supernatants were precipitated by adding 0.4 volumes of 50% Polyethylene glycol 6000 (Sigma Aldrich) in 375 mM NaCl to the samples, storing 30 min at 4° C. and centrifugation 1500 g at 4° C. In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the result can be obtained by means of:
Preferably, the result is obtained by means of real time quantitative PCR.
In another preferred embodiment of the first aspect of the invention or any of its preferred embodiments, the method is carried out in vitro using a sample from the human subject, and wherein at the time of taking the sample from the human subject, the human subject is not being treated for cardiomyopathy.
In another preferred embodiment of the first aspect of the invention or any of its preferred embodiments, miRNA expression is normalized.
A second aspect of the invention relates to a method for classifying a human subject into one of two groups, wherein group 1 comprises the subjects that can be identified by means of the method according to the first aspect of the invention, and wherein group 2 represents the remaining subjects.
A third aspect of the invention relates to a pharmaceutical composition comprising a therapeutic agent suitable for treating a human subject from group 1 that can be identified by means of the method of claim 11.
A fourth aspect of the invention refers to a method for the differential diagnosis of acute myocardial infarction from acute myocarditis in a human subject suspected of suffering from any of these pathologies, wherein the method comprises using, as an indicator, the expression levels, obtained from a biological sample isolated from the human subject, of miR-721, and obtaining a result of the method by comparing the expression levels of said miRNA with a reference value or with the expression levels of a control, wherein an increased expression of miR-721 is indicative of acute myocarditis and the absence of expression is indicative of acute myocardial infarction.
In a preferred embodiment of the fourth aspect of the invention, illustrative non-limiting examples of said samples include different types of tissue samples, as well as samples of biological fluids, such as blood, serum, plasma, cerebrospinal fluid, peritoneal fluid, and feces.
Preferably, said samples are blood, serum or plasma biological samples.
In another preferred embodiment of the fourth aspect of the invention or of any of its preferred embodiments, the biological sample is selected from the group consisting of blood, serum or plasma and such biological sample is treated with a lysis agent such as a detergent composition or chaotropic agents and/or physical lysis (e.g. Ultrasounds, vigorous shaking), capable of disrupting the membrane of the exosomes so that such exosomes, in particular exosomes from Th17 cells, liberate their content to the exterior and thus liberate miRNAs such as miR-721.
In another preferred embodiment of the fourth aspect of the invention or of any of its preferred embodiments, the biological sample is selected from the group consisting of blood, serum or plasma and such biological sample is treated so that the exosomal fraction of the Th17 cells is obtained and the determination of the expression levels of miR-721 is performed in such exosomal fraction by using a lysis agent, such as a detergent composition or chaotropic agents and/or physical lysis (e.g. Ultrasounds, vigorous shaking), capable of disrupting the membrane of the exosomes, so that such exosomes liberate their content to the exterior and thus liberate miRNAs such as miR-721.
In another preferred embodiment of the fourth aspect of the invention or of any of its preferred embodiments, the result can be obtained by means of:
Preferably, the result is obtained by means of real time quantitative PCR.
In another preferred embodiment of the fourth aspect of the invention or any of its preferred embodiments, miRNA expression is normalized.
A fifth aspect of the invention refers to a method of treatment of a human subject suspected of suffering from acute myocardial infarction or from acute myocarditis, wherein said method comprises performing the differential diagnosis according to the fourth aspect of the invention or according to any of its preferred embodiments, wherein if there is an increased expression of miR-721 in the biological sample, the patient is treated for acute myocarditis and wherein if there is an absence of expression of miR-721 in the biological sample, the patient is treated for acute myocardial infarction.
In the context of the present invention, suitable treatments for acute myocarditis are limited, mainly being heart transplantation and mechanical assistance devices. Particularly, three major approaches have been considered for the treatment of acute myocarditis:
In the context of the present invention, suitable treatments for the initial therapy of acute myocardial infarction should be directed toward restoration of perfusion as soon as possible to salvage as much of the jeopardized myocardium as possible. This may be accomplished through medical or mechanical means, such as percutaneous coronary intervention (PCI), or coronary artery bypass graft (CABG) surgery.
Although the initial treatment of the different types of acute coronary syndrome (ACS) may appear to be similar, it is very important to distinguish between whether the patient is having an ST-elevation MI (STEMI) or a non-STEMI (NSTEMI), because definitive therapies differ between these two types of MI. Particular considerations and differences involve the urgency of therapy and the degree of evidence regarding different pharmacologic options.
Specific prehospital care includes the following:
Management of ST-elevation myocardial infarction (MI) (STEMI) relies on two essential and key components: rapid recognition and timely reperfusion. Therefore, systems must be placed and implemented to prevent delay in management of patients who develop STEMI. Minimizing delays has been associated with improved overall outcomes as well as reduced mortality and long-term morbidity. Primary percutaneous intervention (PCI) is defined as an emergent percutaneous coronary intervention in the setting of STEMI, without previous fibrinolytic treatment. It is the preferred reperfusion strategy in patients with STEMI. Fibrinolysis is an important reperfusion strategy, particularly in settings where primary PCI cannot be offered to STEMI patients within the recommended timelines. The benefit of fibrinolytic therapy in patients with STEMI is well established, with the largest benefit seen when administered early (within 12 hours after symptomatic onset) and in patients with the highest cardiovascular risk.
Anticoagulant agents are an important adjunctive therapy for reperfusion therapy regardless of the strategy chosen (ie, whether it is primary PCI or fibrinolysis therapy). Different anticoagulation agents are available; the utility of each agent depends on the clinical context, taking into account the method of reperfusion.
All patients with STEMI should receive an empiric loading dose of aspirin (150.5 to 325 mg) as early as possible and prior to reperfusion, regardless of the reperfusion method. A lifelong maintenance dose of (75 to 81 mg) daily should be prescribed to all patients after STEMI.
A sixth aspect of the invention, relates to a kit or device comprising at least one or more oligonucleotides capable of hybridizing with any one of the miRNAs defined in the first or fourth aspects of the invention, preferably for hybridizing with miR-721 and/or miR-155, more preferably for hybridizing with SEQ ID NO 4, means for detecting said hybridization, and a lysis agent as defined above. Preferably, said kit or device additionally comprises a polyT oligonucleotide primer and the oligonucleotide(s) are optionally immobilized in stains on a surface, preferably on the surface of a microarray.
As used herein, “means for detecting said hybridization” is understood as labeled complementary DNA, RNA, modified nucleic acid strand (i.e. probe) to localize a miRNA sequence in a biological samples such as a blood, serum or plasma sample or in a portion or section of tissue, in the entire tissue or cells.
A seventh aspect of the invention relates to the in vitro use of the kit as defined in the sixth aspect of the invention or of a kit or device comprising at least one or more oligonucleotides capable of hybridizing with any one of the miRNAs defined in any of the first or fourth aspects of the invention, for detecting or diagnosing or for obtaining data useful for diagnosing a cardiomyopathy in a human subject. Preferably, said kit or device additionally comprises a polyT oligonucleotide primer and the oligonucleotide(s) are optionally immobilized in stains on a surface, preferably on the surface of a microarray. More preferably, the use of said kits or devices is for detecting or diagnosing or for obtaining data useful for diagnosing a myocarditis, preferably an acute myocarditis, acute myocardial infarction or for diagnosing Takotsubo syndrome in a human subject.
An eight aspect of the invention refers to a nucleotide sequence consisting of SEQ ID NO 4.
An ninth aspect of the invention refers to a fully complementary (100% complementary) DNA, RNA or modified nucleic acid strand (i.e. probe) capable of hybridizing with miRNA sequence SEQ ID NO 4, in particular with no mismatches.
A preferred embodiment of the eighth or ninth aspect of the invention refers to a genetic construct, such as a DNA sequence, capable of transcribing miRNA sequence SEQ ID NO 4.
The following examples help to illustrate but do not limit the present invention.
miRNA-721 was first described in a paper on mouse embryon brain tissue, in which the expression thereof during central nervous system embryogenesis was studied (Wheeler et al., FEBS Letters 2006). In this paper, miRNA was identified as miRY and was subsequently entered in the miR Base as mir-721. This miRNA is located in chromosome 5 within the non-coding region of CUX1 gene. miR-721 belongs to the family of miRNAs miR-130/301/721 (
To study this microRNA in depth and its role in regulating inflammation and therefore in cardiovascular diseases, miRNA was cloned into expression vectors to conduct loss and gain of function assays, as well as for the functional validation of the possible target genes of this miRNA. Since the assays must essentially be conducted in primary cells, retroviral and lentiviral expression vectors have been tested, because they are the most efficient tools for gene expression in mammalian cells given their high ability of being integrated into the host cell genome.
1.2. Cloning miR-721 and Sponge Sequences
For the cloning were designed several flanking sequences of immature miR-721 sequence, of around 50, 75 and 100 base pairs (in both 5′ and 3′ directions), in order to assure correctly processing immature miRNA to mature miRNA, because a duly folded secondary structure is required in order to have mature and functional miRNA (
Furthermore, the sponge system is used to silence miR-721, consisting in the overexpression of the complementary sequence of miRNA mature in a murine lentiviral vector (pCDH-CMV-MCS-EF1-copGFP vector), which acts like a buffer blocking the functional molecules of the miRNA of interest. This construction contains 4 complementary sequences separated by spacers having between 4 and 5 base pairs (
Lentiviral particles are produced by transfection (calcium phosphate method) of HEK293 cell line with the plasmid in which the sequence of interest of the present invention is expressed together with the lentiviral polymerase and envelope plasmids. The lentivirus are collected from the culture supernatant and concentrated by means of ultracentrifugation. The virus stock is kept at −80° C. until use (
1.3. miR-721 Expression in Mouse Primary Cells
These assays were carried out in mouse primary cells. miRNA-721 is expressed in T lymphocytes, fundamentally in Th17 cells, and CD69 expression limits miR-721 levels in these cells (
However, since Th17 cells are hard to obtain and keep in culture, mouse lung endothelial cells (MLEC) are used to conduct these assays. Like in CD69 WT mice naive CD4 cells, in these cells it is found that miR-721 expression is not detected in MLECs from CD69WT animals. Furthermore, MLEC stimulation with PMA/Iono or TNFα does not salve miR-721 expression in CD69WT mice (
1.4. Validation of Target Genes of miR-721
A single target gene validated as a target for miR-721, Meox-2 gene, has been described. The family of miRNAs miR-130/301/721 is a positive regulator of induced pluripotent stem cell (iPSC) generation by means of Meox-2 gene repression (Pfaff et al., EMBO reports 2011). The 3″UTR end of Meox-2 gene has a complementary sequence with respect to the family miR-130/301/721, whereby these miRNAs regulate expression thereof (
In addition to Meox-2, complementary sequences in the 3″UTR end of CD69, CUX1 and Aryl Hydrocarbon Receptor (AHR) genes (
To validate expression of these genes, qPCR was performed in MLECs from CD69WT and KO mice. With this technique it was found that in the presence of miR-721 in CD69KO MLECs, Meox-2, CUX1 and AHR gene transcription is completely inhibited and they are only expressed after activation with strong unspecific stimuli (PMA/Iono) angiogenesis specific stimuli (TNFα), whereas CD69WT MLECs, which does not express miR-721, express detectable levels of these 3 genes in baseline conditions and much higher levels after activation with the stimuli (
To perform these assays, MLEC infection conditions had to be fine-tuned, for which purpose it is fundamental to know the titration of the virus and to infect with a multiplicity of infection (MOI) that is sufficient for infecting all the cells of the plate, but which in turn does not endanger cell viability. Several concentrations of lentiviral particles were tested and an MOI=5 (
Once these conditions were fine-tuned, in vitro assays were performed to validate Meox-2, AHR and CUX1 genes as miR-721 targets, by both overexpressing miR-721 and silencing it with Sponge sequences (
As miR-721 is a novel micro-RNA and has never been defined in human genome, we performed a multiple species sequence alignment of the murine miR-721 and defined the predictive sites for monkey and human miR-721 sequences (
According to these results, CD69 will act as a miR-721 repressor. In turn, miR-721 will be expressed both in Th17 and in endothelial cells and represses Meox-2, AHR and CUX1 genes expression, and therefore new vessel formation processes (
2.1. In Vivo Experiments for Validating the Role of miR-721 in the Th17 Cell Effector Function
Since the study of miR-721 function in the experimental autoimmune myocarditis (EAM) model requires very extensive experiments and working highly inaccessible tissue, the myocardium, a mouse lower limb muscle mouse ischemia model that is widely used in research to study the peripheral arterial disease (PAD) has been developed in the laboratory. To that end, the animals are subjected to a surgery explained next: a 5 mm incision is made in the inguinal area of the left leg allowing exposure of the femoral nerve, vein and artery. The connective tissue is carefully separated, and the vein, artery and nerve are dissected by means of separating the arterial adventitia layer close to the area distal to the femoral artery bifurcation. Once the three components are separated, the femoral artery is ligated by carefully passing thereunder a suture thread and making two surgical knots close to one another. The femoral artery is cut between the two knots that are made. The incision that is made will be sewn up after washing with physiological saline at a moderate temperature of 37° C. Femoral artery occlusion will allow the blood to flow towards the collateral artery, allowing new blood vessel formation (neovascularization) with the subsequent re-irrigation of the leg. The adductor muscle reperfusion monitoring process is carried out by means of analysis by laser Doppler (
Inflammation and autoimmune anomalies play a crucial role in heart and vascular failure progression. Among other molecules, microRNAs have been proposed as one of the most innovative tools for the development of therapies against inflammatory and cardiovascular diseases. A number of papers relate T helper 17 (Th17) cells and the regulatory T (Treg) cells with cardiovascular diseases such as acute coronary syndrome or congestive heart failure. Furthermore, studies conducted in mice after femoral artery ischemia show that Th17 and Tregs modulate post-ischemic neovascularization in an antagonistic manner, revealing a crucial role of those cells in cardiovascular diseases such as peripheral arterial disease (PAD). However, the role of miRNAs derived from these cells is completely unexplored in this field, leaving a window open to the development of new diagnostic and therapeutic tools. This topic is being approached in the laboratory by means of the analysis of the CD69 leukocyte antigen as a Th17/Treg balance regulator because this molecule can be used as a connection link between cardiac inflammation mediated by Th17 cells and heart failure. Results have shown that there are at least 80 miRNAs that are differentially expressed in CD69-deficient Th17 cells compared to WT. Of all these miRNAs, this study focuses on miR-721 because its possible target genes are extremely relevant both in the biology of Th17/Treg cells and in cardiovascular diseases, including among them SOCS-1, AHR, STAT-3, RORγt or Meox-2. With the exception of Meox-2, these miRNA/target gene pairs have never been validated or described in Th17/Treg cells or in cardiovascular diseases. Furthermore, high expression of these miRNAs has been found in lymphoid cells, heart cells and serum from mice with autoimmune myocarditis or after femoral artery ischemia, as well as in patients with cardiomyopathies.
Therefore, femoral ischemia or PAD experiments are based on the hypothesis that CD69KO mice will have exacerbated Th17 responses and high miR-721 expression. In this model, it is described that Th17 cells positively regulate processes of arteriogenesis (Hata et al., Cardiovascular Research 2011), and that Treg cells do so in a negative manner, i.e., inhibiting the process of angiogenesis (Zouggari et al., Circulation 2009).
Taking the foregoing into account, if the hypothesis of the present invention is true, the revascularization process after ischemia should be enhanced in CD69KO animals versus WT animals, because miR-721 will act on Meox-2 and CUX1 genes, angiogenesis inhibitors.
2.2. Study of miR-721's Role and its Target Gene Meox-2 in Neovascularization after Femoral Artery Ischemia
A study of Th17 and Treg cell kinetics in blood after inducing femoral artery ischemia was conducted. Once compared with the control mice, a progressive and significant increase in Th17 cells in blood during the 7 days following surgery can be seen. However, even though a peak in Treg is observed after 24 hours, Treg levels drop to almost baseline levels during day 7 of the experiment (
The increase in Th17 response 7 days after ischemia coincides with an increase of up to 40 times in miR-721 expression in the serum of these animals (
2.3. Expression of the miR-721 Target Gene, Meox-2, is Inhibited after Inducing Ischemia
In endothelial cells from the brain of patients with Alzheimer's disease, it has been demonstrated that Meox-2 silencing stimulates angiogenesis (Wu et al., Nature Medicine 2005), demonstrating that this gene exerts an inhibitory function on angiogenesis in endothelial cells. In the Meox-2 experiments for the present invention, it is induced in adductor muscle tissue 7 days after femoral artery ischemia, which indicates that this gene is activated in this tissue after ischemia (
To verify Meox-2 inhibition in the endothelial compartment in these animals, immunofluorescence analysis of Meox-2 expression in CD31+ endothelial cells in the adductor muscle after 7 days of femoral ischemia is conducted (
In this analysis it is observed that Meox-2 expression is inhibited in CD31+ endothelial cells after ischemia (
In a preliminary study, revascularization after femoral artery ischemia in the right leg of CD69 WT and CD69KO mice of the BALB/c strain was studied, leaving the left leg as a control. Adductor muscle reperfusion monitoring was carried out by means of laser Doppler for one month after ischemia. According to the initial hypothesis, where CD69KO mice are more efficient in Th17 responses, it was found that the recovery kinetics (reperfusion ratio) of CD69KO animals is much faster compared to CD69WT animals (
CD69KO mice have virtually recovered from ischemia 7 days after surgery, which coincides in these animals with a very significant increase in draining lymph nodes (popliteal) of Th17 cells (
2.4. miR-721 Generated by Th17 Cells Exerts its Effector Function on Target Genes in Endothelial Cells in Revascularization Processes after Ischemia
Above it was described that MLEC endothelial cells of CD69 WT animals do not express miR-721 in homeostasis conditions or after activation with different stimuli, so the role of this miRNA in the lymphocyte compartment of the mouse must be studied. To that end, chimeras have been formed by irradiating BL/6 CD69 WT mice reconstituted with a mixture of bone marrow (BM) from Rag2−/−γc−/− animals that were lacking in precursors for T, B and NK cells, and from BL/6 CD69WT or CD69KO animals, at a 3:1 ratio, respectively. In these mixed chimeras, the entire myeloid compartment belongs to Rag2−/−γc−/− animals and is therefore WT, whereas the lymphoid compartment is generated from BL/6 BM, being CD69KO or WT in each case. Furthermore, these chimeras do not express miR-721 in the endothelium because all of the non-lymphoid tissue of the reconstituted animals is WT phenotype. It can therefore be expected that the miRNA-721 that is detected will be produced by the Th17 cells, and not by the endothelial cells. After inducing ischemia, it is observed that the chimeras reconstituted with BM BL/6 CD69KO have a more efficient revascularization process than the BM BL/6 CD69WT chimeras (
miR-721 levels as well as levels of the target genes Meox-2 and CUX1 have also been studied in these mice. Like in BALB/c mice, miR-721 is induced 7 days after ischemia in the BM BL/6 CD69KO chimeras but not in the BM BL/6 CD69WT chimeras (
While Meox-2 and CUX1 gene expression is completely inhibited in the muscle tissue from BM BL/6 CD69KO chimeras, BM BL/6 CD69WT chimeras express high levels of these genes after 7 days of ischemia. This data indicates that high miR-721 expression in the serum of CD69KO chimeras is correlated to complete inhibition of its Meox-2 and CUX1 gene targets in muscle tissue (
According to the results obtained, CD69-deficient naive T CD4+, T regulator and TH17 lymphocytes have changed the expression pattern of various microRNAs. The CD69 molecule limits the severity of cardiac dysfunction in the model of experimental autoimmune myocarditis (EAM) by means of controlling TH17 responses. To that end, this model is used to investigate the role of the previously mentioned miRNAs in this disease. The murine model of EAM is induced by means of immunization with a peptide derived from the α-myosin heavy chain (MyHCα) specific for heart tissue emulsified with CFA (Complete Freund's Adjuvant), which causes effector TH17 cell activation and differentiation. 21 days after immunization, the mice are in the acute phase of the disease, presenting inflammation in the myocardium and anti-cardiac myosin antibodies in serum. The chronic phase appears after 56 days, characterized by fibrosis and degeneration of the myocardium and, accordingly, the development of cardiomyopathies.
To validate the model of EAM with a non-invasive method, the immune response generated in the acute phase of EAM was characterized 21 days after immunization with the MyHCα peptide. To that end, an intravenous fluorescent probe detecting activated elastase and therefore neutrophils and inflammation foci was injected, and the mice were examined by means of fluorescence molecular tomography (FMT). With this technique, it was confirmed that CD69KO mice presented greater inflammation in the heart than WT mice (
3.1. miRNA Expression Levels in the Heart
To investigate miRNA expressions during the acute phase of the model of EAM, after examining inflammation of the myocardium by means of fluorescence molecular tomography on day 21, the mice were sacrificed and the heart was extracted (
Analysis of miRNA expression levels present in the heart of the mice during the acute phase of EAM showed a significant increase in miR-721 expression in the heart of sick WT and CD69KO mice with respect to the control mice. In contrast, no significant differences in miR-155 expression levels in the hearts of the animals were found (
3.2. miRNA Expression Levels in Serum
For the purpose of finding a microRNA detectable in blood that can be used as a diagnostic biomarker, as marker of the severity of myocarditis and/or as a therapeutic target of the disease, the miRNA expression pattern in the serum of mice with experimental autoimmune myocarditis was studied. To that end, blood was extracted from the mice during the acute phase of EAM and miRNA levels present in the serum were analyzed by means of quantitative PCR. The result obtained was a significant increase in miR-721 and miR-155 in the serum of sick mice with respect to the control animals. Furthermore, it was found that CD69KO mice with myocarditis showed greater miR-721 expression in serum than WT mice treated with the peptide MyHC animals and greater than the controls, suggesting that this miRNA could in fact be related to an increase in TH17 and therefore related to the severity of the disease (
Myocarditis is characterized by inflammatory cell infiltration into the myocardium. After the acute process, the loss of cardiomyocytes and the development of fibrosis and necrosis in the myocardium take place, ventricle remodeling, permanent dysfunction of the ventricular wall, dilated cardiomyopathy, heart failure and arrhythmias being able to arise. For the purpose of studying the role of T CD4+ lymphocytes in the development of the disease in humans and the potential thereof as therapeutic targets, these populations in the blood of patients with myocarditis and dilated cardiomyopathies were analyzed in comparison with healthy donors. Furthermore, the microRNA expression levels detected in the animal model of experimental autoimmune myocarditis in the blood plasma of patients and control subjects were measured to find a detectable miRNA in blood that could be used as a diagnostic biomarker and/or as a therapeutic target of the disease.
To quantify the percentage of total T CD4+ lymphocytes circulating in the blood of patients and controls, lymphocytes were isolated from EDTA-anticoagulated peripheral blood by means of a density gradient, and their membrane markers were analyzed by flow cytometry. The analysis showed a statistically significant increase in the proportion of total T CD4+ cells in the blood of patients with acute myocarditis or Takotsubo with respect to that of healthy subjects (
To analyze the percentages of TH17 cells, the lymphocytes were isolated from peripheral blood by means of a density gradient, activated with anti-OKT3, restimulated with PMA, ionomycin and brefeldin and labeled with anti-CD4 and anti-IL-17. Analysis by flow cytometry showed that in total T CD4+ cells, the percentages of TH17 lymphocytes in the blood of patients with chronic myocarditis or Takotsubo cardiomyopathy are similar to those of healthy controls, whereas patients with acute myocarditis have a proportion of TH17 lymphocytes in blood that is significantly higher than that of healthy volunteers (
Analysis of the percentages of regulatory T cells present in the blood of patients with myocarditis and dilated cardiomyopathies comprised the study of total Treg cells, naive Treg cells and memory Treg cells. To that end, the lymphocytes extracted from the blood were activated with OKT3, and the CD4+ CD25+ FOXP3+ (total Treg cells), CD4+ CD25+ FOXP3+ CD45RA+ CD45R0− (naive Tregs) and CD4+ CD25+ FOXP3+ CD45RA− CD45RO+ (memory Tregs) populations were analyzed. The result obtained was that patients with Takotsubo do not present statistically significant differences in relation to the percentages of regulatory T cells in peripheral blood with respect to healthy volunteers. However, patients with both acute and chronic myocarditis present percentages of total Treg cells that are significantly lower than those of control subjects, probably due to a significant reduction of the memory Treg cell population in these patients, because the naive Treg cell population is not affected in these patients. (
4.4. miRNA Expression Levels in Blood Plasma
Finally, for the purpose of validating this study in patients with cardiopathies, the miRNA expression levels detected in the murine model of EAM, in the blood plasma of healthy subjects and patients with acute myocarditis, chronic myocarditis and Takotsubo syndrome were analyzed by extracting total RNA and quantifying the expression thereof by means of qPCR. The trend that was observed was an increase in miR-155 expression levels, and especially in miR-721 expression levels, in patients with cardiomyopathies with respect to healthy subjects, this being higher in patients with acute myocarditis than in those patients in the chronic phase of the disease, and particularly in patients with Takotsubo (
The tables at the end of the present specification illustrate the amounts of sample of used in the present experiments to detect miRNA in the serum of mice with ischemia, myocarditis or in patients with myocarditis, and the comparison of the fold induction between these samples.
5.1. miRNA Expression in Mice after Myocarditis and AMI Induced by Coronary Descending Artery Ligation (LAD).
Based on our previous results in the EAM model and a small cohort of 14 patients with different cardiomyopathies (acute and chronic myocarditis and Takotsubo) and healthy subjects, we found that miR-721 and miR-155 microRNAs derived from Th17 cells and Treg cells respectively, are present in the development of Myocarditis. We have identified and cloned the human miR-721 that we detected with the murine probe for miR-721, moreover we have identified Th17 cells as main producers of miR-721 in mice. As Th17 and Tregs are pivotal in Myocarditis development we found high levels of these miRNAs, when the symptoms appear in patients with Myocarditis compared to Healthy volunteers (
We have analyzed the Th17 cells in mice after coronary descending artery ligation or LAD simulating an Acute Myocardial infarction. We have found that Th17 cells appear in the blood of the mice after two weeks of the ischemic event (when the symptoms appear) and this is consistent with elevated miR-721 levels 15 days after the infarct (
All these data suggest that Myocarditis mice could be diagnose by presence of miR-721 in plasma or serum, whereas AMI mice do not.
Some Acute Myocarditis can mimic AMI symptoms in the first hours and therefore we proposed miR-721 detection in blood as a bona-fide diagnostic tool for Myocarditis patients.
5.2. miRNA Profiling in the Exosomal Fraction of Th17 Cells.
Because of the fact that miR-721 is found in serum of myocarditis mice and patients we estimated it could be coated into exosomes.
So far, the content of selected miRNAs was analyzed by qPCR in exosomes isolated from plasma after PEG (polyethylene glycol) precipitation. We first analyzed the content of miR-721 within exosomes secreted from EAM lymph node cells in mice with moderate myocarditis (WT) and severe myocarditis (CD69−/−) as described in Cruz-Adalia et al, Circulation 2010. First, we collected dLN from wild type and CD69−/− mice 6 days after EAM induction and we cultured them for 48 h in an exosome-free medium (
In this setting we proved that miR-721 is expressed in the exosomal fraction of EAM draining lymph nodes (
We have recruited patients aged from 20 to 80 years.
Patients with Myocarditis:
we will recruit patients of acute myocarditis with at least one of the following parameters: chest pain, elevation of the ST segment, elevated Troponin T or I, low ejection fraction. Although we have also included in the viral cardiomyopathy, Chagas and Takotsubo study and dilated heart diseases (chronic myocarditis).
Patients with AMI:
The patients will be divided into three groups: 1) non-ST elevation acute coronary syndrome (NSTACS) group, confirmed by the lack of ST segment elevation and a significant rise of creatine kinase MB and troponin I levels, unstable angina confirmed by chest pain at rest with definite ischemic proof including ST segment changes and/or T-wave inversion, and angiographic evidence of coronary artery stenosis (>70%)), 2) chronic stable angina (CSA) group; inclusion criteria, effort angina (without a previous history of unstable angina or myocardial infarction) and angiographic evidence of coronary artery stenosis (>70%), and 3) chest pain syndrome (CPS) group, inclusion criterion, chest pain with no accompanying electrocardiographic changes, coronary artery stenosis, or coronary spasm. None of the patients will be currently treated with anti-inflammatory drugs such as non-steroidal anti-inflammatory drugs, steroids, etc.
5.4. Data Base of Clinical Parameters from the Patients.
The anonymous samples received from the different hospital were listed in a data base were the clinical data (Heart function: ECG & Echocardiography, Heart damage: TPI, CK-MB . . . , Magnetic Resonance Imaging with gadolinium enhancement, Dyslipidemia and others) of the patients was specified. An example of the clinical parameters and a summary of the data is represented in
5.5. The Following Blood Samples Parameters have been Analyzed:
—Analysis of Th17 and Tregs in Patients.
Analysis has been performed from at least 2 ml of blood. 1 ml for plasma analysis and 1 ml for peripheral blood leukocutes (PBLs) analysis after Ficoll treatment.
PBLs from healthy donors or patients with Myocarditis or AMI were collected and analyzed by flow cytometry to analyze the proportions in circulating Treg and Th17, Th1 and Th22 cells. Peripheral blood CD4+CD25+FOXP3+CD45RO-CD45RA+naïve Treg (nTreg) cells and CD4+CD25+FOXP3+CD45RO+CD45RA− memory Treg (mTreg) cells in CVD patients has been compared to healthy volunteers.
The response of the innate immune system seems to work the same way in both cardiomyopathies. Myocarditis and AMI patients have lower levels of monocytes and higher levels of granulocytes compared to healthy volunteers (
—miRNA Profiling in Plasma Samples of Patients.
miRNA expression levels were analyzed in plasma from patients by qPCR with exiqon probes for the indicated miRNAs. The microRNA content was evaluated by real-time quantitative-PCR. The data were first normalized versus an artificial microRNA (UniSp5, Ambion) equally added to each sample before the RNA isolation to minimize the effect of differences in the RNA purification yield among samples, following the standard method of 2−ΔCt. Hsa-miR-423-3p was proposed as the most stable miRNA among our samples by two freely-accessible online softwares: NormFinder and GeNorm. UniSp5-normalized samples were then normalized versus hsa-miR-423-3p. Taking the healthy volunteers as the control group, the fold increase in the normalized miRNA levels in the AMI and myocarditis patients was then calculated to evaluate the differences among groups (
Plasma samples from Myocarditis patients have significantly increased levels of miR-721 compared to healthy volunteers whereas plasma samples from AMI patients do not, indicating that miR-721 is a good biomarker for Myocarditis patients (
—miRNA Profiling in the Exosomal Fraction of Plasma Samples from Patients.
We analyzed miR-721 expression into exosomas from plasma samples from healthy volunteers and AMI and Myocarditis patients. With the number of samples analyzed until today, our data suggest that there is increased expression of miR-721 in the exosomal fraction of patients with myocarditis compared to the other groups (
With the data obtained from mice and patients with AMI and Myocarditis we proposed miR-721 as a bona-fide biomarker for Myocarditis diagnosis exclusive to other ischemic cardiomyopathies as Acute Myocardial Infarction. Along myocarditis progression myocardial damage released heart antigens and a specific memory Th17 and Regulatory T cells arises within the first weeks of disease progression. At the moment of the acute symptoms of Myocarditis the Th17 response has taken place and the levels of this population are enhanced in blood. On the contrary, the acute symptoms of the AMI patients takes place in the first hours after the coronary artery ischemia, at that moment no Th17 is developed against heart antigens. The different nature of these two cardiomyopathies will allow us to differentially diagnose them by analyzing the levels of miR-721 in the plasma samples. We have identified miR-721 as exclusively expressed in Th17 cells that is released to the plasma into exosomes from Myocarditis patients, compared to AMI patients and healthy volunteers. The diagnostic approach is outlined in
WT and CD69−/− double reporter (FoxP3-RFP, IL-17-eGFP) mice in C57BL/6 background were used for ex-vivo polarization assays and Th17/Treg/Th22 progression analysis during EAM. BALB/c mice were used as a background for the mixed bone marrow chimeras. All animals maintained at Animal facility of CNIC. All animal procedures were approved by the ethics committee of the Comunidad Autónoma de Madrid and were conducted in accordance with institutional guidelines that comply with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.
Mice were immunized subcutaneously on days 0 and 7 with 100 μg/0.2 mL MyHC-α peptide (MyHC-α614-629) Ac-RSLKLMATLFSTYASADR-OH emulsified 1:1 in complete Freund adjuvant (1 mg/mL; H37Ra; Difco Laboratories, Detroit, Mich.). Animals were sacrificed on day 6 to extract draining lymph nodes for ex-vivo culture or on either days 20-21 or 50-52 to analyze the acute and chronic phase of the disease respectively.
Single cell suspension prepared from mouse axillar and inguinal lymph nodes in FACS buffer (PBS, 0.5% BSA, 1 μM EDTA, 0.1% NaN3). Peripheral Blood Multinucleated Cells isolated from mouse or human blood samples using Ficoll-Isopaque (density=1.121 g/ml) gradient centrifugation. Cell suspensions were stained with fluorochrome-conjugated either human or mouse specific antibodies against CD4, CD3, CD25, CD45RO, CD45RA, IL-17a, IL-22, IFNγ and FoxP3 obtained from eBiosciences. For intrancellular staining, cells were stimulated with 50 ng/ml phorbolmyristate acetate (PMA) and 750 ng/ml ionomycin (P+I) plus brefeldin A (1 μg/ml) in complete culture medium for 3 hrs. Cells were analyzed by BD FACSCanto/FACSFortessa Flow Cytometers. Ex-vivo culture and Treg/Th17 cell isolation. Cell suspensions from lymph nodes were cultured (2×107 cells/nil) during 48 h in exosome-free TexMACS media (Miltenyl Biotech) with MyHC peptide (10 ug/ml) and IL-23 (10 ng/ml). Afterwards, sorting for FoxP3RFP and IL-17eGFP using the BD FACs Aria II cell sorter/iCyt Synergy 4L cell sorter. Sorted cells were cultured again in TexMACS media 48 h with MyHC peptide and either IL-2 for Foxp3+ cells (Treg) or IL-23 for IL-17+ cells (Th17).
MiRNAs were extracted using miRNeasy mini kit (Qiagen) and was screened for purity and concentration in a Nanodrop-1000 Spectrophotometer (Thermo Scientific). Analysis of miRNA-721, sno-135, sno-202, let-7a, let-7b and let-7d was done using TaqMan MicroRNA Reverse Transcription Kit and individual TaqMan MicroRNA probes for retrotranscription. TaqMan Universal PCR Master Mix was used for Real-time quantitative PCR (Applied Biosystem). Analysis of miRNA expression was done using SDS software (Applied Biosystems) and relative expression checked by 2-Δ ΔCT method. Sno-135 and sno-202 were used as endogenous control for serum, cell and tissue samples; and let-7a, let-7b and let-7d for isolated exosomal fractions. Real-time quantitative Polymerase Chain Reaction (RT-qPCR) was performed on ABI Prism 7900HT 384 (Applied Biosystems).
Supernatants from pre-sorted lymph nodes cells or post-sorted Tregs/Th17 were collected after 48 h. Briefly, rest of cells were pelleted (2000 rpm for 10 min). For miRNA isolation, exosomes were precipitated by adding 0.4 volumes of 50% Polyethylene glycol 6000 (Sigma Aldrich) in 375 mM NaCl to the samples, storing 30 min at 4° C. and centrifugation 1500 g at 4° C.
Mice were anaesthetized by inhalation of isoflurane/oxygen (1.25%/98.75%). For echocardiography, mice were examined by a 30 MHz transthoracic echocardiography probe and images were obtained with Vevo 770 (VisualSonics, Toronto, Canada) before EAM induction and during acute and chronic phases. From these images, left ventricle (LV) function was estimated by ejection fraction obtained from the M mode. For Magnetic Resonance Imaging with T1 and T2 mapping, late gadolinium enhancement was used via intravenous injection. Up to 13 slices of each heart were imaged and myocardial water content was evaluated by averaging 3-5 ROI (brightness) values all along each myocardial slice and normalizing with the brightness of the gadolinium in the lumen of both left and right ventricles.
Hearts were fixed in 10% phosphate-buffered formalin and embedded in paraffin. Sections were cut and stained with Haematoxilin-eosin to determine the level of inflammation and with Masson's trichrome to detect collagen deposition. Histological analysis was performed on a Nikon Eclipse 90i microscope. H&E stained sections were scored for inflammation as follows: 0, no inflammatory infiltrates; 1, small foci of inflammatory cells; 2, larger foci of >100 inflammatory cells; 3, 10-30% of a cross section occupied by inflammatory cells; 4, >30% of a cross section occupied (Cruz-Adalia et al, 2010).
All the variables are expressed as Mean±Standar Deviation of the Mean (SEM). Statistical analysis was performed assessing first for the normality of the distributions using Kolmogórov-Smirnov test. If normal, unpaired Student t-test for two groups and one-way ANOVA analysis for multiple comparisons. If not normal distributions, U-Mann-Whitney test for two groups analysis and Kruskal-Wallis test for multiple comparisons. All the analyses were performed with GraphPad Prism 6.0 software.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15197221.3 | Dec 2015 | EP | regional |
| 15382596.3 | Dec 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/079335 | 11/30/2016 | WO | 00 |
