P120 A novel population of classical monocytes is expanded in CD and defined by an upregulation of genes relating to mononuclear cell differentiation and response to microbes

Abstract

Abstract Background In Crohn's disease (CD), differentiation of classical blood monocytes to intestinal macrophages in the mucosa generates inflammatory rather than regulatory cells. Microbial products and cytokines can systemically reprogramme monocytes via effects on myelopoiesis. We hypothesised that in CD transcriptional changes occur in monocytes prior to their recruitment to the intestine which predispose to pro-inflammatory signatures in the mucosa. Here, we used single cell RNAsequencing (scRNAseq) to explore monocyte heterogeneity and identify CD-associated changes in gene expression profiles. Methods PBMCs were isolated from newly diagnosed CD patients (n=10) and matched healthy controls (HC, n=10). scRNAseq was performed using the 10x Genomics Chromium 3’ assay at a target capture rate of 10,000 cells/sample and sequencing at ~50,000 reads/cell. A dataset was generated using Cell Ranger and following unsupervised clustering (using Seurat) the CD14+ and CD16+ monocyte populations were selected. Data were further analysed using Seurat, DeSEQ2, g:Profiler, GSEA and Cytoscape. Results In addition to non-classical and intermediate monocytes, five transcriptionally distinct clusters of classical monocytes were identified in both CD patients and controls. One cluster (‘cluster 3’) was significantly increased in abundance in CD; other clusters were present at similar frequency in health and CD. Cluster 3 was defined by gene ontology (GO) pathways related to response to bacteria, cell migration, lymphocyte proliferation and response to oxidative stress. In CD compared with health, the most upregulated genes included those encoding EGR (early growth response) proteins 1-3 that play a key role in the functional plasticity of monocytes, and the key inflammatory cytokine IL-1β, as well as cytokines and regulators of their signalling (e.g. CXCL2/3, SOCS3). Overall, in Cluster 3, GO pathways positively enriched in CD related to microbial response signalling, canonical WNT signalling and mononuclear cell differentiation. Conclusion Classical monocytes are heterogeneous and include a novel subset that is expanded in CD. In this population, the expression of genes and pathways related to functional plasticity, response to microbes and inflammatory activity are enhanced in CD. These data support a systemic reprogramming of monocytes in CD that predisposes to inflammatory activity upon recruitment to the intestine.