Role of oxidative stress on diesel-enhanced influenza infection in mice

Abstract

Numerous studies have shown that air pollutants, including diesel exhaust (DE), reduce host defenses, resulting in decreased resistance to respiratory infections. This study sought to determine if DE exposure could affect the severity of an ongoing influenza infection in mice, and examine if this could be modulated with antioxidants. BALB/c mice were treated by oropharyngeal aspiration with 50 plaque forming units of influenza A/HongKong/8/68 and immediately exposed to air or 0.5 mg/m3 DE (4 hrs/day, 14 days). Mice were necropsied on days 1, 4, 8 and 14 post-infection and lungs were assessed for virus titers, lung inflammation, immune cytokine expression and pulmonary responsiveness (PR) to inhaled methacholine. Exposure to DE during the course of infection caused an increase in viral titers at days 4 and 8 post-infection, which was associated with increased neutrophils and protein in the BAL, and an early increase in PR. Increased virus load was not caused by decreased interferon levels, since IFN-β levels were enhanced in these mice. Expression and production of IL-4 was significantly increased on day 1 and 4 p.i. while expression of the Th1 cytokines, IFN-γ and IL-12p40 was decreased. Treatment with the antioxidant N-acetylcysteine did not affect diesel-enhanced virus titers but blocked the DE-induced changes in cytokine profiles and lung inflammation. We conclude that exposure to DE during an influenza infection polarizes the local immune responses to an IL-4 dominated profile in association with increased viral disease, and some aspects of this effect can be reversed with antioxidants.

Figure 1

Exposure to DE enhances influenza titers and IFN-β expression. A) Viral titers were quantified in lung homogenates as TCID₅₀ on day 1, 4, 8, and 14 p.i. B) IFN-β mRNA expression was quantified in lung RNA by RT-PCR. Values are normalized to β-actin and expressed as relative quantification. *significantly different from air exposed influenza infected mice (p < 0.05, n = 11).

Figure 2

Exposure to DE enhances influenza induced pulmonary inflammation. BAL was obtained day 1, 4, 8, and 14 p.i. A) Neutrophil counts per ml of BAL. B) Protein concentration (μg/ml) in BAL. C) Pathology scores of mouse lung sections were stained with H & E and visualized and scored using light microscopy in tissues collected on day 4 p.i. Left column is representative of 100× magnification and right column is representative of 400× magnification. *significantly different from air exposed mice (p < 0.05, n = 8 for uninfected; n = 11 for influenza infected).

Figure 3

Exposure to DE enhances influenza induced pulmonary responsiveness (PR). PR measured by Buxco systems at day 1, 4, 8, and 14 p.i., reported as PenH values with increasing doses of aerosolized methacholine. *significantly different from air and air/flu exposed mice (p < 0.05, n = 6).

Figure 4

Exposure to DE during an influenza infection increases the message of Interleukin 4 (IL-4) and decreases the expression of IFN-γ and IL-12p40 cytokines. IL-4, IFN-γ and IL-12p40 gene expression was analyzed in lung RNA on day 1, 4, 8, and 14 p.i. IL-4 protein production was measured in BAL by ELISA at day 1, 4, 8, and 14 p.i. A) Levels of IL-4 mRNA were quantified in lung homogenates by real-time RT-PCR and normalized to levels of β-actin in mice exposed to air or DE during an influenza infection. B) Levels of IL-4 protein were measured in BAL. C) IFN-γ mRNA; D) IL-12p40 subunit mRNA. *significantly different from air or air/flu exposed mice (p < 0.05; n = 11 for influenza infected).

Figure 5

Antioxidants can increase glutathione (GSH) and decrease hemeoxygenase 1 (HO-1) expression during DE enhanced influenza infection. A) Levels of GSH were quantified in PCA lung homogenates by HPLC and expressed as nmol of GSH per gram of lung tissue. Graph is representative of day 4 p.i. B) Levels of HO-1 mRNA were quantified in lung homogenates by real-time RT-PCR and normalized to levels of β-actin in mice exposed to air on day 4 p.i. (p < 0.05, n = 6).

Figure 6

Antioxidants have no effect on DE enhanced influenza titers but decrease DE enhanced pulmonary inflammation. A) Viral titers were quantified in lung homogenates as TCID₅₀ on day 1 and 4 p.i. *significantly different from air/flu (p < 0.05, n = 6). B) Neutrophil counts per ml of BAL on day 4 p.i. C) PR measured by Buxco systems at day 1 p.i. reported as PenH values with increasing doses of aerosolized methacholine. *significantly different from saline controls (p < 0.05, n = 6).

Figure 7

Antioxidants decrease Interleukin 4 (IL-4) and increase the expression of IFN-γ during a DE-enhanced influenza infection. IL-4 and IFN-γ gene expression was analyzed in lung RNA on day 4 p.i. A) Levels of IL-4 mRNA were quantified in lung homogenates by real-time RT-PCR and normalized to levels of β-actin in mice exposed to air on day 4 p.i. B) IFN-γ on day 4 p.i. *significantly different from saline control (p < 0.05, n = 6).