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* = Presenting author

P021 Inflammatory monocytes are key regulators of colonic inflammation in murine and human IBD

G.-R. Jones*1, P. Cook2, A. MacDonald2, T. Fenton2, A. Kelly2, M. Travis2, S. Campbell3

1University of Edinburgh, Gastroenterology, Edinburgh, United Kingdom, 2University of Manchester, Department of Immunology, Manchester, United Kingdom, 3University of Manchester / Manchester Royal Infirmary, Department of Gastroenterology, Jean Mcfarlane Building; School for Nursing, Manchester, United Kingdom

Background

Murine intestinal macrophages (MΦ) are dependent on re-population from Ly6CHi blood monocytes, whose differentiation in the intestine corresponds with marked change in phenotype. Ly6CHi monocytes are pro-inflammatory, secreting significant IL-1β, whereas MΦ are tolerant to their local microenvironment. This balance is disrupted in ileal Crohn’s disease, suggesting the balance of monocytes and MΦ is important in tissue inflammation. It is unclear whether this balance is disrupted in colonic inflammatory bowel disease (IBD) with the role of MΦ in monocyte recruitment poorly understood.

Methods

Murine colon lamina propria (LP) cells and whole blood was isolated from day 6 2% dextran sulphate sodium (DSS)-treated C57Bl/6 mice or drinking water controls and stained with an antibody cocktail (Ly6C, Ly6G, CD11b, CD11c, F4/80, MHC-II, and SiglecF). Cells were cultured ex vivo with GolgiStop (LP) +/- 1μ g/g LPS (blood). Cells were stained intracellularly for IL-1β and acquired using a BD Fortessa. For microarray analysis, monocytes and MΦ were purified by flow cytometric sorting from day 6 DSS-treated mice and RNA isolated for analysis by IlluminaRef6 microarray.

Patients attending for colonoscopy at Manchester Royal Infirmary were consented for endoscopic biopsy collection. Samples were obtained from healthy controls (n = 4), quiescent IBD (n = 10), and active IBD (n = 6) as defined by the endoscopic assessment of disease activity. 
LP cells were then isolated and stained with an antibody cocktail including CD14, HLA-DR, CD11c, and CD64.

Results

LP monocytes were the largest IL-1β+ myeloid population, by total number and per cell production, with significantly greater IL-1β production from LPS-stimulated blood monocytes, in cells isolated from DSS-treated mice versus controls.

The ratio of LP monocytes: MΦ was significantly increased in DSS-treated mice versus controls. Active colonic IBD was similarly associated with significant accumulation of CD14Hi monocyte-like versus CD14Low MΦ cells compared with both quiescent IBD and healthy controls. Microarray analysis of colon LP monocytes and MΦ from DSS-treated mice revealed significant up regulation (> og 2-fold change, adj, p < 0.01) of MΦ chemokine pathways including Ccl3, Ccl7, Ccl8 and Cxcl1 expression.

Conclusion

The balance of the monocyte-MΦ axis is disrupted in colitis favouring recruitment of monocytes. Murine blood monocytes appear ‘primed’ in inflammation even before egress into tissue sites, and represent the dominant IL-1β+ LP population in murine colitis. Monocyte differentiation into MΦ favours further damaging monocyte and neutrophil recruitment in DSS colitis. Understanding the priming signals to blood monocytes and blocking their recruitment may help limit colonic inflammation in IBD.