MOLECULAR CHARACTERIZATION OF RESPONSE TO ETROLIZUMAB AND ANTI-TNF REVEALS TREATMENT REFRACTORY ULCERATIVE COLITIS IS ASSOCIATED WITH ABUNDANCE OF RESIDUAL NEUTROPHIL SUBSETS AND ACTIVE FIBROBLAST POPULATIONS

Background: HIBISCUS I/II assessed the efficacy of etrolizumab versus adalimumab with a 10-week induction regimen in patients with ulcerative colitis (UC). We sought to characterize gene expression changes associated with clinical outcomes (Mayo clinical score/MCS) to infer mechanisms of action and resistance pathways to biologic treatment.
Methods: Bulk RNA-seq analysis was performed on colonic biopsies taken from the most inflamed segments, pre- and post-induction, in UC patients enrolled in HIBISCUS I/II. A scRNA-seq atlas was generated using paired inflamed-uninflamed colonic biopsies in an independent UC cohort (n=20), and cell-cell interaction and pseudo-time trajectory analyses were applied. Differentially expressed genes identified in HIBISCUS I/II were mapped to discrete cell subsets using the scRNA-seq atlas.
Results: Significant reductions in ITGAE expression and genes encoding for dendritic cells, cytotoxic CD8 T cells, and CD8 (gd) T cells were observed in patients treated with etrolizumab but not adalimumab. Applying cell signatures derived from an independent single-cell dataset provided more resolution to confirm reductions in subsets of tissue infiltrating neutrophils, monocyte-derived macrophages, inflammatory fibroblasts, and increases in colonic epithelial cells. These changes were significant in association with clinical outcomes, including MCS remission, and common to both etrolizumab and adalimumab treatments.
Pseudo-time trajectory analyses revealed four different neutrophil states in order of maturity: PADI4 Hi, OSM Hi, MX1 Hi, and CXCR4 Hi. Less mature neutrophils expressed higher levels of proteases (MMP9, LYZ), inflammatory cytokines (CXCL1, IL1B, OSM), and cytokine or chemokine receptors (LTB4R, CXCR1, CXCR2). More mature neutrophils expressed higher levels of CXCL2, TNF-a, CXCR4, and angiogenic factors such as VEGFA. We found stronger and more abundant predicted cytokine and chemokine interactions in the less mature neutrophil populations in the inflamed tissue. OSM: OSMR and IL1B: IL1R1 signaling localized strongly to CXCR2-expressing neutrophils compared to more mature CXCR4Hi neutrophils with fewer, though specific interactions, including TNFSF14:LTBR. Changes in less mature neutrophils were more closely associated with non-remission in etrolizumab and adalimumab-treated patients. In contrast, changes in mature CXCR4 Hi neutrophils were not associated with clinical outcomes.
Conclusions: Our data provide longitudinal gene signatures associated with etrolizumab and adalimumab response in patients with UC and reveal single-cell resolution of the cell types and their interactions that may underlie these findings. We also reveal novel insights into neutrophil states and their signaling pathways that may represent resistance mechanisms in UC patients resistant to biologic therapies.