SPP1+ Macrophages Involved in Juvenile Xanthogranuloma via PPARG Signaling Pathway

Juvenile xanthogranuloma (JXG) is the most common non-Langerhans cell histiocytic disorder in children. However, the pathogenesis remains unclear.

Lesional and adjacent normal (NOR) skin samples were obtained from patients with histologically confirmed JXG. Each biopsy sample was processed for either bulk or single-cell RNA sequencing (scRNA‑seq). Differentially expressed genes (DEGs) in the bulk transcriptome were identified and subjected to functional enrichment analyses. ScRNA-seq data were analyzed using the Seurat software for cell clustering and annotation. Single-cell regulatory network inference and clustering (SCENIC) and regulon‑module analyses were performed to quantify the differences in regulatory activity between JXG and NOR. Cell-cell communication networks were inferred using the CellChat software. Transcription factor (TF) regulons corresponding to pathway‑focused DEGs were delineated, and their activity and cellular distribution were assessed using the AUCell algorithm. Macrophage developmental trajectories and trajectory-associated genes were inferred using Monocle 3. Immunohistochemistry (IHC) was performed to examine the distribution and expression of SPP1 and related markers in JXG and similar histiocyte-related disorders. Gene set enrichment analysis (GSEA) was used to compare functional differences between SPP1+ and SPP1- macrophage subsets.

A total of 4,656 DEGs were identified, including 2,209 upregulated and 2,447 downregulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed significant enrichment of the cytokine-cytokine receptor interaction (CCRI) pathway, comprising 158 CCRI-related DEGs. These DEGs revealed a complex immune profile in JXG, characterized by a predominance of pro-inflammatory cytokines despite the presence of M2-like features. Among the immune cell clusters, the M6 regulon module exhibited higher activity in JXG lesions than in NOR lesions. CellChat analysis revealed robust crosstalk between immune and non-immune cells, with SPP1‑mediated signaling being significantly upregulated in JXG. Almost all CCRI-DEG-specific TF regulons were highly and concordantly expressed in the macrophage subsets. Pseudotime analysis resolved two major macrophage branches, and the distribution of the trajectory-associated gene SPP1 closely mirrored that of the CCRI-DEG-specific TF regulons. IHC analysis indicated that SPP1 is an important marker for distinguishing JXG from other similar disorders. GSEA further showed that SPP1+ macrophages displayed activation of the PPARG signaling pathway, suppression of adaptive immune responses, and involvement in lipid homeostasis and functional regulation.

Cytokine-mediated interactions within macrophages play a pivotal role in the pathogenesis of JXG microenvironment, with SPP1+ macrophages showing a close association with pathogenesis, potentially via PPARG-mediated modulation of immune and metabolic homeostasis.