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Nutritional Immunology
Research Communication

Iron-Deficient Mice Fail to Develop Autoimmune Encephalomyelitis

Department of Nutrition, The Pennsylvania State University, 126 South Henderson, University Park, PA 16802

²To whom correspondence should be addressed. E-mail: mxc69{at}psu.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Determinations of the effects of iron status on the immune system are complicated by the fact that microorganisms and immune cells both utilize iron. To determine the role of iron in immune function, we utilized a model [experimental autoimmune encephalomyelitis (EAE)] in which a strong antigen-specific CD4+ T-cell response develops in the absence of infection. EAE is an autoimmune disease frequently used as a model for the human disease multiple sclerosis (MS). EAE was induced in B10.PL mice fed low iron (1 mg/kg), normal iron (10 mg/kg) or high iron (160 mg/kg) diets that were replete in all other nutrients. Liver iron measurements verified iron status, i.e., low iron mice had 1.9 µmol/g tissue, normal iron mice, 3.27 µmol/g tissue and high iron mice, 5.35 µmol/g tissue. EAE symptoms were most severe in normal iron mice, and EAE did not develop in low iron mice. The incidence of EAE was 71% in normal iron mice, 62% in iron-overloaded mice and 0% in iron-deficient mice. Two of seven mice in the normal iron group developed severe EAE and were euthanized. None of the iron-overloaded mice developed severe EAE. Other measures of EAE severity were similar in the normal and iron-overloaded mice. The data suggest that iron deficiency provides protection from the development of EAE and that iron excess with its potential contribution to free radical formation was not an important factor. The mechanism of EAE inhibition in iron-deficient mice likely involves the delivery and metabolism of iron for optimal CD4+ T-cell development.

KEY WORDS: • iron • experimental autoimmune encephalomyelitis • multiple sclerosis • mice

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). One of the most useful models of this disease is murine experimental autoimmune encephalomyelitis (EAE). EAE is an in vivo test of CD4+ T-cell development and function. CD4+ T-cell depletion results in the failure of EAE to develop (1). Furthermore, if CD4+ T cells are transferred from a paralyzed mouse to a naïve mouse, all of the symptoms of EAE develop (1). In particular, transferring T-helper 1 cells [responsible for interleukin (IL)-2, INF- and tumor necrosis factor (TNF)- production], which recognize CNS proteins from paralyzed mice to naïve mice causes EAE to develop (1,2). The EAE model presents the opportunity to look at the development of a strong, CD4-driven immune response in the absence of any infection.

The effects of iron deficiency and iron overload on immune function include alterations in cell-mediated, antibody-mediated and innate immunity [reviewed by Weis (3)]. Iron is a crucial element in many metabolic pathways. Studies of the effects of iron on immunity are complicated by the reciprocal regulation of iron metabolism by the immune system. In fact, chronic inflammation has been shown to result in anemia, presumably due to immune-induced alterations of iron usage (4). Although anemia has been shown to occur in people with chronic inflammation, including autoimmune diseases, the effect of iron deficiency anemia on the autoimmune response and severity of disease has not been studied (3,4).

Iron deficiency anemia and HIV infection are major public health problems in developing countries. Iron deficiency has been associated with decreased immunity and higher morbidity from infectious diseases (5–8). Conversely, iron supplementation has been shown to increase progression and mortality in HIV-infected people (9). The increased rate of mortality after iron supplementation of HIV positive individuals is likely a result of the increased severity of opportunistic infections (9). Prokaryotic as well as eukaryotic cells require iron for their growth and function (9). Therefore, iron availability affects the balance between the growth of microbial pathogens and the effectiveness of the immune response. A number of human pathogens including bacteria, protozoa and fungi have been shown to use iron as a growth factor (9). It is unclear what effect iron supplementation (especially in individuals with normal iron status) has on immune function. The role of iron status in immune function is unclear.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Mice.

Male B10.PL mice (n = 24) were obtained from the Jackson Laboratory (Bar Harbor, ME) at 4 wk of age. Upon arrival, the mice were divided into three experimental groups (n = 8/group). At the time of immunization, the mice were 10 wk old. The protocols used were approved by The Pennsylvania State University Animal Care and Use Committee, Protocol #A314 41–01.

Diets.

All mice were fed synthetic diets made in the laboratory (10–12). Diets were fed immediately upon arrival. Before and during the experiment, the mice were fed a diet formulated to be identical for each experimental group except for the iron content. The three experimental groups were designated low iron (1 mg ferrous sulfate/kg diet), normal iron (10 mg ferrous sulfate/kg diet) and high iron (160 mg ferrous sulfate/kg diet).

Hematology and iron analysis.

At 42 d postimmunization, the mice were killed by CO₂ asphyxiation. Hemoglobin was measured colorimetrically by the cyanmethemoglobin method (Sigma-Aldrich, St. Louis, MO) from blood collected by heart puncture via a heparinized syringe. Hematocrit was determined by centrifugation of blood collected into heparinized microcapillary tubes (VWR, West Chester, PA). Plasma was collected by centrifugation at 10,000 x g for 10 min, then frozen at -70°C until it was analyzed for serum iron and total iron-binding capacity (TIBC) by established procedures (13). Transferrin saturation was calculated as serum iron/TIBC x 100. Liver nonheme iron was determined by the standard colorimetric technique described by Bothwell et al. (14). Total iron content of whole-brain homogenates was determined according to our standard laboratory method using acid digestion and analysis with atomic absorption spectrophotometry (15,16). To confirm iron status of the mice before immunization, blood was collected from the tail of each mouse for hemoglobin and hematocrit determinations.

EAE disease induction.

The mice were used for experiments at 10 wk of age or after consuming the diets for 6 wk. Myelin basic protein (MBP) was isolated, purified and the EAE immunizations were done exactly as described (17,18). Briefly, the MBP was dissolved in 0.1 mol/L acetic acid at a concentration of 8 g/L. Mice were immunized subcutaneously with 400 µg of MBP emulsified in an equal volume of complete Freund’s adjuvant (total volume 0.1 mL) (Difco Laboratories, Detroit, MI) containing Mycobacterium tuberculosis H37 Ra. Additionally, the mice were given an intraperitoneal injection of 200 ng of pertussis toxin (LIST Biological Laboratories, Campbell, CA) resuspended in sterile saline on the day of immunization and 2 d after immunization. The mice were scored daily by three independent observers who were unaware of treatment using the EAE scoring system: 0 = normal; 1 = limp tail; 2 = paraparesis with a clumsy gait; 3 = hind limb paralysis; 4 = hind and forelimb paralysis; 5 = moribund. Because the EAE score of 1 or a limp tail is difficult to determine unambiguously, only mice with an EAE severity score 2 were included in the incidence values. In addition, the day of EAE onset was determined on the basis of the first day a mouse developed an EAE score 2. Mice that developed severe EAE (score of 5) would not have survived and were euthanized at this time. At 42 d postimmunization, the remaining mice were killed by CO₂ asphyxiation. Blood was collected by heart puncture via a heparinized syringe for iron analyses.

Cell cultures and cytokine ELISA.

At 42 d post-EAE induction, all of the remaining mice were killed. Axillary, brachial and inguinal lymph nodes (LN) were obtained from all mice. The LN were collected and then disrupted manually using a pair of forceps and a 21-gauge needle to obtain single-cell suspensions. Two experiments were done using cells from groups of 3–4 mice each. The final concentration of LN cells was 10¹⁰ cells/L. Cell culture medium was RPMI-1640 (Sigma) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 2 mmol/L L-glutamine (Sigma), 0.05 mmol/L ß2-mercaptoethanol (Sigma), 4 g/L sodium bicarbonate (EA Science, Gibbstown, NJ). Cells were cultured in RPMI-C with 50 mg/L MBP. Supernatants were collected 48 h later for cytokine analysis by ELISA using the kits from PharMingen (San Diego, CA). ELISA were used to determine the levels of IL-2, IL-4, IL-5, INF- and TNF-. The limits of detection were IL-2, 800 µg/L; IL-4, 2000 µg/L; IL-5, 4000 µg/L; INF-, 2000 µg/L; and TNF-, 5200 µg/L.

Statistics.

Groups of age-matched male mice (n = 7–8) were used for each experiment. Data were subjected to one-way ANOVA with diet as the between-group variable. Type three sums of squares was used for the ANOVA of Fig. 2A where the variances were greatly unequal. The Students Newman-Keuls multiple comparison analyses were used to analyze the differences in the means of values from the three diet groups. A two-sample test for binomial proportions was used for analysis of the proportions shown in Table 2. Data were analyzed using StatView (SAS Institute, Cary, NC).

View larger version (22K): [in this window] [in a new window]   FIGURE 2 Myelin basic protein (MBP)-specific IL-2 (A) and INF- (B) secretion in cells from mice induced to develop experimental autoimmune encephalomyelitis (EAE) and fed low, normal or high iron diets. Values are means ± SD from n = 3–4 mice. One representative of two experiments is shown. Bars with different letters differ, P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The body weights of mice fed low, normal or high iron diets were not different before, during or at the end of the experiment (data not shown). Consumption of the low iron diet for 4 wk significantly decreased hemoglobin concentration (137 ± 4 g/L, low; 167 ± 5 g/L, normal; and 171 ± 2 g/L high) and the hematocrit (0.46 ± 0.01, low; 0.50 ± 0.02, normal; and 0.53 ± 0.02, high) compared with both the normal and high iron groups. After 12 wk, mice fed low iron diets had significantly lower hematocrit, serum iron, percentage transferrin saturation and higher TIBC than did the normal and high iron mice (Table 1). Liver nonheme iron concentrations differed among the groups, verifying iron status at the end of the experiment (Fig. 1A). Brain iron concentrations from the normal and high iron groups did not differ but both were significantly greater than in the low iron group (Fig. 1B).

View larger version (19K): [in this window] [in a new window]   FIGURE 1 Liver and brain iron concentrations in mice fed low, normal or high iron diets for 12 wk: (A) liver iron concentrations and (B) brain iron concentrations. Values are means ± SEM, n = 7–8. Bars with different letters differ, P < 0.05.

Immunization with MBP resulted in 0, 71 and 62% incidences of EAE in the low, normal and high iron diet groups, respectively (Table 2). Low iron mice did not develop symptoms of EAE. Severe EAE (peak score of 5) developed in two of the normal iron mice (Table 2). The EAE severity was so great in these mice that they were euthanized before the end of the experiment. High iron mice developed an intermediate level of EAE with a peak severity of 3 (data not shown and Table 2). The day of EAE onset was determined for each mouse that developed an EAE severity score of 2 (Table 2). The day of EAE onset did not differ between normal and high iron groups and was much later in the low iron group (Table 2).

Immune response.

MBP-specific cytokine secretion was measured in cultures from the lymphocytes of low, normal and high iron mice 42 d after immunization. The IL-4, IL-5 and TNF- concentrations were below the level of detection in all samples. Lymphocytes from low iron mice secreted significantly more IL-2 than lymphocytes from normal or high iron mice, which did not differ from each other (Fig. 2A). In contrast, INF- secretion was lower in the high iron group than in the other two groups, which did not differ (Fig. 2B).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
EAE did not develop in iron-deficient mice. This suggests a profound impairment in the development of CD4+ T cells in iron deficiency. Perhaps the high prevalence of low iron status (and other nutritional deficiencies) explains why autoimmune diseases such as MS have such a low incidence in developing countries. The absence of autoimmunity in the mice fed low iron diets suggests that all immune responses, which depend on CD4+ T cell induction, would also be compromised during iron deficiency. This includes infectious immunity, CD4+ T-cell function in HIV-positive/AIDS-negative individuals and the response of a person to a vaccine. In fact, a small study in institutionalized elderly subjects showed that individuals with low iron levels were significantly less responsive to an influenza vaccine (19). The development of the CD4+ T-cell response is impaired during iron deficiency.

Iron chelation therapy with desferrioxamine reduced the cellular iron status of SJL mice with EAE and also reduced the clinical severity of symptoms (20). The authors suggested that iron chelation removed free iron radicals, which in turn decreased reactive oxygen species and damage in the CNS (20). Our data offer an alternative explanation for the result. Iron chelation would decrease the iron available to generate CD4+ T cells. Our data point to a crucial role of iron in the development and function of CD4+ T cells, which cause paralysis and symptoms of EAE to develop. In the absence of adequate iron, the T cells would not mature, find their way to the CNS and induce symptoms of EAE. Of course, it is possible that both free radicals and CD4+ T-cell function are reduced when iron is low.

EAE was of intermediate severity in mice fed excess iron. The differences between normal iron and high iron mice were of a more subtle nature than those between either group and the low iron group. The only marker of iron status that differed between the normal iron and high iron groups was the liver concentrations of iron, which showed that mice fed high amounts of iron had more liver iron than mice fed less iron. The mice fed high iron diets had less severe EAE than the normal iron group. The maximum EAE severity score was 3 in the high iron group vs. 5 in the normal iron group. Similarly, 29% of the normal iron mice developed severe EAE and were euthanized, whereas none of the high iron mice developed severe EAE. The data suggest that high levels of iron were associated with a reduction in the induction of CD4+ T cells.

Lymphocytes from iron-deficient mice produced high levels of antigen-specific IL-2 compared with lymphocytes from normal iron or high iron mice. IL-2 production was measured 42 d after immunization with MBP and was associated with the failure of iron-deficient mice to develop EAE. IL-2 is a growth factor for T cells and in IL-2 deficient knockout (KO) mice, autoimmune disease develops (21). The autoimmune disease in IL-2 KO mice is due to an absence of regulatory T cells characterized by the presence of CD25 or the IL-2 receptor (21). Iron-deficient mice may have a greater frequency of CD25+ T cells, which is associated with the protection of these mice from EAE. Future research should investigate the role of iron in the development of CD25+ regulatory T cells.

The INF- response was significantly lower in lymphocytes from mice consuming high iron compared with the other two groups. Lymphocytes from all three groups of mice secreted high amounts of INF-. There does not appear to be an obvious association between the INF- response and the severity of EAE in these mice. Perhaps the timing of the analyses for INF- was not optimal. Normal iron mice developed severe EAE; mice with severe EAE (score of 5) would not survive and were euthanized immediately. In addition, this model of EAE is characterized by periods of paralysis and then partial recovery. By 42 d postimmunization, the EAE disease severity had peaked and the mice had partially recovered. In fact, all of the remaining mice had only mild EAE at this time point (range of severity scores 0–2). The mice were kept for 42 d to determine whether the iron-deficient group would eventually develop EAE, but they did not.

The failure of iron-deficient mice to develop EAE is impressive. Many of the pharmaceutical approaches to inhibiting EAE are less effective than iron deficiency. Of course, iron deficiency anemia and the associated morbidity preclude the translation of this research to humans. The data in EAE established that in the absence of infection, iron status has dramatic effects on CD4+ T-cell function. The high iron mice developed an intermediate form of EAE, more severe than low iron fed mice and less severe than normal iron mice. The differences between normal and high iron mice in measures of iron status as well as EAE severity were of a more subtle nature. The data suggest that both low and high levels of dietary iron impair CD4+ T-cell function. In the absence of infection, normal iron status is required for optimal CD4+ T-cell function.

	FOOTNOTES

 
¹ Funded by the National Sciences Foundation, Research Experiences for Undergraduates, DBI-0097697.

³ Abbreviations used: CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; KO, knockout; LN, lymph node; MBP, myelin basic protein; MS, multiple sclerosis; TIBC, total iron-binding capacity; TNF, tumor necrosis factor.

Manuscript received 7 March 2003. Initial review completed 24 April 2003. Revision accepted 19 May 2003.

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