Environmental Cadmium Enhances Lung Injury by Respiratory Syncytial Virus Infection

Abstract

Cadmium (Cd) is a naturally occurring environmental toxicant that disrupts mitochondrial function at occupational exposure levels. The impacts of Cd exposure at low levels through dietary intake remain largely uncharacterized. Human respiratory syncytial virus (RSV) causes severe morbidity, which can require hospitalization and result in death in young children and elderly populations. The impacts of environmental Cd exposure on the severity of RSV disease are unknown. Herein, we used a mouse model to examine whether Cd pre-exposure at a level of dietary intake potentiates pulmonary inflammation on subsequent infection with RSV. Mice were given Cd or saline in drinking water for 28 days. Subsets of these mice were infected with RSV at 5 days before the end of the study. Cd pre-exposure caused relatively subtle changes in lung; however, it elevated the IL-4 level and altered metabolites associated with fatty acid metabolism. After RSV infection, mice pre-exposed to Cd had elevated lung RSV titer and increased inflammation, as measured by histopathology, immune cell infiltration, cytokines, and chemokines. RSV infection after Cd pre-exposure also caused widespread perturbation in metabolism of glycerophospholipids and amino acids (Trp, Met, and Cys, branched-chain amino acids), as well as carnitine shuttle associated with mitochondrial energy metabolism. The results show that Cd burden by dietary intake potentiates RSV infection and severe disease with associated mitochondrial metabolic disruption.

Figure 1

Low-level cadmium (Cd) burden elevated lung histopathology and viral titers after respiratory syncytial virus (RSV) challenge. A–C: Collected lung tissues from individual mouse from all four groups [vehicle control (Cont), Cd only, RSV, and Cd + RSV] at day 5 after RSV challenge (28 days of Cd administration) were examined for histopathology after staining with hematoxylin and eosin (A), periodic acid-Schiff (PAS; B), and hematoxylin and Congo red (H&CR; C). Insets: Magnified eosinophilic cell spots positively stained with H&CR. Arrowheads indicate inflamed airways, blood vessel, and eosinophil granulocytes. D: RSV lung viral titers at day 5 after infection. E–G: Images taken by microscopy were used for quantification, as follows: inflammation score using a 1 to 10 scoring system, where 1 indicates minimal pathology and 10 indicates maximum pathology for the airways, interstitial spaces, and blood vessels (E); % of PAS-positive airway mucus expression (F); and H&CR-positive eosinophil cells counted under the microscopic field (G). Statistical significances were performed by one-way analysis of variance. Data are expressed as means ± SEM. n = 8 for all groups (D–G). ^∗P < 0.05 versus RSV only; ^†P < 0.05. Scale bars= 100 μm (A–C). Original magnification, ×100 (A–C). PFU, plaque-forming unit.

Figure 2

Cadmium (Cd)–promoted infiltration of monocytes and lymphocytes in lung. The lung tissues (A–C) and bronchoalveolar lavage (BAL) fluid (D–F) from individual mice were collected from individual mouse of each group (Figure 1). Monocytes (F4/80⁺CD11c⁻CD11b⁺Ly6C^hiSiglecF⁻) and CD4 (CD3, CD4) and CD8 (CD3, CD8) lymphocytes were analyzed by flow cytometry, and quantified cells are shown in bar graphs using an unpaired two-tailed t-test. Error bars indicate means ± SEM of cell counts from individual animals (A–F). n = 8 per group (A–F). ^∗P < 0.05 versus control (cont); ^†P < 0.05 versus respiratory syncytial virus (RSV) only group.

Figure 3

Cadmium (Cd) intake enhanced proinflammatory cytokine levels in the lung after respiratory syncytial virus (RSV) infection. Inflammatory cytokines were determined in lung lysates and bronchoalveolar lavage (BAL) fluids and analyzed for interferon (IFN)-γ (A and E), IL-1β (B and F), IL-6 (C and G), and IL-4 (D and H) quantitation by corresponding cytokine enzyme-linked immunosorbent assay kits. Data are expressed as means ± SEM (A–H). n = 8 per group (A–H). ^∗P < 0.05 versus control (cont).

Figure 4

Cadmium (Cd) pre-exposure enhanced chemokine regulated on activation, normal T cells expressed and secreted (RANTES), and keratinocyte cytokine (KC) production after respiratory syncytial virus (RSV) infection. Chemokines were determined in lung lysates and bronchoalveolar lavage (BAL) fluid, as described in Materials and Methods, and analyzed for KC (A and C) and RANTES (B and D) quantification by enzyme-linked immunosorbent assay. Data are expressed as means ± SEM (A–D). n = 8 per group (A–D). ^∗P < 0.05 versus control (cont); ^†P < 0.05 versus RSV only.

Figure 5

Lung (A) and bronchoalveolar lavage (BAL) fluid (C) metabolic features that differed between cadmium (Cd) + respiratory syncytial virus (RSV) and RSV treatment (limma test, P < 0.05) allow group separation by two-way unsupervised hierarchical clustering analysis and are shown to be enriched in metabolic pathways (B and D) by Mummichog analysis. Numbers in metabolic pathways (B and D) indicate the overlap size (ie, number of significant metabolites relative to the total number of metabolites detected in the pathway). n = 8 mice per group (A and C).

Figure 6

Network structure of metabolome interacting with inflammatory markers in mice treated with cadmium (Cd; A), respiratory syncytial virus (RSV; B), and Cd + RSV (C). Association between nine inflammatory markers (pink or purple circle) and the metabolome (11,341 features; gray squares) from mouse lung are visualized at |ρ| ≥ 0.7, showing a distinct IL-4 and metabolite hub in mouse lung treated with Cd or Cd + RSV. Red edge indicates positive association; blue indicates negative association. Details of metabolite annotation can be found in Supplemental Table S2. Not shown: control mice with low Cd showed no network correlation of Cd and IL-4. n = 8 (A–C).

Figure 7

Network structure of metals interacting with inflammatory markers through common metabolic associations in mouse lung treated with cadmium + respiratory syncytial virus. Association among 9 inflammatory markers (circle), 11 metals (squares), and the metabolome (11,341 features; data not shown) from mouse lung (|ρ| > 0.8) were integrated using xMWAS. The number of commonly associated metabolites between two elements (metal or marker) was reflected by the width of edges connecting the two elements, showing a strong interaction of ¹¹⁴Cd, ⁷⁷Se, and ⁶⁴Zn with IL-4 but not with other inflammation markers. Metals that did not share metabolite association with IL-4 were excluded from the network. Details of metabolites in community (C1 to C4) and annotation of metabolites can be found in Supplemental Table S3. For simplicity, isotopic information of metals are omitted (see Materials and Methods for details). n = 8. IFN-γ, interferon-γ; KC, keratinocyte cytokine; RANTES, regulated on activation, normal T cells expressed and secreted.

Figure 8

Proposed schematic diagram: cadmium (Cd)–potentiated inflammation by respiratory syncytial virus (RSV) infection. Low-dose Cd–potentiated RSV infection caused lung inflammation and injury by dysregulating metals, disrupting mitochondrial metabolism, stimulating oxidative stress, and elevating proinflammatory cytokines, chemokines, and infiltration of immune cells. KC, keratinocyte cytokine; RANTES, regulated on activation, normal T cells expressed and secreted; TrxR2, thioredoxin reductase 2.