Iron Deficiency Impairs Intra-Hepatic Lymphocyte Mediated Immune Response

Abstract

Hepatic expression of iron homeostasis genes and serum iron parameters predict the success of immunosuppression withdrawal following clinical liver transplantation, a phenomenon known as spontaneous operational tolerance. In experimental animal models, spontaneous liver allograft tolerance is established through a process that requires intra-hepatic lymphocyte activation and deletion. Our aim was to determine if changes in systemic iron status regulate intra-hepatic lymphocyte responses. We used a murine model of lymphocyte-mediated acute liver inflammation induced by Concanavalin A (ConA) injection employing mice fed with an iron-deficient (IrDef) or an iron-balanced diet (IrRepl). While the mild iron deficiency induced by the IrDef diet did not significantly modify the steady state immune cell repertoire and systemic cytokine levels, it significantly dampened inflammatory liver damage after ConA challenge. These findings were associated with a marked decrease in T cell and NKT cell activation following ConA injection in IrDef mice. The decreased liver injury observed in IrDef mice was independent from changes in the gut microflora, and was replicated employing an iron specific chelator that did not modify intra-hepatic hepcidin secretion. Furthermore, low-dose iron chelation markedly impaired the activation of isolated T cells in vitro. All together, these results suggest that small changes in iron homeostasis can have a major effect in the regulation of intra-hepatic lymphocyte mediated responses.

Fig 1. Iron and haematological parameters in mice fed with an iron-deficient diet.

Mice were fed for 3 weeks with an iron deficient (IrDef) or iron replete (IrRepl) diet. Bar plots display mean and SEM. Abbreviations: RBC (red blood cells), Hb (Hemoglobin), Plts (platelets), WBC (white blood cells).

Fig 2. Influence of iron levels on the immunophenotype of intra-hepatic (A) and spleen (B) leukocytes.

Mice were fed for 3 weeks with an iron deficient (IrDef) or iron replete (IrRepl) diet. Bar plots show the frequency (mean and SEM) of specific leukocyte subsets in the liver (A) and spleen (B), as assessed by flow cytometry after excluding dead cells.

Fig 3. Iron deficiency results in attenuated immune-mediated hepatitis following ConA administration.

(A) Serum levels of alanine aminotransferase (ALT; IU/mL) before, 6 hours and 12 hours after administration of ConA in mice fed for 3 weeks with an iron deficient (IrDef) or iron replete (IrRepl) diet. (B) Cytokine levels in serum samples collected before, 6 hours and 12 hours after the administration of ConA. (C) Transcript levels of Il4, Il6 and TNFα in liver tissue samples collected before and 12 hours after the administration of ConA. (D) Representative liver histology (200x) at 12 hours after the administration of ConA (upper panel) and histologic evaluation of necrosis and infiltration (lower panel).

Fig 4. ConA-induced T and NKT lymphocyte activation is reduced in iron deficiency.

(A) Intra-hepatic frequency of CD3⁺ expressing NK1.1 and CD3⁺NK1.1^- at baseline, 3 hours and 6 hours after ConA injection. (B) Intra-hepatic frequency of IFNγ production by NKT cells and T cells, defined as CD3⁺NK1.1⁺ and CD3⁺NK1.1^-, respectively, (left panel) and representative staining of IFNγ production by NKT cells (right panel) (C) Frequency of splenic CD3⁺ NK1.1^- T cells and the percentage of these ones producing IFNγ.

Fig 5. Iron chelation impairs CD4⁺ T cell activation and proliferation in vitro.

(A) Percentages of CD3⁺CD4⁺ cells from splenocytes undergoing in at least one division are represented with representative fluorescent histogram in the lower part. Splenocytes were incubated 96 hours in presence of ConA (0.1mg/mL) and in presence of low doses of iron chelator (HPO CP182, 5μM or DFO, 10μM). (B) Percentages of CD3⁺CD4⁺ cells from isolated naive CD4⁺ cells undergoing in at least one division and (C) CD25 MFI are represented are displayed with representative fluorescent histogram in the lower part. Isolated CD4⁺ naïve T cells incubated 5 days in presence of anti-CD3/CD28 plate-bound antibody (2μg each). Un means unstimulated cells.

Fig 6. The inhibitory effects of iron deficiency in ConA-induced hepatitis are independent from changes in gut microbiome and hepcidin levels.

(A) ALT serum levels (IU/mL) before, and 12 hours after the administration of ConA to mice fed for 3 weeks with an iron deficient (IrDef) or iron replete (IrRepl) diet, in the presence or absence of a 4-antibiotic (Atb) cocktail. (B) Relative expression of Hamp in liver tissue samples from mice receiving a 3 day-course of HPO CP28 (20 nmoles, daily) compared to control mice receiving PBS (p = 0.48). (C) ALT serum levels 12 hours after ConA challenge in mice treated with HPO CP28 or with PBS (p = 0.041). (D) ALT serum levels 12 hours after ConA challenge in mice pre-treated two hours before with a single intraperitoneal injection of 100μg of mouse hepcidin or sterile PBS (p = 0.84). Bar plots display mean and SEM.