圆锥角膜（Keratoconus, KC）是一种以角膜变薄和圆锥形变为特征的进行性角膜疾病，主要累及角膜中央或旁中央区域，导致患者近视加重和不规则散光，严重影响视力。流行病学研究显示，KC的全球患病率存在显著地域差异，每10万人中约0.2至4790例不等，其中20至30岁人群发病率最高。传统治疗中，晚期KC主要依赖角膜移植手术。然而，KC的病因尚未完全阐明，现有研究表明遗传易感性、过敏、哮喘及揉眼等因素可能与其发病相关，因此需要深入探索KC的病理机制。

本研究采用10× Visium平台对5例KC患者和3例对照角膜样本进行空间转录组测序，并结合单细胞RNA测序数据，系统解析了角膜上皮和基质细胞的空间分布特征及基因表达模式。研究发现，角膜样本可明确分为上皮和多种基质亚群，其中KC角膜中央区域呈现显著的炎症信号激活，而周边区域则表现为炎症抑制状态。通过基因本体（GO）和基因集富集分析，我们发现在KC患者中，上皮及基质细胞中涉及白细胞迁移、细胞粘附和免疫调控等通路的差异表达基因（DEGs）具有明显的区域特异性表达特征，这为KC炎症过程的空间异质性提供了分子层面的证据。

为进一步阐明免疫细胞在KC发病中的作用，本研究对单细胞转录组数据进行了整合分析，成功鉴定了树突状细胞（DCs）、单核细胞/巨噬细胞及T细胞等免疫细胞亚群。结果显示，尽管KC与对照角膜中免疫细胞的总体数量无显著差异，但DCs呈现活化状态，其分泌的IL-1β可能通过调控角膜基质细胞中基质金属蛋白酶（MMPs）的表达，促进细胞外基质降解，从而导致角膜变薄和前凸。细胞通讯分析进一步验证了这一机制，表明在KC角膜中，虽然整体细胞间通讯减弱，但DCs与基质细胞之间的免疫信号显著增强，尤其是通过IL-1β、CXCL2及CCL22等信号通路介导的基质重塑过程。

为验证上述发现，本研究开展了体外细胞共培养实验和动物模型研究。在体外实验中，利用LPS激活的DCs处理角膜基质细胞，结果显示MMP-1、MMP-2和MMP-3的表达显著上调，且基质降解能力明显增强。在小鼠KC模型中，通过col II和LPS联合处理成功诱导出KC样病理变化，表现为角膜曲率增大和厚度下降。针对这一病理过程，研究采用MyD88二聚化抑制剂ST2825进行干预，结果表明ST2825能够有效抑制IL-1β信号传导，降低MMPs表达和细胞外基质降解，从而改善KC样病理特征。

尽管传统观点认为KC是一种非炎症性疾病，但本研究首次在空间转录组层面综合分析揭示了KC角膜中存在显著的区域性炎症差异：中央角膜炎症激活而周边角膜呈现炎症抑制状态。这一发现不仅深化了对KC发病机制的理解，也为临床早期诊断和干预提供了新的视角，提示应关注角膜不同区域的炎症状态。基于体内外实验的验证结果，抗炎治疗，尤其是针对IL-1β通路的靶向干预，有望成为KC辅助治疗的新策略。

Keratoconus (KC) is a progressive corneal disorder characterized by corneal thinning and conical deformation, primarily affecting the central or paracentral regions, leading to increased myopia and irregular astigmatism, which significantly impairs visual acuity. Epidemiological data indicate substantial global variations in KC prevalence, ranging from 0.2 to 4,790 cases per 100,000 individuals, with an annual incidence rate of 1.5 to 25 cases per 100,000 individuals. The highest incidence is typically observed in individuals aged 20 to 30 years. Traditionally, advanced KC has relied on corneal transplantation as a treatment; however, recent advancements, such as corneal collagen cross-linking (CXL) and novel methods utilizing graphitic carbon nitride, have been employed to enhance corneal biomechanical stability. Despite these developments, the etiology of KC remains unclear, with genetic predisposition, allergies, asthma, and eye rubbing identified as potential risk factors, underscoring the urgent need to elucidate the pathophysiological mechanisms of KC. 

In this study, corneal samples from five KC patients and three controls were analyzed using the 10× Visium platform for spatial transcriptomic sequencing, integrated with single-cell RNA sequencing (scRNA-seq) data to investigate the spatial distribution and gene expression of corneal epithelial and stromal cells. The results revealed that corneal samples were categorized into epithelial and multiple stromal subpopulations, with inflammatory signaling markedly activated in the central KC cornea, while inflammatory suppression was observed in the peripheral region. Gene Ontology (GO) and gene set enrichment analyses indicated that differentially expressed genes (DEGs) involved in leukocyte migration, cell adhesion, and immune regulation exhibited region-specific expression patterns in KC, providing molecular evidence for the spatial heterogeneity of inflammation in KC. 

To further explore the role of immune cells in KC pathogenesis, single-cell transcriptomic data were analyzed, identifying immune cell subpopulations such as dendritic cells (DCs), monocytes/macrophages, and T cells. Although no significant differences in the overall immune cell numbers between KC and control corneas were observed, DCs were found to be in an activated state, with IL-1β secretion potentially regulating matrix metalloproteinase (MMP) expression in corneal stromal cells, thereby promoting extracellular matrix degradation and contributing to corneal thinning and protrusion. Cell-cell communication analysis further supported this hypothesis, demonstrating that, despite a general reduction in intercellular signaling in KC corneas, immune signaling between DCs and stromal cells was significantly enhanced, particularly through IL-1β, CXCL2, and CCL22 pathways, promoting stromal remodeling. 

To validate these findings, in vitro co-culture and in vivo animal model experiments were conducted. In cell experiments, corneal stromal cells treated with LPS-activated DCs exhibited significantly upregulated MMP-1, MMP-2, and MMP-3 expression, along with enhanced matrix degradation capacity. In a murine KC model, combined treatment with collagen II and LPS induced KC-like pathological changes, characterized by increased corneal curvature and reduced thickness. To target this pathological process, the MyD88 dimerization inhibitor ST2825 was administered, effectively suppressing IL-1β signaling, reducing MMP expression and extracellular matrix degradation, and subsequently mitigating KC-like pathological features. 

Although KC has traditionally been considered a non-inflammatory disease, this study is the first to comprehensively analyze KC corneas at both the spatial transcriptomic and single-cell levels, revealing significant regional inflammatory heterogeneity, with inflammation activation in the central cornea and suppression in the periphery. This finding not only deepens the understanding of KC pathogenesis but also suggests that early diagnosis and intervention should focus on inflammation status in different corneal regions. Based on in vitro and in vivo validation, anti-inflammatory therapy, particularly targeting the IL-1β pathway, holds promise as a potential adjunctive treatment strategy for KC.