Dermatomyositis With or Without Anti-Melanoma Differentiation-Associated Gene 5 Antibodies
2016; Elsevier BV; Volume: 186; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2015.11.010
ISSN1525-2191
AutoresYves Allenbach, Gaëlle Leroux, Xavier Suárez‐Calvet, Corinna Preuße, Eduard Gallardo, B. Hervier, Aude Rigolet, Anne‐Sophie Moreau, Debora Pehl, Nicolas Limal, Peter Hufnagl, Norman Zerbe, Alain Meyer, Jessie Aouizerate, Y. Uzunhan, Thierry Maisonobe, Hans‐Hilmar Goebel, Olivier Benveniste, Werner Stenzel, A. Hot, A. Grados, N. Schleinitz, Laure Gallet, Nathalie Streichenberger, Philippe Petiot, É. Hachulla, David Launay, H. Devilliers, M. Hamidou, Divy Cornec, Boris Bienvenu, Vincent Langlois, H. Lévesque, Aurélien Delluc, Laurent Drouot, Jean‐Luc Charuel, F. Jouen, Norma B. Romero, O. Dubourg, Sarah Léonard-Louis, Anthony Béhin, Pascal Laforêt, T. Stojkovic, B. Eymard, N. Costedoat‐Chalumeau, Emmanuelle Salort‐Campana, Anne Tournadre, Lucile Musset, Brigitte Bader‐Meunier, Isabelle Koné‐Paut, Jean Sibilia, Laurent Servais, Olivier Fain, C. Larroche, Elizabeth Diot, Benjamin Terrier, Raphaël De Paz, Antoine Dossier, Dominique Ménard, Chafika Morati, M Roux, Xavier Ferrer, J. Martinet, Sophie Besnard, Rémi Bellance, P. Cacoub, David Saadoun, Laurent Arnaud, B. Grosbois, S. Herson, Olivier Boyer,
Tópico(s)Muscle Physiology and Disorders
ResumoThe anti-melanoma differentiation-associated gene 5 (MDA5) autoantibody is specifically associated with dermatomyositis (DM). Nevertheless, anti–MDA5+-patients experience characteristic symptoms distinct from classic DM, including severe signs of extramuscular involvement; however, the clinical signs of myopathy are mild or even absent. The morphological and immunological features are not yet described in adulthood. Data concerning the pathophysiology of anti-MDA5 DM are sparse; however, the importance of the interferon (IFN) type I pathway involved in DM has been shown. Our aim was to define morphological alterations of the skeletal muscle and the intrinsic immune response of anti–MDA5-positive DM patients. Immunohistological and RT-PCR analysis of muscle biopsy specimens from anti-MDA5 and classic DM were compared. Those with anti-MDA5 DM did not present the classic features of perifascicular fiber atrophy and major histocompatibility complex class I expression. They did not show significant signs of capillary loss; tubuloreticular formations were observed less frequently. Inflammation was focal, clustering around single vessels but significantly less intense. Expression of IFN-stimulated genes was up-regulated in anti-MDA5 DM; however, the IFN score was significantly lower. Characteristic features were observed in anti-MDA5 DM and not in classic DM patients. Only anti-MDA5 DM showed numerous nitric oxide synthase 2–positive muscle fibers with sarcoplasmic colocalization of markers of regeneration and cell stress. Anti–MDA5-positive patients demonstrate a morphological pattern distinct from classic DM. The anti-melanoma differentiation-associated gene 5 (MDA5) autoantibody is specifically associated with dermatomyositis (DM). Nevertheless, anti–MDA5+-patients experience characteristic symptoms distinct from classic DM, including severe signs of extramuscular involvement; however, the clinical signs of myopathy are mild or even absent. The morphological and immunological features are not yet described in adulthood. Data concerning the pathophysiology of anti-MDA5 DM are sparse; however, the importance of the interferon (IFN) type I pathway involved in DM has been shown. Our aim was to define morphological alterations of the skeletal muscle and the intrinsic immune response of anti–MDA5-positive DM patients. Immunohistological and RT-PCR analysis of muscle biopsy specimens from anti-MDA5 and classic DM were compared. Those with anti-MDA5 DM did not present the classic features of perifascicular fiber atrophy and major histocompatibility complex class I expression. They did not show significant signs of capillary loss; tubuloreticular formations were observed less frequently. Inflammation was focal, clustering around single vessels but significantly less intense. Expression of IFN-stimulated genes was up-regulated in anti-MDA5 DM; however, the IFN score was significantly lower. Characteristic features were observed in anti-MDA5 DM and not in classic DM patients. Only anti-MDA5 DM showed numerous nitric oxide synthase 2–positive muscle fibers with sarcoplasmic colocalization of markers of regeneration and cell stress. Anti–MDA5-positive patients demonstrate a morphological pattern distinct from classic DM. Dermatomyositis (DM) is a group of idiopathic inflammatory myopathies defined by skin rash, proximal weakness, and specific pathological muscle features.1Hoogendijk J.E. Amato A.A. Lecky B.R. Choy E.H. Lundberg I.E. Rose M.R. Vencovsky J. de Visser M. Hughes R.A. 119th ENMC international workshop: trial design in adult idiopathic inflammatory myopathies, with the exception of inclusion body myositis, 10–12 October 2003, Naarden, The Netherlands.Neuromuscul Disord. 2004; 14: 337-345Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar Characteristically, these encompass the following: perivascular inflammation in the perimysium, perifascicular fiber atrophy, sarcolemmal major histocompatibility complex (MHC) class I staining with a perifascicular enhancement, and microvascular pathology, including capillary enlargement, capillary loss, deposition of membrane attack complex (C5b-9) on vascular endothelial cells, and tubuloreticular inclusions in endothelial cells.1Hoogendijk J.E. Amato A.A. Lecky B.R. Choy E.H. Lundberg I.E. Rose M.R. Vencovsky J. de Visser M. Hughes R.A. 119th ENMC international workshop: trial design in adult idiopathic inflammatory myopathies, with the exception of inclusion body myositis, 10–12 October 2003, Naarden, The Netherlands.Neuromuscul Disord. 2004; 14: 337-345Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 2De Visser M. Emslie-Smith A.M. Engel A.G. Early ultrastructural alterations in adult dermatomyositis: capillary abnormalities precede other structural changes in muscle.J Neurol Sci. 1989; 94: 181-192Abstract Full Text PDF PubMed Scopus (102) Google Scholar The pathophysiology of the disease is not fully understood. Hence, on the basis of results of transcriptome analysis of skeletal muscle, it has been emphasized that interferon (IFN) pathways seem to play an important role.3Greenberg S.A. Pinkus J.L. Pinkus G.S. Burleson T. Sanoudou D. Tawil R. Barohn R.J. Saperstein D.S. Briemberg H.R. Ericsson M. Park P. Amato A.A. Interferon-alpha/beta-mediated innate immune mechanisms in dermatomyositis.Ann Neurol. 2005; 57: 664-678Crossref PubMed Scopus (454) Google Scholar, 4Salajegheh M. Kong S.W. Pinkus J.L. Walsh R.J. Liao A. Nazareno R. Amato A.A. Krastins B. Morehouse C. Higgs B.W. Jallal B. Yao Y. Sarracino D.A. Parker K.C. Greenberg S.A. Interferon-stimulated gene 15 (ISG15) conjugates proteins in dermatomyositis muscle with perifascicular atrophy.Ann Neurol. 2010; 67: 53-63Crossref PubMed Scopus (121) Google Scholar Also, a specific vasculopathy associated with molecular mechanisms of hypoxia has been described in detail.5Gitiaux C. Kostallari E. Lafuste P. Authier F.J. Christov C. Gherardi R.K. Whole microvascular unit deletions in dermatomyositis.Ann Rheum Dis. 2013; 72: 445-452Crossref PubMed Scopus (37) Google Scholar Nevertheless, DM encompasses an inhomogeneous group of disorders because a large spectrum of different extramuscular manifestations can be observed, and the muscle involvement is variable, even absent sometimes: the so-called amyopathic DM.6Sontheimer R.D. Dermatomyositis: an overview of recent progress with emphasis on dermatologic aspects.Dermatol Clin. 2002; 20: 387-408Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar Different DM-specific autoantibodies have been reported so far, and it has been postulated that their presence is associated with homogeneous clinical subgroups of DM.7Hamaguchi Y. Kuwana M. Hoshino K. Hasegawa M. Kaji K. Matsushita T. Komura K. Nakamura M. Kodera M. Suga N. Higashi A. Ogusu K. Tsutsui K. Furusaki A. Tanabe H. Sasaoka S. Muro Y. Yoshikawa M. Ishiguro N. Ayano M. Muroi E. Fujikawa K. Umeda Y. Kawase M. Mabuchi E. Asano Y. Sodemoto K. Seishima M. Yamada H. Sato S. Takehara K. Fujimoto M. Clinical correlations with dermatomyositis-specific autoantibodies in adult Japanese patients with dermatomyositis: a multicenter cross-sectional study.Arch Dermatol. 2011; 147: 391-398Crossref PubMed Scopus (235) Google Scholar Anti-melanoma differentiation-associated gene 5 (MDA5; official symbol IFIH1) is one of them, and the presence of anti-MDA5 antibodies (MDA5+) delineates a clinical syndrome characterized by mild or absent muscle weakness [with mild or no increase of creatine kinase levels (ie, the prototype of amyopathic DM)]. Extramuscular manifestations are predominant, including severe interstitial lung disease and atypical but characteristic skin lesions with skin ulcers, palmar papules, and/or mechanic's hands.7Hamaguchi Y. Kuwana M. Hoshino K. Hasegawa M. Kaji K. Matsushita T. Komura K. Nakamura M. Kodera M. Suga N. Higashi A. Ogusu K. Tsutsui K. Furusaki A. Tanabe H. Sasaoka S. Muro Y. Yoshikawa M. Ishiguro N. Ayano M. Muroi E. Fujikawa K. Umeda Y. Kawase M. Mabuchi E. Asano Y. Sodemoto K. Seishima M. Yamada H. Sato S. Takehara K. Fujimoto M. Clinical correlations with dermatomyositis-specific autoantibodies in adult Japanese patients with dermatomyositis: a multicenter cross-sectional study.Arch Dermatol. 2011; 147: 391-398Crossref PubMed Scopus (235) Google Scholar Thus, MDA5+ patients harbor a characteristic clinical phenotype, at variance from the classic form of DM, raising the question of the role of anti-MDA5 antibodies, and the involvement of separate underlying molecular mechanisms. It appeared that there are ethnic variations in frequency of anti-MDA5 positivity because most of the cases have been described from Asian populations; however, the associated clinical phenotype in different ethnicities has to be further explored.8Hall J.C. Casciola-Rosen L. Samedy L.A. Werner J. Owoyemi K. Danoff S.K. Christopher-Stine L. Anti-MDA5-associated dermatomyositis: expanding the clinical spectrum.Arthritis Care Res Hoboken. 2013; 65: 1307-1315Crossref PubMed Scopus (193) Google Scholar To date, muscle pathology of 11 MDA5+ children has been analyzed in comparison to MDA5− juvenile DM patients,9Tansley S.L. Betteridge Z.E. Gunawardena H. Jacques T.S. Owens C.M. Pilkington C. Arnold K. Yasin S. Moraitis E. Wedderburn L.R. McHugh N.J. UK Juvenile Dermatomyositis Research GroupAnti-MDA5 autoantibodies in juvenile dermatomyositis identify a distinct clinical phenotype: a prospective cohort study.Arthritis Res Ther. 2014; 16: R138Crossref PubMed Scopus (124) Google Scholar but extensive illustrations have not been reported in adult patients, nor have molecular pathways, including immune parameters, been analyzed. Herein, we hypothesized that specific morphological features and specific molecular pathways occur in MDA5+ patients' skeletal muscles, in accordance with the unique phenotype of MDA5+ patients, which is at variance from classic DM patients. We demonstrate that none of the MDA5+ patients presented the characteristic morphology of classic DM (perifascicular fiber atrophy, characteristic vasculopathy, or important perivascular inflammation). The expression of IFN-stimulated genes was up-regulated, but at lower levels compared with DM patients. In addition, only MDA5+ patients showed focal nitric oxide synthase 2 (NOS2) positive fibers. NOS2 expression colocalized with markers of regeneration and markers of cell stress, suggesting a protective mechanism in the skeletal muscle. Ten muscle biopsy specimens from MDA5+ patients (n = 9; one patient had two muscle biopsy specimens) were identified in four French university hospitals involved in this retrospective study. Patients were diagnosed on the basis of the positivity of qualitative commercial dot blot assay, performed according to the manufacturer's recommendations (D-tek, Mons, Belgium). Muscle biopsy specimens (n = 7) from DM patients (n = 7), diagnosed on the basis of morphological and clinical European Neuromuscular Centre criteria,1Hoogendijk J.E. Amato A.A. Lecky B.R. Choy E.H. Lundberg I.E. Rose M.R. Vencovsky J. de Visser M. Hughes R.A. 119th ENMC international workshop: trial design in adult idiopathic inflammatory myopathies, with the exception of inclusion body myositis, 10–12 October 2003, Naarden, The Netherlands.Neuromuscul Disord. 2004; 14: 337-345Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar were included as controls. The serum of the classic DM controls was tested negative for anti-MDA5 antibodies. Furthermore, the serum of all patients was also tested for presence of Jo-1, anti–PL-7, anti–PL-12, anti–signal recognition particle, and anti-PM/Scl and anti–Mi-2 autoantibodies using commercial kits (D-tek). For quantitative real-time PCR, gene expression in DM samples was normalized to results obtained in normal muscle [ie, skeletal muscle biopsy specimens without any light microscopic or ultrastructural abnormalities (n = 7)]. The following patients' characteristics were documented: epidemiological characteristics, presence of muscle weakness, creatine kinase level, presence of typical skin rash of DM (heliotrope periorbital edema; violaceous papules or macules, scaly if chronic, at metacarpopharyngeal and interpharyngeal joints, and other bony prominences; and erythema of chest, neck, and upper back),1Hoogendijk J.E. Amato A.A. Lecky B.R. Choy E.H. Lundberg I.E. Rose M.R. Vencovsky J. de Visser M. Hughes R.A. 119th ENMC international workshop: trial design in adult idiopathic inflammatory myopathies, with the exception of inclusion body myositis, 10–12 October 2003, Naarden, The Netherlands.Neuromuscul Disord. 2004; 14: 337-345Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar and presence of interstitial lung disease.10Saketkoo L.A. Mittoo S. Huscher D. Khanna D. Dellaripa P.F. Distler O. et al.Connective tissue disease related interstitial lung diseases and idiopathic pulmonary fibrosis: provisional core sets of domains and instruments for use in clinical trials.Thorax. 2014; 69: 428-436Crossref PubMed Scopus (87) Google Scholar All frozen samples were analyzed in the Department of Neuropathology (Charité-Universtitätsmedizin, Berlin, Germany), following standardized procedures. Cryostat sections (7 μm thick) of muscle biopsy specimens were used. The following stains were performed: hematoxylin and eosin, periodic acid–Schiff, acid phosphatase, non-specific esterase, and Gömöri trichrome. Immunohistochemical analysis included using the following antibodies: anti-MHC class I (mouse, clone W6/32; Dako, Glostrup, Denmark), anti-MHC class II (mouse, clone C3/43; Dako), anti-CD3 (rabbit, polyclonal; Dako), anti-CD4 (rabbit, clone SP35; Zytomed, Berlin, Germany), anti-CD8 (mouse, clone C8/144B; Dako), anti-CD68 (mouse, clone EBM11; Dako), anti-CD79a (rabbit, polyclonal; GeneTex, Irvine, CA), anti-CD20 (clone 144B; Dako), anti–C5b-9 complex (mouse, clone aE11; Dako), anti–IFN-stimulated gene 15 (ISG15; rabbit, polyclonal; Abcam, Cambridge, UK), retinoic acid–inducible gene 1 (RIG-I; mouse, 2M6F10; Thermo Scientific, Rockford, IL), anti-neural cell adhesion molecule (clone ERIC-1; AbD Serotec, Kidlington, UK), anti-NOS2 (rabbit, polyclonal; GeneTex), anti-heat shock protein (HSP) 70 (mouse, clone 3A3; Abcam), and anti-blood dendritic cell antigen 2 (mouse, clone 10E6.1; Millipore, Schwalbach, Germany). An automated slide staining system (BenchMark XT; Ventana Medical Systems, Tucson, AZ) was used. Utrophin (mouse, clone DRP3/20C5; Leica Microsystems, Wetzlar, Germany) staining was used to visualize endomysial capillaries. Ultrastructural analysis was performed after fixation in 2.5% glutaraldehyde for 48 hours at 4°C, post fixation in 1% osmium tetroxide, and embedding of the muscle tissue in araldite. Ultrathin sections were stained with uranyl acetate and lead citrate. A P902 electron microscope (Zeiss, Oberkochem, Germany) was used to analyze the specimens. All slides were retrospectively analyzed blinded to the antibody status by three myopathologists (W.S., Y.A., H.-H.G.). The presence or absence of each item is mentioned in the study. For quantitative analysis, slides were digitized using Pannoramic 250 (3DHistech, Budapest, Hungary) at a resolution of 0.244 μm per pixel. Whole slide images were processed using a virtual microscope on the basis of ImageJ software version 6.1.1 (NIH, Bethesda, MD; http://imagej.nih.gov/ij).11Zerbe N. Hufnagl P. Schlüns K. Distributed computing in image analysis using open source frameworks and application to image sharpness assessment of histological whole slide images.Diagn Pathol. 2011; 6: S16Crossref PubMed Scopus (31) Google Scholar The density of immune cells was calculated after counting positive cells from the whole area of the biopsy specimen. For capillary analysis, a sufficient number of randomly selected detail images at ×100 magnification allowed coverage of 50% to 60% of the whole cross section of the biopsy. For each sample, the number of capillaries and the minimum Feret diameters were measured automatically. The number of entire cross sections of muscle fibers was counted manually. These data permitted us to calculate the capillary/fiber ratio and capillary density.12Engel A. Franzini-Armstrong C. ed 2. Myology, Basic and Clinical. Vol 1. McGraw-Hill Medical Publishing, New York, NY1994Google Scholar For RNA extraction from the muscle tissue samples, RT-PCR and quantitative PCR were used with the same methods as described before.13Preusse C. Goebel H.H. Held J. Wengert O. Scheibe F. Irlbacher K. Koch A. Heppner F.L. Stenzel W. Immune-mediated necrotizing myopathy is characterized by a specific Th1-M1 polarized immune profile.Am J Pathol. 2012; 181: 2161-2171Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Briefly, the expression levels of gene transcripts were analyzed by quantitative real-time RT-PCR using the 5′-nuclease technology on an ABI PRISM 7900HT Sequence Detection System, and the human TaqMan predeveloped assay reagents (both from Applied Biosystems, Darmstadt, Germany). The assay identification probes are as follows: 2′-5′-oligoadenylate synthetase 1 (OAS1), Hs00973637_m1; OAS3, Hs00196324_m1; MDA5, Hs01070332_m1; myxovirus (influenza virus) resistance 1 (MX1), Hs00895608_m1; RIG-I, Hs00204833_m1; IFN-γ (IFNG), Hs00989291_m1; and STAT1, Hs01013989_m1. The relative abundance of target transcripts was normalized to the expression levels of the endogenous control gene: phosphoglycerate kinase 1 (PGK1), Hs99999906_m1. For each probe, individual (MDA5+ and classic DM patients) data were expressed as relative quantification value. The relative quantification value is equal to 2−ΔΔCt (ie, normalized fold-change relative to the mean expression for the seven healthy controls combined). As previously described, the median fold-change in expression of six IFN-stimulated genes (ISGs) was given for each patient's biopsy specimen, defining an IFN score.14Rice G.I. Forte G. Szynkiewicz M. Chase D.S. Aeby A. Abdel-Hamid M.S. et al.Assessment of interferon-related biomarkers in Aicardi-Goutières syndrome associated with mutations in: a case-control study.Lancet Neurol. 2013; 12: 1159-1169Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar Immunofluorescence analysis was performed as previously described.13Preusse C. Goebel H.H. Held J. Wengert O. Scheibe F. Irlbacher K. Koch A. Heppner F.L. Stenzel W. Immune-mediated necrotizing myopathy is characterized by a specific Th1-M1 polarized immune profile.Am J Pathol. 2012; 181: 2161-2171Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Briefly, sections were blocked with the appropriate serum and incubated with the previously mentioned primary antibodies. The secondary antibody was added for 1 hour. The same protocol was repeated with a second primary antibody and an appropriate secondary antibody for double immunofluorescence staining. Images were taken with the Zeiss Observer.Z1 microscope (immunofluorescence), Axiovision software version 4 (Zeiss), or the Olympus (Hamburg, Germany) BX50 microscope, the digital camera DP25, and cell D software version 5.1. Agreement from the French Ministry of Research (AC 2008-87) was obtained for the collection, analysis, storage, and reuse. All samples were taken for diagnostic purposes. We conducted the research after consideration and approval of the research ethics committee of the Pitié-Salpêtrière Hospital (Paris, France). Categorical variables are reported as numbers and/or percentages and were compared using a Fisher's exact test. Quantitative variables are reported as means ± SD and compared using the Mann-Whitney test. P < 0.05 was considered significant. Kruskal-Wallis one-way analysis of variance was applied to analyze quantitative differences of mRNA transcripts, using Bonferroni correction of the post hoc tests. GraphPad Prism software version 6.00 (GraphPad Software, La Jolla, CA) was used for analysis. Patients' characteristics are summarized in Table 1. Patients were mainly female and were aged 40 to 49 years when the muscle biopsy specimen was obtained. Age and sex ratio were not different in MDA5+ versus classic DM groups. The extramuscular phenotype was different because most MDA5+ patients experienced interstitial lung disease, whereas interstitial lung disease was absent in controls. In addition, signs of myopathy were more frequent and/or more intense in classic DM patients. Only one quarter of MDA5+ patients experienced verifiable muscle weakness, and only 55% of them had increased creatine kinase levels. All DM patients presented typical skin manifestations, whereas most MDA5+ DM patients experienced characteristic skin lesions uncommon in classic DM (none of the classic DM patients presented atypical skin lesions, such as palmar papules, mechanic's hands, and/or skin ulcers) (Table 1). None of the DM patients harbored anti-synthetase syndrome, two classic DM patients were anti–Mi-2 antibody positive, and none of MDA5+ patients had myositis-specific antibodies other than anti-MDA5.Table 1Patient CharacteristicsCriterionMDA5+Classic DMP valuesAge, years∗Data presented as means ± SD.44 ± 17.747 ± 12.10.6Female, %77851Muscle weakness281000.003CK level, IU/L∗Data presented as means ± SD.517 ± 8562693 ± 34460.03Interstitial lung disease, %6700.01Atypical skin lesions, %9000.001The atypical skin lesions correspond to presence of palmar papules, mechanic's hands, and/or skin ulcers.CK, creatine kinase; DM, dermatomyositis; MDA, melanoma differentiation-associated protein.∗ Data presented as means ± SD. Open table in a new tab The atypical skin lesions correspond to presence of palmar papules, mechanic's hands, and/or skin ulcers. CK, creatine kinase; DM, dermatomyositis; MDA, melanoma differentiation-associated protein. One of the main characteristics of DM pathology is the presence of a characteristic vasculopathy. To assess signs of vasculopathy, we first tested the presence of C5b-9 deposition on endomysial capillaries (Figure 1A). Characteristic C5b-9 deposition was regularly (85%) detected in classic DM, but this was not the case for MDA5+ patients (30%, P = 0.049). Capillary rarefaction is a further feature of vasculopathy in DM. Indeed, capillary density was low in classic DM patients (277.9 ± 49.5 per mm2), and it was significantly higher (340 ± 52.3 per mm2, P = 0.05) in MDA5+ patients (Figure 1, B and C). Focal capillary rarefaction was only rarely noted in MDA5+ patients (Figure 1B). The capillary/fiber ratio was slightly elevated in MDA5+ patients (1.2 ± 0.3) compared with classic DM (1.1 ± 0.2); however, it did not reach statistical significance (P = 0.36) (Figure 1D). Enlarged capillaries, another sign of vasculopathy, were commonly observed in classic DM patients; however, they could only occasionally be observed in MDA5+ patients (Figure 1B). Indeed, the mean percentage of enlarged capillaries (>10 μm diameter) was high in classic DM patients (13.9% ± 6.8%) and was only 5.7% ± 3.2% in MDA5+ patients (P = 0.006) (Figure 1, E and F). In addition, all classic DM patients had tubuloreticular inclusions within vascular endothelial cells (Figure 1G), whereas they were detected in barely 50% of MDA5+ patients (P = 0.04). Together, these results show that typical vasculopathy, as found in DM, is not a characteristic feature of MDA5+ patients. The presence of perifascicular fiber atrophy is one of the most characteristic signs of DM. This pattern was not observed in MDA5+ patient biopsy specimens (P = 0.003) (Figure 2A), except in one case in which a focal mild perifascicular atrophy was noted next to a perivascular infiltrate (data not shown). In contrast, all but one classic DM patient showed the typical perifascicular atrophy (P = 0.003) (Figure 2B). In the same line, in MDA5+ patients, MHC-I expression was essentially focal or absent (Figure 2C), only one patient showed a mild diffuse expression. On the contrary, a diffuse MHC class I expression with reinforcement in perifascicular regions, which is a characteristic feature of DM, was regularly met in classic DM (P = 0.004) (Figure 2D). By definition, DM is a myopathy with inflammatory infiltrates mainly occurring in the perivascular and perimysial areas. All but one MDA5+ patient harbored inflammatory infiltrates; however, the topographic distribution of these infiltrates differed considerably from classic DM patients. In MDA5+ patients, infiltrates were clustering focally in the perimysium near a single vessel (Figure 3A), whereas in classic DM, as expected, inflammation was detected multifocally, leading to a more homogeneous inflammatory aspect (Figure 3B). Macrophages were the predominant immune cells in MDA5+ patients and essentially accumulated only focally in the perimysium as well (Figure 3, C and D). Along that line, cellular density of T cells (CD3+ cells) was significantly elevated in classic DM (35.8 ± 30 cells per mm2), whereas T cells were rare in MDA5+ patients (3.6 ± 3.1 cells per mm2; P < 0.0001) (Figure 3E). Among T cells, CD8+ T cells represented 7.7 ± 7.1 cells per mm2 in classic DM patients, and were significantly lower in MDA5+ patients (1.3 ± 2.5 cells per mm2; P < 0.01). CD4+ T cells were detectable more frequently in classic DM (22.4 ± 5.7 cells per mm2 versus 2.9 ± 2.9 cells per mm2; P < 0.001) (Figure 3, F and G). CD20+ B-cell density was elevated in classic DM (8.2 ± 6.3 cells per mm2), but it was significantly lower in MDA5+ patients (2.1 ± 2.8 cells per mm2; P < 0.02) (Figure 3H). Few plasma cells were present in MDA5+ and classic DM patients, with no significant difference (0.3 ± 0.6 and 1.0 ± 1.2 cells per mm2; P = 0.1; data not shown). We next compared immune pathways in MDA5+ and classic DM patients and first tested the IFN pathway. We analyzed the expression of six ISGs: OAS1, ISG15, OAS3, MX1, RIG-I, and IFIH1. All these genes were considerably up-regulated in both groups compared with normal muscle with high relative quantification values (Figure 4A). Nevertheless, the up-regulation of ISGs was less important in MDA5+ patients than in classic DM, and the IFN score was significantly lower in MDA5+ patients (165.8 ± 138.2 versus 590.2 ± 141.5; P < 0.004) (Figure 4B). To identify cells involved in the IFN pathway, we first searched for the presence of plasmacytoid dendritic cells (pDCs; stained by anti-blood dendritic cell antigen 2), which are known to secrete a large amount of IFN-α. Only occasional pDCs were observed in a minority of MDA5+ and classic DM patients (Supplemental Figure S1). On the other hand, we observed that muscle fibers expressed proteins of the ISG group at different levels. RIG-I and ISG15 were regularly detectable in classic DM, on skeletal muscle fibers in the perifascicular region (Figure 4, C and E). On the contrary, only 50% of MDA5+ patients showed a variable degree of RIG-I staining (Figure 4D), and none of the MDA5+ patients exhibited detectable ISG15 immunoreactivity in muscle fibers (Figure 4F); however, some immune cells showed a positive immunostaining (Figure 4F). Together, these results show that the IFN pathway was also activated to a lesser degree in MDA5+ patients compared with classic DM patients. The IFN-associated cytokines are considered strong inducers of STAT1, which is required for type 1 helper T-cell (Th1) activation as part of the adaptive immune response.15Oestreich K.J. Weinmann A.S. Transcriptional mechanisms that regulate T helper 1 cell differentiation.Curr Opin Immunol. 2012; 24: 191-195Crossref PubMed Scopus (71) Google Scholar We tested STAT1 gene expression and found increased levels of STAT1 transcription in MDA5+ specimens but less important compared with classic DM patient biopsy specimens (Figure 5A). These results are consistent with the increase of gene expression of the Th1 cytokine IFNG that we observed in both groups, which again was less pronounced in specimens from MDA5+ patients (Figure 5B). To further investigate Th1 immunity, we also tested NOS2 expression characterizing macrophage activation in a Th1 environment.16Sica A. Mantovani A. Macrophage plasticity and polarization: in vivo veritas.J Clin Invest. 2012; 122: 787-795Crossref PubMed Scopus (3876) Google Scholar We detected numerous NOS2+ mononuclear infiltrates in both groups, but they were more abundant in the classic DM patients (Figure 5, B and C). Strikingly, however, specimens from MDA5+ patients showed a strong NOS2+ immune reaction on the sarcoplasm of myofibers; conversely, classic DM patients did not (P < 0.003) (Figure 5B). In MDA5+ patients, numerous NOS2+ fibers were detectable in fascicles adjacent to perimysial vessels with inflammatory infiltrates (Figure 5D). Because NOS2 positivity was a striking feature in MDA5 patients' biopsy specimens only, we hypothesized a protective, and not a detrimental, effect of NOS2 in the skeletal muscle. Therefore, we tested for the presence of chaperone proteins associated with protection. Indeed, NOS2+ fibers in MDA5+ patient biopsy specimens strongly colocalized with HSP70 (Figure 5E). In addition, colocalization with neural cell adhesion molecule was also observed (Figure 5F); as mentioned above, classic DM (MDA5−) patients did not show NOS2-positive fibers (no colocalization, data not shown). In classic DM, however, only single fibers were NOS2 positive, whereas fibers in the perifascicular region were HSP70 positive and no colocalization was detectable (Figure 5G). In this study, addressing morphological and immunological aspects in MDA5+ patients, we show that muscle pathology and the local immune response differ from classic DM patients. The main finding of this study is that we observed a characteristic specific NOS2 expression on the sarcoplasm of HSP70-positive muscle fibers in MDA5+ DM patients, which was absent in classic DM patients. Conversel
Referência(s)