Final Progress Report
Proposal No. NICE-04
Principal Investigator: Lloyd Mayer, M.D.
Applicant Organization: Mount Sinai School of Medicine (New York, New York, U.S.A.)
Project Title: Define bacterial antigens affecting epithelial cell phenotype and function
Period of Award: January 1, 2003 - December 31, 2003
The goal of our project was to define factors (bacterial, cellular and soluble) that regulate CD1d expression on intestinal epithelial cells. We published with Dr. Richard Blumberg that Hsp110 increases CD1d expression on T84 cells. This molecule is present in stool, is induced by bacteria/bacterial products and may account in part for the biased expression of CD1d on colonocytes (colonocytes > small bowel enterocytes; right colon > left colon). This latter finding is part of a manuscript that is being submitted to the Journal of Immunology.
We further studied the role of T cells and cytokines in CD1d expression, using the T84 monolayer system. Basal, but not apical, culture of mitogen-activated peripheral blood T cells (PHA) induced the expression of CD1d. This was regulated at the transcriptional level (measured mRNA by real time PCR) and occurred within 48 hours of co-culture. Unactivated T cells had no effect. This induction of CD1d mRNA appears to be differentiation stage dependent. Nonpolarized nondifferentiated T84 cells expressed less CD1d mRNA than polarized epithelial monolayers. This would be in accord with ex vivo data generated by us (CD1d is not expressed on the surface of crypt epithelial cells, but is expressed on surface colonocytes - CD1d mRNA displays the opposite pattern).
Since mitogen-activated T cells do not represent a physiologic process, we moved towards the co-culture of T84 cells with freshly isolated lamina propria T cells from normal controls as well as patients with inflammatory bowel disease (IBD). LPLs (normal) failed to promote T84 differentiation (barrier function measured by increased transepithelial resistance) and failed to enhance expression of CD1d. They did signal the epithelium, inducing the phosphorylation of Akt. Thus, while the LPL did not appear to regulate CD1d, it was at least interacting with and promoting the activation of the epithelium.
Regulation of CD1d may occur by both cognate and noncognate interactions. Given the positive effect of PHA-activated T cells on CD1d expression by T84 cells, we next attempted to define the cytokines that might be involved in this process. PHA-activated T cells were analyzed for an array of cytokines by Riboquant cytokine RNAse protection assays. Increases in a number of cytokines were noted, especially IFNγ, TGFβ, IL6 and IL2. These cytokines were then tested alone or in combination in the T84 monolayer system. None of these cytokines promoted CD1d mRNA or protein expression. We then attempted the reverse experiment where PHA-stimulated T cell supernatants were added basolaterally to a T84 monolayer in the presence or absence of neutralizing Abs to specific cytokines. Abs to IFNγ and TGFβ markedly attenuated (but did not completely abrogate) CD1d expression induced by the mixture of cytokines. Given the findings with recombinant cytokines described above, these data suggest that IFNγ works to enhance CD1d expression in conjunction with another as yet unidentified cytokine. We are currently screening for candidates using other blocking mAbs.
Summary:
The efforts of this project have resulted in two papers and one novel area of research that has served as the basis for an R01 application. CD1d regulation is an important area, as this non-classical class I molecule is involved in regulating both innate and adaptive immunity in the gut.
Lay Summary:
The immune system in the intestine is regulated very differently than the immune system in the rest of the body. We have focused on the lining cells of the intestine, epithelial cells, and asked how they contribute to regulation of immunity in the intestine. We have shown that bacteria in the intestine promote an environment that shuts down immune responses.