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© 2008 American Society for Nutrition J. Nutr. 138:849-855, May 2008

Nutritional Immunology

Copper Deficiency Increases the Virulence of Amyocarditic and Myocarditic Strains of Coxsackievirus B3 in Mice1

Allen D. Smith*, Sebastian Botero and Orville A. Levander

Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, USDA, Beltsville, MD 20705

* To whom correspondence should be addressed. E-mail: allen.smith{at}ars.usda.gov.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Methods
 Results
 Discussion
 LITERATURE CITED
 
Deficiency in several trace elements, including copper and selenium, is associated with increased levels of oxidative stress. Copper deficiency also has been shown to impair immune function. Previous work by others demonstrated that passage of an amyocarditic or myocarditic strain of coxsackievirus B3 (CVB3) through selenium- or vitamin E-deficient mice led to increased cardiac pathology. To determine whether a copper deficiency would similarly alter the pathogenesis of CVB3 infections, Swiss outbred dams and their litters were fed copper-deficient diets from birth and received either deionized water or water with 0.315 mmol/L copper as copper sulfate. At 4 wk of age, copper-adequate or -deficient male and female offspring were infected with an amyocarditic or myocarditic strain of CVB3. Heart titers were elevated at d 3 and 7 postinfection in copper-deficient mice infected with the myocarditic CVB3 strain (CVB3/20) but only at d 7 in deficient mice infected with the amyocarditic CVB3 strain (CVB3/0) compared with copper-adequate controls. Copper-deficient mice infected with either strain of CVB3 had increased cardiac pathology compared with copper-adequate controls. Genomic sequences of viruses isolated from copper-adequate and -deficient mice were identical. Heart cytokine expression was elevated in copper-deficient CVB3-infected mice compared with infected controls. Circulating CVB3-specific IgG2a but not IgM levels were decreased in copper-deficient mice. Thus, copper deficiency is associated with an increased inflammatory response but decreased acquired immune response to CVB3 infection that results in increased cardiac pathology, presumably due to increased viral load.


   Introduction
TOP
 ABSTRACT
 Introduction
Methods
 Results
 Discussion
 LITERATURE CITED
 
Copper deficiency is associated with increased oxidative stress in both in vivo and in vitro studies. Increased superoxide concentrations were detected in copper-deficient embryos (1). Copper deficiency has been reported to increase lipid peroxidation (2), and early and advanced glycation end products have been found in copper-deficient rats (3). The expression of an enzyme upregulated by oxidative stress, heme oxygenase-1, is increased by copper deficiency (4), and mitochondria from copper-deficient rats have increased mitochondrial hydrogen peroxide formation (5). Protein carbonyls, an indicator of oxidative modification, were found in mitochondria from copper-deficient HL-60 cells (6) and in erythrocyte membrane proteins (7).

Copper deficiency has also been shown to affect immune function. Copper deficiency is known to cause neutropenia (8) and impair the respiratory burst of neutrophils and macrophages (9,10). Impaired phagocytic activity in copper-deficient rats infected with Salmonella typhymurium was associated with higher mortality and decreased survival times (11). NK cell-killing activity is also impaired by copper deficiency (8).

Copper deficiency affects acquired immunity in addition to innate immunity. Copper-deficient mice produced fewer splenic antibody-producing cells in response to sheep red blood cells (12) and copper-deficient rats have decreased serum antibody titers to sheep red blood cells (13). Copper deficiency alters splenic lymphoid subsets, including CD4+ cells (13,14) and their proliferative response to mitogens (15,16), which can be restored by copper repletion (17). The T-cell defects appear to be due to decreased interleukin (IL)2 mRNA and protein production (15,18,19). Thus, copper deficiency has been demonstrated to alter both innate and acquired immunity.

Deficiencies in other micronutrients, including selenium and vitamin E, are also associated with increased oxidative stress (20–24) and alterations in immune function (5,25–29). Passage of coxsackievirus B3 (CVB3)2 or influenza virus through selenium-deficient mice resulted in increased pathology that was associated with specific changes to the viral genomes (30–32). It was proposed that oxidative stress was responsible for the selection of more virulent strains of virus. Therefore, we were interested in determining if a deficiency in copper would also lead to increased pathology in mice infected with CVB3 and if there was a common mechanism induced by increased oxidative stress.


   Methods
TOP
 ABSTRACT
 Introduction
 Methods
Results
 Discussion
 LITERATURE CITED
 
    Mice. Late-term pregnant female Swiss mice were obtained from the NCI-Frederick Animal Production Area and housed individually in micro-isolator cages with free access to food and deionized water. All dams received a modification of the AIN-76A-based copper-deficient diet (Harland Teklad) as described in Prohaska (33) that contained 0.2 mg/kg diet of selenium, 50 IU/kg diet of vitamin E, [DL-{alpha} tocopheryl acetate (500 IU/g) 0.1 g/kg], and water containing 0.315 mmol/L (20 mg/L) copper as copper sulfate. The copper-deficient diet prepared as described contained ~0.5 mg/kg diet of copper by analysis (34). Upon giving birth, one-half of the dams and their litter continued receiving copper-supplemented water while the other one-half were given only deionized water. Mice continued to receive their respective treatments for the remainder of the experiment. At 4–5 wk of age, mice of both sexes were infected with CVB3 as described below. All animal experiments were approved by the Beltsville Area Animal Care and Use Committee.

    Liver copper determination. To determine whether the dietary regimen produced a copper deficiency, liver samples from copper-adequate and copper-deficient mice of both sexes were processed as previously described (35). Copper concentrations were determined by atomic absorption spectroscopy and were expressed as µg copper/g wet liver.3

    Infection and sample collection. At 4–5 wk of age, male and female mice were infected by intraperitoneal injection with either 105 tissue culture infectious dose 50 (TCID50) of an amyocarditic strain, CVB3/0 (36), or a myocarditic strain, CVB3/20 (37) of virus. On d 3 and 7 postinfection, mice were killed and various organs aseptically removed. Organs were divided into at least 2 pieces, one of which was frozen for determination of viral load. Another piece was fixed in 10% formalin for histological analysis or snap frozen in liquid N2 for gene expression studies. Similar sections from each organ were taken for each method of analysis.

    Measurement of heart cytokine expression. RNA was extracted from the heart using Trizol reagent (Invitrogen) according to the manufacturer's recommendations (38). The methods for cDNA synthesis, determination of RNA integrity, and real-time PCR conditions were as previously described (39). Then 50 ng cDNA/reaction was used for PCR amplification and amplification [threshold cycle (Ct)] was measured on an iCycler Real-Time PCR Detection system (Bio-Rad). To evaluate the effects of treatment, the mean of Ctcontrol was subtracted from the mean of Cttreatment. This value is defined as {Delta}Ct. The relative fold-increase or -decrease was then calculated as 2–{Delta}Ct (40). Data are reported as the mean fold-change in gene expression ± SEM normalized to gene expression levels in copper-adequate uninfected mice.

    Determination of viral heart titers and pathology. Tissues were processed to determine viral load as previously described (41) and data are expressed as the TCID50 per g of tissue. Titers reported in the tables and figures are the means and SEM of log-transformed data and include estimated titers for samples that were below the measurable limit of the assay used. Estimated titers are calculated titers that represent the lowest detectable titer for each sample due to inherent limitations of the assay and were included in the data for statistical purposes. Formalin-fixed tissues from d 7 postinfection were processed for hematoxylin and eosin staining. Sections were evaluated semiquantitatively (scale of 0–4.0, with 4.0 being the most severe pathology) for the relative degree (from heart to heart) of tissue necrosis and cellular infiltration. We combined pathology scores from 2 separate experiments with each virus.

    Virus passage experiments. Virus obtained from copper-adequate or copper-deficient mouse hearts was passed 1 time through HeLa-H1 cells to obtain sufficient quantities of virus for sequencing and for inoculation into copper-adequate mice. Mice were given 105 TCID50 of individual virus isolates. On d 7 postinfection, the mice were killed, and one-half of each heart was saved for determining the viral load and the other one-half was fixed in buffered formalin for subsequent processing and hematoxylin and eosin staining.

    Sequencing of the viral genome. Viral RNA from individual isolates was obtained by extraction with Trizol following the manufacturer's recommendations (Invitrogen) and reverse transcribed with Superscript II kit and random primers. Six sets of primers comprising ~40% of the total viral genome were used for PCR amplification and the resulting PCR products were separated by gel electrophoresis, purified using the QIAGEN PCR purification kit, sequenced using the BigDye Terminator mix V3.1 (Applied Biosystems), purified with Edge Biosystem Performa columns, and analyzed on a 3100 Applied Biosystems DNA sequencer. The sequences obtained were compared with published sequences for CVB3/0 and CVB3/20.

    Statistical analysis. Unless otherwise indicated, data obtained from male and female mice were combined for all variables tested. For viral titer data, only within-day comparisons were made using a t test. Gene expression data were analyzed using ANOVA with a post hoc multiple comparisons analysis using SigmaStat program (SPSS). Data were transformed as necessary to pass the normality and equal variance tests for statistical analysis or were analyzed using the Sheffé's F test using StatView (SAS Institute) or an ANOVA was performed on ranks. A P-value of <0.05 was considered significant. Values in the text are means ± SEM.


   Results
TOP
 ABSTRACT
 Introduction
 Methods
 Results
Discussion
 LITERATURE CITED
 
    Indices of copper status. Mice born from dams that did not receive copper postpartum had less liver copper (1.55 ± 0.06 µg/g) than mice from dams receiving copper in their drinking water (5.26 ± 0.14 µg/g) (P < 0.001). The liver copper concentrations for adequate and deficient mice are similar to previously published values (42,43) using the same experimental conditions. Similarly, previous work has demonstrated that the dietary regimen employed herein that reduces liver copper concentrations also altered biochemical indicators of copper deficiency in the heart, including decreased cytochrome c oxidase activity, increased heart weight (33), and reduced copper levels in the heart (44,45). Heart weight was greater in copper-deficient mice (162 ± 11 mg) than in controls (100 ± 2 mg).

    Heart viral titers. We determined the viral load in hearts from copper-adequate and -deficient mice infected either with CVB3/0 or CVB3/20. Heart viral titers in copper-deficient mice infected with the myocarditic strain, CVB3/20, were elevated on d 3 (P < 0.05) and 7 postinfection (P < 0.001; Fig. 1A). Heart viral titers also were elevated at d 7 (P < 0.001) postinfection in mice infected with the amyocarditic strain, CVB3/0 (Fig. 1B) and in 1 of 2 experiments on d 3. In addition, whereas all copper-deficient mice infected with CVB3/0 had measurable titers, a large percentage of copper-adequate mice had heart titers at or below the level of detection at d 7, indicating that clearance of CVB3/0 was being delayed in the copper-deficient mice.


Figure 1
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  		FIGURE 1  Heart viral titers are elevated in copper-deficient mice infected with a myocarditic (CVB3/20, A) or amyocarditic (CVB3/0, B) strain of CVB3. Copper-adequate or -deficient mice infected with either CVB3/0 or CVB3/20 were killed 3 or 7 d postinfection. Panel A data were generated from hearts of male CVB3/20-infected mice and panel B from hearts of male and female CVB3/0-infected mice. Values are means ± SEM, n = 3-12. Data from d 3 and 7 were analyzed separately. Shown in parentheses in panel B is the fraction of mice with a measurable titer at d 7. Mice in all other groups had measurable titers. *Different from infected copper-adequate mice killed on that day, P < 0.05.

 
    Effect of copper deficiency on anti-CVB3 antibody production. CVB3-specific IgM and IgG2a (associated with a Th1 response) antibody production was measured by ELISA. CVB3-specific IgM production was unaffected by copper deficiency (data not shown). In sharp contrast, there was a large decrease in CVB3-specific Ig2a production at 7 d postinfection (Fig. 2).


Figure 2
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  		FIGURE 2  Copper deficiency decreases the CVB3-specific serum IgG2a antibody response. Copper-adequate or -deficient mice infected with CVB3 were killed 7 d postinfection and blood was collected. The resulting serum was tested in an ELISA using purified CVB3 as antigen- and class-specific secondary antibodies. Data are means ± SD, n = 4-5. *Different from infected copper-adequate mice killed on that day, P < 0.05.

 
    Effect of copper deficiency on CVB3-induced heart pathology. CVB3/20 is a myocarditic strain of CVB3 that normally induces lesions in the heart characterized by destruction of myocytes, a cellular infiltrate, and calcification. The amyocarditic strain, CVB3/0, typically causes few if any lesions in the heart. Upon necropsy, white lesions were noted on hearts from copper-deficient mice infected with CVB3/20. Hematoxylin and eosin-stained heart sections from both copper-adequate and -deficient mice infected with either CVB3/20 or CVB3/0 were evaluated for the extent of virus-induced cardiac pathology (scale 0–4). Cardiac pathology was greater in copper-deficient CVB3/20-infected mice (3.2 ± 0.2) than in infected controls (0.9 ± 0.1) (P < 0.001) and in copper-deficient CVB3/0-infected mice (1.0 ± 0.2) than in infected controls (0.07 ± 0.05) (P < 0.001). CVB3/20 produced discrete lesions in limited regions of the heart in copper-adequate mice (Fig. 3F), but the lesions were larger, more necrotic, and involved much larger regions of the heart in copper-deficient mice (Fig. 3G,H). Although the lesions were much smaller and fewer in the CVB3/0-infected copper-deficient mice (Fig. 3D,E) compared with CVB3/20-infected mice, copper-adequate mice infected with CVB3/0 typically did not have any cardiac lesions (Fig. 3C). To determine whether the increased pathology was due to changes in the virus rather than copper-induced alterations in the host response, virus isolates obtained from hearts of copper-adequate or -deficient mice infected with either CVB3/0 or CVB3/20 were used to infect copper-adequate mice. No differences were observed in the heart viral titers or the degree of cardiac pathology in mice receiving virus from copper-deficient mice vs. copper-adequate mice (data not shown). This result suggested that the virus had not changed as a result of growth in copper-deficient mice.


Figure 3
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  		FIGURE 3  Photomicrographs of CVB3-induced cardiac pathology in copper-adequate or -deficient mice. Hearts from copper-adequate or -deficient mice infected with CVB3 were killed 7 d postinfection, fixed in neutral buffered formalin, and paraffin embedded. Five-micrometer sections were cut and stained with hematoxylin and eosin. Shown are representative sections from each group. (A) Uninfected copper deficient; (B) uninfected copper adequate; (C) CVB3/0-infected copper adequate; (D) CVB3/0-infected copper deficient; (E) CVB3/0-infected copper-deficient mouse showing more severe pathology; (F) CVB3/20-infected copper adequate; (G) CVB3/20-infected copper deficient; (H) CVB3/20-infected copper-deficient mouse showing more severe pathology that is present throughout the heart. Arrows point to areas of high cellular infiltrate. Arrowheads point to areas of necrosis. All photomicrographs are at 40 times magnification.

 
To confirm this observation, the original CVB3/0 stock and virus isolated from hearts of 3-4 copper deficient or copper adequate mice infected with CVB3/0 had specific regions of their viral genome sequenced. The viral genomic regions chosen for sequencing had previously been shown to undergo mutation in response to host selenium or vitamin E deficiency. Two changes were observed in the sequence of the CVB3/0 stock used to inoculate the mice compared with the published CVB3/0 sequence (36) (nucleotide 598, T/C and nucleotide 830, G/A). In addition, nucleotide 234 changed from a C to a T in virus isolated from hearts of both copper-adequate and -deficient mice, indicating that this change was not diet related but due to some other factor (Table 1). This contrasts with the 6 nucleotide changes found in CVB3/0 isolates that were obtained from selenium- or vitamin E-deficient mice (30,31), of which only position 234 was changed in copper-adequate and -deficient mice. We have observed that the change at position 234 from a C to a T occurs easily upon passing CVB3/0 through adult mice (A. D. Smith, unpublished data) and this was also seen by Gay et al. (46). Gay et al. (46) also identified several other positions within the CVB3/0 sequence that changed in virus isolated from hearts of old mice but not young adult mice. Two of these changes were the previously noted changes at nucleotide positions 598 (T/C) and 830 (G/A) that were present in our CVB3/0 stock. None of the other nucleotide changes observed in aged mice (46) were seen in virus isolated from copper-adequate or -deficient mice. Thus, copper deficiency does not appear to induce the same type of genomic changes as those observed with selenium- or vitamin E-deficient or aged mice.


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  		TABLE 1 Nucleotide comparison between CVB3/0, CVB3/20, and CVB3/0 isolated from hearts of copper-adequate (Cu ad) or -deficient mice (Cu def)

 
    Effect of copper deficiency on cytokine mRNA expression in heart tissue. The effect of copper deficiency on cytokine expression in the heart was evaluated by real-time PCR. Cytokine expression in the heart of uninfected copper-deficient mice did not change compared with uninfected copper-adequate mice (data not shown). Expression of the housekeeping gene RPL32 was not affected by diet or infection. Cytokines associated with a Th1 immune response, interferon (IFN){gamma} and IL-12p40, were upregulated by CVB3/20 infection in both copper-adequate and -deficient mice (Table 2) compared with uninfected copper-adequate mice. The expression of these cytokines in CVB3/20-infected copper-deficient mice was significantly higher than observed in CVB3/20-infected copper-adequate mice. In addition, the expression of tumor necrosis factor (TNF)-{alpha}, IL-6, IL-2, FoxP3, and IL-13 were also higher in CVB3/20-infected copper-deficient compared with infected copper-adequate mice and there were significant diet x infection interactions for a number of cytokines (Table 2). In CVB3/0-infected mice, IFN{gamma} and IL-12p40 were upregulated only in copper-deficient mice, whereas IL-10 expression was upregulated in both copper-deficient and -adequate mice.


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  		TABLE 2 Copper deficiency alters gene expression levels in mice infected with CVB3/0 or CVB3/20 at d 7 postinfection1

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Methods
 Results
 Discussion
LITERATURE CITED
 
Here, we report that copper deficiency increases the virulence of both amyocarditic and myocarditic strains of CVB3 in mice but is not associated with changes in the viral genome that were previously reported for CVB3/0-infected selenium- or vitamin E-deficient mice (30). Although 3 nucleotide changes from the published CVB3/0 sequence were observed, 2 of these (positions 598 and 830) were present in the CVB3/0 viral stock used to inoculate the mice and the 3rd (position 234) was observed in viral isolates from both copper-adequate and -deficient mice, indicating that the change was not caused by copper deficiency. Therefore, passage of CVB3 through copper-deficient mice does not result in the appearance of an altered and more virulent CVB3 strain as was observed in selenium- or vitamin E-deficient, or aged, mice.

The reasons for the lack of changes to the viral genome with copper deficiency, considering the fact that copper, selenium, and vitamin E deficiency all result in increased oxidative stress, are unclear. The mechanism for accumulation of mutations in CVB3 with selenium or vitamin E deficiency is unknown but has been proposed to be the result of oxidative stress exerting a selective force that results in the appearance of a "new" virus either by selecting a preexisting quasi-species variant alone or in combination with an increased viral mutation rate. One possibility for the lack of appearance of mutant viruses in this study is that selenium and copper deficiency cause oxidative stress in different cellular compartments. Copper deficiency decreases the activity of the mitochondrial enzyme cytochrome-c oxidase (5,33,47), increased oxidative stress in mitochondria of HL-60 cells (6), and increases heme oxygenase-1 expression and mitochondrial hydrogen peroxide formation (4). Selenium deficiency causes a dramatic decrease in cytoplasmic glutathione peroxidase, the activity of which was unchanged in HL-60 cells grown in a low-copper medium (6) or in copper-deficient rat heart (33). CVB3 replication is known to occur in the cytoplasm [reviewed in (48)], thus raising the possibility that oxidative stress produced in the cytoplasm may have a greater impact on the accumulation of mutations in CVB3 than oxidative stress generated in other cellular compartments. However, these results do suggest that copper deficiency affects the host's response to the viral infection rather than changes to the virus.

The increased pathology observed in copper-deficient CVB3-infected mice was associated with increased viral load in the hearts of copper-deficient mice. We have observed increased CVB3-induced cardiac pathology associated with increased viral replication in the heart with aurothiomalate- or mercury chloride-treated mice (41,49). Others also have reported that cardiac pathology is associated with increased heart viral replication (50–53). Increased viral load in the heart indicates that the copper-deficient mice were not able to control viral replication as efficiently as copper-adequate mice.

The increased viral replication and heart pathology were associated with increased cytokine production. Our results are consistent with previous studies demonstrating cytokine expression, including IFN{gamma}, IL-1β, IL-2, IL-6, IL-10, and TNF{alpha} in cardiac tissue from CVB3-infected mice (54–56). We also show here that FoxP3 expression is upregulated by CVB3 infection. Whereas cytokines associated with a Th1 response were elevated in both copper-adequate and -deficient mice, the expression in CVB3/20 infected copper deficient mice were markedly higher, as was the heart pathology. The proinflammatory cytokine, TNF{alpha}, was only upregulated in CVB3/20-infected copper-deficient mice and likely reflects the robust inflammatory response in hearts from copper-deficient mice. Cytokine expression in hearts of the amyocarditic strain CVB3/0 was not as high and the number of heart lesions was lower in CVB3/0-infected copper-deficient mice. These results suggest that elevated cytokine levels correlate with cardiac damage and cellular infiltration in hearts from CVB3-infected mice.

Leipner et al. (56) showed that expression of TNF{alpha}, IL-6, IL-10, and IL-12p40 increased as soon as viral RNA became detectable in the heart, indicating that these cytokines are being produced by cardiac myocytes, whereas IFN{gamma} and IL-2 expression correlated with the onset of inflammatory cell infiltration. Therefore, increased IFN{gamma} and IL-2 expression in CVB3/20-infected copper-deficient mice greater than that in copper-adequate mice may reflect the increased inflammatory response present in the deficient mice, whereas increased expression of the other cytokines may reflect the increased viral replication in copper-deficient mice. Furthermore, although cytokine expression early in infection may be important for coordinating a response, evidence suggests that continued cytokine stimulation, especially by TNF{alpha}, can lead to continued immunological stimulation and increased tissue damage (57–59).

Copper deficiency has been shown to impair T-cell–mediated immunity [reviewed in (60)] and antigen-specific antibody production (8,12,13). In agreement with these results, we also found that antigen-specific IgG2a antibody production was significantly inhibited by copper deficiency. Antigen-specific IgM production, which is not T-cell or IL-2 dependent, was not affected by copper deficiency. Previous work has demonstrated that CVB3-specific antibody production was necessary for effective control and clearance of CVB3 infections (56,61,62). Clearance correlated with a strong viral-specific IgG response (56,62). Thus, the increased viral load in copper deficiency is likely due to the lack of antigen-specific IgG2a production. In the absence of effective acquired immune response to control viral replication, the innate immune response may remain highly activated, leading to a more proinflammatory state with increased tissue pathology. A recent study (63) provided evidence in support of a modulating effect of adaptive immune cells on the innate immune response. The elevated expression of FoxP3, a marker for Treg cells (64), in hearts from infected copper-deficient mice may reflect an attempt to control immune pathology similar to Treg cell control of the severity of herpes simplex virus-induced inflammatory lesions (65).

The results presented here demonstrate that copper deficiency increased CVB3 replication in the heart and increased cardiac pathology. In addition, copper-deficient mice infected with the myocarditic CVB3/20 strain had increased cytokine gene expression. CVB3-specific IgG2a but not IgM production was significantly reduced in copper-deficient mice and this reduction is likely responsible for enhanced viral replication. Passage of the amyocarditic CVB3/0 strain through copper-deficient mice did not result in the appearance of a more virulent strain of CVB3, as has been observed with selenium- or vitamin E-deficient (30,31,66,67) or aged mice (46). Thus, whereas a deficiency in selenium or copper can induce oxidative stress, copper deficiency does not select for more virulent strains of virus but appears to impair specific antibody production and increase proinflammatory gene expression, which leads to higher viral load and subsequently enhanced pathology.


   FOOTNOTES
 
1 Author disclosures: A. D. Smith, S. Botero, and O. A. Levander, no conflicts of interest. Back

2 Abbreviations used: Ct, threshold cycle; CVB3, coxsackievirus B3; IFN, interferon; IL, interleukin; TCID50, tissue culture infectious dose 50; TNF, tumor necrosis factor. Back

3 To convert µg copper to µmol, multiply by .0157. Back

Manuscript received 24 December 2007. Initial review completed 7 January 2008. Revision accepted 7 March 2008.


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 Introduction
 Methods
 Results
 Discussion
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