Experimental hemochromatosis due to MHC class I HFE deficiency: immune status and iron metabolism

Abstract

The puzzling linkage between genetic hemochromatosis and histocompatibility loci became even more so when the gene involved, HFE, was identified. Indeed, within the well defined, mainly peptide-binding, MHC class I family of molecules, HFE seems to perform an unusual yet essential function. As yet, our understanding of HFE function in iron homeostasis is only partial; an even more open question is its possible role in the immune system. To advance on both of these avenues, we report the deletion of HFE alpha1 and alpha2 putative ligand binding domains in vivo. HFE-deficient animals were analyzed for a comprehensive set of metabolic and immune parameters. Faithfully mimicking human hemochromatosis, mice homozygous for this deletion develop iron overload, characterized by a higher plasma iron content and a raised transferrin saturation as well as an elevated hepatic iron load. The primary defect could, indeed, be traced to an augmented duodenal iron absorption. In parallel, measurement of the gut mucosal iron content as well as iron regulatory proteins allows a more informed evaluation of various hypotheses regarding the precise role of HFE in iron homeostasis. Finally, an extensive phenotyping of primary and secondary lymphoid organs including the gut provides no compelling evidence for an obvious immune-linked function for HFE.

Figure 1

Generation of an HFE deficient mouse strain. (A) Structure of the endogenous locus and the targeting construct. HFE exons are noted, as is the probe used for screening. As indicated, exons encoding the α1 and α2 domains are replaced by pgk-neo^r in the mutant animals. H, HindIII; B, BglII. (B) Representative Southern blot of DNA from mutant and control mice. Genomic DNA was digested with HindIII, and the blot was hybridized with the 5′ probe indicated in A; the genotypes of the animals are shown above. (C) Northern blot analysis of kidney and liver RNA from +/+, +/o, and o/o mice. Twenty micrograms of total RNA was run in each lane, and filters were hybridized with an HFE cDNA (spanning exons 2–5). The filter was stripped and hybridized with a hypozanthine phosphoribosyl transferase probe as a loading control.

Figure 2

Iron storage in hepatic cells from +/+ and o/o mice fed on different diets. Light micrographs of a periportal liver section from +/+ controls on a standard diet (A) and after 2 weeks of iron-rich feeding (B). The white areas are peripheral vessels. Although no iron staining is visible in A, parenchymal iron accumulation in (B) is represented by small blue dots in the cells around the periportal vessel. C and D show periportal sections from o/o animals on iron-adequate and iron-rich diets, respectively. Pearl’s staining shows marked parenchymal iron accumulation in C and to an even higher extent in D. In all cases, iron accumulation decreases with increasing distance from the periportal area. Pearl’s Prussian blue staining. (A and B, ×1,000; C and D, ×400).

Figure 3

HFE-deficient mice harbor normal number of various T cell subpopulations. Shown is a representative FACS analysis of lymphocytes isolated from HFE^o/o and HFE^+/+ littermates (13 weeks of age). CD4/CD8 staining of thymocytes and lymph node cells is shown above and αβ (TCRβ)/γδTCR staining of IELs below. Total cell numbers isolated from each mouse were similar, and the percentages of relevant populations are noted.