Abstract Background Approximately 30% of Crohn’s disease (CD) patients develop a refractory form, usually marked by persistent inflammation resistant to conventional therapies. The cellular interactions and genetic factors driving this resistance remain poorly understood, limiting effective treatment options Methods we performed single-cell RNA sequencing (scRNA-seq) on colonic and ileal biopsies from 45 CD patients (13 treatment-naïve, 17 in remission, 15 difficultto-treat) and 10 healthy controls. To dissect interaction changes beyond general inflammation, we first compared treatment-naïve (TN) and remission (RM) CD to catch the inflammation-associated alterations. Based on this, we adjusted for inflammation effects when analyzing difficult-totreat (DTT) CD patients, comparing inflammation-corrected DTT with remission to identify DTT-specific changes, calculated as Ratio = (DTT-(TN-RM))/RM. Transcriptomic data were further integrated with whole-exome sequencing (WES) in 27 families with refractory CD to identify mechanisms to treatment resistance. Results Our study comprises 289,272 high-quality single-cell transcriptomes from 55 gut samples. Subclustering of five immune, stromal and epithelial cell compartments yielded 49 distinct cell states. Notably, our analysis revealed substantial heterogeneity in disease state between the colon and ileum, both in cellular composition and in the expression patterns of CD risk genes (Figure 1). Then, distinct tissue-specific mechanisms were evident (Figure 2). In the colon, DTT-specific increases were observed in the fibroblast to ILC-like cell interactions. Notably, the PVR–CD226 axis was uniquely enriched in DTT, with PVR and CD226 highly expressed in inflammatory fibroblasts and ILC-like cells, respectively, leading to elevated TNF expression and enhanced TNF-α production and secretion. In the ileum, DTT-specific increases were identified in HES1+ epithelial cell to ILC-like cell interactions. The IL7–IL7R axis was uniquely enriched in DTT patients, with IL7R highly expressed in ILC-like cells, accompanied by increased IL7-mediated activation of the JAK–STAT signaling pathway. In addition, our WES analysis identified mutations in TTN and RNF213, which contribute to DTT by modulating the above mechanisms in the colon and ileum, respectively. Our findings indicate that DTT CD, often involving both colon and ileum, responds poorly to monotherapy but improves markedly with combined anti-TNFα and JAK inhibition. Conclusion This study revealed distinct regional drivers of difficultto-treat CD, highlight the need for combined treatment with anti-TNFα and JAK inhibition to achieve improved outcomes in difficult-to-treat CD. Conflict of interest: Wang, Wei: No conflict of interest Shen, Mengping: No conflict of interest Lin, Jue: No conflict of interest Zhang, Qi: No conflict of interest Zhu, Ruixin: No conflict of interest Zhi, Min: No conflict of interest
Wang et al. (Thu,) studied this question.