Study subjects
Biopsies from the pectoralis of MG patients were collected during thymectomies (Marie Lannelongue Hospital, Le Plessis Robinson). RNA was extracted, and the quality controlled as described below. When the RNA was not high quality, it was excluded. Finally, three positive MG patients with common features were included in the microarray experiments. These patients were young (24-, 26-, and 27-year-old) females, positive for anti-AChR antibodies (6, 10, and 27 nM), with a generalized form disease (IIB in 2 cases, and IIIB in 1 case), and untreated by corticosteroids. Since muscle controls from age- and sex-matched individuals were not available at the time of the microarray experiments, a pool of RNA from muscle biopsies from healthy adults (reference HT1008) was provided by Origene Technology (Rockville, MD, USA).
In order to test the IL-6 protein levels in the muscles, another set of muscle biopsies was collected from SPMG patients (12–49 years old; 6 females and 2 males, from Ia–IVb) and control patients (17–51 years old; 5 females and 2 males). The muscle biopsies from MG patients were collected during thymectomy when the patients were in a clinical stable status, and they were rarely very severely affected. Only one patient had a very severe form (IVb). The muscles from the control patients were collected during cardiac surgery, enabling sampling of the same muscle (pectoralis) as in the MG patients. None of the control patients had other skeletal muscle diseases. Thymus histologies from these MG patients ranged from normal (1) to involuted thymus (1) and follicular hyperplasia (4).
For the effects of sera on IL-6 production, 6 SPMG patients (6 females; age range: 18–34 years, 1 with MG severity IIA, 4 with IIB, and 1 with IIIA) and 6 seronegative MG (SNMG) patients (5 females and 1 male; age range: 19–55 years, 3 with MG severity I, 2 with IIB, and 1 with IIIB) were included. SNMG patients were also negative for anti-MuSK antibodies. Patients on corticosteroid treatment were excluded from the study. Sera from 6 healthy controls, aged 18–45 years, were obtained from the French blood bank. Sera from 6 patients suffering from other muscle diseases were also used: 2 with glycogen storage disease, type III, 1 fascio-scapulo-humeral dystrophy, 1 desminopathy, 1 neuroectodermosis, and 1 spinal amyotrophy.
Ethics and consent
All the procedures were approved by the local ethics committee “Comité de Protection des Personnes (CPP)”, Kremlin-Bicêtre, France (agreement number 06–018). The muscle biopsies were obtained from MG and non-MG patients who signed an informed consent.
Animals and antigen preparation
Female Lewis rats aged 6–7 weeks were obtained from the Animal Breeding Center of The Weizmann Institute of Science, and were maintained in the Institute’s animal facility. Female C57Bl/6 J mouse aged 5 weeks were obtained from Janvier laboratories and acclimatized one week in the animal facility (University Pierre and Marie Curie) prior to immunization. All of the experiments in this study were performed according to the institutional guidelines for animal care. Torpedo AChR was purified from the electric organ of Torpedo californica by affinity chromatography, as previously described [17].
Induction and clinical evaluation of EAMG
To induce EAMG, rats were immunized once in both hind footpads via a subcutaneous injection of Torpedo AChR (40 μg/rat) emulsified in complete Freund’s adjuvant (CFA) supplemented with additional non-viable Mycobacterium tuberculosis H37RA (0.5 mg/rat; Difco Laboratories, Detroit, MI, USA). The control rats were immunized with CFA and H37RA. Clinical signs of EAMG were monitored on alternate days for 8–10 weeks following disease induction, as previously described [15].
Six-week female mice were immunized by subcutaneous injections in both hind footpads and in the back with Torpedo AChR (30 μg/mouse) emulsified in CFA supplemented with H37RA (1 mg/mouse). Control mice were immunized with CFA and H37RA. Approximately 30 days later, the mice received a subcutaneous boost in the back of the same amount of TAChR in CFA, without additional H37RA; the control mice received a similar boost. The mice were monitored for muscle force and weakness every 10 days. A global score based on the animals’ weights, grip force, and ability to remain on an inverted grid was calculated to quantify their clinical state. Each of these three parameters was graded on a scale of 0–3 to yield a final score on 9, where 0 corresponded to healthy mice and 9 corresponded to severely affected mice.
Microarray experiments
Strategy of the microarray
We adopted a strategy previously used for MG thymus analysis using pools of thymic tissues from homogeneous groups of patients [13,18]. Many of the deregulated genes identified by this approach were then validated in biological studies, such as CXCL13 [19], IFNs [12], and CCL21 [14]. By using pools of muscle tissue instead of individual tissue, we focused our analysis on the primary common changes instead of individual changes. This strategy was validated by our biostatistian (GC). Another advantage of using pools is the ability to perform several technical replicates (quadruplicates in the current study), which is impossible with individual tissue given limitations of both tissue and money. Indeed, performing technical replicates is important to strengthen the results since manipulation of a high number of normalized data can lead to a significant rate of false-negative results.
GeneChip probing and analysis
Rat muscle samples
Muscle samples were harvested from rats when they reached a clinical score of 2 [15]. Since the disease is induced in the hind legs, the thigh muscles that are also affected were used for the extraction of total RNA using the RNeasy midi kit (Qiagen GmbH, Hilden, Germany). Two RNA samples were used for each group, and each sample consisted of a pool from three individual rats.
The GeneChip RG-U34A arrays (Affymetrix, Santa Clara, CA, USA) containing probes for 8000 rat genes and 1000 ESTs were used to screen and quantify the mRNA transcript level in rat thigh muscle samples. Probing and analysis of these samples were performed at the Weizmann Institute microarray unit, as previously described in the literature [15]. Genes showing a fold change greater than 2 were selected for further evaluation.
Human muscle samples
Total RNA from muscles of MG patients or from muscle controls (Origene Technology) was extracted using the Trizol reagent (Gibco, Paisley, Scotland) and purified, as previously described in the literature [18]. The sample concentration and purity was first assessed using the NanoDrop spectrophotometer. Then the quality control to assess the sample integrity was checked on an Agilent Bioanalyser (Massy, France). For microarray analysis. only high quality RNAs with RIN (RNA integrity number ) higher than 7, in a scale ranging from from 1 (totally degraded RNA) to 10 (completely intact RNA) were used. Twenty μg of total muscle RNA was labeled with cyanine 5 or cyanine 3 using the direct labeling protocol of Agilent optimized for their cDNA chips, as previously described [19]. For each array, the control muscle RNA was crossed with RNA from MG muscle and these comparisons were conducted in quadruplicate. All of the procedures have been detailed elsewhere [19,13]. Briefly, the labeled cDNA was hybridized overnight onto the human 1 cDNA arrays from Agilent (G4100A; 12,814 unique clones) and scanned using a 428 Affimetrix scanner (MWG Biotech). The images were analyzed with a GenePix pro V4.0 (Axon Instruments). The raw data were then corrected by a non-linear transformation (the Lowess algorithm) using a TIGR Microarray Data Analysis System (http://www.tm4.org/midas.html). A statistical tool “Significance analysis of microarrays (SAM)” was used to identify the gene hit lists that were differentially expressed in human muscle MG compared with control muscle [20].
Expression analysis
The gene hit lists established in parallel in humans and rats were then submitted to two bioinformatic resources, as previously described in the literature [13]. These two resources provide different types of information:
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1.
GOTree Machine (GOTM) is a web-based platform for interpreting microarray data or other interesting gene sets using Gene Ontology hierarchies (http://bioinfo.vanderbilt.edu/webgestalt/) [21]. Statistical analysis with relatively enriched gene numbers can suggest biological areas that warrant further study. GOTM generates a GOTree, a tree-like structure to navigate the gene ontology directed acyclic graph for input gene sets. GOTM reports enrichments that are statistically significant, as determined by a hypergeometric test.
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2.
Ingenuity Pathways Analysis identifies pathways that are overrepresented (https://analysis.ingenuity.com/pa Ingenuity Mountain View, CA, USA) [22,23].
Quantitative real-time PCR
Quantitative real-time PCR (Q-RT-PCR) on rat and mouse samples was performed using a LightCycler (Roche diagnostic) apparatus, as previously described in the literature [15]. Each sample was run in duplicate and the mean values were used for calculations. The expression levels of β-actin and GAPDH were monitored in all rat samples and were found to be similar. The primers were as follows: rat IL-6 forward: 5′-ctagtgcgttatgcctaag-3′, IL-6 reverse: 5′-ccatctggctaggtaaca-3′; rat IL-6R forward: 5′-ctgaatagagatgcccgt-3′, IL-6R reverse: 5′-gtcactcgcgtaaacc-3′; rat GAPDH forward: 5′-ccaaggagtaagaaaccc-3′. GAPDH reverse: 5′-ggtgcagcgaactttat-3′; rat β-actin forward: 5′-tactgccctggctcctagca-3′; β-actin reverse: 5′-tggacagtgaggccaggatag-3; mouse IL-6 forward: 5′-agttgccttcttgggactga-3′; IL-6 reverse: 5′-tccacgatttcccagagaac-3′; mouse IL-6R forward: 5′-agggtgtctgcttcctgcta-3′; IL-6R reverse: 5′-catctgaggccactcagtca-3′; mouse Rpl32 forward: 5′-caccagtcagaccgatatgtgaaaa-3′; Rpl32 reverse: 5′-tgttgtcaatgcctctgggttt-3′.
Cell culture
For the cellular experiments, we used immortalized cultures of human myoblasts. These cells, called LHCNM2, were previously derived from the pectoralis major muscle of a 41-year-old male Caucasian heart-transplant donor and immortalized by introduction of human telomerase and cyclin-dependent protein kinase 4 [24]. The cells were cultured in medium containing four parts Dulbecco’s modified Eagle’s medium (DMEM; 4.5 mg/ml glucose) and one part medium 199, supplemented with 20% fetal bovine serum. For the differentiation studies, the cells were trypsinized, counted, and 50,000 cells were plated on to a 48-well plate. After 6–7 days, when the cells had become confluent, the proliferation medium was removed and replaced with DMEM (Gibco) supplemented with 10 mg/ml bovine insulin (Sigma-Aldrich, Saint-Quentin Fallavier France) and 100 mg/ml of human apo-transferrin (Sigma-Aldrich), as previously described in the literature [24].
Cell stimulation
The LHCNM2 cells were plated in 48-well plates (50,000 cells/well). After 24 h, the medium was replaced with a medium containing the MG patient sera (diluted 1/100 in the regular medium) or monoclonal antibodies directed against AChR. Four different antibodies were used: mAb 198 (IgG2a isotype), mAb 35 (IgG1 isotype), mAb 155 (IgG2a isotype) [25], and anti-AChR (IgG1 isotype) from Acris Antibodies GmbH (reference SM1445). The rat IgG2a and IgG1 isotype controls (clones 54447 and 43414) were purchased from R&D Systems, Inc. (Minneapolis, MN). The results were normalized to the control sera or the relevant isotype control, respectively. The monoclonal antibodies and their isotype controls were used at a concentration of 3 μg/ml.
Cell proliferation
Cell proliferation was assessed by flow cytometry using Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) dye, as previously described in the literature [26]. Briefly, LHCNM2 cells were treated with CFSE (5 μM/1 × 106 cells) (Molecular Probes, Interchim, France) for 10 min at 37°C. After washings, the cells were seeded in 24-well plates, allowed to attach for 24 h, and then incubated with monoclonal Abs. The cells were then collected after 24, 48, and 72 h incubation and acquired on a FACScalibur (BD Biosciences, Le Pont de Claix, France). The cytometry analysis was completed using Flowjo software Tree Star, Inc. (Ashland, OR, USA).
ELISA assays
Enzyme-linked immunosorbant assay (ELISA) was performed on culture supernatants, as well as on muscle extracts to measure their IL-6 content. The frozen muscle biopsies from patients were thawed and homogenized in extraction buffer (Tris–HCl pH8 20 mM, NaCl 137 mM, glycerol 10%, NP-40 1%, EDTA 2 mM) supplemented with proteinase inhibitor cocktail (Complete, Roche, France). The homogenates were then centrifuged and the supernatant was kept at −80°C until analysis.
All of the reagents for the ELISA were from Immunotools (Friesoythe, Germany) and used according to the manufacturer’s instructions. Briefly, the plates were coated overnight with the anti-IL-6 antibody at 4°C and washed 5 times. The aspecific sites were blocked for 1 h at 37°C. The plates were washed 5 times and then samples were added, incubated for 1 h at 37°C, and washed 5 more times. The biotinylated IL-6 antibody was added, incubated 1 h at 37°C, and washed 5 times. Streptavidin-HRP was incubated 20 min at room temperature, and washed 5 times. TMB substrate was allowed to incubate for 5 min before adding the stop solution. Optical density (OD) was read at 450 nm in an MRX Revelation ELISA plate reader (Dynex Technologies, Inc. Chantilly, VA, USA). A standard curve was obtained with serial dilutions of IL-6 and the results were expressed in pg/ml. The cytokine concentration was calculating using the mean of the duplicate measurements for each sample.
Analysis of Akt phosphorylation
Akt phosphorylation was assessed by a Western Blot. The LHCN myotubes and myoblasts were treated for two days with AChR antibodies or control isotype. On the day of the analysis (differentiation day 7 (D7) for the myotubes), they were rinsed and incubated in serum-free medium for 3 h, before being treated with insulin for 10 min. The LHCN cells were rinsed with phosphate buffered saline and the proteins were then extracted with an extraction buffer (Tris HCl pH8 20 mM, NaCl 137 mM, glycerol 10%, NP-40 10%, EDTA 2 mM) supplemented with antiprotease and antiphosphatase mixes (Complete Mini and PhosSTOP, respectively, Roche, France). Cellular debris was eliminated by centrifugation at 14,000 rpm for 20 min at 4°C. The protein extracts were kept at −80°C prior to analysis.
The proteins were thawed on ice, denatured in Laemmli buffer at 95°C for 5 min, separated by polyacrylamide gel electrophoresis (Precise Tris-Hepes gels, Pierce, France), and transferred to a nitrocellulose membrane. The protein transfer was evaluated using Ponceau staining. The membrane was saturated with 5% of milk proteins in Tris buffered saline with 0.05% Tween (TBST), incubated with anti-pAkt (Ser 473) rabbit antibody (dilution 1/500 to 1/1000) (Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C, washed three times in TBST, incubated for one hour in secondary antibody (anti-rabbit HRP, 1/10000, in 1% of milk in TBST), washed three times in TBST, and revealed with electrochemiluminescence (ECL) Prime on autoradiography films (Amersham, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The process of immunodetection was repeated with an anti-Akt antibody (Cell Signaling Technology) to assess total Akt. Band intensities were evaluated using Fiji Is Just ImageJ.
Statistical analysis
For each data set, the normality of the samples was tested using three normality tests (Kolmogorov-Smirnov, D’Agostino and Pearson omnibus, and Shapiro-Wilk). If the samples were characterized by a Gaussian distribution, the significance of the results was analyzed using a Student’s t-test or ANOVA (more than 2 groups). Otherwise, nonparametric tests were used: Mann–Whitney for comparison of 2 groups and Kruskal-Wallis non-parametric ANOVA for comparison of 3 groups or more. All analyses were done using GraphPad software (GraphPad, San Diego, CA, USA).