研究背景

皮肤分枝杆菌疾病是由分枝杆菌感染皮肤引起的一组疾病，包括皮肤结核、麻风及皮肤非结核分枝杆菌感染。近年来皮肤分枝杆菌感染的发病率呈增长趋势，已成为威胁我国公众健康的问题之一。朗汉斯巨细胞（ Langhans giant cells，简称LGCs ）是分枝杆菌感染性肉芽肿的标志性特征，是宿主免疫防御与病原体持续存活之间动态博弈的关键参与者。然而，LGCs的形成机制及其抗分枝杆菌功能仍未完全阐明。

研究目的

系统解析MFGE8调控LGCs形成的机制通路，阐明LGCs抗分枝杆菌功能的相关免疫学机制，并探索LGCs的细胞治疗效果。

研究方法

1. LGCs模型构建与基因筛选

（1） 体外诱导纯化：体外用GM-CSF&IL-4诱导小鼠骨髓单核巨噬细胞并过滤后得到纯化的LGCs；

（2） 差异基因鉴定：通过单细胞测序技术比较LGCs与巨噬细胞的基因表达谱，筛选出关键差异基因MFGE8和OC-STAMP；

（3） 基因表达验证：应用RT-qPCR、ELISA、免疫荧光和免疫组化等技术，在体内外鼠源和人源LGCs中验证MFGE8和OC-STAMP的表达。

2. LGCs形成机制研究

（1） 时序表达分析：通过RT-qPCR和ELISA检测MFGE8、OC-STAMP在LGCs形成过程中的动态表达；

（2） 基因功能验证：采用siRNA干扰和基因敲除鼠模型，系统评估MFGE8、OC-STAMP对LGCs形成率的影响及上下游调控关系；

（3） 信号通路解析：结合RNA-seq、ChIP-qPCR和Western blot技术，揭示GM-CSF/IL-4调控MFGE8表达的信号通路；

（4） 分子机制验证：通过RNA-seq联合PI3K/AKT通路抑制剂实验，阐明MFGE8通过OC-STAMP调控LGCs形成的分子机制。

3. LGCs的抗菌功能机制与治疗评估

（1） 抗菌功能检测：应用流式细胞术和涂菌计数法评估LGCs对分枝杆菌的杀伤效能；

（2） 免疫机制研究：整合Time-lapse成像、PCR Array、ELISA、WB等技术解析LGCs的抗菌免疫机制；

（3） 治疗模型构建：建立小鼠足垫海分枝杆菌感染模型，使用慢病毒构建的GFP-LGCs进行局部移植及疗效评估；

（4） 疗效分析指标：检测治疗后病原载量、肉芽肿病理变化及炎症因子谱，综合评价LGCs的治疗效果。

研究结果

1. LGCs形成的分子调控机制

（A） MFGE8与OC-STAMP在LGCs中的表达水平显著高于巨噬细胞；

（B） MFGE8与OC-STAMP在LGCs形成过程中表达逐渐升高，敲低或抑制两分子后LGCs的形成均减少；

（C） GM-CSF&IL-4诱导M0细胞的RNA-seq时序分析及体外实验表明，二者通过增强JAK2/STAT5表达及磷酸化水平，进而上调MFGE8的表达；

（D） MFGE8敲低后，GM-CSF&IL-4诱导的RNA-seq时序分析结合体外实验表明，PI3K/AKT通路磷酸化水平被抑制，进而导致OC-STAMP表达下调及LGCs形成减少。

2. LGCs的抗菌功能特性

（1） LGCs保留巨噬细胞标记F4/80，同时高表达M1型标记CD86及树突状细胞标记CD11C MHC II，CD206（M2型）无显著差异；

（2） LGCs与海分枝杆菌共孵育后细菌载量显著减少；

（3） Time-Lapse显示LGCs吞噬分枝杆菌后出现细胞质肿胀、内容物泄漏等焦亡特征；

（4） 焦亡相关基因IL-1β、Caspase-1、GSDMD等表达水平在体外感染分枝杆菌的LGCs及皮损中的LGCs上显著上调。

3.   LGCs的治疗潜力

相比PBS治疗组，将 LGCs移植至小鼠皮肤分枝杆菌感染足垫后，局部组织中的细菌载量降低，组织病理中感染性肉芽肿的厚度减少。LGC治疗在初始阶段可诱导局部IL-1β等促炎细胞因子的释放，而在后期可促进IL-10等调节性细胞因子的产生。

研究结论

GM-CSF和IL-4通过JAK2/STAT5通路上调MFGE8的表达，而后MFGE8进一步通过PI3K/AKT通路上调OC-STAMP的表达促进LGCs的形成；LGCs在清除分枝杆菌过程中激活了细胞焦亡通路；局部移植LGCs可显著降低感染负荷并重塑免疫平衡，为皮肤分枝杆菌感染提供新的治疗策略。

Background
Cutaneous mycobacterial infections, including cutaneous tuberculosis, leprosy, and nontuberculous mycobacterial infections, are caused by mycobacterial skin infections. In recent years, the incidence of cutaneous mycobacterial infections has increased, posing a public health concern. Langhans giant cells (LGCs), a pathological hallmark of Mycobacterium-induced infectious granulomas, serve as critical participants in the dynamic interplay between host immune defense and pathogen persistence. However, their formation mechanisms and direct antimycobacterial functions remain to be fully elucidated.
Objective 
This study aimed to systematically investigate the MFGE8-regulated signaling pathways driving LGCs formation, elucidate the immunological mechanisms of their antimycobacterial function, and evaluate their therapeutic potential.
Methods
 1. LGCs Model Construction and Gene Screening
(1) In vitro induction and purification: Purified LGCs were obtained by in vitro induction of mouse bone marrow-derived mononuclear macrophages with GM-CSF and IL-4, followed by filtration.
(2) Differential gene identification: Single-cell sequencing compared gene expression profiles between LGCs and macrophages, identifying MFGE8 and OC-STAMP as key differentially expressed genes.
(3) Gene validation: RT-qPCR, ELISA, immunofluorescence, and immunohisto-chemistry validated MFGE8 and OC-STAMP expression in murine and human LGCs.
2. Mechanisms of LGCs Formation
(1) Dynamic expression analysis: RT-qPCR and ELISA assessed temporal changes in MFGE8 and OC-STAMP during LGCs formation.
(2) Functional validation: siRNA knockdown and gene knockout models evaluated the roles of MFGE8 and OC-STAMP in LGCs formation and their regulatory hierarchy.
(3) Signaling pathway analysis: Integration of RNA-seq, ChIP-qPCR, and Western blot analyses revealed the signaling pathway through which GM-CSF/IL-4 regulates MFGE8 expression.
(4) Molecular mechanism verification: RNA-seq and PI3K/AKT pathway inhibitors confirmed MFGE8 regulates OC-STAMP to drive LGCs formation.
3. Antimicrobial Function and Therapeutic Evaluation
(1) Antimicrobial assay: Flow cytometry and bacterial colony counting assessed LGCs’ mycobacterial-killing capacity.
(2) Immune mechanism study: Time-lapse imaging, PCR Array, ELISA and Western blot explored LGCs’ antimicrobial immune mechanisms.
(3) Therapeutic model: A Mycobacterium marinum-infected mouse footpad model was established, and GFP-labeled LGCs were locally transplanted for efficacy evaluation.
(4) Efficacy analysis: post-treatment pathogen load, granuloma pathology, and inflammatory cytokine profiles were analyzed.
Results
1. Molecular Regulation of LGCs Formation
(1) MFGE8 and OC-STAMP expression in LGCs was significantly higher than in macrophages.
(2) During LGCs formation, the expression of MFGE8 and OC-STAMP progressively increased. Knockdown or inhibition of either molecule reduced LGCs formation.
(3) Time-course RNA-seq analysis and in vitro experiments on GM-CSF&IL-4-induced M0 cells demonstrated that these cytokines enhance JAK2/STAT5 expression and phosphorylation levels, thereby upregulating MFGE8 expression.
(4) Following MFGE8 knockdown, RNA-seq time-course analysis combined with in vitro experiments under GM-CSF&IL-4 induction revealed that PI3K/AKT pathway phosphorylation levels were suppressed, resulting in downregulated OC-STAMP expression and reduced LGCs formation.
2. Antimicrobial Function of LGCs
(1) LGCs retained the macrophage marker F4/80 but exhibited reduced expression compared to M0 cells, while showing upregulated expression of the M1-type marker CD86 and dendritic cell marker CD11C MHC II, with no significant difference in the M2-type marker CD206.
(2) Co-culture of LGCs with Mycobacterium marinum significantly reduced bacterial load.
(3) Time-lapse imaging revealed pyroptotic features in LGCs post-bacterial phagocytosis, including cytoplasmic swelling and leakage of cellular contents.
(4) Pyroptosis-related genes (IL-1β, Caspase-1, GSDMD) were significantly upregulated in LGCs infected with mycobacteria in vitro and in LGCs from skin lesions.
3. Therapeutic Efficacy of LGCs
Compared to the PBS-treated group, local transplantation of LGCs into the footpads of mice with cutaneous mycobacterial infection led to a reduction in bacterial load within local tissues, decreased thickness of infectious granulomas in histopathological analysis。 LGCs treatment initially induced the release of pro-inflammatory cytokines (IL-1β) at early stages, while promoting the production of regulatory cytokines (IL-10) in later phases.
Conclusion
GM-CSF and IL-4 upregulate MFGE8 expression through the JAK2/STAT5 pathway, which subsequently activates OC-STAMP via the PI3K/AKT pathway to drive LGCs formation. LGCs demonstrate mycobactericidal activity concomitant with pyroptosis activation in infected models. Local LGCs transplantation effectively reduces infection burden and restores immune balance, offering a novel therapeutic strategy for cutaneous mycobacterial infections.