Host-Viral Interactions: Role of Pattern Recognition Receptors (PRRs) in Human Pneumovirus Infections

pathogen

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

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

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

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

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

Induction of hMPV-specific neutralizing antibodies in the absence of TLR4.

<p>TLR4^−/− and WT mice were infected with hMPV and sacrificed weekly to determine antibody titers by a plaque reduction neutralization assay. The lower detection limit for this assay is 2 log₂ serum dilution. At 6 weeks p.i. mice were challenged with hMPV and neutralizing antibody titers were determined 1 week later. Data are expressed as mean of four mice/group and represents one of two independent experiments.</p