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High YKL-40 serum levels and its expression in the muscle tissues of patients with antisynthetase syndrome

High YKL-40 serum levels and its expression in the muscle tissues of patients with antisynthetase... Background: The protein chitinase-3-like-1 (YKL-40) is rarely analyzed in patients with myositis. Therefore, we aimed to evaluate YKL-40 serum levels; correlate them with laboratory and clinical parameters, disease status, and treatment schemes; and analyze the YKL-40 expression in the muscle tissues of patients with antisynthetase syndrome (ASSD). Methods: This cross-sectional single-center study included 64 adult patients with ASSD who were age-, gender-, and ethnicity-matched to 64 healthy control individuals. Their YKL-40 serum levels were analyzed using the Enzyme-Linked Immunosorbent Assay (ELISA) kit method, while YKL-40 expression in muscle tissues was analyzed using an immunohistochemical technique. Disease status was assessed using the International Myositis Assessment and Clinical Studies Group (IMACS) set scores. Results: The patients’ mean age was 44.8 ± 11.8 years, and median disease duration was 1.5 (0.0–4.0) years. These patients were predominantly female (82.8%) and Caucasian (73.4%). Most patients had stable disease. The median YKL-40 serum level was significantly higher in patients with ASSD when compared to the healthy individuals: 538.4 (363.4–853.1) pg/mL versus 270.0 (201.8–451.9) pg/mL, respectively; P < 0.001. However, YKL-40 serum levels did not correlate with any clinical, laboratory, disease status, or therapeutic parameters (P > 0.050), except tumor necrosis factor alpha (TNF-α) serum levels (Spearman’s correlation, rho = 0.382; P = 0.007). YKL-40 was highly expressed by inflammatory cells found in muscle biopsy specimens. Conclusions: High YKL-40 serum levels were observed in patients with ASSD and correlated positively with TNF-α serum levels. Moreover, YKL-40 was expressed by the inflammatory cells of the muscle tissue. Keywords: Antisynthetase syndrome, Chitinase, Inflammatory myopathy, Muscle biopsy, Myositis Introduction The protein chitinase-3-like-1 (YKL-40) is a glycopro- Antisynthetase syndrome (ASSD) is a rare systemic tein secreted by inflammatory cells, including macro- autoimmune myopathy characterized by muscle, joint, phages and neutrophils [5, 6]. YKL-40 is associated with and pulmonary involvement, as well as by the presence several physiological processes, such as inflammation, of fever, “mechanic’shands,” and Raynaud’s phenomenon cell proliferation, angiogenesis, tissue fibrosis, and tissue [1–4]. In the laboratory, the ASSD is characterized by the remodeling [7]. YKL-40 also participates in a wide range presence of anti-aminoacyl-tRNA synthetase autoanti- of inflammatory responses, also stimulating the produc- bodies, such as anti-Jo-1 [1, 2]. tion of pro-inflammatory cytokines (e.g., IL-6, IL-18, and tumor necrosis factor alpha [TNF-α]), which are capable * Correspondence: samuel.shinjo@usp.br of increasing YKL-40 serum levels as a feedback mech- Division of Rheumatology, Faculdade de Medicina FMUSP, Universidade de anism [8, 9]. Sao Paulo, Av. Dr. Arnaldo, 455, 3° andar, sala 3184 - Cerqueira César. CEP: 01246-903, Sao Paulo, Brazil © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Carboni et al. Advances in Rheumatology (2021) 61:44 Page 2 of 10 High YKL-40 serum levels have been described in A commercially available line blot test kit (Myositis osteoarthritis [10] and in several systemic autoimmune Profile Euroline Blot test kit, Euroimmun, Lübeck, diseases, such as rheumatoid arthritis [11–14], giant cell Germany) was used to identify anti-aminoacyl-tRNA arteritis [15], systemic sclerosis [16, 17], Takayasu’s arteritis synthetase autoantibodies (anti-Jo-1, anti-EJ, anti-OJ, [18], and systemic autoimmune myopathies [19–21]. anti-PL-7, anti-PL-12, and anti-Ro-52). The assessment In rheumatoid arthritis, YKL-40 serum levels are not was performed according to previously established only increased but also correlate positively with the methods [22]. The immunological patterns evaluated disease activity [11–13]. However, YKL-40 is not were antinuclear antibodies (ANA) detected by an indir- associated with radiographic progression or predictive ect immunofluorescence technique on HEp-2 cells. values for disease remission [14]. In patients with We excluded pregnant patients and those with other systemic sclerosis, YKL-40 serum levels correlate systemic autoimmune diseases (overlap syndrome), mainly with joint involvement [16], lung involvement chronic or acute infection, neoplasia-associated myositis, (fibrosis or reduction in the diffusing capacity for car- and uncontrolled and serious comorbidities (e.g., liver dis- bon monoxide [DLCO]), digital articular deformities ease, nephropathy, and heart disease). Observing the internal through cutaneous retraction, and reduced patient sur- protocol of our service, a screening for neoplasms and other vival [17]. High YKL-40 serum levels are also described lung diseases was performed in all cases of myopathies. in patients with dermatomyositis and polymyositis, The following pre-standard and pre-parameterized correlating with the disease activity and severity and data were collected from the patients’ interviews: demo- with pulmonary involvement [19–21]. graphic information (current age, gender, ethnicity), clin- However, no studies have assessed YKL-40 serum ical information (disease duration; muscle, pulmonary, levels exclusively in patients with ASSD, which was the and joint involvement; Raynaud’s phenomenon; presence primary motivation for this study. We aimed to correlate of fever; and “mechanic’s hands”), laboratory findings YKL-40 serum levels with clinical and laboratory param- (serum levels of CPK, lactate dehydrogenase, aspartate eters, disease status, and therapeutic schemes, as well aminotransferase, alanine aminotransferase), disease sta- as to analyze the YKL-40 expression in muscle tissue tus, treatment (immunosuppressive, immunomodulatory, specimens from patients with ASSD. and glucocorticoid therapies), and comorbidities. More- over, on the interview day, blood samples (20 mL) were taken for laboratory analysis after an eight-hour fasting. Patients and methods Current clinical and laboratory disease status was This cross-sectional single-center study took place from assessed by applying the following questionnaires and 2017 to 2019 and included 66 adult patients with ASSD International Myositis Assessment & Clinical Studies followed up at the outgoing clinic of our service. All pa- Group (IMACS) scores: Myositis Disease Activity tients fulfilled the modified ASSD criteria classification Assessment Visual Analogue Scales (MYOACT) [23, 24], proposed by Connors et al. [1], but with modifications Manual Muscle Testing (MMT-8) [25], overall disease to increase its specificity, including the presence of at assessment by a physician and by the patient using a least two of the following three manifestations: muscle, visual analogue scale (VAS) [26], and the Health Assess- joint, and lung involvement; and in addition to persistent ment Quality (HAQ) [27]. fever; Raynaud’s phenomenon; “mechanic’s hands;” and Pulmonary assessment was performed using chest CT the presence of serum anti-aminoacyl-tRNA synthetase images. The following parameters were investigated: autoantibody. incipient pneumopathy, ground-glass opacities with or Muscle involvement was defined as the presence of without bronchiectasis, pulmonary nodules, and pul- progressive and proximal limb muscle weakness, in- monary fibrosis (honeycombing areas) [1, 28, 29]. All creased serum levels of muscle enzymes, such as creatine chest CT images were analyzed independently by the phosphokinase (CPK), myopathic pattern on electro- two authors of the present study, who are experienced neuromyography, and muscle biopsy compatible with in connective tissue diseases (RCSC and GLBP). In case inflammatory myopathy. Joint involvement was defined of disagreement between them, the images were dis- as non-deforming and non-erosive arthritis or arthralgia. cussed with radiology groups from our institute. Additionally, as part of the internal protocol of our Chest CT images obtained during a period ranging from service, patients underwent computed tomography (CT) six months before to six months after the patients’ partici- chest scans at disease onset and during follow-up. In this pation were included in this study. When possible, the study, lung involvement was defined as the presence of pulmonary assessment was supplemented by a pulmonary incipient pneumopathy, pulmonary nodules, ground-glass function test that considered the forced expiratory volume opacities with or without bronchiectasis, and pulmonary in one second (FEV1), forced vital capacity (FVC), FEV1/ fibrosis (e.g., honeycombing areas). FVC ratio, and DLCO. Carboni et al. Advances in Rheumatology (2021) 61:44 Page 3 of 10 The patients with ASSD were gender-, age-, and clinical and demographic characteristics were expressed ethnicity-matched to healthy control individuals as mean ± standard deviation (SD), for continuous vari- (patients’ relatives or employees of our service) selected ables, or as frequencies and percentages (%), for categor- for convenience. All laboratory tests were performed on ical variables. The median (interquartile 25th to 75th) the control group, and they were asked to answer all was calculated for continuous variables that did not have interview questions, except those related to the disease a normal distribution. Comparisons between patient and data. control parameters were made using the Mann-Whitney For the cytokine assessment, the material was test and Student t-test for continuous variables, while centrifuged at 3000 rpm for 10 min immediately after the Fisher’s exact test or chi-square test were used to as- blood collection (< 30 min), and the sera were aliquoted sess categorical variables. Spearman’s correlation was and stored at − 80 °C for further cytokine analysis. The used for analyzing the correlation (rho) between con- YKL-40 quantification was performed using a specific kit tinuous variables and YKL-40 values. Values between 0 (Human CHI3L1 ELISA kit, RayBiotech, USA) and and ± 0.333 were considered weak; values between ± processed following the manufacturer’s protocol. The 0.333 and ± 0.666 were considered moderate; and those interferon gamma (IFN-γ)and TNF-α tests were per- between ±0.666 and ± 1.000 were considered strong.” formed using the LUMINEX 100/200x xMAP technology The relation between YKL-40 values and other variables (Millipore, USA), as described elsewhere [30]. (identified as the presence or absence of a certain char- Additionally, the YKL-40 expression and location were acteristics) was performed using the Student t-test. To assessed in muscle tissues of three patients with ASSD, analyze the factors that together influence YKL-40, a whose biopsies were taken from the vastus lateralis multiple regression was performed considering all vari- muscle upon diagnosis. Sequential 5-μm thick frozen ables with P value < 0.10 as explanatory and the value of sections were first stained by hematoxylin-eosin and, YKL-40 as dependent. A value of P < 0.05 was adopted then, immunohistochemistry was performed. For immu- to indicate statistical significance. All analyses were per- nohistochemical analysis, monoclonal antibodies (CD4, formed using the statistical software SPSS version 22.0 CD8, CD68, and YKL-40: Abcam, USA) were used. (Chicago, IL, USA). Frozen specimens were fixed for 10 min in acetone at 4 °C. Endogenous peroxidase was blocked using 1% Results H O in absolute methanol three times for 10 min. After Sixty-six patients with ASSD were initially included in 2 2 rinsing the specimens in phosphate buffered saline (PBS) the present study. Two patients were excluded, one due (0.01 M, pH 7.4) for five minutes, these were incubated to overlap and the other due to neoplasia-associated in fetal serum in a wet chamber for one hour at 37 °C. myositis. No patients had acute or chronic infection, Primary antibodies were diluted in PBS and albumin were pregnant, or had severe and decompensated co- from 1% bovine serum was applied in wet chamber at morbidities. Therefore, 64 patients with ASSD were 37 °C overnight. Next, slides were washed in PBS and a evaluated and age-, gender-, and ethnicity-matched to secondary mouse biotinylated solution (StreptABCom- 64 healthy controls (Fig. 1). plex/HRP) was applied for 30 min at 37 °C and rinsed in The mean age of the 64 patients with ASSD and of the PBS. Then, the prepared StreptABComplex/HRP com- 64 control individuals was 44.8 ± 11.8 years and 42.6 ± plex was applied and incubated for 30 min at 37 °C. After 10.4 years, respectively (P = 0.278), with a predominance rinsing in PBS and incubation, the reactions were visual- of females (82.8% versus 82.8%, P > 0.999), and Caucasians ized using a solution of chromogenic substrate (3,3′-di- (73.4% versus 73.4%, P > 0.999) in both groups. The aminobenzidine tetrahydrochloride) for peroxidase. After median ASSD disease duration was 1.5 (0.0–4.0) years. a final rinse, hematoxylin counterstaining was performed. The initial and cumulative clinical manifestations, The slides were then mounted and covered with an autoantibody panels, and comorbidities of patients with aqueous-based mounting medium. All muscle specimens ASSD are shown in Table 1. were prepared at the same time as a batch. Human amyg- The current disease status of patients with ASSD at dala was used as a positive control, while a muscle speci- the time of inclusion in this study, lung parameters, and men from a patient with ASSD, but whose biopsy results treatments are shown in Table 2. were normal (without infiltrate inflammation), was used YKL-40 serum levels were significantly higher in pa- as a negative control. Inflammatory cell phenotyping tients with ASSD when compared to the control group: (CD4, CD8, CD68, and YKL-40) was analyzed in the 538.4 (363.4–853.1) pg/mL versus 270.0 (201.8–451.9) endomysium, perimysium, and pericapillary (endomysium pg/mL, respectively; P < 0.001 (Table 2). Additionally, a and perimysium) areas in 10 fields (200x magnification). significant increase in TNF-α levels was observed in pa- For statistical analysis, the Kolmogorov-Smirnov test tients with ASSD compared to the control group: 48.4 was used to assess each parameter distribution. The (31.0–66.3) pg/mL versus 39.6 (29.3–49.0) pg/mL; P = Carboni et al. Advances in Rheumatology (2021) 61:44 Page 4 of 10 Fig. 1 Study flowchart. ASSD: antisynthetase syndrome 0.011. IFN-γ serum levels were comparable in both Table 1 Cumulative clinical manifestations, autoantibody groups. panels, initial creatine phosphokinase serum levels, and Concerning demographic, clinical, laboratory, treata- comorbidities of the patients with antisynthetase syndrome ment, and comorbidity parameters, YKL-40 serum levels Antisynthetase syndrome n =64 had correlation with patients’ age (rho = 0.294; P = Cumulative clinical manifestations 0.040), TNF-α (rho = 0.382; P = 0.007), and current use Muscle involvement 61 (95.3) of prednisone (P < 0.047) - Tables 3 and 4. However, in Joint involvement 59 (92.2) multivariate analysis, only TNF-α levels had significant Lung involvement 57 (89.1) factor to predict the YKL-40 values (Table 5). Concerning the immunohistochemical analysis, YKL- Ground-glass 46 (71.9) 40 was expressed by inflammatory cells. Figure 2 shows Incipient pneumopathy 40 (62.5) a muscle biopsy specimen from a patient with ASSD. Pulmonary fibrosis 14 (21.9) Pulmonary nodules 11 (17.2) Discussion “Mechanic’s hands” 57 (89.1) To the best of our knowledge, this study is the first to Raynaud’s phenomenon 50 (78.1) show the relevance of YKL-40 in patients with ASSD. Fever 46 (71.9) High YKL-40 serum levels were observed in patients Autoantibody panels with ASSD and correlated positively with TNF-α serum Antinuclear factor 52 (81.3) levels. Besides, YKL-40 was expressed by inflammatory Anti-Ro-52 28 (43.8) cells in muscle tissues. Although ASSD is a rare disease, this study employed Anti-tRNA aminoacyl transferase 64 (100.0) strict inclusion criteria to increase the specificity in the Anti-Jo-1 52 (81.3) selection of the involved patients. In this context, a Anti-PL-12, anti-PL-7 or anti-EJ 12 (18.7) significant sample was included and paired by age, Anti-OJ 0 gender, and ethnicity with healthy individuals, thus Initial creatine phosphokinase (U/L) 3015 (632–8463) allowing for greater accuracy in comparative outcomes. Comorbidities Finally, information on patients was based on parame- Systemic arterial hypertension 22 (34.4) terized and pre-standardized data to ensure reliable Diabetes mellitus 12 (18.8) study data. Hypothyroidism 3 (4.7) High YKL-40 expression is described in the pathogen- esis of a number of diseases, and its utility as a bio- Acute myocardial infarction 3 (4.7) marker has been the subject of several studies [8–18]. Stroke 1 (1.6) Biologically, YKL-40 can stimulate angiogenesis and a Data are expressed as mean ± standard deviation (SD); median (interquartile 25th - 75th) or frequency (%) wide range of responses, such as inflammation, tissue Carboni et al. Advances in Rheumatology (2021) 61:44 Page 5 of 10 Table 2 Demographic, current disease status, pulmonary involvement, treatment, and the cytokines serum levels of the patients with antisynthetase syndrome. Laboratory profiles of the control group ASSD Control P (n = 64) (n = 64) IMACS set scores MMT-8 (0–80) 78 (71–80) – Patient’ VAS (0–10) 4.0 (2.0–6.0) – Physician’ VAS (0–10) 3.0 (0.0–5.0) – MYOACT (0–60) 4.0 (0.5–2.5) – HAQ (0.00–3.00) 0.67 (0.13–1.38) – CPK (U/L) 208 (98–597) 106 (78–161) < 0.001 LDH (U/L) 352 (243–590) 346 (312–345) 0.937 AST (U/L) 24 (17–37) 20 (16–23) 0.009 ALT (U/L) 24 (16–57) 18 (13–25) 0.030 Lung involvements Ground-glass 46 (71.9) – Incipient pneumopathy 40 (62.5) – Pulmonary fibrosis 14 (21.9) – Pulmonary nodules 11 (17.2) – FVC (% predict), n = 39 62.5 (48.0–73.5) – FEV1 (% predicted), n = 39 64 (49–77) – FEV1/FVC ratio, n = 39 0.90 (0.93–1.08) – DLCO (% predicted), n = 39 61.0 (37.8–73.0) – Treatment Prednisone Current use 46 (71.9) – Dose (mg/day) 10 (0–40) – Immunosuppressive drugs 47 (73.4) – Azathioprine 23 (35.9) – Mycophenolate mofetil 11 (17.2) – Methotrexate 14 (21.9) – Antimalarial 4 (6.3) – Leflunomide 2 (3.1) – Cyclosporine 3 (4.7) – Cyclophosphamide 1 (1.7) – Rituximab 16 (25.0) – Cytokines YKL-40 (pg/mL) 538.4 (363.4–853.1) 270.0 (201.8–451.9) < 0.001 IFNγ (pg/mL) 0.02 (0.00–1.89) 0.02 (0.00–0.17) 0.876 TNFα (pg/mL) 48.4 (31.0–66.3) 39.6 (29.3–49.0) 0.011 Data are expressed as mean ± standard deviation (SD); median (interquartile 25th - 75th) or frequency (%) ALT alanine aminotransferase; ASSD antisynthetase syndrome; AST aspartate aminotransferase; CPK creatine phosphokinase; DLCO diffusion of carbon dioxide; FEV1 forced expiratory volume in one second; FVC forced vital capacity; HAQ Health Assessment Questionnaire; IFN interferon; IMACS: International Myositis Assessment and Clinical Studies Group; LDH lactate dehydrogenase; MMT-8 Manual Muscle Testing; MYOACT Myositis Disease Activity Assessment Visual Analogue Scales; TNF tumor necrosis factor; VAS Visual Analog Scale; YKL chitinase-3-like-1 protein remodeling, and allergic reactions. These responses are YKL-40 is seldom described in studies regarding sys- often mediated by other cytokines (e.g., IL-6, IL-18, and temic autoimmune myopathies [19–21]. In this context, TNF-α), which can regulate the YKL-40 secretion [8, 9]. Hozumi et al. [19] showed high YKL-40 serum levels in Carboni et al. Advances in Rheumatology (2021) 61:44 Page 6 of 10 Table 3 Correlation between YKL-40 serum levels and cases with associated interstitial lung disease. Addition- continuous variables (demographic, clinical, laboratory and lung ally, patients with neoplasm associated myositis were parameters, and disease status) of the patients with included. antisynthetase syndrome Gao et al. [20] also observed high YKL-40 serum levels rho P in patients with dermatomyositis and polymyositis, Demographic data which correlated positively with interstitial lung disease and with the disease activity and severity evaluated via Age 0.294 0.040 MYOACT, C-reactive protein, ferritin, and erythrocyte Clinical parameters sedimentation rates. However, the authors did not Fever 0.070 0.631 analyze anti-aminoacyl-tRNA synthetase autoantibodies Raynaud phenomenon 0.144 0.324 in their patients. “Mechanic’s hands” −0.045 0.757 Jiang et al. [21] observed a positive correlation between Muscle involvement −0.219 0.131 YKL-40 serum levels and patients with clinically amyo- pathic dermatomyositis and positive anti-MDA-5, espe- Joint involvement −0.190 0.192 cially in those with severe interstitial lung disease. Laboratory parameters In our study, high YKL-40 serum levels were also ob- TNF-α level 0.382 0.007 served in patients with ASSD. However, YKL-40 did not IFN-γ level 0.125 0.395 correlate with any clinical and laboratory parameters, Lung involvement disease status, or therapeutic schemes. There was also FVC (% predict) 0.018 0.930 no correlation with any pulmonary parameters (e.g., pa- tient’s lung symptoms, chest CT images, and pulmonary FEV1 (% predicted) 0.089 0.667 function tests). Since this was a cross-sectional analysis, FEV1/FVC ratio −0.066 0.758 a prospective analysis of these parameters would be DLCO (% predicted) 0.533 0.091 interesting. Disease status To analyze disease status, Hozumi et al. [19] used vari- MMT-8 0.037 0.801 ables such as ferritin, KL-6 (Krebs von den Lungen-6), Patient’ VAS −0.173 0.239 SP-D (surfactant protein-D), PaO , and DLCO, while Jiang et al. [21] used ferritin, CPK, T CD3+ cell count, Physician’ VAS −0.234 0.110 C-reactive protein, FVC, and DLCO. In contrast, we MYOACT −0.077 0.614 used the IMACS set scores, which validated instruments HAQ 0.166 0.266 for clinical evaluation in systemic autoimmune myop- Creatine phosphokinase 0.131 0.371 athies [23–27]. Lactate dehydrogenase 0.138 0.460 Unlike limited studies regarding YKL-40 in myositis, a Aspartate aminotransferase 0.033 0.854 limitation of our study was the fact that we did not spe- cifically analyze the YKL-40 expression in lung biopsy Alanine aminotransferase 0.034 0.866 specimens. Nonetheless, the YKL-40 expression analyzed Treatment in muscle specimens reinforces the hypothesis that YKL- Prednisone: dose (mg/day) 0.217 0.139 40 expression is systemic, not being limited to lung DLCO diffusion of carbon dioxide; FEV1 forced expiratory volume in one involvement. second; FVC forced vital capacity; HAQ Health Assessment Questionnaire; IFN interferon; MMT-8 Manual Muscle Testing; MYOACT Myositis Disease Activity YKL-40 can be expressed and secreted by various Assessment Visual Analogue Scales; TNF tumor necrosis factor; VAS Visual types of cells, including macrophages, neutrophils, and Analog Scale chondrocytes. Its expression is regulated by several cyto- kines, which mediate the proliferation, tissue remodel- patients with dermatomyositis and polymyositis with ing, and production of other inflammatory mediators. In associated interstitial lung disease, and YKL-40 serum ASSD and systemic autoimmune inflammatory disease, levels were associated with a worse prognosis. Further- the elevated YKL-40 serum levels might derive come more, the authors correlated YKL-40 levels negatively from systemic inflammatory cells or local cells (e.g., with DLCO and arterial oxygen pressure (PaO ), find- blood, muscle, skin, lungs). This was the first study to ing a high expression of YKL-40 in alveolar macro- demonstrate the increased expression of YKL-40 in the phages and bronchial epithelial cells. Notably, the inflammatory cells of muscle tissue obtained from a pa- authors found that 42% of their patients had positive tient with ASSD. anti-aminoacyl-tRNA synthetase autoantibodies. There- As previously mentioned, YKL-40 is regulated by vari- fore, they might have included patients with ASSD and ous pro-inflammatory cytokines [8, 9]; therefore, we not necessarily pure dermatomyositis and polymyositis assessed the TNF-α and IFN-γ serum levels. Carboni et al. Advances in Rheumatology (2021) 61:44 Page 7 of 10 Table 4 Relation between YKL-40 serum levels and others variables (demographic, clinical, laboratory and lung parameters, disease status, and comorbidities) of the patients with antisynthetase syndrome Group 1 Group 2 P value n Mean ± SD n Mean ± SD Demographic data Gender (female) 53 628.5 ± 363.6 11 691.3 ± 461.0 0.658 Ethnicity (Caucasian) 47 572.5 ± 316.7 17 793.2 ± 468.2 0.111 Clinical parameters Fever 18 579.4 ± 343.8 46 664.3 ± 394.0 0.484 Raynaud phenomenon 14 536.6 ± 361.8 50 677.4 ± 382.7 0.255 “Mechanic’s hands” 7 658.6 ± 334.6 57 637 ± 389.2 0.891 Muscle involvement 3 980.2 ± 178.7 61 625.6 ± 379.2 0.198 Joint involvement 5 973.5 ± 602.1 59 610.4 ± 347.5 0.066 Laboratory parameters Antinuclear factor 12 685.7 ± 303.5 52 628.4 ± 398.4 0.674 Anti-Ro-52 35 663.9 ± 406.5 28 638.5 ± 348.0 0.818 Anti-Jo-1 11 728.4 ± 455.9 53 617.4 ± 359.7 0.414 Anti-PL-7 60 619.1 ± 377.6 4 876.1 ± 351.5 0.197 Anti-PL-12 60 627.2 ± 360.9 4 784.8 ± 595.5 0.431 Anti-EJ 61 653.7 ± 380.8 3 320.4 ± 52.2 0.227 Lung involvement Ground-glass 18 658.2 ± 348.1 46 634.2 ± 392.6 0.851 Incipient pneumopathy 24 695.9 ± 387.1 40 607.6 ± 376.4 0.437 Pulmonary fibrosis 50 586.2 ± 335.7 14 826.2 ± 471.8 0.063 Treatment Prednisone (current use) 17 483.3 ± 222.6 46 684.2 ± 406.3 0.047 Immunosuppressives/biologicals Azathioprine 43 618.9 ± 354.7 21 676.5 ± 425.2 0.613 Mycophenolate mofetil 54 648.4 ± 383.8 10 603.1 ± 375.2 0.750 Methotrexate 52 606.3 ± 338.1 12 790.3 ± 521.9 0.337 Antimalarial 61 645.3 ± 385.8 3 516.7 ± 91.7 0.643 Leflunomide 62 642.1 ± 382.5 2 544.0 ± 0 0.801 Cyclosporine 62 650.8 ± 381.1 2 388.7 ± 272.8 0.343 Cyclophosphamide 63 644.0 ± 381.8 1 451.9 ± 0 0.621 Rituximab 48 690.0 ± 364.8 16 537.0 ± 397.9 0.188 Comorbidities Systemic arterial hypertension 42 590.4 ± 379.9 22 742.5 ± 366.9 0.190 Diabetes mellitus 52 645.1 ± 395.0 12 610.0 ± 283.2 0.823 Hypothyroidism 61 628.0 ± 381.0 3 922.5 ± 205.8 0.286 Acute myocardial infarction 61 641.6 ± 382.6 3 567.2 ± 0 0.848 Stroke 63 645.2 ± 381.0 1 391.6 ± 0 0.513 SD standard deviation High TNF-α serum levels were observed in patients study, in which a positive correlation was found between with ASSD, similar to what occurs in other systemic YKL-40 and TNF-α serum levels. autoimmune myopathies [31, 32]. TNF-α can induce Although IFN-γ is involved in the ASSD pathophysi- YKL-40 production [8]. These data corroborated our ology, we did not find high cytokine serum levels in the Carboni et al. Advances in Rheumatology (2021) 61:44 Page 8 of 10 Table 5 Multivariate analysis Coefficient Standard error Lower Upper P value CI (95%) CI (95%) (Constant) 374.3 105.6 161.6 587.0 0.001 TNF α level 5.4 1.8 1.7 9.0 0.005 R = 0.163 CI confidence interval present study. IFN-γ has a transient effect, being quickly The present study had some limitations. First, spirom- cleared in the bloodstream [31, 33]. Therefore, it is etry was not performed in all patients because of various difficult to detect. In the pathophysiology of systemic internal circumstances, such as patients with difficulty in autoimmune myopathies, the participation of Th1 and performing spirometry (dyspnea) and lack of available Th2 cells is described as follows: IFN-γ is produced spirometry equipment at patient selection, even with by initially present Th1 cells and, then, Th1 cells these tests being carried out only in a later phase of the regulate Th2 cells using other inflammatory cytokines, ASSD diagnosis and treatment (> six months). Second, including YKL-40 [34, 35]. the degree of dyspnea, six-minute walking distance, and Fig. 2 Expression of YKL-40 in a muscle specimen from a patient with antisynthetase syndrome. Positive immunohistochemistry reaction of (A) YKL-40, (B) YKL-40*; (D) CD68, (E) CD4, and (F) CD8. Magnification is 200x or 400x*. As a negative control of YKL-40 (C), a specimen from a patient with antisynthetase syndrome, but without infiltrate inflammation Carboni et al. Advances in Rheumatology (2021) 61:44 Page 9 of 10 oxygen saturation were not analyzed. Third, the analysis 4. González-Gay MA, Montecucco C, Selva-O’Callaghan A, Trallero-Aragua E, Molberg O, Andersson H, et al. Timing of onset affects arthritis presentation of pneumopathy in CT was only descriptive, since there pattern in antisyntethase syndrome. Clin Exp Rheumatol. 2018;36(1):44–9. is no criterion in sytemic autoimmune myopathies that 5. Hakala BE, White C, Recklies AD. Human cartilage gp-39, a major secretory quantifies the degree of lung involvement. product of articular chondrocytes and synovial cells, is a mammalian member of a chitinase protein family. J Biol Chem. 1993;268(34):25803–10. https://doi.org/10.1016/S0021-9258(19)74461-5. Conclusions 6. De Ceuninck F, Gaufillier S, Bonnaud A, Sabatini M, Lesur C, Pastoureau P. This study showed high YKL-40 serum levels in patients YKL-40 (cartilage gp-39) induces proliferative events in cultured chondrocytes and synoviocytes and increases glycosaminoglycan synthesis with ASSD, and these correlated positively with TNF-α in chondrocytes. Biochem Biophys Res Commun. 2001;285(4):926–31. serum levels. Although other studies have shown the https://doi.org/10.1006/bbrc.2001.5253. expression of YKL-40 in lungs of patients with various 7. Lee CG, Da Silva CA, Dela Cruz CS, Ahangari F, Ma B, Kang MJ, et al. Role of chitin and chitinase/chitinase-like proteins in inflammation, tissue systemic autoimmune myopathies, we have shown high remodeling, and injury. Annu Rev Physiol. 2011;73(1):479–501. https://doi. YKL-40 levels in inflammatory muscle tissue cells. org/10.1146/annurev-physiol-012110-142250. 8. Prakash M, Bodas M, Prakash D, Nawani N, Khetmalas M, Mandal A, et al. Abbreviations Diverse pathological implications of YKL-40: answers may lie in ‘outside-in’ ALT: Alanine aminotransferase; ANA: Antinuclear antibodies; signaling. Cell Signal. 2013;25(7):1567–73. https://doi.org/10.1016/j.cellsig.2 ASSD: Antisynthetase syndrome; AST: Aspartate aminotransferase; CO: Carbon 013.03.016. monoxide; CPK: Creatine phosphokinase; CT: Computed tomography; 9. Kazakova MH, Batalov AZ, Mateva NG, Sarafian VS. YKL-40 and cytokines-a FEV1: Forced expiratory volume in one second; FVC: Forced vital capacity; new diagnostic constellation in rheumatoid arthritis? Folia Med. 2017;59(1): HAQ: Health Assessment Quality; HE: Hematoxylin-eosin; IMACS: International 37–42. https://doi.org/10.1515/folmed-2017-0013. Myositis Assessment and Clinical Studies Group; LDH: Lactate 10. Huang K, Wu LD. YKL-40: a potential biomarker for osteoarthritis. J Int Med dehydrogenase; MMT: Manual Muscle Testing; MYOACT: Myositis Disease Res. 2009;37(1):18–24. https://doi.org/10.1177/147323000903700102. Activity Assessment Visual Analogue Scales; PBS: Phosphate buffered saline; 11. Johansen JS, Stoltenberg M, Hansen M, Florescu A, Horslev-Petersen K, Rho: Correlation; TNF: Tumor necrosis factor; VAS: Visual analogue scale; YKL- Lorenzen I, et al. Serum YKL-40 concentrations in patients with rheumatoid 40: Protein chitinase-3-like-1 arthritis: relation to disease activity. Rheumatology (Oxford). 1999;38(7):618– 26. https://doi.org/10.1093/rheumatology/38.7.618. Acknowledgments 12. Jafari-Nakhjavani MR, Ghorbanihaghjo A, Bagherzadeh-Nobari B, Malek- Not applicable. Mahdavi A, Rashtchizadeh N. Serum YKL-40 levels and disease characteristics in patients with rheumatoid arthritis. Caspian J Int Med. 2019; Authors’ contributions 10:92–7. All authors contributed equally to write and review the manuscript. The 13. Väänänen T, Vuolteenaho K, Kautiainen H, Nieminen R, Mottonen T, author(s) read and approved the final manuscript. Hannonen P, et al. NEO-RACo Study Group. Glycoprotein YKL-40: A potential biomarker of disease activity in rheumatoid arthritis during Funding intensive treatment with csDMARDs and infliximab. Evidence from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) #2014/ randomised controlled NEO-RACo trial. PLoS One. 2017;12:e0183294. 09079–1, Conselho Nacional de Desenvolvimento Científico e Tecnológico 14. Peltomaa R, Paimela L, Harvey S, Helve T, Leirisalo-Repo M. Increased level (CNPq) #303379/2018–9 and Faculdade de Medicina da USP - SP to SKS. of YKL-40 in sera from patients with early rheumatoid arthritis: a new marker for disease activity. Rheumatol Int. 2001;20(5):192–6. https://doi.org/1 Availability of data and materials 0.1007/s002960100115. Not applicable. 15. Johansen JS, Baslund B, Garbarsch C, Hansen M, Stoltenberg M, Lorenzen I, et al. YKL-40 in giant cells and macrophages from patients with giant cell Declarations arteritis. Arthritis Rheum. 1999;42(12):2624–30. https://doi.org/10.1002/1529- 0131(199912)42:12<2624::AID-ANR17>3.0.CO;2-K. Ethics approval and consent to participate 16. Montagna GL, D’Angelo S, Valentini G. Cross-sectional evaluation of The study was approved by the local ethics committee (CAAE: YKL-40 serum concentrations in patients with systemic sclerosis. 89386618.0.0000.0068) and all participants signed the informed consent Relationship with clinical and serological aspects of disease. form. Rheumatolology. 2003;30:2147–51. 17. Nordenbaek C, Johansen JS, Halberg P, Wiik A, Garbarsch C, Ullman S, et al. Consent for publication High serum levels of YKL-40 in patients with systemic sclerosis are Not applicable. associated with pulmonary involvement. Scand J Rheumatol. 2005;34(4): 293–7. https://doi.org/10.1080/03009740510018598. Competing interests 18. Sun Y, Kong X, Wu S, Ma L, Yan Y, Lv P, et al. YKL-40 as a new biomarker of All authors declare that they have no conflicts of interest. disease activity in Takayasu arteritis. Int J Cardiol. 2019;293:231–7. https://doi. org/10.1016/j.ijcard.2019.06.058. Received: 11 December 2020 Accepted: 21 June 2021 19. Hozumi H, Fujisawa T, Enomoto N, Nakashima R, Enomoto Y, Suzuki Y, et al. Clinical utility of YKL-40 in polymyositis/dermatomyositis-associated interstitial lung disease. J Rheumatol. 2017;44(9):1394–401. https://doi.org/1 References 0.3899/jrheum.170373. 1. Connors GR, Christopher-Stine L, Oddis CV, Danoff SK. Interstitial lung 20. Gao MZ, Wei YY, Xu QW, Ji R, Han Z-J, Jian T-W, et al. Elevated serum YKL- disease associated with the idiopathic inflammatory myopathies: what 40 correlates with clinical characteristics in patients with polymyositis or progress has been made in the past 35 years? Chest. 2010;138(6):1464–74. dermatomyositis. Ann Clin Biochem. 2019;56(1):95–9. https://doi.org/10.11 https://doi.org/10.1378/chest.10-0180. 77/0004563218786979. 2. Cavagna L, Trallero-Araguás E, Meloni F, Cavazzana I, Rojas-Serrano J, Feist E, 21. Jiang L, Wang Y, Peng Q, Shu X, Wang G, Wu X, et al. Serum YKL-40 level is et al. Influence of antisynthetase antibodies specificities on antisynthetase associated with severity of interstitial lung disease and poor prognosis in syndrome clinical spectrum time course. J Clin Med. 2019;8(11):2013. https:// dermatomyositis with anti-MDA5 antibody. Clin Rheumatol. 2019;38(6): doi.org/10.3390/jcm8112013. 1655–63. https://doi.org/10.1007/s10067-019-04457-w. 3. Shinjo SK, Levy-Neto M. Anti-Jo-1 antisynthetase syndrome. Rev Bras 22. Cruellas MG, Viana V dos S, Levy-Neto M, FHC DS, Shinjo SK. Myositis- Reumatol. 2010;50(5):492–500. https://doi.org/10.1590/S0482-5004201 specific and myositis-associated autoantibody profiles and their clinical 0000500003. Carboni et al. Advances in Rheumatology (2021) 61:44 Page 10 of 10 associations in a large series of patients with polymyositis and dermatomyositis. Clinics. 2013;68:909–14. 23. Rider LG, Giannini EH, Harris-Love M, Joe G, Isenberg D, Pilkington C, et al. International myositis assessment and clinical studies group. Defining clinical improvement in adult and juvenile myositis. J Rheumatol. 2003;30(3): 603–17. 24. Rider LG, Feldman BM, Perez MD, Rennebohm RM, Lindsley CB, Zemel LS, et al. Development of validated disease activity and damage indices for the juvenile idiopathic inflammatory myopathies: I. physician, parent, and patient global assessments. Juvenile dermatomyositis disease activity collaborative study group. Arthritis Rheum. 1997;40(11):1976–83. https://doi. org/10.1002/art.1780401109. 25. Harris-Love MO, Shrader JA, Koziol D, Pahlajani N, Jain M, Smith M, et al. Distribution and severity of weakness among patients with polymyositis, dermatomyositis, and juvenile dermatomyositis. Rheumatology (Oxford). 2009;48(2):134–9. https://doi.org/10.1093/rheumatology/ken441. 26. Miller FW, Rider GL, Chung YL, et al. International Myositis Outcome Assessment Collaborative Study Group. Proposed preliminary core set measures for disease outcome assessment in adult and juvenile idiopathic inflammatory myopathies. Rheumatology (Oxford). 2001;40:1262–73. 27. Bruce B, Fries JF. The Stanford health assessment questionnaire: dimensions and practical applications. Health Qual Life Outcomes. 2003;1(1):20. https:// doi.org/10.1186/1477-7525-1-20. 28. Waseda Y, Johkoh T, Egashira R, Sumikawa H, Saeki K, Watanabe S, et al. Antisynthetase syndrome: pulmonary computed tomography findings of adult patients with antibodies to aminoacyl-tRNA synthetases. Eur J Radiol. 2016;85(8):1421–6. https://doi.org/10.1016/j.ejrad.2016.05.012. 29. Debray MP, Borie R, Revel MP, Naccache J-M, Khalil A, Toper C, et al. Interstitial lung disease in anti-synthetase syndrome: initial and follow-up CT findings. Eur J Radiol. 2015;84(3):516–23. https://doi.org/10.1016/j.ejrad.2 014.11.026. 30. Sada KE, Yamasaki Y, Maruyama M, Sugiyama H, Yamamura M, Maeshima Y, et al. Altered levels of adipocytokines in association with insulin resistance in patients with systemic lupus erythematosus. J Rheumatol. 2006;33(8): 1545–52. 31. Salomonsson S, Lundberg IE. Cytokines in idiopathic inflammatory myopathies. Autoimmunity. 2006;39(3):177–90. https://doi.org/10.1080/0891 32. Lundberg I, Brengman JM, Engel AG. Analysis of cytokine expression in muscle in inflammatory myopathies, Duchenne dystrophy, and non-weak controls. J Neuroimmunol. 1995;63(1):9–16. https://doi.org/10.1016/0165-572 8(95)00122-0. 33. Gallay L, Mouchiroud G, Chazaud B. Interferon-signature in idiopathic inflammatory myopathies. Curr Opin Rheumatol. 2019;31(6):634–42. https:// doi.org/10.1097/BOR.0000000000000653. 34. Hou C, Baba-Amer Y, Bencze M, Relaix F, Authier FJ. The effect of interferon- gamma on skeletal muscle cell biology. Med Sci. 2018;34:35–8. 35. Isoda K, Kotani T, Takeuchi T, Kiboshi T, Hata K, Ishida T, et al. Comparison of long-term prognosis and relapse of dermatomyositis complicated with interstitial pneumonia according to autoantibodies: anti-aminoacyl tRNA synthetase antibodies versus antimelanoma differentiation-associated gene 5 antibody. Rheumatol Int. 2017;37(8):1335–40. https://doi.org/10.1007/s002 96-017-3729-y. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Advances in Rheumatology Springer Journals

High YKL-40 serum levels and its expression in the muscle tissues of patients with antisynthetase syndrome

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Abstract

Background: The protein chitinase-3-like-1 (YKL-40) is rarely analyzed in patients with myositis. Therefore, we aimed to evaluate YKL-40 serum levels; correlate them with laboratory and clinical parameters, disease status, and treatment schemes; and analyze the YKL-40 expression in the muscle tissues of patients with antisynthetase syndrome (ASSD). Methods: This cross-sectional single-center study included 64 adult patients with ASSD who were age-, gender-, and ethnicity-matched to 64 healthy control individuals. Their YKL-40 serum levels were analyzed using the Enzyme-Linked Immunosorbent Assay (ELISA) kit method, while YKL-40 expression in muscle tissues was analyzed using an immunohistochemical technique. Disease status was assessed using the International Myositis Assessment and Clinical Studies Group (IMACS) set scores. Results: The patients’ mean age was 44.8 ± 11.8 years, and median disease duration was 1.5 (0.0–4.0) years. These patients were predominantly female (82.8%) and Caucasian (73.4%). Most patients had stable disease. The median YKL-40 serum level was significantly higher in patients with ASSD when compared to the healthy individuals: 538.4 (363.4–853.1) pg/mL versus 270.0 (201.8–451.9) pg/mL, respectively; P < 0.001. However, YKL-40 serum levels did not correlate with any clinical, laboratory, disease status, or therapeutic parameters (P > 0.050), except tumor necrosis factor alpha (TNF-α) serum levels (Spearman’s correlation, rho = 0.382; P = 0.007). YKL-40 was highly expressed by inflammatory cells found in muscle biopsy specimens. Conclusions: High YKL-40 serum levels were observed in patients with ASSD and correlated positively with TNF-α serum levels. Moreover, YKL-40 was expressed by the inflammatory cells of the muscle tissue. Keywords: Antisynthetase syndrome, Chitinase, Inflammatory myopathy, Muscle biopsy, Myositis Introduction The protein chitinase-3-like-1 (YKL-40) is a glycopro- Antisynthetase syndrome (ASSD) is a rare systemic tein secreted by inflammatory cells, including macro- autoimmune myopathy characterized by muscle, joint, phages and neutrophils [5, 6]. YKL-40 is associated with and pulmonary involvement, as well as by the presence several physiological processes, such as inflammation, of fever, “mechanic’shands,” and Raynaud’s phenomenon cell proliferation, angiogenesis, tissue fibrosis, and tissue [1–4]. In the laboratory, the ASSD is characterized by the remodeling [7]. YKL-40 also participates in a wide range presence of anti-aminoacyl-tRNA synthetase autoanti- of inflammatory responses, also stimulating the produc- bodies, such as anti-Jo-1 [1, 2]. tion of pro-inflammatory cytokines (e.g., IL-6, IL-18, and tumor necrosis factor alpha [TNF-α]), which are capable * Correspondence: samuel.shinjo@usp.br of increasing YKL-40 serum levels as a feedback mech- Division of Rheumatology, Faculdade de Medicina FMUSP, Universidade de anism [8, 9]. Sao Paulo, Av. Dr. Arnaldo, 455, 3° andar, sala 3184 - Cerqueira César. CEP: 01246-903, Sao Paulo, Brazil © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Carboni et al. Advances in Rheumatology (2021) 61:44 Page 2 of 10 High YKL-40 serum levels have been described in A commercially available line blot test kit (Myositis osteoarthritis [10] and in several systemic autoimmune Profile Euroline Blot test kit, Euroimmun, Lübeck, diseases, such as rheumatoid arthritis [11–14], giant cell Germany) was used to identify anti-aminoacyl-tRNA arteritis [15], systemic sclerosis [16, 17], Takayasu’s arteritis synthetase autoantibodies (anti-Jo-1, anti-EJ, anti-OJ, [18], and systemic autoimmune myopathies [19–21]. anti-PL-7, anti-PL-12, and anti-Ro-52). The assessment In rheumatoid arthritis, YKL-40 serum levels are not was performed according to previously established only increased but also correlate positively with the methods [22]. The immunological patterns evaluated disease activity [11–13]. However, YKL-40 is not were antinuclear antibodies (ANA) detected by an indir- associated with radiographic progression or predictive ect immunofluorescence technique on HEp-2 cells. values for disease remission [14]. In patients with We excluded pregnant patients and those with other systemic sclerosis, YKL-40 serum levels correlate systemic autoimmune diseases (overlap syndrome), mainly with joint involvement [16], lung involvement chronic or acute infection, neoplasia-associated myositis, (fibrosis or reduction in the diffusing capacity for car- and uncontrolled and serious comorbidities (e.g., liver dis- bon monoxide [DLCO]), digital articular deformities ease, nephropathy, and heart disease). Observing the internal through cutaneous retraction, and reduced patient sur- protocol of our service, a screening for neoplasms and other vival [17]. High YKL-40 serum levels are also described lung diseases was performed in all cases of myopathies. in patients with dermatomyositis and polymyositis, The following pre-standard and pre-parameterized correlating with the disease activity and severity and data were collected from the patients’ interviews: demo- with pulmonary involvement [19–21]. graphic information (current age, gender, ethnicity), clin- However, no studies have assessed YKL-40 serum ical information (disease duration; muscle, pulmonary, levels exclusively in patients with ASSD, which was the and joint involvement; Raynaud’s phenomenon; presence primary motivation for this study. We aimed to correlate of fever; and “mechanic’s hands”), laboratory findings YKL-40 serum levels with clinical and laboratory param- (serum levels of CPK, lactate dehydrogenase, aspartate eters, disease status, and therapeutic schemes, as well aminotransferase, alanine aminotransferase), disease sta- as to analyze the YKL-40 expression in muscle tissue tus, treatment (immunosuppressive, immunomodulatory, specimens from patients with ASSD. and glucocorticoid therapies), and comorbidities. More- over, on the interview day, blood samples (20 mL) were taken for laboratory analysis after an eight-hour fasting. Patients and methods Current clinical and laboratory disease status was This cross-sectional single-center study took place from assessed by applying the following questionnaires and 2017 to 2019 and included 66 adult patients with ASSD International Myositis Assessment & Clinical Studies followed up at the outgoing clinic of our service. All pa- Group (IMACS) scores: Myositis Disease Activity tients fulfilled the modified ASSD criteria classification Assessment Visual Analogue Scales (MYOACT) [23, 24], proposed by Connors et al. [1], but with modifications Manual Muscle Testing (MMT-8) [25], overall disease to increase its specificity, including the presence of at assessment by a physician and by the patient using a least two of the following three manifestations: muscle, visual analogue scale (VAS) [26], and the Health Assess- joint, and lung involvement; and in addition to persistent ment Quality (HAQ) [27]. fever; Raynaud’s phenomenon; “mechanic’s hands;” and Pulmonary assessment was performed using chest CT the presence of serum anti-aminoacyl-tRNA synthetase images. The following parameters were investigated: autoantibody. incipient pneumopathy, ground-glass opacities with or Muscle involvement was defined as the presence of without bronchiectasis, pulmonary nodules, and pul- progressive and proximal limb muscle weakness, in- monary fibrosis (honeycombing areas) [1, 28, 29]. All creased serum levels of muscle enzymes, such as creatine chest CT images were analyzed independently by the phosphokinase (CPK), myopathic pattern on electro- two authors of the present study, who are experienced neuromyography, and muscle biopsy compatible with in connective tissue diseases (RCSC and GLBP). In case inflammatory myopathy. Joint involvement was defined of disagreement between them, the images were dis- as non-deforming and non-erosive arthritis or arthralgia. cussed with radiology groups from our institute. Additionally, as part of the internal protocol of our Chest CT images obtained during a period ranging from service, patients underwent computed tomography (CT) six months before to six months after the patients’ partici- chest scans at disease onset and during follow-up. In this pation were included in this study. When possible, the study, lung involvement was defined as the presence of pulmonary assessment was supplemented by a pulmonary incipient pneumopathy, pulmonary nodules, ground-glass function test that considered the forced expiratory volume opacities with or without bronchiectasis, and pulmonary in one second (FEV1), forced vital capacity (FVC), FEV1/ fibrosis (e.g., honeycombing areas). FVC ratio, and DLCO. Carboni et al. Advances in Rheumatology (2021) 61:44 Page 3 of 10 The patients with ASSD were gender-, age-, and clinical and demographic characteristics were expressed ethnicity-matched to healthy control individuals as mean ± standard deviation (SD), for continuous vari- (patients’ relatives or employees of our service) selected ables, or as frequencies and percentages (%), for categor- for convenience. All laboratory tests were performed on ical variables. The median (interquartile 25th to 75th) the control group, and they were asked to answer all was calculated for continuous variables that did not have interview questions, except those related to the disease a normal distribution. Comparisons between patient and data. control parameters were made using the Mann-Whitney For the cytokine assessment, the material was test and Student t-test for continuous variables, while centrifuged at 3000 rpm for 10 min immediately after the Fisher’s exact test or chi-square test were used to as- blood collection (< 30 min), and the sera were aliquoted sess categorical variables. Spearman’s correlation was and stored at − 80 °C for further cytokine analysis. The used for analyzing the correlation (rho) between con- YKL-40 quantification was performed using a specific kit tinuous variables and YKL-40 values. Values between 0 (Human CHI3L1 ELISA kit, RayBiotech, USA) and and ± 0.333 were considered weak; values between ± processed following the manufacturer’s protocol. The 0.333 and ± 0.666 were considered moderate; and those interferon gamma (IFN-γ)and TNF-α tests were per- between ±0.666 and ± 1.000 were considered strong.” formed using the LUMINEX 100/200x xMAP technology The relation between YKL-40 values and other variables (Millipore, USA), as described elsewhere [30]. (identified as the presence or absence of a certain char- Additionally, the YKL-40 expression and location were acteristics) was performed using the Student t-test. To assessed in muscle tissues of three patients with ASSD, analyze the factors that together influence YKL-40, a whose biopsies were taken from the vastus lateralis multiple regression was performed considering all vari- muscle upon diagnosis. Sequential 5-μm thick frozen ables with P value < 0.10 as explanatory and the value of sections were first stained by hematoxylin-eosin and, YKL-40 as dependent. A value of P < 0.05 was adopted then, immunohistochemistry was performed. For immu- to indicate statistical significance. All analyses were per- nohistochemical analysis, monoclonal antibodies (CD4, formed using the statistical software SPSS version 22.0 CD8, CD68, and YKL-40: Abcam, USA) were used. (Chicago, IL, USA). Frozen specimens were fixed for 10 min in acetone at 4 °C. Endogenous peroxidase was blocked using 1% Results H O in absolute methanol three times for 10 min. After Sixty-six patients with ASSD were initially included in 2 2 rinsing the specimens in phosphate buffered saline (PBS) the present study. Two patients were excluded, one due (0.01 M, pH 7.4) for five minutes, these were incubated to overlap and the other due to neoplasia-associated in fetal serum in a wet chamber for one hour at 37 °C. myositis. No patients had acute or chronic infection, Primary antibodies were diluted in PBS and albumin were pregnant, or had severe and decompensated co- from 1% bovine serum was applied in wet chamber at morbidities. Therefore, 64 patients with ASSD were 37 °C overnight. Next, slides were washed in PBS and a evaluated and age-, gender-, and ethnicity-matched to secondary mouse biotinylated solution (StreptABCom- 64 healthy controls (Fig. 1). plex/HRP) was applied for 30 min at 37 °C and rinsed in The mean age of the 64 patients with ASSD and of the PBS. Then, the prepared StreptABComplex/HRP com- 64 control individuals was 44.8 ± 11.8 years and 42.6 ± plex was applied and incubated for 30 min at 37 °C. After 10.4 years, respectively (P = 0.278), with a predominance rinsing in PBS and incubation, the reactions were visual- of females (82.8% versus 82.8%, P > 0.999), and Caucasians ized using a solution of chromogenic substrate (3,3′-di- (73.4% versus 73.4%, P > 0.999) in both groups. The aminobenzidine tetrahydrochloride) for peroxidase. After median ASSD disease duration was 1.5 (0.0–4.0) years. a final rinse, hematoxylin counterstaining was performed. The initial and cumulative clinical manifestations, The slides were then mounted and covered with an autoantibody panels, and comorbidities of patients with aqueous-based mounting medium. All muscle specimens ASSD are shown in Table 1. were prepared at the same time as a batch. Human amyg- The current disease status of patients with ASSD at dala was used as a positive control, while a muscle speci- the time of inclusion in this study, lung parameters, and men from a patient with ASSD, but whose biopsy results treatments are shown in Table 2. were normal (without infiltrate inflammation), was used YKL-40 serum levels were significantly higher in pa- as a negative control. Inflammatory cell phenotyping tients with ASSD when compared to the control group: (CD4, CD8, CD68, and YKL-40) was analyzed in the 538.4 (363.4–853.1) pg/mL versus 270.0 (201.8–451.9) endomysium, perimysium, and pericapillary (endomysium pg/mL, respectively; P < 0.001 (Table 2). Additionally, a and perimysium) areas in 10 fields (200x magnification). significant increase in TNF-α levels was observed in pa- For statistical analysis, the Kolmogorov-Smirnov test tients with ASSD compared to the control group: 48.4 was used to assess each parameter distribution. The (31.0–66.3) pg/mL versus 39.6 (29.3–49.0) pg/mL; P = Carboni et al. Advances in Rheumatology (2021) 61:44 Page 4 of 10 Fig. 1 Study flowchart. ASSD: antisynthetase syndrome 0.011. IFN-γ serum levels were comparable in both Table 1 Cumulative clinical manifestations, autoantibody groups. panels, initial creatine phosphokinase serum levels, and Concerning demographic, clinical, laboratory, treata- comorbidities of the patients with antisynthetase syndrome ment, and comorbidity parameters, YKL-40 serum levels Antisynthetase syndrome n =64 had correlation with patients’ age (rho = 0.294; P = Cumulative clinical manifestations 0.040), TNF-α (rho = 0.382; P = 0.007), and current use Muscle involvement 61 (95.3) of prednisone (P < 0.047) - Tables 3 and 4. However, in Joint involvement 59 (92.2) multivariate analysis, only TNF-α levels had significant Lung involvement 57 (89.1) factor to predict the YKL-40 values (Table 5). Concerning the immunohistochemical analysis, YKL- Ground-glass 46 (71.9) 40 was expressed by inflammatory cells. Figure 2 shows Incipient pneumopathy 40 (62.5) a muscle biopsy specimen from a patient with ASSD. Pulmonary fibrosis 14 (21.9) Pulmonary nodules 11 (17.2) Discussion “Mechanic’s hands” 57 (89.1) To the best of our knowledge, this study is the first to Raynaud’s phenomenon 50 (78.1) show the relevance of YKL-40 in patients with ASSD. Fever 46 (71.9) High YKL-40 serum levels were observed in patients Autoantibody panels with ASSD and correlated positively with TNF-α serum Antinuclear factor 52 (81.3) levels. Besides, YKL-40 was expressed by inflammatory Anti-Ro-52 28 (43.8) cells in muscle tissues. Although ASSD is a rare disease, this study employed Anti-tRNA aminoacyl transferase 64 (100.0) strict inclusion criteria to increase the specificity in the Anti-Jo-1 52 (81.3) selection of the involved patients. In this context, a Anti-PL-12, anti-PL-7 or anti-EJ 12 (18.7) significant sample was included and paired by age, Anti-OJ 0 gender, and ethnicity with healthy individuals, thus Initial creatine phosphokinase (U/L) 3015 (632–8463) allowing for greater accuracy in comparative outcomes. Comorbidities Finally, information on patients was based on parame- Systemic arterial hypertension 22 (34.4) terized and pre-standardized data to ensure reliable Diabetes mellitus 12 (18.8) study data. Hypothyroidism 3 (4.7) High YKL-40 expression is described in the pathogen- esis of a number of diseases, and its utility as a bio- Acute myocardial infarction 3 (4.7) marker has been the subject of several studies [8–18]. Stroke 1 (1.6) Biologically, YKL-40 can stimulate angiogenesis and a Data are expressed as mean ± standard deviation (SD); median (interquartile 25th - 75th) or frequency (%) wide range of responses, such as inflammation, tissue Carboni et al. Advances in Rheumatology (2021) 61:44 Page 5 of 10 Table 2 Demographic, current disease status, pulmonary involvement, treatment, and the cytokines serum levels of the patients with antisynthetase syndrome. Laboratory profiles of the control group ASSD Control P (n = 64) (n = 64) IMACS set scores MMT-8 (0–80) 78 (71–80) – Patient’ VAS (0–10) 4.0 (2.0–6.0) – Physician’ VAS (0–10) 3.0 (0.0–5.0) – MYOACT (0–60) 4.0 (0.5–2.5) – HAQ (0.00–3.00) 0.67 (0.13–1.38) – CPK (U/L) 208 (98–597) 106 (78–161) < 0.001 LDH (U/L) 352 (243–590) 346 (312–345) 0.937 AST (U/L) 24 (17–37) 20 (16–23) 0.009 ALT (U/L) 24 (16–57) 18 (13–25) 0.030 Lung involvements Ground-glass 46 (71.9) – Incipient pneumopathy 40 (62.5) – Pulmonary fibrosis 14 (21.9) – Pulmonary nodules 11 (17.2) – FVC (% predict), n = 39 62.5 (48.0–73.5) – FEV1 (% predicted), n = 39 64 (49–77) – FEV1/FVC ratio, n = 39 0.90 (0.93–1.08) – DLCO (% predicted), n = 39 61.0 (37.8–73.0) – Treatment Prednisone Current use 46 (71.9) – Dose (mg/day) 10 (0–40) – Immunosuppressive drugs 47 (73.4) – Azathioprine 23 (35.9) – Mycophenolate mofetil 11 (17.2) – Methotrexate 14 (21.9) – Antimalarial 4 (6.3) – Leflunomide 2 (3.1) – Cyclosporine 3 (4.7) – Cyclophosphamide 1 (1.7) – Rituximab 16 (25.0) – Cytokines YKL-40 (pg/mL) 538.4 (363.4–853.1) 270.0 (201.8–451.9) < 0.001 IFNγ (pg/mL) 0.02 (0.00–1.89) 0.02 (0.00–0.17) 0.876 TNFα (pg/mL) 48.4 (31.0–66.3) 39.6 (29.3–49.0) 0.011 Data are expressed as mean ± standard deviation (SD); median (interquartile 25th - 75th) or frequency (%) ALT alanine aminotransferase; ASSD antisynthetase syndrome; AST aspartate aminotransferase; CPK creatine phosphokinase; DLCO diffusion of carbon dioxide; FEV1 forced expiratory volume in one second; FVC forced vital capacity; HAQ Health Assessment Questionnaire; IFN interferon; IMACS: International Myositis Assessment and Clinical Studies Group; LDH lactate dehydrogenase; MMT-8 Manual Muscle Testing; MYOACT Myositis Disease Activity Assessment Visual Analogue Scales; TNF tumor necrosis factor; VAS Visual Analog Scale; YKL chitinase-3-like-1 protein remodeling, and allergic reactions. These responses are YKL-40 is seldom described in studies regarding sys- often mediated by other cytokines (e.g., IL-6, IL-18, and temic autoimmune myopathies [19–21]. In this context, TNF-α), which can regulate the YKL-40 secretion [8, 9]. Hozumi et al. [19] showed high YKL-40 serum levels in Carboni et al. Advances in Rheumatology (2021) 61:44 Page 6 of 10 Table 3 Correlation between YKL-40 serum levels and cases with associated interstitial lung disease. Addition- continuous variables (demographic, clinical, laboratory and lung ally, patients with neoplasm associated myositis were parameters, and disease status) of the patients with included. antisynthetase syndrome Gao et al. [20] also observed high YKL-40 serum levels rho P in patients with dermatomyositis and polymyositis, Demographic data which correlated positively with interstitial lung disease and with the disease activity and severity evaluated via Age 0.294 0.040 MYOACT, C-reactive protein, ferritin, and erythrocyte Clinical parameters sedimentation rates. However, the authors did not Fever 0.070 0.631 analyze anti-aminoacyl-tRNA synthetase autoantibodies Raynaud phenomenon 0.144 0.324 in their patients. “Mechanic’s hands” −0.045 0.757 Jiang et al. [21] observed a positive correlation between Muscle involvement −0.219 0.131 YKL-40 serum levels and patients with clinically amyo- pathic dermatomyositis and positive anti-MDA-5, espe- Joint involvement −0.190 0.192 cially in those with severe interstitial lung disease. Laboratory parameters In our study, high YKL-40 serum levels were also ob- TNF-α level 0.382 0.007 served in patients with ASSD. However, YKL-40 did not IFN-γ level 0.125 0.395 correlate with any clinical and laboratory parameters, Lung involvement disease status, or therapeutic schemes. There was also FVC (% predict) 0.018 0.930 no correlation with any pulmonary parameters (e.g., pa- tient’s lung symptoms, chest CT images, and pulmonary FEV1 (% predicted) 0.089 0.667 function tests). Since this was a cross-sectional analysis, FEV1/FVC ratio −0.066 0.758 a prospective analysis of these parameters would be DLCO (% predicted) 0.533 0.091 interesting. Disease status To analyze disease status, Hozumi et al. [19] used vari- MMT-8 0.037 0.801 ables such as ferritin, KL-6 (Krebs von den Lungen-6), Patient’ VAS −0.173 0.239 SP-D (surfactant protein-D), PaO , and DLCO, while Jiang et al. [21] used ferritin, CPK, T CD3+ cell count, Physician’ VAS −0.234 0.110 C-reactive protein, FVC, and DLCO. In contrast, we MYOACT −0.077 0.614 used the IMACS set scores, which validated instruments HAQ 0.166 0.266 for clinical evaluation in systemic autoimmune myop- Creatine phosphokinase 0.131 0.371 athies [23–27]. Lactate dehydrogenase 0.138 0.460 Unlike limited studies regarding YKL-40 in myositis, a Aspartate aminotransferase 0.033 0.854 limitation of our study was the fact that we did not spe- cifically analyze the YKL-40 expression in lung biopsy Alanine aminotransferase 0.034 0.866 specimens. Nonetheless, the YKL-40 expression analyzed Treatment in muscle specimens reinforces the hypothesis that YKL- Prednisone: dose (mg/day) 0.217 0.139 40 expression is systemic, not being limited to lung DLCO diffusion of carbon dioxide; FEV1 forced expiratory volume in one involvement. second; FVC forced vital capacity; HAQ Health Assessment Questionnaire; IFN interferon; MMT-8 Manual Muscle Testing; MYOACT Myositis Disease Activity YKL-40 can be expressed and secreted by various Assessment Visual Analogue Scales; TNF tumor necrosis factor; VAS Visual types of cells, including macrophages, neutrophils, and Analog Scale chondrocytes. Its expression is regulated by several cyto- kines, which mediate the proliferation, tissue remodel- patients with dermatomyositis and polymyositis with ing, and production of other inflammatory mediators. In associated interstitial lung disease, and YKL-40 serum ASSD and systemic autoimmune inflammatory disease, levels were associated with a worse prognosis. Further- the elevated YKL-40 serum levels might derive come more, the authors correlated YKL-40 levels negatively from systemic inflammatory cells or local cells (e.g., with DLCO and arterial oxygen pressure (PaO ), find- blood, muscle, skin, lungs). This was the first study to ing a high expression of YKL-40 in alveolar macro- demonstrate the increased expression of YKL-40 in the phages and bronchial epithelial cells. Notably, the inflammatory cells of muscle tissue obtained from a pa- authors found that 42% of their patients had positive tient with ASSD. anti-aminoacyl-tRNA synthetase autoantibodies. There- As previously mentioned, YKL-40 is regulated by vari- fore, they might have included patients with ASSD and ous pro-inflammatory cytokines [8, 9]; therefore, we not necessarily pure dermatomyositis and polymyositis assessed the TNF-α and IFN-γ serum levels. Carboni et al. Advances in Rheumatology (2021) 61:44 Page 7 of 10 Table 4 Relation between YKL-40 serum levels and others variables (demographic, clinical, laboratory and lung parameters, disease status, and comorbidities) of the patients with antisynthetase syndrome Group 1 Group 2 P value n Mean ± SD n Mean ± SD Demographic data Gender (female) 53 628.5 ± 363.6 11 691.3 ± 461.0 0.658 Ethnicity (Caucasian) 47 572.5 ± 316.7 17 793.2 ± 468.2 0.111 Clinical parameters Fever 18 579.4 ± 343.8 46 664.3 ± 394.0 0.484 Raynaud phenomenon 14 536.6 ± 361.8 50 677.4 ± 382.7 0.255 “Mechanic’s hands” 7 658.6 ± 334.6 57 637 ± 389.2 0.891 Muscle involvement 3 980.2 ± 178.7 61 625.6 ± 379.2 0.198 Joint involvement 5 973.5 ± 602.1 59 610.4 ± 347.5 0.066 Laboratory parameters Antinuclear factor 12 685.7 ± 303.5 52 628.4 ± 398.4 0.674 Anti-Ro-52 35 663.9 ± 406.5 28 638.5 ± 348.0 0.818 Anti-Jo-1 11 728.4 ± 455.9 53 617.4 ± 359.7 0.414 Anti-PL-7 60 619.1 ± 377.6 4 876.1 ± 351.5 0.197 Anti-PL-12 60 627.2 ± 360.9 4 784.8 ± 595.5 0.431 Anti-EJ 61 653.7 ± 380.8 3 320.4 ± 52.2 0.227 Lung involvement Ground-glass 18 658.2 ± 348.1 46 634.2 ± 392.6 0.851 Incipient pneumopathy 24 695.9 ± 387.1 40 607.6 ± 376.4 0.437 Pulmonary fibrosis 50 586.2 ± 335.7 14 826.2 ± 471.8 0.063 Treatment Prednisone (current use) 17 483.3 ± 222.6 46 684.2 ± 406.3 0.047 Immunosuppressives/biologicals Azathioprine 43 618.9 ± 354.7 21 676.5 ± 425.2 0.613 Mycophenolate mofetil 54 648.4 ± 383.8 10 603.1 ± 375.2 0.750 Methotrexate 52 606.3 ± 338.1 12 790.3 ± 521.9 0.337 Antimalarial 61 645.3 ± 385.8 3 516.7 ± 91.7 0.643 Leflunomide 62 642.1 ± 382.5 2 544.0 ± 0 0.801 Cyclosporine 62 650.8 ± 381.1 2 388.7 ± 272.8 0.343 Cyclophosphamide 63 644.0 ± 381.8 1 451.9 ± 0 0.621 Rituximab 48 690.0 ± 364.8 16 537.0 ± 397.9 0.188 Comorbidities Systemic arterial hypertension 42 590.4 ± 379.9 22 742.5 ± 366.9 0.190 Diabetes mellitus 52 645.1 ± 395.0 12 610.0 ± 283.2 0.823 Hypothyroidism 61 628.0 ± 381.0 3 922.5 ± 205.8 0.286 Acute myocardial infarction 61 641.6 ± 382.6 3 567.2 ± 0 0.848 Stroke 63 645.2 ± 381.0 1 391.6 ± 0 0.513 SD standard deviation High TNF-α serum levels were observed in patients study, in which a positive correlation was found between with ASSD, similar to what occurs in other systemic YKL-40 and TNF-α serum levels. autoimmune myopathies [31, 32]. TNF-α can induce Although IFN-γ is involved in the ASSD pathophysi- YKL-40 production [8]. These data corroborated our ology, we did not find high cytokine serum levels in the Carboni et al. Advances in Rheumatology (2021) 61:44 Page 8 of 10 Table 5 Multivariate analysis Coefficient Standard error Lower Upper P value CI (95%) CI (95%) (Constant) 374.3 105.6 161.6 587.0 0.001 TNF α level 5.4 1.8 1.7 9.0 0.005 R = 0.163 CI confidence interval present study. IFN-γ has a transient effect, being quickly The present study had some limitations. First, spirom- cleared in the bloodstream [31, 33]. Therefore, it is etry was not performed in all patients because of various difficult to detect. In the pathophysiology of systemic internal circumstances, such as patients with difficulty in autoimmune myopathies, the participation of Th1 and performing spirometry (dyspnea) and lack of available Th2 cells is described as follows: IFN-γ is produced spirometry equipment at patient selection, even with by initially present Th1 cells and, then, Th1 cells these tests being carried out only in a later phase of the regulate Th2 cells using other inflammatory cytokines, ASSD diagnosis and treatment (> six months). Second, including YKL-40 [34, 35]. the degree of dyspnea, six-minute walking distance, and Fig. 2 Expression of YKL-40 in a muscle specimen from a patient with antisynthetase syndrome. Positive immunohistochemistry reaction of (A) YKL-40, (B) YKL-40*; (D) CD68, (E) CD4, and (F) CD8. Magnification is 200x or 400x*. As a negative control of YKL-40 (C), a specimen from a patient with antisynthetase syndrome, but without infiltrate inflammation Carboni et al. Advances in Rheumatology (2021) 61:44 Page 9 of 10 oxygen saturation were not analyzed. Third, the analysis 4. González-Gay MA, Montecucco C, Selva-O’Callaghan A, Trallero-Aragua E, Molberg O, Andersson H, et al. Timing of onset affects arthritis presentation of pneumopathy in CT was only descriptive, since there pattern in antisyntethase syndrome. Clin Exp Rheumatol. 2018;36(1):44–9. is no criterion in sytemic autoimmune myopathies that 5. Hakala BE, White C, Recklies AD. Human cartilage gp-39, a major secretory quantifies the degree of lung involvement. product of articular chondrocytes and synovial cells, is a mammalian member of a chitinase protein family. J Biol Chem. 1993;268(34):25803–10. https://doi.org/10.1016/S0021-9258(19)74461-5. Conclusions 6. De Ceuninck F, Gaufillier S, Bonnaud A, Sabatini M, Lesur C, Pastoureau P. This study showed high YKL-40 serum levels in patients YKL-40 (cartilage gp-39) induces proliferative events in cultured chondrocytes and synoviocytes and increases glycosaminoglycan synthesis with ASSD, and these correlated positively with TNF-α in chondrocytes. Biochem Biophys Res Commun. 2001;285(4):926–31. serum levels. Although other studies have shown the https://doi.org/10.1006/bbrc.2001.5253. expression of YKL-40 in lungs of patients with various 7. Lee CG, Da Silva CA, Dela Cruz CS, Ahangari F, Ma B, Kang MJ, et al. Role of chitin and chitinase/chitinase-like proteins in inflammation, tissue systemic autoimmune myopathies, we have shown high remodeling, and injury. Annu Rev Physiol. 2011;73(1):479–501. https://doi. YKL-40 levels in inflammatory muscle tissue cells. org/10.1146/annurev-physiol-012110-142250. 8. Prakash M, Bodas M, Prakash D, Nawani N, Khetmalas M, Mandal A, et al. Abbreviations Diverse pathological implications of YKL-40: answers may lie in ‘outside-in’ ALT: Alanine aminotransferase; ANA: Antinuclear antibodies; signaling. Cell Signal. 2013;25(7):1567–73. https://doi.org/10.1016/j.cellsig.2 ASSD: Antisynthetase syndrome; AST: Aspartate aminotransferase; CO: Carbon 013.03.016. monoxide; CPK: Creatine phosphokinase; CT: Computed tomography; 9. Kazakova MH, Batalov AZ, Mateva NG, Sarafian VS. YKL-40 and cytokines-a FEV1: Forced expiratory volume in one second; FVC: Forced vital capacity; new diagnostic constellation in rheumatoid arthritis? Folia Med. 2017;59(1): HAQ: Health Assessment Quality; HE: Hematoxylin-eosin; IMACS: International 37–42. https://doi.org/10.1515/folmed-2017-0013. Myositis Assessment and Clinical Studies Group; LDH: Lactate 10. Huang K, Wu LD. YKL-40: a potential biomarker for osteoarthritis. J Int Med dehydrogenase; MMT: Manual Muscle Testing; MYOACT: Myositis Disease Res. 2009;37(1):18–24. https://doi.org/10.1177/147323000903700102. Activity Assessment Visual Analogue Scales; PBS: Phosphate buffered saline; 11. Johansen JS, Stoltenberg M, Hansen M, Florescu A, Horslev-Petersen K, Rho: Correlation; TNF: Tumor necrosis factor; VAS: Visual analogue scale; YKL- Lorenzen I, et al. Serum YKL-40 concentrations in patients with rheumatoid 40: Protein chitinase-3-like-1 arthritis: relation to disease activity. Rheumatology (Oxford). 1999;38(7):618– 26. https://doi.org/10.1093/rheumatology/38.7.618. Acknowledgments 12. Jafari-Nakhjavani MR, Ghorbanihaghjo A, Bagherzadeh-Nobari B, Malek- Not applicable. Mahdavi A, Rashtchizadeh N. Serum YKL-40 levels and disease characteristics in patients with rheumatoid arthritis. Caspian J Int Med. 2019; Authors’ contributions 10:92–7. All authors contributed equally to write and review the manuscript. The 13. 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Journal

Advances in RheumatologySpringer Journals

Published: Jul 5, 2021

Keywords: Antisynthetase syndrome; Chitinase; Inflammatory myopathy; Muscle biopsy; Myositis

References