Title : Variation of ferroportin expression in IBD patient intestinal macrophages
Abstract:
Background:
Hepcidin and ferroportin are master regulators of systemic iron homeostasis in mammals. Hepcidin is encoded for by the HAMP gene and is released by hepatocytes to regulate systemic iron levels. Thereafter, hepcidin frequently affects duodenal cells for dietary iron transport or splenic red pulp macrophages to facilitate iron recycling. However, earlier work from Bessman Lab has shown that hepcidin also affects local tissue iron in both the cecum and colon during states of intestinal inflammation and may further have an impact on the observed recovery (1). Additionally, mouse experiments using 3.5% DSS in water to induce an IBD model have shown that oral gavage hepcidin leads to significant weight recovery when compared to control. When this experiment was repeated in mice that contained hepcidin-resistant macrophages, the results were no longer significant, indicating that the observed therapeutic effect relied on the inhibition of macrophage ferroportin. Accordingly, we sought to stain colon sections from human IBD samples to see if there was an increase in ferroportin expression across colonic macrophages.
Methods:
In preparation of staining, the slides were deparaffinized and subsequently run through an antigen retrieval step. Slides were then placed with a blocker solution in addition to a primary co-stain for ferroportin using Alexa 647 and macrophages using CD68. The following day, the sections received secondary stain with Rabbit Anti-Alexa488 and tertiary stain with Goat Anti-Rabbit Alexa 647. The slides were then mounted with a mounting medium containing DAPI. The slides were imaged using a Nikon A1R Spectral Confocal Microscope and visualized through NIS-Elements software.
Results:
In healthy patient samples, there was no apparent overlap of ferroportin and macrophages found in the images that were stained. Additionally, there was little positive ferroportin staining to be seen across the section overall. Rather, the areas that were positive for ferroportin were distinct from those that were positive for macrophage staining as well. In comparison, the IBD samples displayed a noticeable overlap between the macrophage and ferroportin stains. This overlap became progressively more apparent in slides that taken from areas significant for some underlying pathology (stenosis, fistula, etc).
Conclusion:
In accordance with earlier work, a phenotype characterized by an increase in intracellular iron efflux via macrophages was observed through the staining results of healthy and IBD human patient samples. This work confirms that macrophage ferroportin may be a tractable therapeutic target for promoting mucosal healing after IBD flares. In the future, we aim to assess to macrophage ferroportin expression in a wider panel of IBD patients to determine which patients are most likely to benefit from ferroportin inhibition, and in further pre-clinical work we aim to define the mechanism by which ferroportin inhibition promotes mucosal healing.
Audience Take Away:
- It will help people understand the physiological processes occurring in an inflammatory state within the intestine.
- It will help people further understand the role of iron metabolism in these diseases.
- May help people in approaching intestinal staining or design of staining experiment.
- Potentially investigate different avenues for therapeutics for IBD patients.
