Antonieta Guerrero-Plata

Activity and Regulation of Alpha Interferon in Respiratory Syncytial Virus and Human Metapneumovirus Experimental Infections

ABSTRACT Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause a similar spectrum of respiratory infections in humans. Classified within the Paramyxoviridae family, Pneumovirinae subfamily, RSV and hMPV present a significant degree of divergence in genome constellation, organization, and protein sequences. RSV has been reported to be a poor inducer of alpha/beta interferons (IFN-α/β) and partially resistant to its antiviral activity. The nature of the innate immune response to hMPV is currently unknown. Herein, an experimental mouse model was used to investigate the interplay between RSV and hMPV infections and IFN-α in the airways. RSV-infected BALB/c mice treated intranasally with either poly-ICLC, a potent inducer of IFN-α, or directly with recombinant IFN-α showed significantly reduced lung viral titers, inflammation, and clinical disease than untreated controls. However, RSV was significantly less sensitive to the antiviral activity of IFN-α than hMPV. Similarly, when the ability to directly induce IFN-α production was assessed, RSV was clearly a weaker inducer of IFN-α than hMPV, as shown by both kinetics and the absolute amount of IFN-α secreted into the bronchoalveolar lavage. To further investigate the putative inhibitory effect of these viruses on IFN-α production, mice were infected for 48 h prior to treatment with poly-ICLC or a specific Toll-like receptor 9 ligand, CpG oligodeoxynucleotides. Strikingly, both poly-ICLC- and CpG-mediated IFN-α production was abrogated by either RSV or MPV infection. These results suggest that a complex interplay between virus-specific and host-mediated responses regulates IFN-α in the lung during infection by members of the Pneumovirinae family.

Human Metapneumovirus Induces a Profile of Lung Cytokines Distinct from That of Respiratory Syncytial Virus

ABSTRACT Lung cytokine and chemokine production by BALB/c mice infected with human metapneumovirus (hMPV) was compared to respiratory syncytial virus (RSV)-infected mice. hMPV infection induced lower levels of the inflammatory cytokines interleukin-1 (IL-1), IL-6 and tumor necrosis factor alpha but was a more potent inducer of granulocyte-macrophage colony-stimulating factor and triggered a more sustained production of the CXC chemokine KC compared to RSV. hMPV was a stronger inducer of both alpha interferon (IFN-α) and IFN-γ responses than RSV. In regard to immunomodulatory cytokines, hMPV failed to induce detectable IL-10 or IL-12p70 but was a potent inducer of IL-12 p40 subunit. The implications for hMPV pathogenesis are discussed.

Differential Activation of Human Monocyte-Derived and Plasmacytoid Dendritic Cells by West Nile Virus Generated in Different Host Cells

ABSTRACT Dendritic cells (DCs) play a central role in innate immunity and antiviral responses. In this study, we investigated the production of alpha interferon (IFN-α) and inducible chemokines by human monocyte-derived dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) infected with West Nile virus (WNV), an emergent pathogen whose infection can lead to severe cases of encephalitis in the elderly, children, and immunocompromised individuals. Our experiments demonstrated that WNV grown in mammalian cells (WNVVero) was a potent inducer of IFN-α secretion in pDCs and, to a lesser degree, in mDCs. The ability of WNVVero to induce IFN-α in pDCs did not require viral replication and was prevented by the treatment of cells with bafilomycin A1 and chloroquine, suggesting that it was dependent on endosomal Toll-like receptor recognition. On the other hand, IFN-α production in mDCs required viral replication and was associated with the nuclear translocation of IRF3 and viral antigen expression. Strikingly, pDCs failed to produce IFN-α when stimulated with WNV grown in mosquito cells (WNVC7/10), while mDCs responded similarly to WNVVero or WNVC7/10. Moreover, the IFN-dependent chemokine IP-10 was produced in substantial amounts by pDCs in response to WNVVero but not WNVC7/10, while interleukin-8 was produced in greater amounts by mDCs infected with WNVC7/10 than in those infected with WNVVero. These findings suggest that cell-specific mechanisms of WNV recognition leading to the production of type I IFN and inflammatory chemokines by DCs may contribute to both the innate immune response and disease pathogenesis in human infections.

Monomeric and Dimeric CXCL8 Are Both Essential for In Vivo Neutrophil Recruitment

Rapid mobilization of neutrophils from vasculature to the site of bacterial/viral infections and tissue injury is a critical step in successful resolution of inflammation. The chemokine CXCL8 plays a central role in recruiting neutrophils. A characteristic feature of CXCL8 is its ability to reversibly exist as both monomers and dimers, but whether both forms exist in vivo, and if so, the relevance of each form for in vivo function is not known. In this study, using a ‘trapped’ non-associating monomer and a non-dissociating dimer, we show that (i) wild type (WT) CXCL8 exists as both monomers and dimers, (ii) the in vivo recruitment profiles of the monomer, dimer, and WT are distinctly different, and (iii) the dimer is essential for initial robust recruitment and the WT is most active for sustained recruitment. Using a microfluidic device, we also observe that recruitment is not only dependent on the total amount of CXCL8 but also on the steepness of the gradient, and the gradients created by different CXCL8 variants elicit different neutrophil migratory responses. CXCL8 mediates its function by binding to CXCR2 receptor on neutrophils and glycosaminoglycans (GAGs) on endothelial cells. On the basis of our data, we propose that dynamic equilibrium between CXCL8 monomers and dimers and their differential binding to CXCR2 and GAGs mediates and regulates in vivo neutrophil recruitment. Our finding that both CXCL8 monomer and dimer are functional in vivo is novel, and indicates that the CXCL8 monomer-dimer equilibrium and neutrophil recruitment are intimately linked in health and disease.

Subversion of Pulmonary Dendritic Cell Function by Paramyxovirus Infections

Lower respiratory tract infections caused by the paramyxoviruses human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are characterized by short-lasting virus-specific immunity and often long-term airway morbidity, both of which may be the result of alterations in the Ag-presenting function of the lung which follow these infections. In this study, we investigated whether hMPV and RSV experimental infections alter the phenotype and function of dendritic cell (DC) subsets that are recruited to the lung. Characterization of lung DC trafficking demonstrated a differential recruitment of plasmacytoid DC (pDC), conventional DC (cDC), and IFN-producing killer DC to the lung and draining lymph nodes after hMPV and RSV infection. In vitro infection of lung DC indicated that in pDC, production of IFN-α, TNF-α, and CCL5 was induced only by hMPV, whereas CCL3 and CCL4 were induced by both viruses. In cDC, a similar repertoire of cytokines was induced by hMPV and RSV, except for IFN-β, which was not induced by RSV. The function of lung pDC was altered following hMPV or RSV infection in vivo, as we demonstrated a reduced capacity of lung pDC to produce IFN-α as well as other cytokines including IL-6, TNF-α, CCL2, CCL3, and CCL4 in response to TLR9 stimulation. Moreover, we observed an impaired capacity of cDC from infected mice to present Ag to CD4+ T cells, an effect that lasted beyond the acute phase of infection. Our findings suggest that acute paramyxovirus infections can alter the long-term immune function of pulmonary DC.

T Lymphocytes Contribute to Antiviral Immunity and Pathogenesis in Experimental Human Metapneumovirus Infection

ABSTRACT Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is a leading cause of lower respiratory tract infections in children, the elderly, and immunocompromised patients. Virus- and host-specific mechanisms of pathogenesis and immune protection are not fully understood. By an intranasal inoculation model, we show that hMPV-infected BALB/c mice developed clinical disease, including airway obstruction and hyperresponsiveness (AHR), along with histopathologic evidence of lung inflammation and viral replication. hMPV infection protected mice against subsequent viral challenge, as demonstrated by undetectable viral titers, lack of body weight loss, and a significant reduction in the level of lung inflammation. No cross-protection with other paramyxoviruses, such as respiratory syncytial virus, was observed. T-lymphocyte depletion studies showed that CD4+ and CD8+ T cells cooperate synergistically in hMPV eradication during primary infection, but CD4+ more than CD8+ T cells also enhanced clinical disease and lung pathology. Concurrent depletion of CD4+ and CD8+ T cells completely blocked airway obstruction as well as AHR. Despite impaired generation of neutralizing anti-hMPV antibodies in the absence of CD4+ T cells, mice had undetectable viral replication after hMPV challenge and were protected from clinical disease, suggesting that protection can be provided by an intact CD8+ T-cell compartment. Whether these findings have implications for naturally acquired human infections remains to be determined.

The monomer‐dimer equilibrium and glycosaminoglycan interactions of chemokine CXCL8 regulate tissue‐specific neutrophil recruitment

Chemokines exert their function by binding the GPCR class of receptors on leukocytes and cell surface GAGs in target tissues. Most chemokines reversibly exist as monomers and dimers, but very little is known regarding the molecular mechanisms by which the monomer‐dimer equilibrium modulates in vivo function. For the chemokine CXCL8, we recently showed in a mouse lung model that monomers and dimers are active and that the monomer‐dimer equilibrium of the WT plays a crucial role in regulating neutrophil recruitment. In this study, we show that monomers and dimers are also active in the mouse peritoneum but that the role of monomer‐dimer equilibrium is distinctly different between these tissues and that mutations in GAG‐binding residues render CXCL8 less active in the peritoneum but more active in the lung. We propose that tissue‐specific differences in chemokine gradient formation, resulting from tissue‐specific differences in GAG interactions, are responsible for the observed differences in neutrophil recruitment. Our observation of differential roles played by the CXCL8 monomer‐dimer equilibrium and GAG interactions in different tissues is novel and reveals an additional level of complexity of how chemokine dimerization regulates in vivo recruitment.

Critical Role of MDA5 in the Interferon Response Induced by Human Metapneumovirus Infection in Dendritic Cells and In Vivo

ABSTRACT Human metapneumovirus (hMPV) is a respiratory paramyxovirus of global clinical relevance. Despite the substantial knowledge generated during the last 10 years about hMPV infection, information regarding the activation of the immune response against this virus remains largely unknown. In this study, we demonstrated that the helicase melanoma differentiation-associated gene 5 (MDA5) is essential to induce the interferon response after hMPV infection in human and mouse dendritic cells as well as in an experimental mouse model of infection. Our findings in vitro and in vivo showed that MDA5 is required for the expression and activation of interferon (IFN) regulatory factors (IRFs). hMPV infection induces activation of IRF-3, and it regulates the expression of IRF-7. However, both IRF-3 and IRF-7 are critical for the production of type I and type III IFNs. In addition, our in vivo studies in hMPV-infected mice indicated that MDA5 alters viral clearance, enhances disease severity and pulmonary inflammation, and regulates the production of cytokines and chemokines in response to hMPV. These findings are relevant for a better understanding of the pathogenesis of hMPV infection.

Cigarette Smoke Condensate Enhances Respiratory Syncytial Virus–Induced Chemokine Release by Modulating NF-kappa B and Interferon Regulatory Factor Activation

Exposure to cigarette smoke is a risk factor contributing to the severity of respiratory tract infections associated with respiratory syncytial virus (RSV). Stimulation of airway epithelial cells by either RSV or cigarette smoke condensate (CSC) has been shown to induce secretion of the proinflammatory chemokines. However, the effect of coexposure of airway epithelial cells to CSC and RSV on inducible chemokine production has not been previously investigated. The results of this study indicate that CSC costimulation significantly increased RSV-induced interleukin-8 (IL-8) and monocyte chemoattactant protein-1 gene and protein expression when compared with each stimulus alone. Promoter deletion studies identified the interferon stimulatory response element (ISRE) of the IL-8 promoter as a critical region responsible for the synergistic increase of IL-8 gene transcription during mixed exposure. CSC costimulation enhanced RSV-induced activation of interferon regulatory factor (IRF)-1 and IRF-7, which bind to the ISRE site. CSC also furthered RSV-induced activation of the transcription factor nuclear factor kappa B (NF-kappaB), as shown by increased NF-kappaB DNA binding to its specific site of the IL-8 promoter and increased NF-kappaB-driven gene transcription. Therefore, our data demonstrate that a combined exposure to CSC and RSV synergistically increases chemokine expression in airway epithelial cells, suggesting that CSC contributes to an exuberant immune response to RSV by stimulating overlapping signal transduction pathways.

Impact and Regulation of Lambda Interferon Response in Human Metapneumovirus Infection

ABSTRACT Human metapneumovirus (hMPV) is a respiratory paramyxovirus that is distributed worldwide and induces significant airway morbidity. Despite the relevance of hMPV as a pathogen, many aspects of the immune response to this virus are still largely unknown. In this report, we focus on the antiviral immune response, which is critical for viral clearance and disease resolution. Using in vitro and in vivo systems, we show that hMPV is able to induce expression of lambda interferon 1 (IFN-λ1), IFN-λ2, IFN-λ3, and IFN-λ4. The induction of IFN-λ expression by hMPV was dependent on interferon regulatory factor 7 (IRF-7) expression but not on IRF-3 expression. Treatment of hMPV-infected mice with IFN-λ reduced the disease severity, lung viral titer, and inflammatory response in the lung. Moreover, the IFN-λ response induced by the virus was regulated by the expression of the hMPV G protein. These results show that type III interferons (IFN-λs) play a critical protective role in hMPV infection. IMPORTANCE Human metapneumovirus (hMPV) is a pathogen of worldwide importance. Despite the relevance of hMPV as a pathogen, critical aspects of the immune response induced by this virus remain unidentified. Interferons (IFNs), including IFN-λ, the newest addition to the interferon family, constitute an indispensable part of the innate immune response. Here, we demonstrated that IFN-λ exhibited a protective role in hMPV infection in vitro and in an experimental mouse model of infection.