背景：肌炎特异性抗体在特发性炎性肌病（idiopathic inflammatory myopathy，IIM）患者的疾病诊断，治疗以及预后判断中发挥重要的价值。合并快速进展性肺间质纤维化（rapid progressive interstitial lung disease，RP-ILD）的IIM患者死亡率高，预后较差。半乳糖凝集素-9（galectin-9，Gal-9）在免疫调节过程中发挥多种功能。本研究分为两部分，第一部分探讨一类IIM特异性抗体——抗Mi-2抗体相关的临床表型及预后；第二部分探讨一类新型IIM相关的血清学标志物——Gal-9在IIM患者中的分布及临床意义。

第一部分 抗Mi-2抗体阳性皮肌炎患者的临床表型及预后研究

目的：探讨抗Mi-2抗体阳性皮肌炎（dermatomyositis，DM）患者的临床特征，病理特征，抗体水平与患者疾病活动度之间的相关性，患者的治疗反应以及预后情况。

方法: 采用ELISA法检测357例DM患者血清中的抗Mi-2β抗体。在横断面研究和纵向研究中分析抗Mi-2β抗体相关的临床特征。

结果：在DM患者中，有40例（11.2%）患者血清抗Mi-2β抗体阳性。抗Mi-2β+ DM患者合并“V”形疹（P = 0.001），披肩征（P = 0.003）和肌无力（P = 0.013）的比例显著升高，但合并ILD（P = 0.005）和恶性肿瘤（P = 0.041）的比例显著下降。抗Mi-2β抗体水平与医师对疾病活动度的整体评估评分（r = 0.33，P = 0.039，n = 40），血清肌酸激酶水平（r = 0.52，P < 0.001，n = 40）呈正相关，与徒手肌力检查评分呈负相关（r = -0.34，P = 0.029，n = 40）。在纵向研究中，抗Mi-2β抗体水平与患者的疾病活动度呈正相关（β = 0.039，P < 0.001，n = 16）。在长期随访中，中位随访时间为44个月，有97.0%的患者出现临床缓解。此外，有26例患者的病程超过2年，其中有61.5%（16/26）的患者在患病过程中没有出现复发。有5例（15.1%）患者病情缓解的时间超过3个月，且停止服用药物。与抗Mi-2β- DM患者相比，抗Mi-2β+ DM患者的死亡率显著降下降（log-rank P = 0.035）。在长期随访中，我们发现并非所有缓解期的患者血清中的抗Mi-2β抗体都转为阴性。

结论：抗Mi-2β抗体阳性患者是DM的一类特殊亚型，该亚型表现为典型的近端肌肉以及皮肤受累，较少合并ILD及恶性肿瘤，治疗效果好，预后好。患者的血清该抗体水平与疾病活动度相关，但在长期随访中，患者血清中的抗体较少转阴。

第二部分Galectin-9在特发性炎性肌病中的分布及临床意义的研究

目的：检测Gal-9在DM患者中的表达；探究Gal-9在DM-ILD发病机制中的作用途径。

方法：用ELISA法检测154例IIM患者和30例健康对照的血清Gal-9水平。采用横断面研究和纵向研究分析患者血清Gal-9水平与临床特征之间的关系。检测Gal-9在IIM患者和健康对照血清和分离的外周血单个核细胞 （peripheral blood monocular cells，PBMCs）中的表达。采用免疫组织化学的方法检测抗黑色素瘤分化相关基因5（melanoma differentiation-associated gene 5，MDA5）抗体阳性DM患者肺组织中Gal-9及其配体Tim-3和CD44的表达。此外，本研究探索了Gal-9对人肺成纤维细胞（MRC-5）的作用。

结果：IIM患者 [median 19.8 ng/mL, 四分位数间距（interquartile range, IQR）10.0-33.6，n = 154] 的血清Gal-9水平显著高于健康对照（median 4.9 ng/mL，IQR 3.5-6.3，n = 30）（P < 0.001）。分层分析显示，DM患者（median 23.7 ng/mL，IQR 12.3-35.9，n = 129）的血清Gal-9水平显著高于免疫介导坏死性肌病（immune-mediated necrotizing myopathy，IMNM）患者（median 7.4 ng/mL，IQR 5.2-10.8，n = 25）（P < 0.001）和健康对照（P < 0.001）。抗MDA5+ DM患者（median 33.8 ng/mL，IQR 21.9-44.7 ng/mL，n = 56）的血清Gal-9水平显著高于抗MDA5- DM患者（median 16.2 ng/mL，IQR 10.0-26.9 ng/mL，n = 73）（P < 0.001）。在抗MDA5+ DM患者中，合并RP-ILD的患者（median 42.4 ng/mL，IQR 34.9-68.5，n = 20）血清Gal-9水平显著高于不合并RP-ILD的患者（median 26.4 ng/mL，IQR 17.0-39.8，n = 36）（P < 0.001），且血清Gal-9水平与RP-ILD的严重程度相关。在横断面研究和纵向研究中，血清Gal-9水平与抗MDA5+ DM患者的疾病活动度呈正相关。抗MDA5+ DM患者PBMCs产生Gal-9的水平显著高于IMNM患者（P = 0.022）和健康对照（P = 0.045）。此外，抗MDA5+ DM患者PBMCs中Gal-9 mRNA的水平与I型干扰素诱导基因MX1（r = 0.659，P = 0.020，n = 12）和IFIH1（r = 0.787，P = 0.002，n = 12）的mRNA水平呈正相关。免疫组化显示，合并RP-ILD的DM患者肺组织中Gal-9和Tim-3的表达增强。体外培养的肺成纤维细胞MRC-5经Gal-9蛋白刺激后，单核细胞趋化蛋白-1的mRNA表达增加（P = 0.024）。

结论：Gal-9是抗MDA5+ DM，特别是合并RP-ILD患者疾病活动度的一个潜在的血清学志物。Gal-9/Tim-3通路的异常表达可能参与了DM-ILD的发病机制。

Background: Myositis specific antibodies play important roles in the diagnosis, treatment and prognosis of patients with idiopathic inflammatory myopathy (IIM). The mortality rate was high and the prognosis was poor in IIM patients with rapidly progressive interstitial lung disease (RP-ILD). Multiple functions of galectin-9 (Gal-9) were found in immune regulation. The first part was to explore the clinical phenotype and prognosis of anti-Mi-2-positive dermatomyositis (DM) patients; the second part was to explore the distribution and clinical significance of a new myositis-related serum biomarker Gal-9 in patients with IIM.

Part I. A study on the clinical features and prognosis of anti-Mi-2 antibodies in DM patients

Objective: To investigate the clinical features, pathological features, the correlation between serum levels of anti-Mi-2 antibodies and disease activity, and prognosis of DM patients carrying anti-Mi-2 antibodies.

Methods: Three hundred and fifty-seven DM patients were included. Serum anti-Mi-2β antibodies were detected by enzyme-linked immunosorbent assays (ELISA). The clinical features associated with anti-Mi-2β antibodies were analyzed in cross-sectional and longitudinal studies.

Results: Forty out of the 357 (11.2%) DM patients were determined positive for anti-Mi-2β antibodies. Anti-Mi-2β-positive DM patients were found to have a higher incidence of V sign (P = 0.001), shawl sign (P = 0.003) and muscle weakness (P = 0.013) compared with patients without anti-Mi-2β antibodies. In addition, they were found to have lower frequency of ILD (P = 0.005) and malignancy (P = 0.041) than patients witout anti-Mi-2β antibodies. The levels of anti-Mi-2β antibodies were positively correlated with physician’s global assessment (PGA) visual analog scale (VAS) scores (r = 0.33, P = 0.039, n = 40), serum creatine kinase levels (r = 0.52, P < 0.001, n = 40) and negatively correlated with manual muscle testing in 8 muscles scores (r = -0.34, P = 0.029, n = 40). The longitudinal study also revealed a positive correlation between anti-Mi-2β antibody levels and PGA VAS scores (β = 0.039, P < 0.001, n = 16). The medium follow-up period was 44 months. Ninety-seven percent of patients got clinical remission. Among 26 patients whose disease course longer than 2 years, sixteen (61.5%) patients were monocyclic, five (15.1%) patients showed complete remission and they were drug-free for more than 3 months. The mortality rate of anti-Mi-2β-positive DM patients was lower than anti-Mi-2β-negative patients by using Kaplan-Meier survival curves (log-rank P = 0.035). Interestingly, in the long-term longitudinal observation, anti-Mi-2β antibodies did not return negative in all patients.

Conclusion: DM patients carrying anti-Mi-2β antibodies were frequently involved in cutaneous and muscle, and had low incidence of ILD and malignancy. In addition, the treatment response and outcome of anti-Mi-2β-positive DM patients were good. The correlation was observed between anti-Mi-2β levels and disease activities, but the anti-Mi-2β levels rarely became negative in the longitudinal study.

Part II. A study on the prevalence and related clinical significance of Gal-9 in IIM patients

Objective: To investigate the expression of Gal-9 in patients with DM and the association between Gal-9 and DM-ILD.

Methods: One hundred and fifty-four patients with IIM and 30 healthy controls (HCs) were included in the current study. The association between serum levels of Gal-9 and clinical features were analyzed both in cross-sectional and longitudinal studies. The expression of Gal-9 in the sera and isolated peripheral blood mononuclear cells (PBMCs) were tested. The expression of Gal-9 and its ligand (T-cell immunoglobulin mucin (Tim)-3 and CD44) was tested in lung tissues from patients carrying anti-melanoma differentiation-associated gene 5 (MDA5) antibodies by immunohistochemistry. The response of human lung fibroblasts (MRC-5) stimulated Gal-9 was tested in vitro.

Results: Significantly differences of serum levels of Gal-9 were observed in IIM patients (median 19.8 ng/mL, interquartile range (IQR) 10.0-33.6, n = 154) than HCs (median 4.9 ng/mL, IQR 3.5-6.3, n = 30) (P < 0.001). Serum levels of Gal-9 were higher in DM patients (median 23.7 ng/mL, IQR 12.3-35.9, n = 129) compared to those in immune-mediated necrotizing myopathy (IMNM) patients (median 7.4 ng/mL, IQR 5.2-10.8, n = 25) and HCs (P < 0.001). Serum levels of Gal-9 were significantly higher in anti-MDA5-positive DM patients (median 33.8 ng/mL，IQR 21.9-44.7 ng/mL, n = 56) than in anti-MDA5-negative DM patients (median 16.2 ng/mL, IQR 10.0-26.9 ng/mL, n = 73) (P < 0.001). Among DM patients carrying anti-MDA5 antibodies, higher serum levels of Gal-9 were observed in patients with RP-ILD (median 42.4 ng/mL, IQR 34.9-68.5, n = 20) than patients without RP-ILD (median 26.4 ng/mL, IQR 17.0-39.8, n = 36) (P < 0.001). In cross-sectional and longitudinal studies, the association was observed between serum levels of Gal-9 and disease activity in DM patients carrying anti-MDA5 antibodies. Higher levels of Gal-9 were produced from PBMCs isolated from DM patients than those from IMNM patients (P = 0.022) and HCs (P = 0.045). Positive correlation was observed between the Gal-9 mRNA levels and type I interferon-inducible genes MX1 (r = 0.659, P = 0.020, n = 12) and IFIH1 (r = 0.787, P = 0.002, n = 12) in PBMCs from DM patients carrying anti-MDA5 antibodies. The expression of Gal-9 and Tim-3 was increased in the lung tissues of DM patients with RP-ILD. The mRNA expression of monocyte chemoattractant protein-1 in MRC-5 fibroblasts stimulated with Gal-9 was up-regulated in vitro (P = 0.024).

Conclusion: Gal-9 might be a biomarker for monitoring disease activity and the severity of RP-ILD in DM patients carrying anti-MDA5 antibodies. Aberrant expression of the Gal-9 and its ligand Tim-3 might be involved in the DM-ILD immunopathogenesis.