中文摘要

目的

类风湿关节炎相关间质性肺病（Rheumatoid arthritis-associated interstitial lung disease, RA-ILD）是一种与RA密切相关的严重并发症，肺部纤维化是其重要特征之一。由于RA-ILD的病理机制尚不完全明确且临床治疗手段有限，迫切需要探索RA-ILD的相关机制，以期为临床干预和个性化治疗提供理论依据。①本研究的目的在于探讨RA-ILD的临床特点，为诊断治疗提供大数据依据；②建立RA-ILD动物模型，为RA-ILD的机制研究提供载体；③多组学分析早期RA-ILD肺组织的病理特点及潜在分子机制，为RA-ILD干预和研究提供新方向；④结合基础和临床，探讨Cd8⁺TEM在RA-ILD肺组织损伤、修复和纤维化进程中的关系；⑤基于RA-ILD动物模型，探讨ILD一线药泼尼松和RA一线药MTX的治疗作用，以期为RA-ILD的临床干预提供实验依据。

材料和方法

通过回顾性队列研究，用大数据分析方法，对国家风湿病数据中心（CRDC）中国RA大数据和中国疾控中心（CDC）数据进行分析，总结RA-ILD有别于无ILD合并的RA（RA-noILD）临床特点。通过建立多种动物模型，用CT影像、病理切片等分析关节炎和间质肺病的特点，筛选具有RA-ILD特点的动物模型。通过单细胞测序、蛋白组测序和空间转录组测序，分析肺组织间的分子差异。通过多组学分析加RA-ILD患者全血样本验证，用流式、qPCR等，分析Cd8⁺TEM在肺纤维化中发挥作用的免疫机制。基于RA-ILD动物模型，用MTX（0.5 mg/kg/周）和泼尼松（6 mg/kg/天）进行治疗，行CT、病理等检查，观察RA和ILD一线药在RA-ILD中的治疗效果。

结果

本研究纳入88,793例RA患者，其中RA-ILD组1,058例，RA-noILD组1,058例（随机匹配）。结果表明，RA-ILD组患者的发病年龄显著较RA-noILD组晚，男性比例较高，病程较长，吸烟或既往吸烟比例也较高。RA-ILD组的死亡率显著高于RA-noILD组，主要死因为肺炎和肺纤维化（占比＞40%）。关节炎的严重程度分析显示，RA-ILD组的关节压痛和肿胀关节数均显著高于RA-noILD组。肺部表现方面，RA-ILD组干咳发生率高达78.92%，KL-6阳性率为48.68%，均显著高于RA-noILD组。实验室检查显示，RA-ILD组的RF、anti-CCP滴度较高，炎症标志物CRP和ESR水平显著升高。治疗分析表明，RA-ILD组的疾病活动度明显高于RA-noILD组，且好转率显著低于RA-noILD组（P＜0.001）。

单纯的胶原诱导关节炎（Collagen-Induced Arthritis, CIA）模型不能形成RA-ILD动物模型。CIA混合博来霉素（bleomycin, BLM）或蛋白脂多糖（ lipopolysaccharide, LPS）的CIABLMS和CIALPSS模型，在1-6月后可观察到CIA评分反复升高，关节炎反复发作，同时组织病理、CT影像观察到肺部病变持续加重。CIABLMS则出现全身多器官纤维化，时间越长纤维化程度越重。综合评估CIALPSS更符合RA-ILD特点。

通过scRNA-seq、蛋白质组学和空间转录组学分析，发现髓系细胞、基质细胞、上皮细胞和淋巴细胞的变化显著。在髓系细胞中，CIALPSS和CIABLMS组的肺泡巨噬细胞、单核细胞和浆细胞样树突细胞增多，富集于抗原递呈、补体激活、T细胞活化等通路；基质细胞中，成纤维细胞和间皮细胞比例升高，纤维化基因上调，涉及ECM重塑、TGF-β信号和血管生成；上皮细胞中AT2细胞增多，相关基因上调，涉及抗原递呈和氧化应激；淋巴细胞中，CIALPSS组T细胞和中性粒细胞活化，CIABLMS组B细胞比例增加，表现出免疫调节差异。蛋白组和空间转录组学验证了这些特点。

RA-ILD模型组中Cd8⁺TEM的比例显著增加，表现出增强的细胞毒性、增殖和趋化能力，尤其是Nkg7、Gzmk、Ccl5等细胞毒性因子和趋化因子的上调。细胞间通讯分析表明，Cd8⁺TEM上的Cx3cr1受体与AT2细胞上的Cx3cl1配体结合，促进Cd8⁺TEM的初步招募；Ccl5和Ccl4等趋化因子通过与Cd8⁺TEM上的Ccr5受体相互作用，形成正反馈回路，进一步促进Cd8⁺TEM的聚集。同时，Ccl5、Ccl4和Ccl3等趋化因子通过与Ccr1和Ccr5受体作用，招募粒细胞、巨噬细胞和树突状细胞，形成免疫热点，导致细胞因子风暴，加剧肺组织损伤并促进肺纤维化进程。空间转录组学和蛋白质组学验证了单细胞结果。通过分析RA-ILD患者血细胞和RA-ILD的肺组织（GSE180139）进一步验证了Cd8⁺TEM在RA-ILD中的机制。

GCs治疗后，关节炎症状在10天内显著缓解，CIA评分迅速下降，但停药后部分鼠发生关节炎复发。治疗过程中，少数鼠出现干咳，特别是在运动后症状加重。CT影像和组织病理学分析表明，治疗组肺部纤维化实变区增多（P＜0.05）。H&E染色、Masson染色、天狼星红和EVG染色进一步证实了炎症细胞浸润和纤维化增加。纤维化相关指标如TIMPI-1和Hydroxyproline升高（P＜0.05），纤维化加剧。安全性评估中，CIABLMS-T组出现肿瘤样赘生物1例，8只大鼠出现血尿，且组织病理显示存在肾脏损害。此外，RA-ILD小鼠模型中的MTX治疗有效改善关节炎，但肺组织损伤较模型组更严重，需要进一步验证这一结果。

结论

①RA-ILD患者年龄较大、男性比例更高、病程更长且吸烟史较多，关节炎更严重，肺功能异常和炎症水平更高，死亡风险显著增加。治疗效果较差，需更个性化和强化治疗以改善预后。②根据模型发病机制、病理状态及疾病特点评估最终认为CIABLMS和CIALPSS与RA-ILD的发病机制、病理特点更相似。推荐运用CIABLMS和CIALPSS作为RA-ILD动物模型。③、④多组学分析揭示了RA-ILD大鼠模型中的多个肺纤维化分子特点及免疫失衡机制，发现和验证了Cd8⁺ TEM在RA-ILD肺纤维化中的关键作用。⑤GCs可有效缓解RA-ILD模型的关节炎，但对RA-ILD的肺纤维化控制效果有限。MTX也可有效治疗关节炎，但在RA-ILD中无明显疗效，甚至可能加重肺损伤。

Abstract

Objective:

Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is a severe complication closely related to rheumatoid arthritis (RA), with pulmonary fibrosis being one of its key features. Due to the unclear pathophysiological mechanisms of RA-ILD and limited clinical treatment options, there is an urgent need to explore its underlying mechanisms to provide a theoretical basis for clinical intervention and personalized therapy. The objectives of this study are as follows: ①To investigate the clinical characteristics of RA-ILD and provide a basis for its diagnosis and treatment. ②To establish an animal model of RA-ILD to facilitate further research. ③To analyze the pathological characteristics and potential molecular mechanisms of early-stage RA-ILD using multi-omics approaches, offering new insights for intervention and research. ④To explore the role of Cd8⁺TEM cells in lung tissue damage, repair, and fibrosis processes in RA-ILD by integrating basic and clinical research. ⑤To assess the therapeutic effects of prednisone, a first-line drug for ILD, and methotrexate (MTX), a first-line drug for RA, based on the RA-ILD animal model, aiming to provide experimental evidence for clinical intervention.

Materials and Methods:

A retrospective cohort study was conducted using big data analysis methods on the China Rheumatology Data Center (CRDC) and the China Centers for Disease Control and Prevention (CDC) databases to compare the clinical characteristics of RA-ILD patients with those of RA patients without ILD (RA-noILD). Various animal models were established, and characteristics of arthritis and interstitial lung disease were analyzed using CT imaging and histopathology to identify models that best replicate RA-ILD.
Multi-omics analyses, including single-cell sequencing (scRNA-seq), proteomics, and spatial transcriptomics, were used to investigate molecular differences in lung tissues. Whole blood samples from RA-ILD patients were analyzed to validate findings from multi-omics studies using flow cytometry and qPCR, focusing on the immunological role of Cd8⁺TEM in pulmonary fibrosis.
Based on the RA-ILD animal model, MTX (0.5 mg/kg/week) and prednisone (6 mg/kg/day) were administered, and their therapeutic effects were evaluated using CT imaging and pathological examinations.

Results:

1.This study included 88,793 RA patients, of which 1,058 were in the RA-ILD group and 1,058 in the RA-noILD group (randomly matched). Analysis showed that the onset age of RA-ILD patients was significantly later than that of the RA-noILD group, with a higher proportion of males, longer disease duration, and higher rates of smoking or former smoking. The mortality rate in the RA-ILD group was significantly higher, mainly due to pneumonia and pulmonary fibrosis (over 40%). The severity of arthritis was higher in the RA-ILD group, with significantly more joints exhibiting tenderness and swelling compared to the RA-noILD group. Pulmonary manifestations included a high incidence of dry cough (78.92%) and a KL-6 positivity rate of 48.68%, both significantly higher than in the RA-noILD group. Laboratory tests revealed higher RF and anti-CCP titers, as well as significantly elevated inflammatory markers (CRP and ESR) in the RA-ILD group. Treatment analysis indicated that the disease activity in the RA-ILD group was higher, with a significantly lower rate of improvement compared to the RA-noILD group (P<0.001).

2.The collagen-induced arthritis (CIA) model alone could not form the RA-ILD model. The CIA mixed with bleomycin (BLM) or lipopolysaccharide (LPS) produced CIABLM and CIALPS models, which exhibited recurring increases in CIA scores, recurrent arthritis flare-ups, and progressively worsening pulmonary lesions observed via pathology and CT after 1-6 months. CIABLM also showed multi-organ fibrosis, which worsened over time. This model was more consistent with RA-ILD features.

3.Single-cell RNA sequencing, proteomics, and spatial transcriptomics revealed significant changes in myeloid cells, stromal cells, epithelial cells, and lymphocytes. In myeloid cells, the CIALPSS and CIABLMS groups showed increased alveolar macrophages, monocytes, and plasmacytoid dendritic cells, with enrichment in antigen presentation, complement activation, and T cell activation pathways. In stromal cells, there was an increase in fibroblasts and mesothelial cells, with upregulation of fibrosis-related genes involved in ECM remodeling, TGF-β signaling, and angiogenesis. In epithelial cells, there was an increase in AT2 cells, with relevant genes upregulated in antigen presentation and oxidative stress pathways. In lymphocytes, the CIALPSS group showed T cell and neutrophil activation, while the CIABLMS group exhibited an increase in B cell proportions, reflecting immune dysregulation. Proteomic and spatial transcriptomic analysis confirmed these findings, identifying specific protein alterations and affected regions.

4.In the RA-ILD model, the proportion of Cd8⁺ TEM cells significantly increased, showing enhanced cytotoxicity, proliferation, and chemotaxis, particularly with upregulation of cytotoxic factors such as Nkg7, Gzmk, and Ccl5. Cell communication analysis revealed that the Cx3cr1 receptor on Cd8⁺ TEM cells interacted with the Cx3cl1 ligand on AT2 cells, promoting the initial recruitment of Cd8⁺ TEM cells. Chemokines like Ccl5 and Ccl4 interacted with the Ccr5 receptor on Cd8⁺ TEM cells, forming a positive feedback loop that further promoted Cd8⁺ TEM cell aggregation. Additionally, Ccl5, Ccl4, and Ccl3 recruited granulocytes, macrophages, and dendritic cells via Ccr1 and Ccr5 receptors, forming immune hotspots, leading to cytokine storms, exacerbating lung tissue damage, and promoting pulmonary fibrosis. Spatial transcriptomics and proteomics confirmed the single-cell results, and RA-ILD patient blood cells and the lung tissue of RA-ILD (GSE180139) further validated some of these mechanisms.

5.After GC treatment, arthritis symptoms significantly alleviated within 10 days, with rapid decreases in CIA scores, but some mice experienced arthritis relapse after discontinuation of treatment. During treatment, a few mice developed dry cough, particularly worsening after exercise. CT and histological analysis indicated an increase in fibrotic consolidation areas in the lung (P<0.05). H&E staining, Masson staining, Sirius red, and EVG staining confirmed inflammation cell infiltration and worsening fibrosis. Fibrosis-related markers such as TIMPI-1 and Hydroxyproline also increased (P<0.05), indicating aggravated fibrosis. In the safety evaluation, the CIABLMS-T group developed tumor-like growths, and some mice exhibited hematuria, suggesting potential renal damage. Additionally, MTX treatment in the RA-ILD mouse model effectively improved arthritis but worsened lung tissue damage compared to the model group, requiring further verification.

Conclusion:

①RA-ILD patients are older, predominantly male, have a longer disease duration, a higher smoking history, more severe arthritis, greater lung dysfunction, and higher systemic inflammation, with significantly increased mortality. More intensive and personalized treatment strategies are needed to improve prognosis. ②The CIABLMS and CIALPSS models closely resemble RA-ILD in terms of pathogenesis and pathology and are recommended as RA-ILD animal models. ③&④Multi-omics analysis identified key molecular features of pulmonary fibrosis and immune dysregulation in RA-ILD. Cd8⁺TEM plays a crucial role in RA-ILD pulmonary fibrosis. ⑤GCs effectively alleviate RA-ILD arthritis but have limited control over pulmonary fibrosis, while MTX may exacerbate lung damage, highlighting the need for alternative treatment strategies.