Prediction of endoscopic post-operative recurrence (POR) in Crohn’s disease (CD) patients following ileocolonic resection (ICR) using clinical risk factors alone has thus far been inadequate. While peripheral blood leukocyte (PBL) DNA methylation has shown promise as a tool for predicting recurrence in cancer, no data in CD patients exists. Therefore, this study explored the association and predictive value of PBL DNA methylation in CD patients following ICR. From a cohort of 117 CD patients undergoing ICR, epigenome-wide PBL methylation profiles from 25 carefully selected patients presenting either clear endoscopic remission (n = 12) or severe recurrence (n = 13) were assessed using the Illumina MethylationEPIC (850K) array. No statistically significant differentially methylated positions (DMPs) or regions (DMRs) associated with endoscopic POR were identified (FDR p ≤ 0.05), further evidenced by the low accuracy (0.625) following elastic net classification analysis. Nonetheless, interrogating the most significant differences in methylation suggested POR-associated hypermethylation in the MBNL1, RAB29 and LEPR genes, respectively, which are involved in intestinal fibrosis, inflammation and wound healing. Notably, we observed a higher estimated proportion of monocytes in endoscopic POR compared to remission. Altogether, we observed limited differences in the genome-wide DNA methylome among CD patients with and without endoscopic POR. We therefore conclude that PBL DNA methylation is not a feasible predictive tool in post-operative CD.

Background: Myeloid cells are critical determinants of the sustained inflammation in Crohn’s Disease (CD). Targeting such cells may be an effective therapeutic approach for refractory CD patients. Bromodomain and extra-terminal domain protein inhibitors (iBET) are potent anti-inflammatory agents; however, they also possess wide-ranging toxicities. In the current study, we make use of a BET inhibitor containing an esterase sensitive motif (ESM-iBET), which is cleaved by carboxylesterase-1 (CES1), a highly expressed esterase in mononuclear myeloid cells. Methods: We profiled CES1 protein expression in the intestinal biopsies, peripheral blood, and CD fistula tract (fCD) cells of CD patients using mass cytometry. The anti-inflammatory effect of ESM-iBET or its control (iBET) were evaluated in healthy donor CD14⁺ monocytes and fCD cells, using cytometric beads assay or RNA-sequencing. Results: CES1 was specifically expressed in monocyte, macrophage, and dendritic cell populations in the intestinal tissue, peripheral blood, and fCD cells of CD patients. ESM-iBET inhibited IL1β, IL6, and TNFα secretion from healthy donor CD14⁺ monocytes and fCD immune cells, with 10- to 26-fold more potency over iBET in isolated CD14⁺ monocytes. Transcriptomic analysis revealed that ESM-iBET inhibited multiple inflammatory pathways, including TNF, JAK-STAT, NF-kB, NOD2, and AKT signaling, with superior potency over iBET. Conclusions: We demonstrate specific CES1 expression in mononuclear myeloid cell subsets in peripheral blood and inflamed tissues of CD patients. We report that low dose ESM-iBET accumulates in CES1-expressing cells and exerts robust anti-inflammatory effects, which could be beneficial in refractory CD patients.

Vedolizumab (VDZ) is a monoclonal antibody approved for the treatment of Crohn's disease (CD). Despite its efficacy, non-response to VDZ is common in clinical practice with no clear understanding of how it manifests. Here, we characterized the cellular repertoire of responders and non-responders to VDZ during treatment. Peripheral blood mononuclear cells (PBMCs) were isolated from CD patients on VDZ treatment that were either steroid-free responder (N = 4) or non-responder (N = 4). Response was defined as ≥3 drop in Simple Endoscopic Score for Crohn's Disease (SES-CD) in combination with a ≥50% reduction in C-reactive protein (CRP) and fecal calprotectin and/or a ≥3 point drop in Harvey-Bradshaw Index (HBI). Single-cell repertoires were characterized using single-cell RNA-sequencing (scRNAseq) and mass cytometry by time of flight (CyTOF). Non-responders to VDZ presented more T cells, but fewer myeloid cells. T cells from non-responders presented lower expression of NFкB signaling inhibitors. A lower relative abundance of plasmacytoid dendritic cells (pDCs) was observed among non-responders. Moreover, non-responder-derived classical monocytes presented lower expression of genes involved in wound-healing and cytokine-cytokine receptor signaling. Taken together, non-response to VDZ during treatment is associated with differences in abundance and expression among T and myeloid cells.

Introduction: There is great current interest in the potential application of DNA methylation alterations in peripheral blood leukocytes (PBL) as biomarkers of susceptibility, progression and treatment response in inflammatory bowel disease (IBD). However, the intra-individual stability of PBL methylation in IBD has not been characterised. Here, we studied the long-term stability of all probes located on the Illumina HumanMethylation EPIC BeadChip array. Methods: We followed a cohort of 46 adult IBD patients (36 Crohn’s disease (CD), 10 ulcerative colitis (UC), median age 44 (IQR: 27-56), 50% female) that received standard care without any intervention at the Amsterdam UMC. Paired PBL samples were collected at two time points with a median 7 (range: 2-9) years in between. Differential methylation and intra-class correlation (ICC) analyses were used to identify time-associated differences and temporally stable CpGs, respectively. Results: Around 60% of all EPIC array loci presented poor intra-individual stability (ICC <0.50); 78.114 (≈9%) showed good (ICC 0.75 – 0.89); and 41.274 (≈5%) excellent (ICC ≥0.90) stability. Focusing on previously identified consistently differentially methylated positions indicated that 22 CD-, 11 UC-, and 24 IBD-associated loci demonstrated high stability (ICC ≥0.75) over time; of these, we observed a marked stability of CpG loci associated to the HLA genes. Conclusion: Our data provide insight into the long-term stability of the PBL DNA methylome within an IBD context, facilitating the selection of biologically relevant and robust IBD-associated epigenetic biomarkers with increased potential for independent validation. These data also have potential implications in understanding disease pathogenesis.

Biological therapeutics are now widely used in Crohn’s disease (CD), with evidence of efficacy from randomized trials and real-world experience. Primary non-response is a common, poorly understood problem. We assessed blood methylation as a predictor of response to vedolizumab (VDZ, anti-a4b7 integrin) or ustekinumab (USTE, anti-IL-12/23p40). We report a two-center, prospective cohort study in which we profiled the peripheral blood DNA methylome of 184 adult male and female CD patients prior to and during treatment with VDZ or USTE in a discovery (n=126) and an external validation cohort (n=58). We defined epigenetic biomarkers that were stable over time and associated with combined clinical and endoscopic response to VDZ or USTE with an area under curve (AUC) of 0.87 and 0.89, respectively. We validated these models in an external cohort yielding an AUC of 0.75 for both VDZ and USTE. These data will now be prospectively tested in a multicenter randomized clinical trial. Graphical