Introduction The inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn’s Disease (CD) occur when failures of immune regulation result in an accumulation of immune effector cells that damage the intestine. A detailed understanding of these processes has heretofore been hampered by difficulties in obtaining phenotypic and functional analyses of the multitude of closely-related immune cells present in both normal and in the diseased intestine. Here we show data from our recently-developed 12-parameter flow cytometric analyses of leukocytes from blood and intestinal biopsies. We anticipate that this approach will enable us to identify immune mechanisms causing or controlling IBD.
Methods After written informed consent was obtained, endoscopic biopsies from the colon and/or terminal ileum were obtained from patients with UC, CD, or from unaffected individuals attending for polyp surveillance colonoscopies. Blood samples were obtained from UC or CD patients attending IBD clinics, or from healthy volunteers. Live single cell suspensions were prepared, and were prepared for flow cytometric analysis, focusing on dendritic cell and T cell populations. Data were analysed using FlowJo software. Differences were analysed by Mann-Whitney or ANOVA, with post tests to assess significance.
Results We have developed novel and reproducible methods for purification of live cells from fresh colonic and ileal biopsies, and for enumerating of T cell and antigen presenting cell populations using 10-colour flow cytometry. We have compared data from IBD patients and healthy controls. Initial analyses of intestinal dendritic cell populations (CD45+ CD14- CD64- CD11c+ MHC class II+) have identified three distinct subsets based on CD103 and SIRPα expression. Our preliminary data indicate that dendritic cells are differently distributed along the intestine. Analyses of intestinal T cells (CD45+ CD3+ CD4+, CD25+ /- CD45RA+/-) have revealed the proportions of naïve, activated, memory, and regulatory T cells expressing the chemokine receptors CCR6, CCR9, CXCR3 and CCR10 in each population.
Conclusion Our novel multi-parameter analyses of live cells prepared from fresh colonic and ileal biopsies enable precise examination of the disease-inducing and effector populations that drive UC and CD. We are using the methods described here to dissect the immunological mechanisms driving inflammation in patients with IBD. We anticipate that our on-going comparisons of each of these immune cell populations in blood and intestinal biopsies from unaffected individuals, CD, and UC patients will reveal important details about pathogenic mechanisms controlling intestinal inflammation.
Disclosure of Interest None Declared.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.