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Background Crohn’s disease often leads to intestinal fibrosis and strictures, with surgery required in over 40% of patients. While neutrophils show promise as therapeutic targets, non-selective inhibition risks immunodeficiency. Here, we aim to systematically investigate the role of neutrophil subclusters in CD fibrosis using single-cell transcriptomics, with multi-level validation in animal models and molecular analyses to identify precise therapeutic targets.Methods Colonic and ileal specimens from CD patients with strictures underwent single-cell RNA and 16S rRNA sequencing to identify neutrophil subsets and translocated bacteria like Clostridium symbiosum. In vitro co-cultures and Inpp5d-/- mouse models with microbial colonization, immune transfer, and antibody blockade validated cellular interactions and mechanisms underlying fibrosis.Results Single-cell profiling revealed that myeloid cells, especially neutrophils, exhibited the highest number of differentially expressed genes and heterogeneity during the progression from normal tissue to ulceration and stricture in CD. Inpp5d-/- mice spontaneously developed ileitis and ileal fibrosis accompanied by massive neutrophil infiltration, which was markedly attenuated by anti-Ly6G antibody treatment. Fine clustering identified a novel HLA-DR+CD74+ neutrophil subset that represented active microbial recognition and response capacity (IDDF2026-ABS-0251 Figure 1). 5R 16S sequencing demonstrated a unique ‘from 0 to 1’ microbial translocation signature in ileal strictures, with Clostridium symbiosum significantly enriched (IDDF2026-ABS-0251 Figure 2). This bacterium strongly induced neutrophil HLA-DR expression and exacerbated ileal inflammation and fibrosis in vivo (IDDF2026-ABS-0251 Figure 3). Adoptive transfer of Clostridium symbiosum-primed HLA-DR+ neutrophils activated ileal fibroblasts. Mechanistically, HLA-DR+ neutrophils possessed a complete antigen presentation machinery and exhibited strong interactions with T cells. Clostridium symbiosum drove T-cell activation in an antigen presentation-dependent manner via these neutrophils, leading to T-cell production of the pro-fibrotic factor AREG (IDDF2026-ABS-0251 Figure 4); blockade of antigen presentation abrogated this effect. Functionally, HLA-DR+ neutrophils upregulated fibroblast activation markers and enhanced migration in a T-cell-dependent manner (IDDF2026-ABS-0251 Figure 5). Neutralization of AREG or its receptor EGFR abolished the pro-fibrotic effect of HLA-DR+ neutrophils on fibroblasts, and in vivo AREG blockade suppressed Clostridium symbiosum-mediated ileal fibrosis (IDDF2026-ABS-0251 Figure 6).Conclusions This study identifies a pivotal HLA-DR+ neutrophil subset with antigen-presenting capacity driving ileal fibrosis, induced by Clostridium symbiosum and mediated via AREG-EGFR signaling, offering novel therapeutic targets for Crohn’s disease fibrosis.Abstract IDDF2026-ABS-0251 Figure 1Abstract IDDF2026-ABS-0251 Figure 2Abstract IDDF2026-ABS-0251 Figure 3Abstract IDDF2026-ABS-0251 Figure 4Abstract IDDF2026-ABS-0251 Figure 5Abstract IDDF2026-ABS-0251 Figure 6