Iron Overload Effects On Immune System Through The Cytokine Secretion By Macrophage
Authors: Matsui Keiko , Ezoe Sachiko , Yokota Takafumi , Ishibashi Tomohiko , Oritani Kenji , Kanakura Yuzuru 
Published: October 2019

Abstract
Iron is essential for almost all organisms. However, free iron can be cytotoxic at high concentration and iron excess can have adverse effects on variety of cells, tissue, and organ functions. In immune systems, many reports have shown the effects of iron, some of which are complex and controversial. Iron deficiency has been reported to be associated with increased susceptibility to infection, but iron overload caused by dietary excess abnormal hemolysis or inherited disorders is also associated with heightened susceptibility to infections. On the other hand, elevated ferritin levels, primarily related to RBC transfusions, have been reported to increase the risk of acute and chronic GVHD in patients received hematopoietic cell transplantation. Although this iron-related risk of GVHD may reflect the organ damage, such as liver, kidney, and pancreas, it also may be caused by imbalance in immune systems.
To exaggerate the effects of iron overload on immune system, we established iron-loaded models in mice. First, as acute iron-loaded model, 10mg of iron dextran, which is even equal to 200U of RCC transfusion in human (define as 200U of iron dextran), was intraperitoneally injected into mice once a day for 18 days. In peripheral blood, T cell and B cell populations were decreased but monocyte/macrophage and neutrophil populations were increased as compared in control mice administered with dextran only. And notably, regulatory T cell (CD4+Foxp3+; Treg) population was significantly reduced in iron loaded mice than control mice (1.40% vs 0.58%). Next, as chronic iron-loaded model, 2U of iron dextran was injected once a week 45 times. Although significant difference was not observed in Treg population in PB, that in spleen was significantly high in iron-loaded mice as compared with that in control mice (3.2% vs 1.7%, p<0.05). From these data, we suspected that the excess of iron reduces Treg population in some organs, thereby effects on immune function.
To clarify the mechanism of Treg-reduction by iron overload, we established an intermediate iron-loaded model, in which 50U of iron dextran was injected into mice once a week for 10 weeks, and following experiments were performed in this model. As previous examinations, Treg population in spleen was reduced in iron-loaded mice (0.86% vs 0.46%). T helper 17 (Th17) cells are recently described lymphocyte subset with common precursors with Treg but with opposing actions to Treg. The difference of Th17 cells could not be detected in spleen because of its too small population, the population of Th17 was increased in small intestine of iron-loaded mice (0.85% vs 1.51%). Treg and Th17 cells can be differentiated from naïve Th cells (CD4+CD62L+) with stimulation of specific cytokines (Treg; TGFβ, IFNγ, and IL4, Th17; TGFβ, IL-6, and IL-1β). We analyzed the direct effects of iron on Treg and Th17 cells during the in vitro differentiation from naïve T cells. Both in Treg and Th17 cell inductions, we could not detect significant differences between cells supplemented with iron dextran and those with dextran only. We also exaggerated the reactive oxide spesies (ROS) accumulated in induced T cell using RedCC1 staining. During the induction of Treg and Th17, although H2O2 supplement increased the ROS accumulation dose dependently, iron dextran-addition did not change the amount of ROS.
Next, we analyzed the serum concentrations of cytokines in iron-loaded mice both in acute and intermediate models. IL-1β, and IL-23 levels were elevated in iron-loaded mice. These data suggested that iron overload reduces Treg cell population and increases Th17 cell population not directly, but through the cytokine secretion from environmental cells. So we evaluated the induction rates of Treg and Th17 cells from naïve T cells under co-culture with monocyte/macrophage. In this condition, Treg induction rate was significantly lower in iron dextran-supplemented cells (48.1% vs 35.3%), and Th17 induction was increased (8.4% vs 18.0%). Furthermore, mRNA expressions of IL-23 and IL-β in macrophages were increased with iron dextran-supplement in in vitro culture.
These data suggest that iron overload changes the balance of Treg and Th17 cells through the proliferation and activation of macrophages, and thereby effects on the immunological condition of some disease, such as GVHD and autoimmune.

Disclosures:
No relevant conflicts of interest to declare.