P0032Preliminary single-nuclei RNA sequencing of creeping fat in Crohn’s disease uncovers adipocyte and immune cell remodelling

Abstract Background Creeping fat (CF), characterized by the expansion of mesenteric adipose tissue (MAT) around the affected intestine, is a hallmark of Crohn’s disease (CD). However, its role in disease pathogenesis remains unclear. An interplay between luminal contents, the intestinal wall and the adjacent mesentery is hypothesized, as gut bacterial translocation drives the formation of CF (1). To extent prior work limited to the stromal vascular fraction, we profiled the complete cellular landscape of CF using single-nuclei RNA sequencing Methods Paired samples from normal MAT and CF were collected from CD patients undergoing an ileocolonic resection for disease complications (n = 4 samples) (Fig. 1A). In-house developed protocols were applied to obtain single-nuclei suspensions, serving as input for 10x Genomics single-nuclei RNA sequencing. Sequencing data were processed using Cell Ranger. Subsequent quality control, normalization, dimensionality reduction, clustering, and differential expression analyses were performed using Seurat, with data integration using Harmony. Cell clusters were annotated using a combination of canonical gene markers and ScType (2). Results After quality control, a total of 16,985 nuclei from mesenteric adipose tissue remained, displaying 1,035 as median number of genes expressed per nucleus. Unsupervised clustering followed by marker-based cell type annotation identified nine distinct cell populations including stromal, immune, and endothelial and stem/progenitor populations, as well as adipocytes (Fig. 1B). Subclustering of the macrophage and adipocyte compartments revealed five adipocyte and three macrophage subtypes (Fig. 1C-D). In CF, this composition shifted toward an expansion of pro-inflammatory macrophages (adj. p 0.0001), alongside an increased proportion of immature and mature adipocytes and a reduction in metabolically active adipocytes (adj. p 0.0001) (Fig. 1E-F). Differential gene expression analyses further revealed a downregulation of lipid metabolic and immune regulatory genes in CF mature adipocytes (adj. p 0.001), whereas metabolically active adipocytes in CF upregulated Major Histocompatibility Complex class II and stress-response transcripts (adj. p 0.001). Conclusion This preliminary study is the first to capture the full mesenteric cellular landscape, including mature adipocytes, at single-nucleus resolution. Despite its pilot nature, the data reveal cell-type-specific transcriptional changes in CF, highlighting adipocyte and macrophage heterogeneity and remodelling. These findings may refine the concept of CF as an active immune-metabolic organ involved in CD pathology, and lay the groundwork for mechanistic insight and future therapeutic exploration. References: (1)Ha CWY, Martin A, Sepich-Poore GD, Shi B, Wang Y, Gouin K, et al. Translocation of Viable Gut Microbiota to Mesenteric Adipose Drives Formation of Creeping Fat in Humans. Cell. 2020;183(3):666–83 e17. (2)Ianevski A, Giri AK, Aittokallio T. Fully-automated and ultra-fast cell-type identification using specific marker combinations from single-cell transcriptomic data. Nat Commun. 2022;13(1):1246. Conflict of interest: Dr. De Greef, Ine: No conflict of interest Skoufos, Georgios: No conflict of interest Mikorska, Antonina: No conflict of interest Geeraerts, Emiel: No conflict of interest Jacobs, Liesl: No conflict of interest Ke, Bo-Jun: No conflict of interest Cuvry, Arno: No conflicts of interest to declare. Derluyn, Louis: No conflict of interest Matteoli, Gianluca: Grant: We are recipient of the opnMe research grant from Boehringer Ingelheim. Bislenghi, Gabriele: no conflict of interest Wolthuis, Albert: No conflict of interest D’Hoore, André Jan Louis: Personal Fees: Takeda, Janssens Guedelha Sabino, João: Speaker’s fees: Lilly, Pfizer, Abbvie, Ferring, Falk, Takeda, Janssen, Fresenius, and Galapagos. Consultancy fees: Takeda, Pfizer, Janssen, Ferring, Fresenius, Abbvie, Galapagos, Celltrion, Pharmacosmos, and Pharmanovia. Research support: Galapagos, Viatris, and Eurogenerics. JS is supported by a Senior Clinical researcher grant from the Research foundation – Flanders. Ferrante, Marc: Research grants from AbbVie, EG Pharma, Celltrion, Janssen, Pfizer, Takeda and Viatris Consultancy fees from AbbVie, AgomAb Therapeutics, Boehringer Ingelheim, Celgene, Celltrion, Eli Lilly, Janssen-Cilag, MRM Health, Merck Sharp and Dohme, Pfizer, Takeda and ThermoFisher Speakers’ fees from AbbVie, Biogen, Boehringer Ingelheim, Dr Falk Pharma, Ferring, Janssen-Cilag, Merck Sharp and Dohme, Pfizer, Takeda, Truvion Healthcare and Viatris Vermeire, Séverine: Grant: AbbVie, Pfizer, Takeda, J&J, Galapagos Personal Fees: AbbVie - AbolerIS Pharma - AgomAb - Alimentiv - Arena Pharmaceuticals - AstraZeneca - Avaxia- BMS - Boehringer Ingelheim - Celgene - CVasThera - Dr Falk Pharma - Ferring - Galapagos - Genentech-Roche - Gilead - GSK - Hospira - Imidomics - Janssen - J&J - Lilly - Materia Prima - MiroBio - Morphic - MrMHealth - Mundipharma - MSD - Pfizer - Prodigest - Progenity - Promakhos Therapeutics - Prometheus - Robarts Clinical Trials - Second Genome - Shire - Surrozen - Takeda - Theravance - Tillots Pharma AG - Zealand Pharma - Other: AbbVie, MSD, Takeda, Ferring, Genentech/Roche, Shire, Pfizer Inc, Galapagos, Mundipharma, Verstockt, Bram: - Research support from AbbVie, Biora Therapeutics, Celltrion, Landos, Pfizer, Sanofi, Sossei Heptares/Nxera and Takeda. - Speaker’s fees from Abbvie, Agomab, Alfasigma, Biogen, Bristol Myers Squibb, Celltrion, Eli Lily, Falk, Ferring, Galapagos, Materia Prima, Johnson and Johnson, Pfizer, Sandoz, Takeda, Tillots Pharma, Truvion and Viatris. - Consultancy fees from Abbvie, Alfasigma, Alimentiv, Anaptys Bio, Applied Strategic, Astrazeneca, Atheneum, BenevolentAI, Biora Therapeutics, Boxer Capital, Bristol Myers Squibb, Domain Therapeutics, Eli Lily, Galapagos, Guidepont, Landos, Merck, Mirador Therapeutics, Mylan, Nxera, Inotrem, Ipsos, Johnson and Johnson, Pfizer, Sandoz, Sanofi, Santa Ana Bio, Sapphire Therapeutics, Sosei Heptares, Takeda, Tillots Pharma and Viatris. - Stock options Vagustim and Thethis Pharma. Voet, Thierry: No conflict of interest Verstockt, Sare: No conflict of interest