Hydroxyl functionalized multi-walled carbon nanotubes modulate immune responses without increasing 2009 pandemic influenza A/H1N1 virus titers in infected mice

Abstract

Growing use of carbon nanotubes (CNTs) have garnered concerns regarding their association with adverse health effects. Few studies have probed how CNTs affect a host's susceptibility to pathogens, particularly respiratory viruses. We reported that exposure of lung cells and mice to pristine single-walled CNTs (SWCNTs) leads to significantly increased influenza virus H1N1 strain A/Mexico/4108/2009 (IAV) titers in concert with repressed antiviral immune responses. In the present study, we investigated if hydroxylated multi-walled CNTs (MWCNTs), would result in similar outcomes. C57BL/6 mice were exposed to 20 μg MWCNTs on day 0 and IAV on day 3 and samples were collected on day 7. We investigated pathological changes, viral titers, immune-related gene expression in lung tissue, and quantified differential cell counts and cytokine and chemokine levels in bronchoalveolar lavage fluid. MWCNTs alone caused mild inflammation with no apparent changes in immune markers whereas IAV alone presented typical infection-associated inflammation, pathology, and titers. The co-exposure (MWCNTs + IAV) did not alter titers or immune cell profiles compared to the IAV only but increased concentrations of IL-1β, TNFα, GM-CSF, KC, MIPs, and RANTES and inhibited mRNA expression of Tlr3, Rig-i, Mda5, and Ifit2. Our findings suggest MWCNTs modulate immune responses to IAV with no effect on the viral titer and modest pulmonary injury, a result different from those reported for SWCNT exposures. This is the first study to show that MWCNTs modify cytokine and chemokine responses that control aspects of host defenses which may play a greater role in mitigating IAV infections.

Keywords: C57BL/6 mouse; Influenza A H1N1 virus; Innate immune responses; Multi-walled carbon nanotubes; Viral titers.

Figure 1.

Characterization of MWCNTs in 1% pluronic. (A) Transmission electron micrographs of MWCNTs (Scale: 100 nm (top) and 20 nm (bottom)). (B) Average hydrodynamic radius (HDR) of MWCNTs at 37 °C for 24 hours. (C) Zeta potential and electrophoretic mobility (EPM) of MWCNTs at 37°C for 24 hours (Mean ± SD). (D) Leached metal concentrations (parts per million, ppm) from MWCNTs in 1% pluronic over 24 hours (Mean ± SD).

Figure 2.

Changes in body weight, virus titers, and viral-specific gene expression by IAV and MWCNTs+IAV. Mice were exposed to either MWCNTs (20 μg per animal) or 1% pluronic on day 0 and then to IAV (3.2 × 10⁴ TCID₅₀ per animal) on day 3. Animals were euthanized on day 7. (A) Body weight and (B) body weight loss were recorded daily. Part of the right lung lobe of was used for quantification of (C) virus titers and (D) mRNA expression of M2 protein (N=11). Viral titer and M2 expression data were logarithm transformed and all data presented as Mean ± SEM. Asterisks (*) indicate statistically significant differences compared to control and different letters above each group indicate statistically significant differences between groups (P<0.05).

Figure 3.

Changes in immune cell profiles in mouse BALF following IAV and MWCNTs+IAV exposure. Mice were exposed to either MWCNTs (20 μg per animal) or 1% pluronic on day 0 and then to IAV (3.2 × 10⁴ TCID₅₀ per animal) on day 3. Animals were euthanized on day 7. BALF fluid was collected to quantify (A) total immune cell number, (B) immune cell percentage, (C) total neutrophils, (D) total lymphocytes, and (E) total macrophages (N=11). Data are presented as Mean ± SEM for cell counts. Different letters above each group indicate statistically significant differences (P<0.05).

Figure 4.

Histopathological changes in lung tissues by IAV and MWCNTs+IAV. Mice were exposed to either MWCNTs (20 μg per animal) or 1% pluronic on day 0 and then to IAV (3.2 × 10⁴ TCID₅₀ per animal) on day 3. Animals were euthanized on day 7. Left lung lobes were perfused with 4% paraformaldehyde and prepared for histopathological analysis in (A) control (100X), (B) MWCNTs alone (100X), (C) MWCNTs alone (400X), (D) IAV alone (100X), and (E) MWCNTs+IAV (100X). “Red arrow” indicates MWCNTs found in macrophages. Six samples per group were measured for volume density of pneumonia (F) and presented as Mean ± SEM. Different letters above each group indicate statistically significant differences (P<0.05).

Figure 5.

Changes in cytokine levels by IAV and MWCNTs+IAV in BALF. Mice were exposed to either MWCNTs (20 μg per animal) or 1% pluronic on day 0 and then to IAV (3.2 × 10⁴ TCID₅₀ per animal) on day 3. Animals were euthanized on day 7 (N=9). Data are presented as Mean ± SEM. Different letters above each group indicate statistically significant differences (P<0.05).

Figure 6.

Changes in chemokine levels by IAV and MWCNTs+IAV in BALF. Mice were exposed to either MWCNTs (20 μg per animal) or 1% pluronic on day 0 and then to IAV (3.2 × 10⁴ TCID₅₀ per animal) on day 3. Animals were euthanized on day 7 (N=9). Data are presented as Mean ± SEM. Different letters above each group indicate statistically significant differences (P<0.05).

Figure 7.

Changes in antiviral and pro-inflammatory gene expression by IAV and MWCNTs+IAV in lung tissue. Mice were exposed to either MWCNTs (20 μg per animal) or 1% pluronic on day 0 and then to IAV (3.2 × 10⁴ TCID₅₀ per animal) on day 3. Animals were euthanized on day 7. Part of the right lung lobes were extracted for RNA isolation. Ten to eleven samples per group were measured for the mRNA expression levels by qPCR and data presented as Mean ± SEM. Different letters above each group indicate statistically significant differences (P<0.05).

Figure 8.

Changes in stress-related gene expression by IAV and MWCNTs+IAV in lung tissue. Mice were exposed to either MWCNTs (20 μg per animal) or 1% pluronic on day 0 and then to IAV (3.2 × 10⁴ TCID₅₀ per animal) on day 3. Animals were euthanized on day 7. Part of the right lung lobes of mice were extracted for RNA isolation. Ten to eleven samples per group were measured for the mRNA expression levels by qPCR and data presented as Mean ± SEM. Different letters above each group indicate statistically significant differences (P<0.05).