吞噬細胞在全身性硬化症間質性肺炎所扮演決定與協調者之角色及功能

Abstract

背景：間質性肺炎（ILD）是全身性硬化症（SSc）死亡的主要原因，全身性硬化症是一種以組織纖維化為特徵的自體免疫疾病。全身性硬化症之間質性肺炎較常見於30-55歲的女性，而特發性肺部纖維化 (IPF) 較常見於 60-75歲的男性。 全身性硬化症間質性肺炎發生早於特發性肺部纖維化，且進展迅速。在過去的文獻中已知帶有FCN1、FABP4、SPP1等細胞特徵之巨噬細胞在肺纖維化的發病機轉中扮演重要的角色，而SPP1巨噬細胞在全身性硬化症間質性肺炎和特發性肺部纖維化中均在肺纖維化的致病機轉上扮演重要角色。我們利用單細胞RNA定序分析方法來辨識全身性硬化症間質性肺炎和特發性肺部纖維化之間的差異，並藉此闡明其不同的發病機制，並提出預防和治療的方向。

方法：我們對 NCBI 基因表現綜合 (GEO) 資料庫 GSE159354 和 GSE212109 進行單細胞RNA定序，並分析健康對照組、全身性硬化症間質性肺炎和特發性肺部纖維化的肺組織切片樣本。主要是透過基因分析與批次校正和註釋細胞類型的整合分析，以區分全身性硬化症間質性肺炎和特發性肺部纖維化患者與健康對照組的比較。我們利用細胞-細胞交互作用推論提出了全身性硬化症間質性肺炎發病機制，並利用SCENIC預測了調節標靶基因的轉錄因子。使用DrugBank資料庫進行轉錄因子基因的藥物標靶預測。

結果：自單核球分化而來的一群巨噬細胞在缺氧壓力下活化MAPK信號通路。由於這群巨噬細胞缺乏ANNEXIN的反饋抑制和全身性硬化症的自體免疫特性，導致肺部纖維化比特發性肺部纖維化更早發生。在最初的肺部組織損傷時，纖維母細胞在SPP1肺泡巨噬細胞的影響下開始活化IL6通路，但IL6似乎與其他發炎和免疫細胞無關。這也許可以解釋為什麼Tocilizumab（一種IL6受體抗體藥物）只能對早期全身性硬化症間質性肺炎患者的肺功能有保護作用。最後，我們確定 BCLAF1，NFE2L2及IRF1是巨噬細胞中MAPK活化的影響轉錄因子。二甲雙胍藥物 (Metformin) 能夠下調 NFE2L2，可作為全身性硬化症間質性肺炎早期預防及治療的候選藥物。

結論：SPP1 肺泡巨噬細胞在全身性硬化症間質性肺炎和特發性肺部纖維化的致病機轉中均具有其重要角色之作用。然而，全身性硬化症間質性肺炎受自體免疫疾病特性和疾病特殊缺氧壓力特性的影響，導致自血液單核球分化而來的一群巨噬細胞所執行的MAPK信號通路持續活化。這可能導致全身性硬化症間質性肺炎之肺纖維化比特發性肺部纖維化更早發生。這些差異可以作為早期預防和治療的潛在研究方向。

Background: Interstitial lung disease (ILD) represents the leading cause of death among patients with systemic sclerosis (SSc), a chronic autoimmune disorder marked by progressive tissue fibrosis. In previous reports on the prevalence of systemic sclerosis, women are at much higher risk for scleroderma than men, with a ratio ranging from 3:1 to 14:1. SSc-related interstitial lung disease (SSc-ILD) predominantly affects females between the ages of 30 and 55, whereas idiopathic pulmonary fibrosis (IPF) is more commonly observed in males aged 60 to 75. Compared to IPF, SSc-ILD tends to develop at a younger age and is characterized by a more rapid progression. Specific macrophage subsets-including FCN1, FABP4, and SPP1 macrophages-have been implicated in fibrotic lung pathology. Notably, SPP1-expressing macrophages are upregulated in both SSc-ILD and IPF, suggesting the shared fibrotic mechanism. We aim to elucidate the differential pathogenic mechanisms between SSc-ILD and IPF, which may underlie the earlier onset and faster progression observed in SSc-ILD, and to identify potential drugs or interventions for early prevention and treatment. Methods: We utilized publicly available single-cell RNA sequencing datasets (GSE159354 and GSE212109) from the NCBI Gene Expression Omnibus (GEO) to analyze lung tissue samples derived from healthy individuals, patients with idiopathic pulmonary fibrosis (IPF), and those with systemic sclerosis-related interstitial lung disease (SSc-ILD). After filtering and batch effect correction, gene expression data were integrated, and cell types were annotated to enable comparison between SSc-ILD and healthy samples. To explore potential mechanisms of SSc-ILD pathogenesis, we inferred cell–cell communication networks and employed SCENIC analysis to identify key transcription factors regulating gene expression. DrugBank Online was used to predict drug–TF interactions, and single-cell level drug repurposing analysis was conducted using the drug2cell Python package, which calculates drug scores based on target gene expression profiles. Results: Under oxidative stress, a specific subset of macrophages initiates activation of the MAPK signaling pathway. In the context of systemic autoimmunity and insufficient inhibitory regulation by annexin, this contributes to an earlier onset and persistent progression of pulmonary fibrosis in SSc-ILD compared to IPF. During the initial stages of lung injury, fibroblasts begin activating the IL6 pathway under the influence of SPP1-producing alveolar macrophages; however, IL6 appears to be unrelated to other inflammatory and immune cells. This may explain why tocilizumab (an anti-IL6-receptor antibody) only preserves lung function in patients with early SSc-ILD. Additionally, we identified the transcription factors BCLAF1, NFE2L2, and IRF1 as key regulators of MAPK activation in macrophages. Among potential therapeutic options, metformin was found to downregulate NFE2L2 and could serve as a repurposed drug candidate. Conclusions: SPP1-producing alveolar macrophages are implicated in the profibrotic processes observed in both IPF and SSc-ILD. In SSc-ILD, however, the interplay of autoimmune responses and oxidative stress appears to sustain MAPK signaling activation in macrophages, potentially contributing to an earlier onset and persistent progression of fibrosis compared to IPF. These distinct pathogenic features highlight important avenues for future research into early intervention and therapeutic strategies.