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Selenium deficiency increases the pathology of an influenza virus infection¹

MELINDA A. BECK^*,2, HEATHER K. NELSON^*, QING SHI, PETER VAN DAEL, EDUARDO J. SCHIFFRIN, STEPHANIE BLUM, DENIS BARCLAY and ORVILLE A. LEVANDER

Departments of
^* Nutrition and
Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7220, USA;
Nestlé Research Center, Lausanne, Switzerland; and
USDA, ARS, Beltsville Human Nutrition Research Center, Beltsville, Maryland, USA

²Correspondence: Department of Pediatrics, 535 Burnett-Womack, CB #7220, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7220, USA. E-mail: melinda_beck{at}unc.edu

SPECIFIC AIM

To determine the effect of a deficiency of the nutritionally essential trace element selenium (Se) in mice infected with influenza virus on lung pathology, viral titers, and the host immune response.

PRINCIPAL FINDINGS

1. Selenium deficiency increases lung pathology in influenza-infected mice
The lungs of selenium-deficient mice had significantly more inflammation at days 4, 6, 10, and 21 after infection than those of Se-adequate controls (Fig. 1 ). Lung pathology peaked at day 6 after infection in both dietary groups. Pathology in the Se-adequate mice began to diminish after day 6, whereas pathology in the Se-deficient mice was still severe even 21 days after infection. The infiltrate in both Se-deficient and Se-adequate mice was characterized as an interstitial pneumonitis, typical for a murine influenza infection.

View larger version (23K): [in this window] [in a new window]   Figure 1. Lung pathology after influenza virus infection in Se-deficient (open bars) and Se-adequate (solid bars) mice. Each bar represents the mean ± the SD of 10 animals. Lung pathology was scored without knowledge of the experimental variables and was graded semiquantitatively according to the relative degree (from lung to lung) of mononuclear cell infiltrate. *Statistically significant difference between groups at that time period (P<0.001).

2. Selenium deficiency induces changes in cellular phenotype of lung infiltrating cells.
Cells recovered from the bronchoalveolar lavage (BAL) fluid of Se-adequate and Se-deficient mice were counted and stained for various cell surface markers in order to further characterize lung histopathology after influenza infection. Stained cells were then analyzed by flow cytometry. The total number of cells recovered from the BAL fluid on days 10 and 21 after infection was significantly higher in the mice fed the Se-deficient diets (8.05±1.0x10⁶ and 3.6±1.4x10⁶) than in the fluid of mice fed the Se-adequate diets (5.1±0.8x10⁶ and 1.3±0.2x10⁶). This finding correlated with the increased histopathology found in the Se-deficient mice during that time.

The phenotype of the infiltrating cells was also assessed for the Se-adequate and Se-deficient mice. Selenium-deficient mice had increased percentages of CD8+ cells and macrophages 5 days after infection compared with Se-adequate mice. However, at day 10 after infection, the percentage of CD8+ cells and, to a lesser extent, CD4+ cells dropped in the Se-deficient mice compared with the Se-adequate mice, thereby suggesting that the immune response was affected by the deficiency in Se.

3. Selenium deficiency has no effect on viral titers
CD8+ T cells are thought to be primarily responsible for the clearance of virus from the lungs of influenza-infected mice. Because the level of CD8+ cells in the lungs of influenza-infected, Se-deficient mice was decreased compared with infected, Se-adequate controls, one might expect increased titers in the Se-deficient mice. However, lung virus titers of Se-deficient and Se-adequate mice were equivalent, although there was wide variation within groups. All mice were able to clear the virus by day 10 after infection.

4. Selenium deficiency has no effect on antibody response
Since a functioning T cell response is required for B cells to produce antibody, a defect in either B or T cell immunity can affect the secretion of virus-specific antibody. To determine whether Se deficiency affected the ability of the host to secrete neutralizing antibody, sera from mice at 4, 5, 14, and 21 days after infection were analyzed for the presence of influenza-specific neutralizing antibody. Since titers were similar in both Se-adequate and Se-deficient mice, there appeared to be no impairment in the ability of B cells to produce antibody.

5. Selenium deficiency alters cytokine and chemokine expression
Differences were found between Se-deficient and Se-adequate mice in the expression of both cytokine and chemokine mRNA in mediastinal lymph nodes during days 4–21 postinfection. The mRNA for IFN- was much less abundant in Se-deficient vs. Se-adequate mice at all time points measured. Similarly, mRNA for IL-2 was also lower in the Se-deficient mice. The levels of mRNA for IL-4 and IL-5 were both decreased at day 4 in Se-deficient mice compared with Se-adequate mice, then increased relative to Se-adequate mice at days 14 and 21. Se-deficient mice had greatly increased levels of mRNA for IL-10 and IL-13 at day 6 postinfection and for IL-4, IL-5, IL-10, and IL-13 at day 14 postinfection when compared with Se-adequate mice. These results suggest that the Se-deficient mice developed a more TH2-like response after influenza infection, whereas the Se-adequate mice displayed a TH1-like response.

Chemokines are important mediators of inflammatory responses and there was an increased expression of chemokine mRNA in the Se-deficient mice beginning at day 6 postinfection, with the greatest increases occurring on days 10 and 14 postinfection (Fig. 2 ). In contrast, chemokine mRNA expression was highest on days 4 and 5 postinfection in the Se-adequate mice and then declined sharply thereafter.

View larger version (34K): [in this window] [in a new window]   Figure 2. Time course of chemokine mRNA expression in the mediastinal lymph nodes of influenza-infected animals. RNA was prepared and chemokine mRNA was detected by a multiprobe RNase protection assay and quantified by image densitometry. Each data point represents a pool from two individual mice repeated three times (total of six mice per time point). Data are expressed as percent change comparing Se-deficient mice with Se-adequate mice (Se-deficient minus Se-adequate divided by Se-adequatex100). Values above 0 indicate the percent increase of mRNA levels of Se-deficient mice compared with Se-adequate mice and values below 0 indicate the percent decrease of mRNA levels from Se-deficient mice compared with Se-adequate mice.

Thus, elevation in chemokine mRNA levels occurred early in the Se-adequate mice, which corresponded to the early increase in lung inflammation of these animals. At later time points, the chemokine response declined in the Se-adequate mice during which the lung pathology also resolved. The Se-deficient mice had increased lung pathology at the later time points, which also corresponded with the later increase in chemokine mRNA in this group.

CONCLUSIONS AND SIGNIFICANCE

The data presented here demonstrate that a dietary deficiency of the nutritionally essential trace element Se increases the lung pathology caused by the influenza virus in mice. Under our conditions, mice fed a diet adequate in Se develop only a relatively mild inflammatory response when infected with influenza A/Bangkok/1/79. However, the inflammation is much more severe in Se-deprived mice. The increased severity of the inflammation is reflected in the increased number of inflammatory cells obtained by BAL as well as the higher pathology score.

These results complement earlier research that showed that Se deficiency induced myocarditis in mice infected with a benign strain of Coxsackie virus. Work by others has indicated that supplementation with Se may be of some benefit in individuals infected with HIV. Taken together, these studies emphasize the general importance of good nutrition in fighting infections and suggest that Se may be of particular benefit against a variety of viral diseases.

As outlined in the schematic diagram (Fig. 3 ), we hypothesize that Se-deficient mice infected with influenza virus develop increased oxidative stress as a result of a lack of GPx activity combined with the viral infection. The Se-adequate mice are also oxidatively stressed, but to a lower extent, due to the activity of GPx. This increased oxidative stress leads to increased NF-B expression, particularly in the Se-deficient mice, leading to enhanced chemokine mRNA expression. In addition, the cytokine pattern in the Se-deficient animals is skewed toward a TH2-like pattern, which results in increased inflammation. Together, the end result is increased lung pathology due to a heightened immune response. Lung inflammation also occurs in the influenza-infected, Se-adequate mice but to a much lesser extent.

View larger version (25K): [in this window] [in a new window]   Figure 3. No caption available.

A second possible explanation for the increased lung pathology observed in the influenza-infected, Se-deficient mice is a change in the virus itself. Previous work showed that the conversion of an avirulent Coxsackie virus to a virulent one in Se-deficient mice was due to a change in the viral genome. Perhaps the genome of the influenza virus likewise changed to a more virulent genotype. This possibility awaits further study.

In conclusion, mice fed a diet deficient in Se suffer much more severe lung pathology after infection with the influenza virus than Se-adequate controls. Although the increased pathology was not associated with an increase in viral titer, it was associated with an increase in the mRNA expression of proinflammatory cytokines and chemokines and a decrease in the expression of antiinflammatory cytokines. Furthermore, the immune response in the infected lung tissue was shifted away from a TH1 response to a TH2 response in the Se-deficient mice. Our work points to the importance of adequate antioxidant defense mechanisms for protection against viral infection and demonstrates that Se-dependent GSH-Px may play an important role during an influenza-induced inflammatory process.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0721fje ; to cite this article, use FASEB J. (April 27, 2001) 10.1096/fj.00-0721fje

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