Abstract
Interleukin-10 receptor deficiency (IL10Rdef) presents as very early onset inflammatory bowel disease (IBD) with an aggressive, Crohn’s-like phenotype characterized by deep ulcers. IL10Rdef myeloid cells express excessive IL1B and several patients have been successfully treated with anti-IL1 therapy. In IBD patients, IL1B is a hallmark of ulcer granulation tissue, the endothelial cell (EC)-rich stroma where wound remodeling occurs. Better understanding of IL1-expressing myeloid cell and tissue EC interactions, through the lens of IL10R deficiency, may shed light on the dynamics affecting ulcer healing in IBD.
Single cell RNA-sequencing (scRNA-seq) was performed on peripheral blood (PB) mononuclear cells of n=8 and intestinal (LP) biopsies of n=7 IL10Rdef patient samples. Ligand-receptor (LR) interactions were assessed using CellChat. Findings were validated in internal and external IBD scRNA-seq datasets of IBD patient samples and murine colitis (including IL10Rdef mice). Immunohistochemistry (IHC) and RNAscope were performed in IBD intestinal samples with ulcers. Human umbilical vein ECs (HUVECs) were treated with IL1B or TGFB1 followed by qPCR and scRNA-seq.
In IL10Rdef samples, IL1B was expressed by 2 clusters of monocytes (PB) and by 1 cluster of DC2s (LP). 3 subsets of ECs were identified, but the IL1 receptor (IL1R1) was robustly expressed only on ACKR1+ (venular) ECs – the niche of immune cell trafficking into tissue. IHC and RNAscope of ulcerated IBD tissue revealed that granulation tissue ECs universally express ACKR1 and IL1R1. In addition to IL1 signaling and EC adhesion pathways (e.g. SELL-MADCAM1) suggesting physical interaction, LR analysis of PB myeloid cells and ACKR1+ ECs highlighted VEGF signaling (the latter uniquely expressed by IL1B+ PB monocytes) as significant at this in silico interface. Compared to those of IBD patients, IL10Rdef ACKR1+ ECs expressed greater markers of cell cycle arrest, including CDKN1A, known (along with VEGF signaling) to control sprouting angiogenesis and possibly endo-mesenchymal transition (endoMT). IL1B incubation of HUVECs resulted in spindle morphology and increased expression of venular identity (e.g. ACKR1, SELE), cell cycle arrest (e.g. CDKN1A), collagen, and myeloid chemotactic markers (e.g. CXCL1).
IL1B, expressed by circulating and tissue myeloid cells, likely plays a role in endothelial rewiring at the site of IBD ulcers. Based on scRNAseq and in vitro data, IL1B may drive the venular (ACKR1+) identity of granulation tissue ECs along with altered metabolism favoring collagen generation and possibly endoMT programs. Considering the fibrotic complications that often accompany IBD ulcers, disruption of IL1 pathway signaling at this site may be a promising strategy for improved wound healing in IBD.
Figure 1. ACKR1+ endothelial cells express the IL1 receptor, localize to granulation tissue, and receive signals from myeloid cells. A. scRNA-seq of IL10Rdef samples (n=8 PB and n=7 LP) UMAP with cellular clusters labeled. B. IL1R1 expression heatmap, with ACKR1+ ECs highlighted. C. IHC of ulcerated IBD tissue reveals ACKR1 expression in granulation tissue endothelial cells. D. LR analysis highlights VEGF signaling by IL1B-expressing PB monocytes to endothelial cells (other signaling such as IL1 and adhesion pathways not shown).
Figure 2. IL1B treatment of HUVECs induces venular identity along with expression of cell cycle arrest, chemotactic factors, and collagen expression. Left panel: scRNA-seq UMAP of HUVECs treated for 7 days with 10ng/ml IL1B or medium-only, colored by treatment group. Right panel: Transcript expression of CDKN1A, CXCL8, ACKR1, SELE, CXCL1, and COL4A1 is highlighted and significantly enriched in the IL1B treated HUVECs.
