DISTINCTIONS IN CD4+ MACROPHAGE BEHAVIOR SIGNIFY CROHN’S DISEASE WITH PERIANAL COMPLICATIONS

Abstract BACKGROUND Perianal fistulizing Crohn’s disease (PCD) is a more severe form of Crohn’s disease affecting a subset of patients with rectal/perianal manifestations. The underlying mechanisms differentiating PCD from Crohn’s Disease without perianal complications (CD) are not clear, and there are no defined prognostic indicators for disease progression. The purpose of this study was to define cellular and molecular factors in the rectal mucosa differentiating PCD from CD that either indicate or explain disease progression. METHODS Rectal mucosal biopsies were collected from consented patients at Children’s Healthcare of Atlanta (CHOA) with PCD (n = 18; 7 inflamed, 11 non-inflamed), CD (n = 25; 10 inflamed, 15 non-inflamed), and controls (n = 7 with no past/present mucosal disease). Mucosal biopsies were either processed for histology by immediate formaldehyde fixation and paraffin embedding (FFPE) or directly protease digested and processed for 10X Genomics 3’ single cell RNA sequencing. Serial FFPE sections were spatially assessed at the protein level by immunofluorescence confocal microscopy for CD4+CD68+ cells with Qpath segmentation and quantification analysis, or at the transcription level with a 500 probe colon gene panel by Xenium from 10X Genomics. Bioinformatic analysis of 10X Genomics platforms was performed using Seurat’s integration and sopa pipelines. RESULTS Single-cell analysis (∼260k cells) identified 27 distinct cell types (Figure1A) and revealed key differences between CD and PCD. Receptor–ligand pair analysis demonstrated that, in PCD, epithelial cells engaged in MHC-II–mediated communication with CD4+ macrophages, in contrast to the conventional interactions with CD4+ T cells observed in CD. (Figure1B) Immunofluorescence staining confirmed the presence of a novel CD4+CD68+ macrophage population in both CD and PCD. (Figure2A-B) Compared to CD, these CD4+CD68+ macrophages in PCD formed dense aggregates, resulting in a significantly greater cluster surface area. (p< 0.001) (Figure2C) Quantitative analysis further demonstrated a markedly higher abundance of CD4+CD68+ macrophages in PCD (p< 0.001). (Figure2D). Xenium spatial transcriptomic analysis not only showed in situ CD4+CD68+ macrophages in both PCD (Figure2E-G) and CD serial tissue sections but also revealed their distinct localization near colonocytes in PCD. This spatial pattern supports single-cell findings showing that MHC-II-mediated communication in PCD predominantly targets epithelial cells, in contrast to B and T cell interactions observed in CD. CONCLUSION The behavior of CD4+CD68+ macrophages in PCD rectal mucosa suggests a distinct immune cell population enriched in PCD that might play a causative role in disease progression. Ongoing spatial and single cell analyses will further define the cellular neighborhoods and mechanistic involvement of these macrophages in PCD.