The practice of vaccination and the use of antibiotics provoked a significant decrease of infectious disease in the spectrum of modern clinical practice. That decrease uncovered simultaneously the appearance of other diseases whose presentation relates mainly to age. HFE-related hemochromatosis is a genetic disorder of iron overload. The combination of some luck, progress in the immunology of transplantation, progress of the understanding of the regulation of iron metabolism, progress in genomics, progress in the development of transgenic mice and in the characterization of adaptive immunity cell populations resulted in a sequence of events responsible for permitting today the early diagnosis and prevention of the iron overload induced clinical manifestations of the disease. Human HFE-related hemochromatosis has been the subject of several reviews by prominent researchers in the field [1
]. In general, emphasis in the pathophysiology of the disease has been focused on the role of hepcidin in iron homeostasis [3
]. Less attention has been directed to the role of HFE in immunology [5
] and to the potential contribution of lymphoid cell numbers to that same pathophysiology [8
In the present paper, under “learning”, we briefly review the evidence sustaining the importance of immunology in HFE-related hemochromatosis, starting by the improbable discovery of its link with HLA to the discovery of the HFE gene as a MHC class I-like gene and its later association with selected immune defects. Under “applying”, we report a 20 year follow up of iron parameters and lymphocyte counts in three children homozygous for the C282Y HFE variant who started to express signs of iron overload in their transition from childhood to adult life. Under “questioning”, we explore the issue of the still unexplained immunological functions of HFE. The paper concludes with a brief overview of its implications for the clinical practice and for the immunological theory.
Learning: A Brief Historical Sequence Where the Improbable Led to Discovery
In 1975 it was improbable to think that HLA could be of clinical importance beyond histocompatibility or diseases with an immunological background. However, Simon thought the improbable and encountered in Fauchet an immunogeneticist open to doing the HLA typing of 20 “idiopathic” hemochromatosis” patients to find 17 with HLA A3 [11
]. The result was the discovery, first published in 1975 in French [11
] and in 1976 in the Lancet paper with the title, “HLA and "non-immunological" disease: idiopathic haemochromatosis” [12
The publications of Simon, Fauchet and coworkers were to provide some background to a postulate published in 1978 saying that the immunological system could have a role in the surveillance of iron toxicity [13
]. That postulate was followed by a series of studies of immunological cell populations in patients with “idiopathic” hemochromatosis [8
], preceding the finding by Feder et al. in 1996 of the hemochromatosis gene as a novel MHC class I-like gene [16
], closing a circle of 21 years from the improbable to the discovery.
Radical changes followed the discovery of the hemochromatosis gene and its association with the C282Y HFE variant. The introduction of genetic testing, combined with Magnetic Resonance Imaging (MRI) as a non-invasive measure of liver iron concentration, the systematic family screening, approaches to disease penetrance from large population studies, all led to a radical change in the clinical presentation and timing of diagnosis well demonstrated by the decreasing frequency of severe liver disease [17
HFE-related hemochromatosis appears thus as a “dream like” age related disease, with a strong genetic marker, reliable and reproducible serum biochemical markers, namely transferrin saturation and serum ferritin, and confirmatory non-invasive tools all enabling diagnosis much before the clinical presentation of the disease.
The surprise, however, arises from the fact that a disease thought of as non-immunological, can be seen inevitably as an immunological disease. Firstly, the gene is an MHC class I like gene in strong linkage with other genes within the MHC cluster [18
]. Secondly, the very early studies of lymphoid cell populations in patients with (at the time) idiopathic hemochromatosis demonstrated abnormally high CD4/CD8 ratios in those with a more severe iron overload [14
]. At that time, it was also demonstrated that after complete iron depletion by repeated phlebotomies, entry of iron measured by changes in transferrin saturation was faster in patients with the highest CD4/CD8 ratios [14
]. Later, a greater importance was attributed to the finding of low numbers of CD8+ T cells associated with the severity of iron overload [9
] and the demonstration that the low CD8+ T lymphocyte numbers in hemochromatosis are due to defects in the most mature effector memory cells [21
]. When those defects were described, they generated some surprise and confusion amongst researchers and clinicians because of the previously existing evidence of iron induced expansions of T lymphocyte populations, namely, relative expansions of CD8+ T lymphocytes, both in experimental models of Fe-citrate injection [22
] and in clinical models of transfusional iron overload [24
]. The reciprocal effect, i.e., that primary immune defects could, in turn, contribute to iron overload, was next confirmed with a number of experimental studies examining and confirming the presence of iron overload in mice deficient in selected [26
] or total lymphocytes [29
] and a more severe phenotype in mice lacking both HFE and β2-microglobulin [30
]. Surprisingly, mice lacking only classical MHC class I molecules also developed iron overload [31
The mechanisms underlying the lymphocyte defects in hemochromatosis are still poorly understood. The most recent evidence points to the possibility of a continuous effect where iron may sustain a constant activation, self-renewal and proliferation of CD8+ cells, and this may eventually lead to exhaustion of the effector memory T cells [32
Regarding the mechanism “how” could lymphocyte defects contribute to iron overload, it is not until 2014 that Pinto and co-workers, in an extensive analysis of the interaction of lymphocytes with non-transferrin bound iron (NTBI), demonstrated that lymphocytes can take in NTBI in vitro [33
] and that in vivo lymphocyte transfer can correct the iron overload of immune-deficient mice [10
], closing thus another circle: From firm but unexplained observations to the demonstration of how lymphoid cell numbers could have a role in the control of iron overload, i.e., by acting as a circulating pool capable of “buffering” NTBI.
Evidence of the inextricable connection between iron homeostasis and the adaptive immune system recently gained a novel impulse with the demonstration that patients with a homozygous p.Tyr20His mutation in the transferrin receptor 1 (TfR1) have a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells [34
]. Besides TfR1, other iron regulatory genes had also been previously found to be critical for lymphocyte activation and function, namely H-ferritin, whose conditional deletion in mice was shown to reduce B and T lymphocyte populations [35
] or hepcidin, whose expression is increased during lymphocyte activation and shown to be necessary for proper lymphocyte proliferation [36
With regard to the cells of the mononuclear/phagocytic system, their role in the recycling of senescent red blood cells provides perhaps the most significant illustration of the close interactions between iron metabolism and the immunological system [24
]. In the case of secondary iron overload, as a result of dyserythropoiesis, hemolysis, or transfusions, macrophages are heavily loaded with iron which is released in the form of low-molecular weight (LMW) iron. This leads not only to increased transferrin saturation but also iron circulating as NTBI that will inappropriately enter tissues and cells [37
]. In HFE-related hemochromatosis, the scenario is somehow different. In spite of the high transferrin saturation and circulating NTBI, as a result of increased iron absorption, little iron is seen in the Kupffer cells and other macrophages, while hepatocytes already show iron overload [2
]. In a previous study of iron release by monocytes after erythrophagocytosis, Moura and co-workers demonstrated that monocytes from hemochromatosis patients released twice as much iron in a LMW form as control cells [38
]. Based on those observations, they proposed for the first time the existence of a basic abnormality in the retention of iron in macrophages and probably from intestinal mucosal cells [38
], a mechanism that is presently well established with the demonstration that hepcidin, which is functionally defective in hemochromatosis, regulates cellular iron efflux by binding to ferroportin and inducing its internalization [3
]. Finally, one may question how the handling of iron in other compartments of the mononuclear/phagocytic system may also impact in their response to other types of toxicity. As an example, increasing evidence suggests that the accumulation of iron in the brain and the consequent microglia activation are hallmarks of neurodegenerative disorders, including Alzheimer’s disease [39
]. However, substantial efforts are still needed to fully understand this and many other aspects of the complex interactions of iron with inflammation and immunity. In our view, HFE hemochromatosis continues to offer a particularly good model to approach new questions.