Scientific Abstract
Proposal No. IBD-0029
Principal Investigator: Adrian Cummins, M.D., Ph.D.
Applicant Organization: The Queen Elizabeth Hospital (Woodville South, Australia)
Project Title: Deficiency of immunoregulatory NK T cells in inflammatory bowel disease
Period of Award: November 1, 2002 - March 31, 2005
Background: Inflammatory bowel disease (IBD) has some features of an autoimmune disease with raised autoantibodies (e.g., p-ANCA) and an increased incidence of other autoimmune diseases. Recent evidence has shown that NK T-cells (Vα24+ VνΦ11/13/8.2+ in man) are deficient in human autoimmune (type 1 diabetes, multiple sclerosis, rheumatoid arthritis, scleroderma) diseases and in animal models (type 1 diabetes in the NOD mouse, lupus erythematosis) of autoimmune disease. NK T-cells have not been investigated before in IBD and therefore their investigation represents a new direction of research.
Hypothesis: Our hypothesis is that NK T-cells are deficient in inflammatory bowel disease. This could contribute to loss of immunoregulation of the gut-associated lymphoid tissue to commensal bacteria. This proposal could be of great significance to IBD, as it opens the possibility of new options for treatment. NK T-cells suppress Th1 immune responses (such as in Crohn’s disease) by promptly producing interleukin-4 (IL-4) and/or by having a cytolytic action on dendritic cells that limit antigen presentation. Our initial work has demonstrated systemic deficiencies of NK (CD161+) cells, NK T-cells (CD161+ CD3+), Vα24+ T-cells, and Vα24+ Vβ11+ T-cells in Crohn’s disease and Vα24+ Vβ11+ T-cells in ulcerative colitis. Thus, there was a deficiency of the CD161+ NK lineage in Crohn’s disease (hence resulting in NK T-cell deficiency), but a more selective deficiency of only immunoregulatory NK T cells in ulcerative colitis.
Aims: The aims of this proposal are: 1) to investigate systemic deficiency of NK T-cells by tetramer staining; 2) to examine mucosal deficiency of Vα24+ mRNA; 3) to assess functional integrity of Vα24+ T-cells by anti-CD3 antibody stimulation and by a-galactosylceramide (a molecule known to specifically stimulate the CD3 receptor of Vα24+ cells) to produce IL-4 and other cytokines; and 4) to investigate the cytotoxic function of NK T-cells in lysing allogeneic monocyte-derived dendritic cells.
Methods and Proposal: Systemic deficiency of NK T-cells will be further investigated in blood from 50 Crohn’s subjects, from 50 subjects with ulcerative colitis and from 50 control subjects (irritable bowel syndrome, screening for colon cancer) using flow cytometry. Mucosal deficiency of Vα24+ mRNA will be investigated in ileal or colonic biopsies from 30 subjects with Crohn’s disease, 30 with ulcerative colitis and 30 control subjects at colonoscopy. mRNA for Vα24+ would be assessed by real time PCR analysis. Systemic Vα24+ cells will be stimulated with anti-CD3 (OKT3) antibody or with a-galactosylceramide and IL-4 intracellular cytokine measured by flow cytometry. Intestinal biopsies will be incubated with either anti-CD3 antibody or a-galactosylceramide, and IL-4 mRNA production assessed by real time PCR. Dendritic cells will be derived from blood monocytes and used as targets for effector Vα24+ cells in a NK T-cell cytotoxicity assay. Vα24+ will be purified by magnetic beads. Our goals by the end of the proposal are to define systemic and mucosal deficiency of NK T-cells in IBD and to assess the functional capacity of residual NK T-cells to produce cytokines and to mediate cytotoxicity of dendritic cells.
Relevance to IBD: The investigation of NK T-cell deficiency is a new direction of research in IBD; the relevance is based on experimental evidence of NK T-cell deficiency in murine models of IBD. Of particular importance is whether residual NK T cells can be stimulated (e.g., by α-galactosylceramide) in humans to replenish NK T-cell deficiency or loss of function. This could open a possible new treatment for IBD.