{
  "abstract": "Introduction Cells of the monocyte-macrophage lineage are key players in systemic sclerosis (SSc) pathogenesis, contributing to inflammation and fibrosis. Moving beyond the simplified M1 vs. M2 dichotomy often used in vitro, our study explores monocyte and macrophages heterogeneity across multiple tissue compartments in SSc using integrated single-cell RNA sequencing (scRNA-seq), validated with AKOYA multiplex staining in situ and an in vitro disease model that closely mimics SSc conditions.Material and Methods Spatial localization of monocytes and macrophages was investigated using AKOYA multiplex histochemistry with markers for activated macrophages (CD206, CD163). SSc activation of blood monocyte-derived macrophages was examined using 5% autologous plasma. Single-cell RNA sequencing was performed on samples from suction blister fluid from lesional forearm skin, peripheral blood mononuclear cells (PBMCs), and skin biopsies from lesional forearm skin of diffuse anti-topoisomerase (ATA)-positive SSc patients (n=15 total samples: 5 blister, 4 PBMC, 6 skin) and healthy volunteers (n=10 total samples: 3 blister, 4 PBMC, 3 skin). Integration across tissue types was performed using scVI for batch correction, followed by gene set enrichment analysis (GSEA), pathway enrichment, and RNA velocity for pseudo-time analysis.Results Spatial analysis revealed CD206+CD163+ double-positive macrophages adjacent to blood vessels in SSc skin, consistent with active monocyte transmigration. In vitro, monocyte-derived populations exhibited significantly increased MRC1 expression (p<0.05) confirming enhanced macrophage activation in SSc ( figure 1).Integrated analysis identified seven distinct macrophage clusters (0-6) with marked tissue-specific distributions. Disease-associated changes revealed coordinated shifts: SSc patients showed increased percentages of cluster 4 in PBMC and blister tissues, exhibiting signatures of apoptotic processes, cellular extravasation, and antigen presentation. Cluster 1, involved in metabolic activation and viral response, showed increased percentages in blister samples from healthy controls compared to SSc. Cluster 3, characterized by IL-1 responses and neutrophil activation, was elevated in blister samples from healthy controls but showed increased representation in skin tissue from SSc patients, indicating tissue redistribution of inflammatory monocytes. Cluster 5 showed increased proportions in SSc blister samples but decreased in diseased skin, suggesting tissue-specific activation patterns. Cluster 6 was notably elevated in SSc skin tissue. RNA velocity analysis revealed impaired monocyte-macrophage differentiation in SSc compared to controls.Conclusions Our integrated multi-tissue analysis reveals functionally distinct monocyte-macrophage populations with disease-specific alterations in SSc. The shift from homeostatic tissue maintenance toward sustained immune activation, coupled with compartment-specific dynamics, highlights the complex immune dysregulation driving SSc pathogenesis.Abstract OC.32 Figure 1Spatial distribution and functional characterization of monocyte-macrophage populations in systemic sclerosis. A) Representative multiplex immunostaining images showing macrophages, blood vessels, and T cells in tissue sections. B) MRC1 expression levels during plasma stimulation in skin samples from healthy controls and SSc patients. C) UMAP visualization of integrated single-cell RNA sequencing data showing eight distinct monocyte-macrophage clusters (0-7) with samples divided by tissue type (PBMC, blister fluid, skin) and disease status (control vs. SSc). D) Proportion plots showing the distribution of each cluster divided by disease status and tissue type, highlighting disease-specific changes in monocyte-macrophage populations across tissue compartments",
  "authors": [
    {
      "affiliations": [
        "UCL, London, UK"
      ],
      "name": "Sandra Maribel Lopez Garces"
    },
    {
      "affiliations": [
        "UCL, London, UK"
      ],
      "name": "Tamara Searle"
    },
    {
      "affiliations": [
        "UCL, London, UK"
      ],
      "name": "Jiaqi Yang"
    },
    {
      "affiliations": [
        "Riptide Bioscience, California, USA"
      ],
      "name": "Henry Lopez"
    },
    {
      "affiliations": [
        "John Hopkins University, Maryland, USA"
      ],
      "name": "Isra Elhussin"
    },
    {
      "affiliations": [
        "UCL, London, UK"
      ],
      "name": "Kristina Clark"
    },
    {
      "affiliations": [
        "John Hopkins University, Maryland, USA"
      ],
      "name": "Clayton Yates"
    },
    {
      "affiliations": [
        "UCL, London, UK"
      ],
      "name": "David Abraham"
    },
    {
      "affiliations": [
        "UCL, London, UK"
      ],
      "name": "Christopher Denton"
    },
    {
      "affiliations": [
        "UCL, London, UK"
      ],
      "name": "Bahja Abdi"
    },
    {
      "affiliations": [
        "UCL, London, UK"
      ],
      "name": "Richard Stratton"
    }
  ],
  "title": "OC.32 Monocyte-derived macrophages in systemic sclerosis: integrated single-cell analysis across tissue compartments",
  "uid": "0c92aaed-0db5-5775-b77a-9f4211643519"
}