背景：分枝杆菌感染性疾病是由结核杆菌、非结核分枝杆菌及麻风杆菌引起的一类全 球性重大公共卫生问题，其核心的组织病理特征为感染性肉芽肿的形成。朗汉斯巨 细胞（Langhans giant cells, LGCs）作为感染性肉芽肿的标志性组分，是宿主与病原 体相互作用的重要参与者，然而其免疫表型特征及动态调控机制仍缺乏系统性研究。 近年来，CXCL1和CXCL2作为趋化因子家族的重要成员，在招募炎性细胞和调节 炎症反应中的作用备受关注，但分枝杆菌感染背景下LGCs是否特异性分泌此类趋 化因子，其分泌所产生的生物学功能，以及病原体触发CXCL1/CXCL2释放的信号 通路机制，仍是尚未解决的科学问题。

目的：系统阐明 LGCs 免疫表型特征，揭示分枝杆菌感染环境中 LGCs 特异性分泌 CXCL1/CXCL2 发挥的生物学作用，以及病原体触发CXCL1/CXCL2释放的具体机 制。

方法：1. LGCs 细胞诱导及分离纯化：采用M-CSF（20 ng/mL）诱导小鼠骨髓单核细 胞分化为M0巨噬细胞；联合GM-CSF（20 ng/mL）与IL-4（10 ng/mL）刺激 3-5 天，诱导M0巨噬细胞融合形成LGCs。通过30 μm 细胞滤网对细胞进行过滤分 离，获得大细胞组分（＞30 μm，LGCs）和小细胞组分（＜30 μm，Macs），并通 过Wright-Giemsa 染色及免疫荧光双重验证其形态特征；随后以MOI=5感染分枝杆 菌，构建LGCs体外感染模型。 2. 转录组测序（RNA sequencing，RNA-seq）：分别收集 LGCs 组、Macs 组及 M0巨噬细胞样本（每组各3例样本），以及海分枝杆菌（Mycobacterium marinum， M. marinum）感染 12 和24小时的LGCs组与未感染对照组（每组各3例样本），提 取总RNA后送RNA-seq。 3. 表型验证：基于转录组差异基因分析筛选出40余个关键表型相关基因，通 过qRT-PCR系统验证其在LGCs中的表达情况。采用qRT-PCR检测LGCs CXCL1、 CXCL2、CXCR2的mRNA表达水平，结合ELISA定量分析细胞上清液中趋化因子 的分泌浓度；利用流式细胞术定量检测LGCs表面CXCR2受体的蛋白表达强度； 进一步通过免疫组化染色技术，在 M. marinum 感染小鼠足垫肉芽肿造模皮肤组织 及人皮肤结核患者皮损样本中，定位验证CXCL1、CXCL2及CXCR2的空间分布 特征。 4. 功能实验：采用Transwell 系统评估LGCs来源CXCL1/CXCL2对M0巨噬 细胞的趋化效应；通过qRT-PCR检测CXCL1/CXCL2-CXCR2轴对促炎因子、抗菌 肽基因表达的影响；采用Wright-Giemsa染色评估CXCR2对巨噬细胞融合的影响。 5. 机制探索：通过TLR2激动剂与阻断剂，以及NF-κB通路抑制剂进行干预 实验，评估分枝杆菌感染对LGCs分泌的 CXCL1/CXCL2调控网络。

结果：1. 相较于 M0巨噬细胞，LGCs展现出显著差异化的免疫分子谱，包括高表达 CXCR2 等趋化因子受体以及CD40、CD80和CD86等共刺激分子；在分枝杆菌感 染后LGCs呈现动态免疫重塑，包括TLR1、TLR2等模式识别受体和共刺激分子等 表达的进一步显著上调。 2. M.marinum 感染后 CXCL1 和CXCL2等趋化因子在LGC中的表达显著高于 M0 巨噬细胞，其受体CXCR2在LGC中的表达亦显著高于M0。分枝杆菌感染可 诱导LGCs中CXCL1、CXCL2、CXCL3和CXCL5等趋化因子在感染后12 h和24 h 持续高表达。 3. M.marinum 感染后 LGCs 分泌的CXCL1/CXCL2 通过CXCR2介导单核巨噬 细胞迁移。 4. M.marinum 感染后 LGCs 分泌的 CXCL1/CXCL2 通过 CXCR2 促进IL-1β 和 IFN-γ 等炎症因子的表达，且CXCL2对促炎因子的激活效应稍强于CXCL1。 5. CXCR2 对巨噬细胞融合有影响，但未见统计学差异。CXCL1/CXCL2及其受 体CXCR2对抗菌肽基因的表达无显著影响。 6. 分枝杆菌感染后通过TLR2 受体和NF-κB 信号通路调控LGCs中CXCL1 和CXCL2的表达与分泌。

结论：首先，本研究首次揭示了LGCs区别于M0巨噬细胞的免疫表型特征，且分枝 杆菌感染可诱导 LGCs 发生免疫表型重塑。其次，分枝杆菌感染后可明显上调 CXCL1 和CXCL2等趋化因子的表达，上述趋化因子不仅可通过CXCR2受体促进 单核巨噬细胞的迁移，还能调节IL-1β和IFN-γ等促炎因子的表达，从而增强感染 部位的免疫应答。此外，本研究还发现分枝杆菌感染后通过TLR2和NF-κB信号 通路调控LGCs中CXCL1和CXCL2的表达与分泌。 

Background:Mycobacterial infectious diseases, caused by Mycobacterium tuberculosis, non tuberculous mycobacteria, and Mycobacterium leprae, represent a major global public health challenge. A core pathological hallmark of these infections is the formation of infectious granulomas. Langhans giant cells (LGCs), as signature components of granulomas, play critical roles in host-pathogen interactions. However, their immunophenotypic characteristics and dynamic regulatory mechanisms remain poorly understood. CXCL1 and CXCL2, key members of the chemokine family, are known to regulate inflammatory cell recruitment and immune responses. Yet, it remains unclear whether LGCs specifically secrete these chemokines during mycobacterial infection, their biological functions, and the signaling pathways underlying pathogen-triggered CXCL1/CXCL2 release.

Objective:This study aims to systematically elucidate the immunophenotypic features of LGCs, investigate the biological roles of LGC-derived CXCL1/CXCL2 in mycobacterial infection, and unravel the mechanisms by which pathogens trigger CXCL1/CXCL2 secretion.

Methods:1. Induction and isolation of LGCs: Mouse bone marrow-derived monocytes were differentiated into M0 macrophages using M-CSF (20 ng/mL), followed by stimulation with GM-CSF (20 ng/mL) and IL-4 (10 ng/mL) for 3–5 days to induce M0 macrophage fusion into Langhans giant cells (LGCs). Cells were filtered through a 30 μm strainer to separate large (>30 μm, LGCs) and small (<30 μm, Macs) fractions. Morphological validation was performed via Wright-Giemsa staining and immunofluorescence. An in vitro LGC infection model was established using mycobacteria at MOI=5. 2. RNA sequencing (RNA-seq): Total RNA was extracted from LGCs, Macs, and M0 macrophages (n=3 per group), as well as Mycobacterium marinum (M. marinum)-infected LGCs at 12/24 hours post-infection and uninfected controls (n=3 per group), followed by RNA-seq. 3. Phenotypic Validation: Over 40 phenotype-associated genes were screened via  transcriptomic analysis. qRT-PCR validated CXCL1, CXCL2, and CXCR2 expression in LGCs. ELISA quantified chemokine secretion, flow cytometry measured CXCR2 surface expression, and immunohistochemistry localized CXCL1/CXCL2/CXCR2 in M. marinum-infected mouse footpad granulomas and human cutaneous tuberculosis lesions. 4. Functional Assays: Transwell systems assessed CXCL1/CXCL2-mediated macrophage chemotaxis. qRT-PCR evaluated the CXCL1/CXCL2-CXCR2 axis on pro-inflammatory cytokines and antimicrobial peptides genes. Wright-Giemsa staining quantified macrophage fusion. 5. Mechanistic Studies: TLR2 agonists/antagonists and NF-κB inhibitors were used to dissect pathways regulating CXCL1/CXCL2 secretion.

Results:1. LGCs exhibited distinct immunophenotypic profiles compared to M0 macrophages, including elevated expression of chemokine receptors (CXCR2) and co-stimulatory molecules (CD40, CD80, CD86). Mycobacterial infection induced dynamic immune remodeling in LGCs, with further upregulation of TLR1/2 and co-stimulatory molecules. 2. CXCL1, CXCL2, CXCL3, and CXCL5 were significantly upregulated in LGCs versus M0 macrophages post-M. marinum infection, peaking at 12/24 hours. CXCR2 expression was also higher in LGCs. 3. Following Mycobacterium marinum infection, CXCL1 and CXCL2 secreted by Langhans giant cells (LGCs) facilitated monocyte-macrophage migration through CXCR2-mediated signaling. 4. Following Mycobacterium marinum infection, CXCL1 and CXCL2 secreted by Langhans giant cells (LGCs) enhanced the expression of pro-inflammatory cytokines, including IL-1β and IFN-γ, through CXCR2 signaling, with CXCL2 exhibiting a marginally stronger activating effect on inflammatory responses compared to CXCL1. 5. CXCR2 showed a trend in promoting macrophage fusion. Neither CXCL1/CXCL2 nor CXCR2 affected antimicrobial peptide gene expression. 6. M. marinum infection regulated CXCL1/CXCL2 secretion via TLR2 and NF-κB pathways.

Conclusion:This study reveals, for the first time, the unique immunophenotype of LGCs and their dynamic remodeling during mycobacterial infection. Pathogen-induced CXCL1/CXCL2 secretion enhances immune responses by recruiting mononuclear macrophages via CXCR2 and amplifying pro-inflammatory cytokine production. Furthermore, TLR2 and NF-κB signaling mediate mycobacterial regulation of CXCL1/CXCL2 in LGCs, providing mechanistic insights into granuloma-associated immune modulation.