目的：瘢痕疙瘩是一种以伤口边缘外持续性瘢痕增生为特征的良性皮肤肿瘤样病变。自然杀伤细胞（Natural killer cells，NK cells）作为天然免疫系统的关键效应细胞，通过分泌干扰素-γ（Interferon-gamma，IFN-γ）、肿瘤坏死因子-α（Tumor Necrosis Factor-alpha，TNF-α）和粒细胞-巨噬细胞集落刺激因子（Granulocyte-Macrophage Colony-Stimulating Factor，GM-CSF）等炎症因子参与细胞毒性作用，同时可分泌双向调节素（Amphiregulin，AREG）促进细胞增殖并抑制凋亡，在肿瘤生长促进和免疫耐受调控等病理生理过程中发挥重要作用。现有研究表明，系统性免疫调节治疗可改善瘢痕疙瘩的临床症状，且该疾病的发病机制和临床转归与局部免疫微环境及全身免疫状态密切相关，提示瘢痕疙瘩并非单纯的局限性皮肤病变。然而，目前关于NK细胞在瘢痕疙瘩发生发展及预后中的作用机制尚未明确。本研究旨在系统探讨NK细胞在瘢痕疙瘩发病机制中的生物学作用及其潜在治疗价值。

方法：收集瘢痕疙瘩病人皮损处 （Lesion，L） 和皮损旁非皮损 （Non-Lesion， NL） 的皮肤消化后进行流式检测，比较L和NL的NK细胞数量和功能差异。通过将NK细胞分别与NL和L的成纤维细胞共培养，细胞因子处理NL的成纤维细胞与NK共培养以及检测NL和L处成纤维细胞产生转化生长因子-β1 （Transforming growth factor- beta 1，TGF-β1） 的水平来研究调控L处NK细胞功能的分子机制。通过四种不同状态的NK细胞的条件培养基与成纤维细胞的短期培养及NK细胞产生的AREG条件培养基长期刺激成纤维细胞研究NK细胞对成纤维细胞的作用。使用人源化NCG小鼠建立瘢痕疙瘩模型，进行AREG与IFNG敲除（Knock out，KO）的NK细胞注射治疗，探究不同状态NK细胞的疗效差异。收集正常人和瘢痕疙瘩病人的外周血单个核细胞（Peripheral blood mononuclear cells，PBMCs），进行流式检测比较正常人和瘢痕疙瘩患者NK细胞的功能差异。进一步使用单细胞RNA测序（single-cell RNA sequencing，scRNA-seq）寻找主要的致病NK细胞亚群。体外实验探究致病的NK细胞亚群的功能及状态。

结果：本实验发现瘢痕疙瘩患者L处NK细胞多于NL，释放IFN-γ和TNF-α降低，释放AREG不变，且L处IFN-γ⁺NK细胞百分比与病情严重程度负相关。与L的成纤维细胞共培养的NK细胞产生IFN-γ降低，进而发现L的成纤维细胞自身释放TGF-β1高于NL，TGF-β1刺激后的成纤维细胞释放更多的TGF-β1抑制L处的NK细胞释放IFN-γ。在皮肤中，NK 细胞产生的 IFN-γ 通过诱导成纤维细胞凋亡和抑制过多的细胞外基质产生，在限制瘢痕疙瘩进展方面发挥着关键作用，而 NK 细胞分泌的 AREG 则对抗这些作用，促进胶原沉积和成纤维细胞活化。AREG KO的NK细胞在瘢痕疙瘩小鼠模型中疗效明显。瘢痕疙瘩患者血液中NK细胞释放IFN-γ和TNF-α降低，释放AREG升高，且IFN-γ⁺NK细胞百分比与病情严重程度负相关，AREG⁺NK细胞百分比与病情严重程度正相关。通过scRNA-seq发现了瘢痕疙瘩患者血中一类干扰素刺激基因 （Interferon-stimulated gene，ISG）阳性NK，瘢痕疙瘩患者中此类NK细胞多于正常人，表达IFN-γ低于其他亚群，干扰素刺激评分最高，NK活化评分最低，耗竭评分最高，一型干扰素通路的基因表达升高。体外验证干扰素β （Interferon-β，IFN-β）短期刺激产生的ISG15⁺NK分泌IFN-γ低于ISG15^- NK，分泌AREG高于ISG15^- NK，瘢痕疙瘩患者中ISG15⁺NK细胞高于正常人，瘢痕疙瘩患者血浆中IFN-β高于正常人。使用IFN-β长时间刺激NK细胞可发生NK细胞耗竭，表现为NK分泌IFN-γ降低，增殖下降，表达T细胞免疫球蛋白和ITIM结构域蛋白（T-cell immunoreceptor with immunoglobulin and ITIM domains，TIGIT）升高，电镜和sea horse发现线粒体形状改变，氧化磷酸化及糖酵解水平降低，敲除一型干扰素通路中的受体和转录因子可逆转耗竭。

结论：这项研究揭示了瘢痕疙瘩患者病变皮肤和血液中 NK 细胞的显著功能变化及其与瘢痕疙瘩发病的密切关联。瘢痕疙瘩病变中 TGF-β 驱动的成纤维细胞进一步抑制了 NK 细胞 IFN-γ 的产生，揭示了复杂而动态的细胞相互作用。在瘢痕疙瘩患者的血液中，出现了一种独特的 NK 细胞亚群，其特点ISG 升高和 IFN-γ 产生减少，这与血浆 IFN-β 水平升高有关。这种升高的 IFN-β 是关键的启动因素，通过受损的线粒体功能和代谢紊乱导致 NK 细胞衰竭。这些发现突出了瘢痕疙瘩相关 NK 细胞功能异常的关键机制，并强调了局部和全身因素在瘢痕疙瘩发病机制中对NK细胞功能紊乱的影响。

Objective: Keloid is a benign skin tumor-like lesion characterized by persistent scar hyperplasia beyond the wound margins. Natural killer cells (NK cells), as key effector cells of the innate immune system, participate in cytotoxic functions by secreting inflammatory cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Additionally, they secrete amphiregulin (AREG) to promote cell proliferation and inhibit apoptosis, playing a critical role in pathophysiological processes such as tumor growth promotion and immune tolerance regulation. Current studies have demonstrated that systemic immunomodulatory therapy can improve the clinical symptoms of keloids, and the pathogenesis and clinical outcomes of this disease are closely associated with the local immune microenvironment and systemic immune status, suggesting that keloids are not merely localized cutaneous disorders. However, the mechanistic role of NK cells in the development, progression, and prognosis of keloids remains unclear. This study aims to systematically investigate the biological functions of NK cells in the pathogenesis of keloids and their potential therapeutic value.

Methods: This study investigates the role of NK cells in keloid pathogenesis by comparing their quantity and functional differences between lesional (L) and adjacent non-lesional (NL) skin tissues from keloid patients. Skin samples were digested and analyzed via flow cytometry. NK cells were co-cultured with fibroblasts derived from both NL and L tissues, and NL fibroblasts were additionally treated with cytokines before co-culture to assess their regulatory effects on NK cell function. The levels of transforming growth factor-beta 1 (TGF-β1) produced by NL and L fibroblasts were measured to elucidate the molecular mechanisms underlying NK cell dysfunction in keloid lesions. To examine the impact of NK cells on fibroblasts, short-term co-culture experiments were performed using conditioned media from four distinct NK cell functional states, while long-term stimulation of fibroblasts was conducted using AREG-enriched NK cell-conditioned media. A humanized NCG mouse model of keloid formation was established to evaluate the therapeutic effects of different NK cell states, including AREG and IFNG knock out (KO) NK cells. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and keloid patients. Comparative functional analysis of NK cells was performed by flow cytometry. Single-cell RNA sequencing (scRNA-seq) was subsequently employed to identify predominant pathogenic NK cell subsets. The functional characteristics and activation states of these disease-associated NK subpopulations were further validated through in vitro experiments.

Results: This study revealed that keloid lesions (L) exhibit a higher abundance of NK cells compared to non-lesional (NL) skin. However, these NK cells demonstrated reduced secretion of IFN-γ and TNF-α, while amphiregulin (AREG) release remained unchanged. Notably, the percentage of IFN-γ⁺ NK cells in L tissue negatively correlated with disease severity. Co-culture experiments demonstrated that NK cells incubated with keloid-derived fibroblasts exhibited diminished IFN-γ production. Further analysis revealed that keloid fibroblasts intrinsically secreted higher levels of TGF-β1 than NL fibroblasts. TGF-β1-stimulated fibroblasts further amplified TGF-β1 secretion, thereby suppressing IFN-γ release by NK cells in L tissue. In skin, NK cell-derived IFN-γ plays a critical role in restraining keloid progression by inducing fibroblast apoptosis and inhibiting excessive extracellular matrix deposition. In contrast, AREG secreted by NK cells counteracts these effects, promoting collagen accumulation and fibroblast activation. AREG KO NK cells demonstrated significant therapeutic efficacy in a keloid mouse model. Peripheral blood analysis of keloid patients showed that NK cells exhibited decreased IFN-γ and TNF-α secretion but elevated AREG release. The percentage of IFN-γ⁺ NK cells inversely correlated with disease severity, whereas AREG⁺ NK cells showed a positive correlation. Single-cell RNA sequencing (scRNA-seq) identified a distinct subset of interferon-stimulated gene (ISG)-positive NK cells in keloid patients. These ISG⁺ NK cells were more abundant in patients than in healthy controls, exhibited lower IFN-γ expression, and displayed the highest interferon-stimulated gene signature, alongside diminished NK activation, elevated exhaustion markers, and upregulated type I interferon (IFN-I) pathway genes. In vitro validation confirmed that short-term IFN-β stimulation induced ISG15⁺ NK cells, which secreted less IFN-γ but more AREG than ISG15⁻ NK cells. Keloid patients exhibited higher frequencies of ISG15⁺ NK cells and elevated plasma IFN-β levels compared to healthy individuals. Prolonged IFN-β stimulation induced NK cell exhaustion, characterized by reduced IFN-γ secretion, impaired proliferation, and increased expression of T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT). Electron microscopy and Seahorse assays revealed mitochondrial morphological alterations, accompanied by diminished oxidative phosphorylation and glycolysis. Genetic ablation of key IFN-I signaling receptors or transcription factors reversed this exhausted phenotype.

Conclusion: This study elucidates significant functional alterations in NK cells from both lesional skin and peripheral blood of keloid patients, highlighting their pivotal role in keloid pathogenesis. We demonstrate that TGF-β-enriched keloid fibroblasts further suppress IFN-γ production by NK cells, revealing intricate cell-cell crosstalk within the keloid microenvironment. Notably, we identified a distinct NK cell subpopulation in keloid patients characterized by elevated interferon-stimulated gene (ISG) signatures and impaired IFN-γ production, which correlated with elevated plasma IFN-β levels. Mechanistically, chronic IFN-β exposure acts as a key initiator driving NK cell exhaustion through mitochondrial dysfunction and metabolic reprogramming. Collectively, these findings uncover fundamental mechanisms underlying NK cell dysfunction in keloid formation, emphasizing how both localized and systemic factors converge to disrupt NK cell-mediated immune regulation in keloid pathogenesis.