研究背景：

掌跖脓疱病（Palmoplantar pustulosis, PPP）是一种以掌跖部位反复出现无菌性脓疱为突出特点的慢性炎症性疾病。既往的研究发现IL17A、IL36γ、IL23等细胞因子在PPP患者皮损和外周血表达水平升高，但靶向上述细胞因子的生物制剂对很多PPP患者疗效并不理想。因此目前PPP的关键致病炎症通路尚不明确。

研究目的：

1. 通过单细胞RNA测序（single-cell RNA sequencing，scRNA-seq）研究PPP皮损内发挥关键致病作用的炎症通路和细胞类型；

2. 对scRNA-seq发现的致病通路和关键细胞因子进行体外实验验证;

3.初步探索（​Janus kinase， JAK）抑制剂治疗中重度PPP患者的疗效和安全性。

研究材料和方法：

1. 本研究纳入了9例中重度PPP患者，收集9块皮损组织样本和5块皮损旁未受累组织（Non-lesion，NL）样本，纳入2例健康受试者收集健康人掌跖皮肤（Healthy control， HC）样本进行scRNA-seq检测；

2. 分析不同样本组之间单细胞层面上细胞构成和基因表达差异，对差异表达基因（Differentially expressed genes，DEGs）进行通路富集分析和上游调控因子分析，对细胞间的配体-受体相互作用进行分析，寻找潜在的致病调控因子和信号通路，并筛选关键致病细胞群；

3. 采用双重免疫荧光和多重免疫荧光技术对单细胞转录组分析发现的关键基因的表达和关键细胞群的分布进行验证；

4. 纳入另外30例中重度PPP患者收集皮损和NL组织，构建皮肤器官培养（Skin organ culture，SOC）模型，结合流式细胞术和免疫荧光染色，验证单细胞转录组分析提示的致病信号通路对皮损角质形成细胞（Keratinocyte，KC）功能的影响；

5. 对本中心既往使用JAK抑制剂托法替布治疗中重度PPP患者的疗效和安全性进行回顾性研究。

研究结果：

1.   对单细胞数据进行聚类分析，并结合标记基因进行细胞类型注释，共得到KC、免疫细胞、血管内皮细胞、成纤维细胞、平滑肌细胞、神经元等主要细胞群。其中，表皮KC和免疫细胞亚群是皮损样本相对NL和HC样本转录表达谱差异最显著的细胞类型；

2.    相较于NL和HC样本，皮损KC中比例升高最明显的KC亚群是棘层 KC II细胞亚群。棘层 KC II亚群相较于NL和HC样本中占主导的棘层KC I亚群呈现出分化水平下降、增殖水平升高的特点。免疫荧光显示皮损样本棘层角质形成细胞相较于NL和HC样本同类细胞存在CK10表达下降，CK14表达升高；皮损表皮Ki67⁺KC数量较NL和HC样本增多；

3. 对皮损表皮KC总群相较于NL和HC表皮KC总群的DEGs进行通路富集分析发现IL6-JAK-信号传导及转录激活蛋白(Signal transducer and activator of transcription, STAT)3通路显著富集，免疫荧光证实皮损表皮pSTAT3⁺KC细胞比例较NL和HC表皮升高。对皮损KC各个亚群分别进行通路富集评分，发现皮损棘层占主导的棘层KC II亚群的IL6-JAK-STAT3富集得分显著高于在NL和HC中占主导的棘层KC I亚群；

4. 对皮损表皮KC中的DEGs进行上游调控因子分析，发现JAK-STAT3信号通路上游的经典细胞因子IL6和抑瘤素M（Oncostatin M，OSM）出现显著富集；受体-配体相互作用分析提示皮损内IL6-IL6受体和OSM-OSM受体之间相互作用显著增强；

5. 多重免疫荧光提示皮损脓疱内CD66b⁺细胞(中性粒细胞)是OSM的主要来源，靠近脓疱边缘的表皮KC内pSTAT3阳性率较远离脓疱边缘的KC升高；

6. 流式细胞术提示STAT3抑制剂和泛JAK抑制剂能显著降低皮损SOC模型中表皮KC中Ki67⁺细胞比例；免疫荧光提示STAT3抑制剂和JAK抑制剂能够提高皮损SOC模型中表皮CK10⁺KC细胞比例；分离皮损组织的真皮和表皮后，用JAK抑制剂直接作用于表皮同样能够降低Ki67⁺KC细胞比例，提高CK10⁺KC细胞比例；JAK抑制剂能够降低皮损KC内中性粒细胞趋化因子CXCL8表达水平；

7. 对皮损SOC模型进行OSM抑制剂和IL6R中和抗体处理，结果显示OSM抑制剂能显著降低皮损表皮KC中Ki67⁺细胞比例，而IL6R中和抗体对皮损KC的Ki67表达无显著影响；OSM抑制剂还能够减少皮损pSTAT3⁺KC比例，提高CK10⁺KC比例；分离NL样本表皮和真皮后，外源OSM因子作用于NL表皮能够提高Ki67⁺KC比例，而JAK抑制剂能够抑制这一现象；

8. 回顾本中心既往接受广谱JAK抑制剂托法替布治疗的8例中重度PPP患者，发现8例患者托法替布治疗12周疗效均达到PPP面积和严重程度评分（PPPASI）下降75%以上，未出现严重不良反应。

结论：

中性粒细胞来源的OSM很可能通过激活JAK-STAT3信号通路诱导KC出现过度增殖、分化受限、趋化因子表达上调等病理性变化，从而在PPP发病过程中发挥重要作用。短期JAK抑制剂治疗对于中重度PPP患者疗效显著。抑制JAK-STAT3信号通路有望成为治疗PPP患者的有效方法。

Background:

Palmoplantar pustulosis (PPP) is a chronic inflammatory skin disease characterized by recurrent sterile pustules on the palms and soles. Previous studies have identified elevated expression of cytokines, such as IL17A, IL36γ, and IL23 in both lesional skin and peripheral blood of PPP patients. However, biologics targeting these cytokines have demonstrated suboptimal efficacy in a substantial proportion of patients. Consequently, the key pathogenic inflammatory pathways driving PPP remain unclear.

Objectives：

To identify the inflammatory pathways and cell types that play critical pathogenic roles in PPP skin lesions with single-cell RNA sequencing (scRNA-seq) analysis;

To validate the functions of pathogenic pathways and key cytokines identified through scRNA-seq analysis via ex vivo experiments;

To evaluate the efficacy and safety of Janus kinase (JAK) inhibitors in treating patients with moderate-to-severe PPP.

Materials and methods：

This study enrolled 9 patients and 2 healthy donors with moderate-to-severe PPP, from whom 9 lesional tissue samples, 5 paired non-lesional (NL) tissue samples, and 3 healthy control (HC) palmoplantar skin samples were collected for scRNA-seq analysis.

Comparative analyses of cellular composition and gene expression profiles at single-cell resolution were performed across sample groups. Differentially expressed genes (DEGs) underwent pathway enrichment analysis and upstream regulatory factor investigation. Ligand-receptor interactions between different cell populations were mapped to identify potential pathogenic regulators, signaling pathways, and critical pathogenic cell subsets.

Dual and multiplex immunofluorescences were employed to validate the spatial expression patterns of key proteins and distribution characteristics of critical cell populations identified through scRNA-seq analysis.

An additional cohort of 30 moderate-to-severe PPP patients provided lesional and NL tissues to establish skin organ culture (SOC) models. These models were combined with flow cytometry and immunofluorescence staining to verify the impact of pathogenic signaling pathways (identified via scRNA-seq) on lesional keratinocyte (KC) function.

A retrospective analysis was conducted to evaluate the efficacy and safety profiles of JAK inhibitor (Tofacitinib) in treating moderate-to-severe PPP patients from our institutional database.

Results:           

Single-cell transcriptomic analysis identified epidermal KC, immune cells, vascular endothelial cells, fibroblasts, smooth muscle cells, and neural cells across lesional, NL, and HC skin samples. Among these, epidermal KCs and immune cell subsets exhibited the most pronounced transcriptional alterations in lesional tissues compared to NL and HC samples. 

Compared to NL and HC samples, the spinous KC II subpopulation showed the most significant proportional increase in lesional KCs. This subpopulation displayed reduced differentiation markers and elevated proliferation markers compared to the dominant spinous KC I subpopulation in NL and HC. Immunofluorescence confirmed decreased CK10 and increased CK14 expression in lesional spinous KCs, along with elevated Ki67⁺ KC numbers in lesional epidermis. 

Pathway enrichment analysis of DEGs in lesional KCs versus NL/HC KCs revealed significant activation of the IL6-JAK- signal transducer and activator of transcription (STAT)3 pathway. Immunofluorescence demonstrated increased proportions of pSTAT3⁺ KCs in lesional epidermis. Subcluster-specific analysis further showed higher IL6-JAK-STAT3 pathway enrichment scores in lesional spinous KC II compared to spinous KC I in NL/HC. 

Upstream regulator analysis of lesional KC DEGs highlighted significant enrichment of IL6 and oncostatin M (OSM), key cytokines upstream of JAK-STAT3 signaling. Ligand-receptor interaction analysis revealed enhanced IL6/IL6R and OSM/OSMR interactions in lesional tissues. 

Multiplex immunofluorescence localized OSM primarily to CD66b⁺ neutrophils within lesional pustules. KCs near pustule margins showed higher pSTAT3 positivity than KCs distal to pustule margins. 

Flow cytometry demonstrated that STAT3 and pan-JAK inhibitor significantly reduced Ki67⁺ KC proportions in lesional skin organ culture (SOC) models. Immunohistochemistry revealed increased CK10⁺ KC proportions after inhibitor treatment. Epidermal isolation experiments confirmed that JAK inhibitors directly suppressed Ki67⁺ KCs and elevated CK10⁺ KCs while reducing CXCL8 expression in lesional KCs. 

OSM inhibition in lesional SOC models reduced Ki67+ and pSTAT3⁺ KCs while increasing CK10⁺ KCs, whereas IL-6R neutralization had no significant effect. Exogenous OSM stimulation of NL epidermis increased Ki67⁺ KCs, an effect reversed by JAK inhibitors. 

Retrospective analysis of 8 moderate-to-severe PPP patients treated with tofacitinib showed ≥75% reduction in PPP Area and Severity Index (PPPASI-75) scores at 12 weeks in all patients, with no severe adverse events. 

Conclusion：

Neutrophil-derived OSM contributes to the pathogenesis of PPP by activating the JAK-STAT3 signaling pathway, thereby driving pathological KC alterations including hyperproliferation, aberrant differentiation, and upregulation of chemokine expression. Short-term JAK inhibitor therapy demonstrated marked efficacy in patients with moderate-to-severe PPP. These findings collectively suggest that targeted inhibition of the JAK-STAT3 signaling pathway represents a promising therapeutic strategy for PPP management.