image_1_Protective Role of Nuclear Factor Erythroid 2-Related Factor 2 Against Respiratory Syncytial Virus and Human Metapneumovirus Infections.PDF

<p>The pathogenesis of respiratory syncytial virus (RSV) infections is characterized by lower airway obstruction driven at great extent by the exuberant production of inflammatory cytokines. We have previously shown that RSV infection in vitro and in vivo results in production of reactive oxygen species along with reduction in the expression of antioxidant enzymes (AOEs), which are involved in maintaining the cellular oxidant–antioxidant balance. These events were associated with the concomitant reduction in nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor that controls AOE expression. The objective of the current study was to establish the role of Nrf2 in shaping innate immune responses, clinical disease, airway inflammation, and viral replication in established experimental models of intranasal RSV and human metapneumovirus (hMPV) infections, by employing mice genetically deficient for the Nrf2 gene. Compared to control wild type (WT), mice genetically deficient in Nrf2 (Nrf2 KO) developed enhanced clinical disease, airway inflammation and pathology, and significantly greater lung viral titers following experimental infection with either RSV or hMPV. In particular, compared to control mice, RSV-infected Nrf2 KO mice lost more body weight and had increased airway obstruction at time points characterized by a remarkable increase in inflammatory cytokines and airway neutrophilia. Airway levels of AOEs and enzymes that regulate synthesis of the endogenous hydrogen sulfide (H₂S) pathway, which we showed to play an important antiviral function, were also decreased in RSV-infected Nrf2 KO compared to WT. In conclusion, these results suggest that Nrf2 is a critical regulator of innate, inflammatory, and disease-associated responses in the airways of mice infected with viruses that are members of the Pneumoviridae family. Importantly, the results of this study suggest that Nrf2-dependent genes, including those controlling the cellular antioxidant and H₂S-generating enzymes and cytokines can affect several aspects of the antiviral response, such as airway neutrophilia, clinical disease, airway obstruction, and viral replication.</p

<i>Paramyxovirus</i> Infection Regulates T Cell Responses by BDCA-1⁺ and BDCA-3⁺ Myeloid Dendritic Cells

<div><p>Respiratory syncytial virus (RSV) and human Metapneumovirus (hMPV), viruses belonging to the family <i>Paramyxoviridae</i>, are the most important causes of lower respiratory tract infection in young children. Infections with RSV and hMPV are clinically indistinguishable, and both RSV and hMPV infection have been associated with aberrant adaptive immune responses. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping adaptive immune responses during infection; however, few studies have examined how interactions of RSV and hMPV with individual mDC subsets (BDCA-1⁺ and BDCA-3⁺ mDCs) affect the outcome of anti-viral responses. To determine whether RSV and hMPV induce virus-specific responses from each subset, we examined co-stimulatory molecules and cytokines expressed by BDCA-1⁺ and BDCA-3⁺ mDCs isolated from peripheral blood after infection with hMPV and RSV, and examined their ability to stimulate T cell proliferation and differentiation. Our data show that RSV and hMPV induce virus-specific and subset-specific patterns of co-stimulatory molecule and cytokine expression. RSV, but not hMPV, impaired the capacity of infected mDCs to stimulate T cell proliferation. Whereas hMPV-infected BDCA-1⁺ and BDCA-3⁺ mDCs induced expansion of Th17 cells, in response to RSV, BDCA-1⁺ mDCs induced expansion of Th1 cells and BDCA-3⁺ mDCs induced expansion of Th2 cells and Tregs. These results demonstrate a virus-specific and subset-specific effect of RSV and hMPV infection on mDC function, suggesting that these viruses may induce different adaptive immune responses.</p></div

Recruitment of inflammatory cells in response to hMPV infection in BAL, lung and lymph nodes in TLR4^−/− mice.

<p>TLR4^−/− and WT mice were either infected with hMPV or mock-infected, and sacrificed at days 1, 2, 3, 5, 7/8 p.i. to collect BAL, lungs and mediastinal lymph nodes (MLN). Total and differential cell counts were determined in the BAL fluid (<b>A</b>). Cells isolated from lungs (<b>B</b>) and MLN (<b>C</b>) were stained with cell-type specific fluorochrome conjugated and processed by flow cytometry. Data are expressed as mean ± SEM of four mice/group and represents one of three independent experiments. *<i>P</i><0.05 when comparing hMPV infected TLR4^−/− mice to infected WT mice.</p

Critical Role of TLR4 in Human Metapneumovirus Mediated Innate Immune Responses and Disease Pathogenesis

<div><p>Human metapneumovirus (hMPV) is one of the main causes of acute respiratory tract infections in children, elderly and immunocompromised patients. The mammalian Toll-like receptors (TLR) were identified as critical regulators of innate immunity to a variety of microbes, including viruses. We have recently shown that hMPV-induced cytokine, chemokine and type I interferon secretion in dendritic cells occurs via TLR4, however, its role in hMPV-induced disease is unknown. In this study, wild-type(WT) and TLR4-deficient mice (TLR4^−/−) were infected with hMPV and examined for clinical disease parameters, such as body weight loss and airway obstruction, viral clearance, lung inflammation, dendritic cell maturation, T-cell proliferation and antibody production. Our results demonstrate that absence of TLR4 in hMPV-infected mice significantly reduced the inflammatory response as well as disease severity, shown by reduced body weight loss and airway obstruction and hyperresponsiveness (AHR), compared to WT mice. Levels of cytokines and chemokines were also significantly lower in the TLR4^−/− mice. Accordingly, recruitment of inflammatory cells in the BAL, lungs, as well as in lymph nodes, was significantly reduced in the TLR4^−/− mice, however, viral replication and clearance, as well as T-cell proliferation and neutralizing antibody production, were not affected. Our findings indicate that TLR4 is important for the activation of the innate immune response to hMPV, however it does play a role in disease pathogenesis, as lack of TLR4 expression is associated with reduced clinical manifestations of hMPV disease, without affecting viral protection.</p></div

Expression of pro-inflammatory cytokine, chemokines and type I IFNs in response to hMPV infection of TLR4-deficient mice.

<p>TLR4^−/− and WT mice were either infected with hMPV or mock infected, and sacrificed at days 1, 2, 3, 5, 7 p.i. to collect BAL fluid. Levels of IL-1α, IL-6, TNF-α, G-CSF, IL-12 p(40), IL-17, Eotaxin, MCP-1, MIP-1β in BAL fluid were measured by Bio-Plex (<b>A</b>). Levels of IFN-α/β (left panel) were measured by ELISA on day 1 p.i., while IFN-γ (right panel) was measured by Bio-Plex at various days p.i. (<b>B</b>). Data are expressed as mean ± SEM of four to six animals/group and represents one of two independent experiments. *<i>P</i><0.05 when comparing hMPV infected TLR4^−/− mice to infected WT mice.</p

RSV, but not hMPV, impairs the ability of BDCA-1⁺ and BDCA-3⁺ mDCs to stimulate CD4⁺ T cell proliferation.

<p>BDCA-1⁺ and BDCA-3⁺ mDC were incubated with RSV, hMPV, UV-hMPV, or media for 24 hours and co-cultured with CFSE-labeled allogeneic CD4⁺ T cells. On day 7, T cell proliferation was measured by examining CFSE expression on live CD4⁺ T cells. (<b>A</b>) Data represent the percentage of proliferating cells from one donor and is representative of data from 3 donors. (<b>B</b>) Bar graphs represent the percent of proliferating CD4⁺ T cells (mean + SEM from 3 donors). * = <i>p≤0.05</i> between co-cultures with treated mDCs as calculated by one-way repeated-measures ANOVA with Tukey post-hoc analysis.</p

Comparison of selected cytokines produced by hMPV-infected BDCA-1⁺ vs. BDCA-3⁺ mDCs.

<p>DC subsets were isolated and incubated with hMPV or media for 40 hours. Cytokine and chemokine levels were measured in cell-free supernatant by multiplex assay. Data represents the mean + SEM of the fold change in cytokine production by infected cells as compared to uninfected cells of the same type from 6 donors. Linear data is reported on a logarithmic scale. The dotted line represents a 2 fold induction from baseline. Black bars = uninfected cells, white bars = hMPV-infected cells. *<i>p≤0.05</i> between BDCA-1⁺ and BDCA-3⁺ mDCs as calculated by 2-way ANOVA with Bonferroni post-hoc test.</p

IFN-α production by RSV and hMPV-infected BDCA-1⁺ and BDCA-3⁺ mDCs.

<p>IFN-α production was assessed by ELISA in the supernatant of (<b>A</b>) BDCA-1<b>⁺</b> and (<b>B</b>) BDCA-3<b>⁺</b> mDCs incubated with RSV, hMPV, UV-RSV, UV-hMPV, or media for 40 hours (mean + SEM from 3 donors). *<i>p≤0.05</i> between treated mDCs as calculated by repeated measures one-way ANOVA with Tukey post-hoc analysis. (<b>C</b>) Comparison of IFN-α production by hMPV-infected BDCA-1<b>⁺</b> vs. BDCA-3<b>⁺</b> mDCs (mean + SEM from 3 donors). * = <i>p≤0.05</i> between BDCA-1<b>⁺</b> and BDCA-3<b>⁺</b> mDCs as calculated by paired t-test.</p

Lung dendritic cell characterization and lymphocyte proliferation in response to hMPV infection in TLR4^−/− mice.

<p>TLR4^−/− and WT mice were mock- or hMPV-infected, and sacrificed at week 1 and 2 p.i. to collect lungs. Single cell suspension was obtained and cDCs population enriched using CD11c-tagged magnetic beads isolation. CD40 and CD86 markers were analyzed in cells positive for CD11c and MHC-II by flow cytometry. Histograms showing the expression of costimulatory molecules (CD40, CD86 and MHCII) in cDC from WT mice (open histograms) and cDC from TLR4^−/− mice (dashed line open histograms), and isotype control (shaded histograms), are shown (<b>A</b>). Graph of baseline expression of costimulatory molecules in lung dendritic cells isolated from WT and TLR4^−/− mice (<b>B</b>). Dendritic cells (CD11c positive cells) were isolated from lungs of hMPV or mock-infected mice either WT or TLR4^−/− at day 7 p.i. and loaded with 10 µg/mL of OVA peptide for 2h prior to coculture with T cells. CD4⁺ T cells isolated from spleen of OT II mice were labeled with CFSE and cocultured with DCs at a ratio of 1∶2 (DC: T). T cell proliferation was measured by CFSE dilution (proliferating CD4⁺ cells have a lower CFSE intensity than non-proliferated control cells). Cultures without antigen served as controls. The bar graph shows the percentage of proliferating (CFSE ^low) T cells among the total CD4⁺ T cell population. Data are expressed as mean ± SEM of four mice/group and represent one of two independent experiments (<b>C</b>).</p

HMPV-induced clinical disease, lung function and viral replication in the absence of TLR4.

<p>TLR4^−/− and wild type (WT) mice were either infected with hMPV (live or UV inactivated) or mock infected. Change in body weight was measured over a period of 12 days. Body weight is expressed as percentage of baseline weight (<b>A</b>). Baseline Penh values were measured over a period of 21 days; post-methacholine challenge airway hyperresponsiveness and airway resistance were measured at day 14 p.i. (<b>B</b>). TLR4^−/− and WT mice were infected with hMPV and sacrificed at days 3, 4, 5 and 7 p.i. to determine viral titers by TCID₅₀ assay. Mice were challenged with hMPV (10⁷ pfu) 6 weeks after primary infection and lung viral titers were determined on day 4 p.i. (peak of viral replication). The lower limit of detection of this assay is 1.5 log₁₀/gram of tissue, represented by the dotted line (<b>C</b>). Data are expressed as mean ± SEM of four to six animals/group. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078849#pone-0078849-g001" target="_blank">Figure 1A and B</a> represents one of three independent experiments and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078849#pone-0078849-g001" target="_blank">Figure 1C</a> represents cumulative data from three independent experiments. *<i>P</i><0.05 when comparing hMPV infected TLR4^−/− mice to WT mice.</p