Final Progress Report
Proposal No. IBD-0253
Principal Investigator: Bobby J. Cherayil, M.D.
Applicant Organization: Massachusetts General Hospital (Charlestown, U.S.A.)
Project Title: Iron-based modulation of cytokine biosynthesis for treatment of intestinal inflammation
Period of Award: November 1, 2008 – February 28, 2011
Summary of Project Aims
The overall goal of the project was to evaluate a novel anti-inflammatory strategy in mouse models of inflammatory bowel disease (IBD). The strategy was based on the use of reagents that inhibited the expression of the iron-regulatory hormone hepcidin that our preliminary studies had implicated in the inflammatory response. Based on our preliminary findings, inhibition of hepcidin expression could have the added advantage of correcting the abnormal iron metabolism and anemia that often accompanies inflammatory conditions. The aim of year 1 of the project was to evaluate the strategy in the piroxicam/IL-10 knock-out model of colitis, and the aim of year 2 was to evaluate it in a T cell transfer colitis model.
Year 1 – We successfully tested the effects of 2 different inhibitors of hepcidin expression in the piroxicam/IL-10 knock-out colitis model: LDN-193189, a small molecule inhibitor of bone morphogenetic protein (BMP)-induced signals required for hepcidin up-regulation, and HJV.Fc, a recombinant fusion protein that inhibits binding of BMPs to their receptor and thereby prevents activation of the signals required for hepcidin up-regulation.
Year 2 – We successfully tested the effects of 3 different inhibitors of hepcidin expression in a T cell-dependent model of colitis in which CD4+CD25- naïve T cells were transferred into lymphocyte-deficient Rag knock-out mice. The 3 inhibitors included LDN-193189, HJV.Fc and an antibody that selectively neutralized BMP6 (the BMP that recent results have shown to be the key inducer of hepcidin in vivo).
Significant Results
a. In both models of colitis, hepcidin was significantly up-regulated following the induction of intestinal inflammation.
b. All 3 of the reagents tested – LDN-193189, HJV.Fc and anti-BMP6 – significantly reduced inflammation-induced up-regulation of hepcidin in either the piroxicam/IL-10 knock-out or T cell transfer colitis models. The extent of hepcidin inhibition was comparable in all 3 cases.
c. Inhibition of hepcidin expression with LDN-193189 or HJV.Fc was associated with decreased severity of intestinal inflammation in the piroxicam/IL-10 knock-out colitis model as indicated by colon histopathology and inflammatory cytokine levels.
d. Inhibition of hepcidin expression with LDN-193189, HJV.Fc or anti-BMP6 was associated with decreased severity of intestinal inflammation in the T cell transfer colitis model as indicated by colon inflammatory cytokine levels. However, there was no appreciable effect on the colon histopathology. This difference from the piroxicam/IL-10 knock-out colitis model suggests that the anti-inflammatory effect of inhibiting hepcidin expression may be more prominent in intestinal inflammation that is driven mainly by innate rather than T cell-dependen mechanisms.
e. In the T cell transfer colitis model, inhibition of hepcidin expression with LDN-193189, HJV.Fc or anti-BMP6 was associated with a significant elevation of serum iron levels.
f. We carried out tissue culture experiments to elucidate the mechanism underlying the anti-inflammatory effect of inhibiting hepcidin expression. We found that peritoneal macrophages isolated from HJV.Fc-treated animals produced lower levels of the pro-inflammatory cytokine TNFα in response to LPS stimulation than control cells. Furthermore, the mechanism of the attenuated inflammatory response of macrophages isolated from mice with low hepcidin levels was related to an abnormality of signaling through the TRAM/TRIF pathway downstream of Toll-like receptor (TLR) 4. This abnormality, the precise details of which we are trying to elucidate, appeared to be caused by low intracellular iron concentrations and impaired translation of certain cytokine mRNAs.
Taken together, our findings provide evidence to support the idea that inhibiting hepcidin expression may represent a viable therapeutic strategy in IBD, with potential benefits on both the severity of intestinal inflammation and on the dysregulated iron metabolism associated with inflammation. Our studies have also shed light on issues of basic immunological interest, including the role of the TRAM/TRIF pathway in TLR4-induced cytokine expression and the effects of altered iron metabolism on this pathway.
Based on preliminary observations suggesting that a small protein called hepcidin might contribute to intestinal inflammation in IBD, this project tested the idea that reducing levels of hepcidin could be of benefit in this disease. We successfully lowered hepcidin levels in 2 different mouse models of IBD using 3 different reagents that we injected into the mice. In all cases, lowering of hepcidin levels was associated with a modest decrease in the severity of intestinal inflammation. We also found that lowering hepcidin levels during colitis raised serum iron concentrations. Thus, our experiments indicate that lowering hepcidin levels may have 2 beneficial effects in IBD – relief from intestinal inflammation and an improvement in serum iron that could help to prevent or treat anemia.