The Human Metapneumovirus Matrix Protein Stimulates the Inflammatory Immune Response In Vitro

Each year, during winter months, human Metapneumovirus (hMPV) is associated with epidemics of bronchiolitis resulting in the hospitalization of many infants. Bronchiolitis is an acute illness of the lower respiratory tract with a consequent inflammation of the bronchioles. The rapid onset of inflammation suggests the innate immune response may have a role to play in the pathogenesis of this hMPV infection. Since, the matrix protein is one of the most abundant proteins in the Paramyxoviridae family virion, we hypothesized that the inflammatory modulation observed in hMPV infected patients may be partly associated with the matrix protein (M-hMPV) response. By western blot analysis, we detected a soluble form of M-hMPV released from hMPV infected cell as well as from M-hMPV transfected HEK 293T cells suggesting that M-hMPV may be directly in contact with antigen presenting cells (APCs) during the course of infection. Moreover, flow cytometry and confocal microscopy allowed determining that M-hMPV was taken up by dendritic cells (moDCs) and macrophages inducing their activation. Furthermore, these moDCs enter into a maturation process inducing the secretion of a broad range of inflammatory cytokines when exposed to M-hMPV. Additionally, M-hMPV activated DCs were shown to stimulate IL-2 and IFN-γ production by allogeneic T lymphocytes. This M-hMPV-mediated activation and antigen presentation of APCs may in part explain the marked inflammatory immune response observed in pathology induced by hMPV in patients

MoDCs and MDMs treated with M-hMPV protein produce high level of cytokines and chemokines respectively.

<p>(A) Supernatants of cells treated for 24 h with elution buffer, LPS 10 ng/mL (positive control) and M-hMPV 0.172, 0.086, 0.034, 0.0172 nM were assayed for the level of IL-8, IL-6, TNF, IL-12p70, IL-1β and IL-10 cytokines by CBA. (B) Supernatant of MDMs treated with elution buffer, LPS 10 ng/mL (positive control), M-hMPV 0.172 and 0.0172 nM were assayed for MIP-1β, RANTES and TNF production by CBA. Bar graph represents concentrations values expressed in ng/mL. Data are represented as means of three experiments ± S.D.</p

M-hMPV treated-MDMs release low level of IFN-α and IFN-β.

<p>MDMs were treated with elution buffer, LPS 10 ng/mL, M-hMPV 0.172 and 0.0172 nM. Supernatants were collected after 24 h and tested for IFN-α and IFN-β production by ELISA. Bar graph represents concentrations values expressed in pg/mL. Data are represented as means of three experiments ± S.D.</p

Marburgvirus Hijacks Nrf2-Dependent Pathway by Targeting Nrf2-Negative Regulator Keap1

Marburg virus (MARV) has a high fatality rate in humans, causing hemorrhagic fever characterized by massive viral replication and dysregulated inflammation. Here, we demonstrate that VP24 of MARV binds Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of nuclear transcription factor erythroid-derived 2 (Nrf2). Binding of VP24 to Keap1 Kelch domain releases Nrf2 from Keap1-mediated inhibition promoting persistent activation of a panoply of cytoprotective genes implicated in cellular responses to oxidative stress and regulation of inflammatory responses. Increased expression of Nrf2-dependent genes was demonstrated both during MARV infection and upon ectopic expression of MARV VP24. We also show that Nrf2-deficient mice can control MARV infection when compared to lethal infection in wild-type animals, indicating that Nrf2 is critical for MARV infection. We conclude that VP24-driven activation of the Nrf2-dependent pathway is likely to contribute to dysregulation of host antiviral inflammatory responses and that it ensures survival of MARV-infected cells despite these responses

Percentage of apoptotic M-hMPV-treated moDCs.

<p>After 24 h of stimulation, M-hMPV-treated moDCs were labeled by DiOC6(3) and propidium iodide prior analysis by flow cytometry. Data are representative of one out of two independent experiments.</p

M-hMPV activated-DCs present antigen to T cells which induce the production of IFN-γ.

<p>moDCs were treated for 24 h with LPS 10 ng/mL and M-hMPV protein 0.344, 0.172, 0.086, 0.034 nM. Cells were harvested after treatment, washed and cultured for 5 days with allogeneic purified T-cells (2×10⁵/well) at a DC/T ratio ranging between 1∶5 and 1∶40. The amount of IFN-γ in the cell-free supernatants of the co-culture was measured by ELISA. Data are represented as means of three experiments ± S.D.</p

M-hMPV bacterial recombinant protein is successfully produced.

<p>(A) SDS-PAGE analysis of M-hMPV recombinant protein was performed after purification by Ni-NTA resin. (B) M-hMPV protein was analyzed by western blot using M-hMPV 1 µg/mL monoclonal antibodies and HCV monoclonal antibody for the negative control together with species-specific secondary antibody goat anti-mouse IgG H+L labeled with alkaline phosphatase.</p

M-hMPV protein binds APCs and is internalized.

<p>(A) Binding experiment. APCs were incubated with increased concentrations of M-hMPV-Fluorescein protein for 20 min at 4°C, washed and then analyzed by FACS. Data are shown as mean fluorescence intensity. (B) Competition experiment. APCs were incubated with M-hMPV-Fluorescein protein 4.1 nM for 20 min at 4°C, washed before addition of increased concentration of unlabeled M-hMPV protein for 20 min at 4°C. Results are shown as % of mean fluorescent intensity of M-hMPV-Fluorescein protein binding. (C) Internalization experiment. Cells were incubated with 1.3 nM M-hMPV-Fluorescein protein during 20 min at 4°C, washed and incubated at different time points at 37°C. Cells were then washed and analyzed. Data are shown as mean fluorescence intensity. (D) Confocal microscopy. Cells were incubated with 0.17 nM M-hMPV protein 30 min at 4°C and then washed and incubated or not at 37°C for 30 min. Observation was realized by confocal microscopy. Data represent one out of two experiments.</p

M-hMPV protein induces maturation of moDCs.

<p>moDCs were treated with M-hMPV protein 0.172 nM (gray filled) and 0.0172 nM (green line), LPS 10 ng/mL (black line), M-hMPV 0.172 nM+polymyxin (orange line), M-hMPV heat treated at 100°C–20 min (pink dotted line) or untreated (iDCs) (gray line) and the phenotype was analyzed after 24 h of treatment. Cells were stained with labeled CD80, CD83, CD86 and CD40 antibodies. Data were acquired by a FACScan and analyzed by CellQuest Pro software. Histograms represent mean fluorescence intensity. Data shown are representative of one out of five independent experiments.</p