Sequential exposure to carbon nanotubes and bacteria enhances pulmonary inflammation and infectivity

Abstract

Carbon nanotubes (CNT), with their applications in industry and medicine, may lead to new risks to human health. CNT induce a robust pulmonary inflammation and oxidative stress in rodents. Realistic exposures to CNT may occur in conjunction with other pathogenic impacts (microbial infections) and trigger enhanced responses. We evaluated interactions between pharyngeal aspiration of single-walled CNT (SWCNT) and bacterial pulmonary infection of C57BL/6 mice with Listeria monocytogenes (LM). Mice were given SWCNT (0, 10, and 40 mug/mouse) and 3 days later were exposed to LM (10(3) bacteria/mouse). Sequential exposure to SWCNT/LM amplified lung inflammation and collagen formation. Despite this robust inflammatory response, SWCNT pre-exposure significantly decreased the pulmonary clearance of LM-exposed mice measured 3 to 7 days after microbial infection versus PBS/LM-treated mice. Decreased bacterial clearance in SWCNT-pre-exposed mice was associated with decreased phagocytosis of bacteria by macrophages and a decrease in nitric oxide production by these phagocytes. Pre-incubation of naïve alveolar macrophages with SWCNT in vitro also resulted in decreased nitric oxide generation and suppressed phagocytizing activity toward LM. Failure of SWCNT-exposed mice to clear LM led to a continued elevation in nearly all major chemokines and acute phase cytokines into the later course of infection. In SWCNT/LM-exposed mice, bronchoalveolar lavage neutrophils, alveolar macrophages, and lymphocytes, as well as lactate dehydrogenase level, were increased compared with mice exposed to SWCNT or LM alone. In conclusion, enhanced acute inflammation and pulmonary injury with delayed bacterial clearance after SWCNT exposure may lead to increased susceptibility to lung infection in exposed populations.

Figure 1.

(A) Experimental protocol of pharyngeal aspiration of C57BL/6 mice with single-walled carbon nanotubes (SWCNT) and/or Listeria monocytogenes (LM). (B) Differential weight gain of C57BL/6 mice pre-exposed to PBS (solid squares), 10 μg/mouse SWCNT (open squares), or 40 μg/mouse SWCNT (circles) 3 days before inoculation with LM. Data represent mean ± SEM obtained from six mice per group. (C–F) Cell profile of BAL samples from C57BL/6 mice after pharyngeal aspiration with SWCNT and/or LM: C, alveolar macrophages; D, PMNs; E, lymphocytes; F, total cells. Open bars represent uninfected animals pre-exposed to PBS (PBS/PBS group); gray bars represent uninfected animals that had received a low dose of SWCNT (SWCNT, 10 μg/mouse/PBS group); solid bars represent uninfected animals that had received a high dose of SWCNT (SWCNT, 40 μg/mouse/PBS group); dotted bars represent infected animals pre-treated with PBS (PBS/LM group); diagonal bars represent infected animals that had received a low dose of SWCNT (SWCNT, 10 μg/mouse/LM group); brick bars represent infected animals that had received a high dose of SWCNT (SWCNT, 40 μg/mouse/LM group). *P < 0.05 versus PBS/PBS control group; ^#P < 0.05 versus PBS/LM-treated animals; ^$P < 0.05 versus SWCNT, 10 μg/mouse/PBS-treated mice; ^βP < 0.05 versus SWCNT, 40 μg/mouse/PBS-treated mice.

Figure 2.

(A) Changes in the level of lactate dehydrogenase activity in the bronchoalveolar lavage (BAL) samples of C57BL/6 mice after pharyngeal aspiration with SWCNT and/or LM. Open bars represent uninfected animals pre-treated with PBS (PBS/PBS group); gray bars represent uninfected animals that had received a low dose of SWCNT (SWCNT, 10 μg/mouse/PBS group); solid bars represent uninfected animals that had received a high dose of SWCNT (SWCNT, 40 μg/mouse/PBS group); dotted bars represent infected animals pre-treated with PBS (PBS/LM group); diagonal bars represent infected animals that had received a low dose of SWCNT (SWCNT, 10 μg/mouse/LM group); brick bars represent infected animals that had received a high dose of SWCNT (SWCNT, 40 μg/mouse/LM group). (B) Level of collagen in the lung of mice after pharyngeal aspiration with SWCNT (40 μg/mouse) and/or LM (10 d after SWCNT exposure). Data represent mean ± SEM obtained from six mice per group. *P < 0.05 versus PBS/PBS control group; ^#P < 0.05 versus PBS/LM-treated animals; ^$P < 0.05 versus SWCNT, 10 μg/mouse/PBS-treated mice; ^βP < 0.05 versus SWCNT, 40 μg/mouse/PBS-treated mice. (C–F) Light micrographs of lung of C57BL/6 mice after pharyngeal aspiration with high dose (40 μg/mouse) of SWCNT and/or LM stained with hematoxylin and eosin at Day 10 of the experiment: C, PBS/PBS group; D, SWCNT/PBS group; E, PBS/LM group; F, SWCNT/LM group. Original magnification: ×200.

Figure 3.

Cytokine profile in BAL of noninfected C57BL/6 mice 3 days after pharyngeal aspiration with SWCNT (40 μg/mouse, solid bars) or PBS (open bars). IFN-γ, IL-13, IL-12p70, IL-10, IL-4, IL-3, TNF-α, and IL-1β were not detected. ND = below the limit of detection. Data represent mean ± SEM obtained from six animals in the SWCNT-treated group and three in the untreated control group. *P < 0.05 versus PBS/PBS group, as determined by unpaired Student's t test with Welch's correction for unequal variances between the two groups.

Figure 4.

Effect of SWCNT pre-exposure on temporal pattern of four representative cytokines in BAL during LM infection. A, CCL-2 (MIP-1α); B, CCL-2 (MCP-2); C, IL-12 p40; D, IL-12 p70. Dotted bar represents uninfected animals pre-treated with PBS (PBS/PBS group). Solid bars represent uninfected animals that had received high dose of SWCNT (SWCNT, 40 μg/mouse/PBS). Solid circles represent infected animals pre-treated with PBS vehicle 3 days before LM administration (PBS/LM group). Open triangles represent infected animals that had received a low dose of SWCNT 3 days before infection (SWCNT, 10 μg/mouse/LM group). Open squares represent infected animals that had received a high dose of SWCNT before infection (SWCNT, 40 μg/mouse/LM group). Data represent mean ± SEM obtained from six animals in each group. *P < 0.05 versus LM/PBS group as assessed by one-way ANOVA and Dunnett's multiple comparisons to the LM alone group.

Figure 5.

(A) Reactive oxygen species production in the BAL cells in response to SWCNT and/or LM exposure. Open bars represent uninfected animals pre-treated with PBS (PBS/PBS group); gray bars represent uninfected animals that had received a low dose of SWCNT (SWCNT, 10 μg/mouse/PBS group); solid bars represent uninfected animals that had received a high dose of SWCNT (SWCNT, 40 μg/mouse/PBS group); dotted bars represent infected animals pre-treated with PBS (PBS/LM group); diagonal bars represent infected animals that had received a low dose of SWCNT (SWCNT, 10 μg/mouse/LM group); brick bars represent infected animals that had received a high dose of SWCNT (SWCNT, 40 μg/mouse/LM group). *P < 0.05 versus PBS/PBS control group; ^$P < 0.05 versus SWCNT, 10 μg/mouse/PBS-treated mice; ^βP < 0.05 versus SWCNT, 40 μg/mouse/PBS-treated mice. (B) Assessment of pulmonary bacterial clearance in lung of C57BL/6 mice after challenge with SWCNT. Open bars represent infected animals pre-treated with PBS (PBS/LM group); gray bars represent infected animals that had received a low dose of SWCNT (SWCNT, 10 μg/mouse/LM group); solid bars represent infected animals that had received a high dose of SWCNT (SWCNT, 40 μg/mouse/LM group). *P < 0.05 versus PBS/LM group; ^#P < 0.05 versus SWCNT, 10 μg/mouse/LM-treated animals.

Figure 6.

Effect of SWCNT on phagocytosis of LM and production of NO· by alveolar macrophages from mouse BAL. (A) Typical fluorescence micrographs of alveolar macrophages pre-treated with SWCNT and co-incubated with LM: a, bright field image; b, red fluorescence image (Nile Red staining of macrophages); c, green fluorescence image (Cell Tracker Green labeled LM); d, overlay of red and green images. (B) Phagocytosis of LM by alveolar macrophages obtained from C57BL/6 mice pre-exposed to SWCNT in vivo. Three hundred macrophages per sample were counted. Data are mean ± SD, n = 3. *P < 0.05 versus control PBS/LM-treated group. Inset: Phagocytosis of LM by alveolar macrophages exposed to SWCNT in vitro. Three hundred macrophages per sample were counted. Data are mean ± SD, n = 3. (C) NO· production by alveolar macrophages obtained from C57BL/6 mice pre-exposed to SWCNT in vivo. Three hundred macrophages per sample were counted. Data are mean ± SD, n = 3. *P < 0.05 versus control PBS/LM-treated group. Inset: NO· production by alveolar macrophages exposed to SWCNT in vitro. Three hundred macrophages per sample were counted. Data are mean ± SD, n = 3.