The Nuclear Protein HMGB1 is Increased in the Stools of IBD Patients and Appears to Be a Novel Marker of Intestinal Inflammation

Abstract

Background The balance between T regulatory cells (Treg) and T helper type 17 (Th17) cells appears important in modulating immune responses in inflammatory bowel disease. CD39 is an immune cell ectonucleotidase, expressed in lipid rafts, which generates immune suppressive adenosine. Genetic polymorphisms of CD39 are noted in IBD and the ectoenzyme has been validated as a marker of memory type Treg. CD161 (killer cell lectin subfamily B, member 1 or NKR-P1A) is a relatively specific surface marker for human Th17 cells and is linked to activation of acid sphingomyelinase that might in turn impact CD39 bioactivity. It is unknown whether there are T cell populations in IBD that express both markers and the putative relevance is unexplored. Aims: To identify dual expression of CD39 and CD161 in blood T cells from patients with IBD, and explore the role of CD161 in regulating immune deviation of CD4+CD39+ T cell populations. Methods The percentage of CD39+CD161+ CD4 T cells in peripheral blood of patients with active or inactive Crohn's disease was determined by FACS. Four phenotypic subtypes of peripheral blood CD4 T cells including CD39+CD161+, CD39+CD161-, CD39-CD161+, and CD39-CD161from healthy donors were sorted by FACS, and stimulated respectively by anti-CD3/CD28 antibodies with with TGF-beta and IL-2 (for Treg cell expansion) or IL-6, IL-23, IL-1beta and TGFbeta (for Th17 cell expansion) for 3 days, and intracellular IL-17 and INF-gamma or FOXP3 were detected by FACS. These four subgroups were treated by ATP or NAD+ for 1 hour, and stained by Annexin V to detect early events of P2X7-mediated apoptosis. Result The percentage of CD39+CD161+ CD4 T cells in peripheral blood of normal controls, active and inactive Crohn's disease patients was 1.26±0.73, 4.55±2.39 and 0.95±0.52%, respectively (p=0.01, for active disease vs. both controls and inactive Crohn's disease). After 3 days, Treg expansion, of FOXP3-positive cells within the CD39+CD161+ group, was less than from the CD39+CD161group (p=0.01). After 3 days In Vitro expansion, the percentages of intracellular IL-17-positive and/or IFN-gamma-positive cells in CD4+CD39+CD161+ populations were higher than other three groups (p=0.01). However, CD39+CD161+ CD4 T cells also exhibited highest levels of NAD+-induced apoptosis (p=0.05 vs. other groups). Conclusions Peripheral blood CD4+CD39+CD161+ T cells are increased in active Crohn's disease. CD161 expression impacts the reciprocal balance of Treg and Th17 cells generated from CD4+CD39+ cells.