Pattern recognition molecule mindin promotes intranasal clearance of influenza viruses

The innate immune response is essential for host defense against microbial pathogen infections and is mediated by pattern recognition molecules recognizing pathogen-associated molecular patterns. Our previous work has demonstrated that the extracellular matrix protein mindin functions as a pattern recognition molecule for bacterial pathogens. In this study, we examined the role of mindin in influenza virus infection. We found that intranasal infection of mindin-deficient mice by influenza virus resulted in dramatically increased virus titers in the lung and intranasal cavity of mutant mice. In contrast, lungs from intratracheally infected mindin-deficient mice contained similar influenza virus titers. We showed that mindin interacted with influenza virus particles directly and that mindin-deficient macrophages exhibited impaired activation after influenza virus infection in vitro. Furthermore, intranasal administration of recombinant mindin significantly enhanced the clearance of influenza virus in wild-type mice. Together, these results demonstrate that mindin plays an essential role in the host innate immune response to influenza virus infection and suggest that mindin may be used as an immune-enhancing agent in influenza infection.     

Increased MDSC accumulation and Th2 biased response to influenza A virus infection in the absence of TLR7 in mice

Toll-like receptors (TLRs) play an important role in the induction of innate and adaptive immune response against influenza A virus (IAV) infection; however, the role of Toll-like receptor 7 (TLR7) during the innate immune response to IAV infection and the cell types affected by the absence of TLR7 are not clearly understood. In this study, we show that myeloid derived suppressor cells (MDSC) accumulate in the lungs of TLR7 deficient mice more so than in wild-type C57Bl/6 mice, and display increased cytokine expression. Furthermore, there is an increase in production of Th2 cytokines by TLR7(-/-) compared with wildtype CD4+ T-cells in vivo, leading to a Th2 polarized humoral response. Our findings indicate that TLR7 modulates the accumulation of MDSCs during an IAV infection in mice, and that lack of TLR7 signaling leads to a Th2-biased response.     

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.     

Antiviral activity of the long chain pentraxin PTX3 against influenza viruses

Proteins of the innate immune system can act as natural inhibitors of influenza virus, limiting growth and spread of the virus in the early stages of infection before the induction of adaptive immune responses. In this study, we identify the long pentraxin PTX3 as a potent innate inhibitor of influenza viruses both in vitro and in vivo. Human and murine PTX3 bound to influenza virus and mediated a range of antiviral activities, including inhibition of hemagglutination, neutralization of virus infectivity and inhibition of viral neuraminidase. Antiviral activity was associated with binding of the viral hemagglutinin glycoprotein to sialylated ligands present on PTX3. Using a mouse model we found PTX3 to be rapidly induced following influenza infection and that PTX3-/- mice were more susceptible than wild-type mice to infection by PTX3-sensitive virus strains. Therapeutic treatment of mice with human PTX3 promoted survival and reduced viral load in the lungs following infection with PTX3-sensitive, but not PTX3-resistant, influenza viruses. Together, these studies describe a novel antiviral role for PTX3 in early host defense against influenza infections both in vitro and in vivo and describe the therapeutic potential of PTX3 in ameliorating disease during influenza infection.     

TLR7 Recognition Is Dispensable for Influenza Virus A Infection but Important for the Induction of Hemagglutinin-Specific Antibodies in Response to the 2009 Pandemic Split Vaccine in Mice

Recognition of pathogen-associated molecular patterns by pattern recognition receptors of the innate immune system is crucial for the initiation of innate and adaptive responses and for immunological memory. We investigated the role of TLR7 in the induction of adaptive immunity and long-term memory following influenza virus infection and vaccination in C57BL/6 mice. During infection with influenza A/PR8/34 virus, the absence of either TLR7 or MyD88 leads to reduced virus-specific antibodies in the serum and antibody-secreting cells in their secondary lymphoid organs, particularly in bone marrow. In spite of this, the absence of TLR7/MyD88 signaling did not impair the production of protective antibodies. Following immunization with the 2009 pandemic inactivated split vaccine, TLR7(-/-) mice had significantly lower levels of germinal center formation, antibody-secreting cells, and circulating influenza virus-specific antibodies than control animals. Consequently, TLR7(-/-) mice failed to develop protective immunological memory upon challenge. Furthermore, the immunogenicity of the split vaccine was likely due to TLR7 recognition of virion RNA, as its removal from the split vaccine significantly reduced the levels of influenza virus-specific antibodies and compromised the vaccine protective efficacy in mice. Taken together, our data demonstrate that TLR7 plays an important role in vaccine-induced humoral immune responses to influenza virus through the interaction with viral RNA present in the split vaccine.     

H5N1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice

Fatal human respiratory disease associated with the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is characterized by severe lung pathology, including pulmonary edema and extensive inflammatory infiltrate. Here, we quantified the cellular immune response to infection in the mouse lung by flow cytometry and demonstrate that mice infected with highly pathogenic (HP) H1N1 and H5N1 influenza viruses exhibit significantly high numbers of macrophages and neutrophils in the lungs compared to mice infected with low pathogenic (LP) viruses. Mice infected with the 1918 pandemic virus and a recent H5N1 human isolate show considerable similarities in overall lung cellularity, lung immune cell sub-population composition, and cellular immune temporal dynamics. Interestingly, while these similarities were observed, the HP H5N1 virus consistently elicited significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages, revealing a potentially critical difference in the pathogenesis of H5N1 infections. Primary mouse and human macrophages and dendritic cells were also susceptible to 1918 and H5N1 influenza virus infection in vitro. These results together indicate that infection with HP influenza viruses such as H5N1 and the 1918 pandemic virus leads to a rapid cell recruitment of macrophages and neutrophils into the lungs, suggesting that these cells play a role in acute lung inflammation associated with HP influenza virus infection.     

The TLR4-TRIF pathway protects against H5N1 influenza virus infection

Prestimulation of the TLR4 pathway with lipopolysaccharide (LPS) protects mice from lethal infection with H5N1 influenza virus. Here, we reveal that the TLR4-TRIF pathway is required for this protective effect by using mice whose TLR4-related molecules were knocked out. Microarray analysis of primary mouse lung culture cells that were LPS pretreated and infected with an H5N1 virus indicated that TLR3 mRNA was upregulated. Primary lung culture cells of TLR3 knockout mice showed no response to LPS pretreatment against H5N1 virus infection, suggesting that TLR3 is also involved in the preventive effect of LPS. Our data suggest that the TLR4-TRIF axis has an important role in stimulating protective innate immunity against H5N1 influenza A virus infection and that TLR3 signaling is involved in this pathway.     

Antiviral activity and increased host defense against influenza infection elicited by the human cathelicidin LL-37

The extensive world-wide morbidity and mortality caused by influenza A viruses highlights the need for new insights into the host immune response and novel treatment approaches. Cationic Host Defense Peptides (CHDP, also known as antimicrobial peptides), which include cathelicidins and defensins, are key components of the innate immune system that are upregulated during infection and inflammation. Cathelicidins have immunomodulatory and anti-viral effects, but their impact on influenza virus infection has not been previously assessed. We therefore evaluated the effect of cathelicidin peptides on disease caused by influenza A virus in mice. The human cathelicidin, LL-37, and the murine cathelicidin, mCRAMP, demonstrated significant anti-viral activity in vivo, reducing disease severity and viral replication in infected mice to a similar extent as the well-characterized influenza virus-specific antiviral drug zanamivir. In vitro and in vivo experiments suggested that the peptides may act directly on the influenza virion rather than via receptor-based mechanisms. Influenza virus-infected mice treated with LL-37 had lower concentrations of pro-inflammatory cytokines in the lung than did infected animals that had not been treated with cathelicidin peptides. These data suggest that treatment of influenza-infected individuals with cathelicidin-derived therapeutics, or modulation of endogenous cathelicidin production may provide significant protection against disease.     

Differential role of TLR- and RLR-signaling in the immune responses to influenza A virus infection and vaccination

The innate immune system recognizes influenza A virus via TLR 7 or retinoic acid-inducible gene I in a cell-type specific manner in vitro, however, physiological function(s) of the MyD88- or interferon-beta promoter stimulator 1 (IPS-1)-dependent signaling pathways in antiviral responses in vivo remain unclear. In this study, we show that although either MyD88- or IPS-1-signaling pathway was sufficient to control initial antiviral responses to intranasal influenza A virus infection, mice lacking both pathways failed to show antiviral responses, resulting in increased viral load in the lung. By contrast, induction of B cells or CD4 T cells specific to the dominant hemagglutinin or nuclear protein Ags respectively, was strictly dependent on MyD88 signaling, but not IPS-1 signaling, whereas induction of nuclear protein Ag-specific CD8 T cells was not impaired in the absence of either MyD88 or IPS-1. Moreover, vaccination of TLR7- and MyD88-deficient mice with inactivated virus failed to confer protection against a lethal live virus challenge. These results strongly suggest that either the MyD88 or IPS-1 signaling pathway is sufficient for initial antiviral responses, whereas the protective adaptive immune responses to influenza A virus are governed by the TLR7-MyD88 pathway.     

Local IL-17A Potentiates Early Neutrophil Recruitment to the Respiratory Tract during Severe RSV Infection

Respiratory syncytial virus (RSV) bronchiolitis triggers a strong innate immune response characterized by excessive neutrophil infiltration which contributes to RSV induced pathology. The cytokine IL-17A enhances neutrophil infiltration into virus infected lungs. IL-17A is however best known as an effector of adaptive immune responses. The role of IL-17A in early immune modulation in RSV infection is unknown. We aimed to elucidate whether local IL-17A facilitates the innate neutrophil infiltration into RSV infected lungs prior to adaptive immunity. To this end, we studied IL-17A production in newborns that were hospitalized for severe RSV bronchiolitis. In tracheal aspirates we measured IL-17A concentration and neutrophil counts. We utilized cultured human epithelial cells to test if IL-17A regulates RSV infection-induced IL-8 release as mediator of neutrophil recruitment. In mice we investigated the cell types that are responsible for early innate IL-17A production during RSV infection. Using IL-17A neutralizing antibodies we tested if IL-17A is responsible for innate neutrophil infiltration in mice. Our data show that increased IL-17A production in newborn RSV patient lungs correlates with subsequent neutrophil counts recruited to the lungs. IL-17A potentiates RSV-induced production of the neutrophil-attracting chemokine IL-8 by airway epithelial cells in vitro. Various lung-resident lymphocytes produced IL-17A during early RSV infection in Balb/c mice, of which a local population of CD4 T cells stood out as the predominant RSV-induced cell type. By removing IL-17A during early RSV infection in mice we showed that IL-17A is responsible for enhanced innate neutrophil infiltration in vivo. Using patient material, in vitro studies, and an animal model of RSV infection, we thus show that early local IL-17A production in the airways during RSV bronchiolitis facilitates neutrophil recruitment with pathologic consequences to infant lungs.     

Evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice

Influenza virus-infected cells undergo apoptosis and become susceptible to phagocytosis by macrophages in vitro, and this leads to the propagation of the virus being inhibited. We previously showed that inhibitors of phagocytosis increased the rate of mortality among influenza virus-infected mice. However, the mode of the phagocytosis of influenza virus-infected cells in vivo has not been investigated. We, in this study, assessed this issue by histochemically analyzing bronchoalveolar lavage cells and lung tissue obtained from C57BL/6 mice infected with influenza A/WSN (H1N1) virus. Both neutrophils and macrophages accumulated in the lung soon after the viral challenge, and either type of cell was capable of phagocytosing influenza virus-infected, apoptotic cells. Changes in the level of phagocytosis and the amount of virus in lung tissue roughly correlated with each other. Furthermore, alveolar macrophages prepared from influenza virus-infected mice showed greater phagocytic activity than those from uninfected mice. The phagocytic activity of macrophages was stimulated in vitro by a heat-labile substance(s) released from influenza virus-infected cells undergoing apoptosis. These results suggested that the level of phagocytosis is augmented both quantitatively and qualitatively in the lung of influenza virus-infected animals so that infected cells are effectively eliminated. Finally, lack of TLR4 caused an increase in the rate of mortality among influenza virus-challenged mice and a decrease in the level of phagocytosis of apoptotic cells in the lung. TLR4 could thus play an important role in the host defense against influenza by positively regulating the phagocytic elimination of infected cells.     

Detrimental contribution of the Toll-like receptor (TLR)3 to influenza A virus-induced acute pneumonia

Influenza A virus (IAV) is the etiological agent of a highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. Recently, we demonstrated, using an in vitro approach, that the pattern recognition Toll-like receptor (TLR)3 plays a key role in the immune response of lung epithelial cells to IAV. In view of these data and the fact that the functional role of TLR3 in vivo is still debated, we designed an investigation to better understand the role of TLR3 in the mechanisms of IAV pathogenesis and host immune response using an experimental murine model. The time-course of several dynamic parameters, including animal survival, respiratory suffering, viral clearance, leukocyte recruitment into the airspaces and secretion of critical inflammatory mediators, was compared in infected wild-type and TLR3(-/-) mice. First, we found that the pulmonary expression of TLR3 is constitutive and markedly upregulated following influenza infection in control mice. Notably, when compared to wild-type mice, infected TLR3-/- animals displayed significantly reduced inflammatory mediators, including RANTES (regulated upon activation, normal T cell expressed and secreted), interleukin-6, and interleukin-12p40/p70 as well as a lower number of CD8+ T lymphocytes in the bronchoalveolar airspace. More important, despite a higher viral production in the lungs, mice deficient in TLR3 had an unexpected survival advantage. Hence, to our knowledge, our findings show for the first time that TLR3-IAV interaction critically contributes to the debilitating effects of a detrimental host inflammatory response.     

Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury

Multiple lung pathogens such as chemical agents, H5N1 avian flu, or SARS cause high lethality due to acute respiratory distress syndrome. Here we report that Toll-like receptor 4 (TLR4) mutant mice display natural resistance to acid-induced acute lung injury (ALI). We show that TLR4-TRIF-TRAF6 signaling is a key disease pathway that controls the severity of ALI. The oxidized phospholipid (OxPL) OxPAPC was identified to induce lung injury and cytokine production by lung macrophages via TLR4-TRIF. We observed OxPL production in the lungs of humans and animals infected with SARS, Anthrax, or H5N1. Pulmonary challenge with an inactivated H5N1 avian influenza virus rapidly induces ALI and OxPL formation in mice. Loss of TLR4 or TRIF expression protects mice from H5N1-induced ALI. Moreover, deletion of ncf1, which controls ROS production, improves the severity of H5N1-mediated ALI. Our data identify oxidative stress and innate immunity as key lung injury pathways that control the severity of ALI.     

Bordetella pertussis infection exacerbates influenza virus infection through pertussis toxin-mediated suppression of innate immunity

Pertussis (whooping cough) is frequently complicated by concomitant infections with respiratory viruses. Here we report the effect of Bordetella pertussis infection on subsequent influenza virus (PR8) infection in mouse models and the role of pertussis toxin (PT) in this effect. BALB/c mice infected with a wild-type strain of B. pertussis (WT) and subsequently (up to 14 days later) infected with PR8 had significantly increased pulmonary viral titers, lung pathology and mortality compared to mice similarly infected with a PT-deficient mutant strain (ΔPT) and PR8. Substitution of WT infection by intranasal treatment with purified active PT was sufficient to replicate the exacerbating effects on PR8 infection in BALB/c and C57/BL6 mice, but the effects of PT were lost when toxin was administered 24 h after virus inoculation. PT had no effect on virus titers in primary cultures of murine tracheal epithelial cells (mTECs) in vitro, suggesting the toxin targets an early immune response to increase viral titers in the mouse model. However, type I interferon responses were not affected by PT. Whole genome microarray analysis of gene expression in lung tissue from PT-treated and control PR8-infected mice at 12 and 36 h post-virus inoculation revealed that PT treatment suppressed numerous genes associated with communication between innate and adaptive immune responses. In mice depleted of alveolar macrophages, increase of pulmonary viral titers by PT treatment was lost. PT also suppressed levels of IL-1β, IL-12, IFN-γ, IL-6, KC, MCP-1 and TNF-α in the airways after PR8 infection. Furthermore PT treatment inhibited early recruitment of neutrophils and NK cells to the airways. Together these findings demonstrate that infection with B. pertussis through PT activity predisposes the host to exacerbated influenza infection by countering protective innate immune responses that control virus titers.     

Galectin-1 binds to influenza virus and ameliorates influenza virus pathogenesis

Innate immune response is important for viral clearance during influenza virus infection. Galectin-1, which belongs to S-type lectins, contains a conserved carbohydrate recognition domain that recognizes galactose-containing oligosaccharides. Since the envelope proteins of influenza virus are highly glycosylated, we studied the role of galectin-1 in influenza virus infection in vitro and in mice. We found that galectin-1 was upregulated in the lungs of mice during influenza virus infection. There was a positive correlation between galectin-1 levels and viral loads during the acute phase of viral infection. Cells treated with recombinant human galectin-1 generated lower viral yields after influenza virus infection. Galectin-1 could directly bind to the envelope glycoproteins of influenza A/WSN/33 virus and inhibit its hemagglutination activity and infectivity. It also bound to different subtypes of influenza A virus with micromolar dissociation constant (K(d)) values and protected cells against influenza virus-induced cell death. We used nanoparticle, surface plasmon resonance analysis and transmission electron microscopy to further demonstrate the direct binding of galectin-1 to influenza virus. More importantly, we show for the first time that intranasal treatment of galectin-1 could enhance survival of mice against lethal challenge with influenza virus by reducing viral load, inflammation, and apoptosis in the lung. Furthermore, galectin-1 knockout mice were more susceptible to influenza virus infection than wild-type mice. Collectively, our results indicate that galectin-1 has anti-influenza virus activity by binding to viral surface and inhibiting its infectivity. Thus, galectin-1 may be further explored as a novel therapeutic agent for influenza.     

Innate immune function of the adherens junction protein p120-catenin in endothelial response to endotoxin

Sepsis-induced acute lung injury is a common clinical disorder in critically ill patients that is associated with high mortality. In this study, we investigated the role of p120-catenin (p120), a constituent of endothelial adherens junctions, in regulating the innate immune function of lungs. In mice in which acute lung injury was induced by i.p. administration of LPS, we observed a rapid decrease in the expression of p120 in lungs. The p120 protein expression was correlated inversely with severity of inflammation. Suppression of p120 expression in lung endothelial cells in mice using small interfering RNA resulted in high sensitivity to endotoxin and greatly increased the mortality compared with controls. Knockdown of p120 also increased the expression of ICAM-1, neutrophil recruitment, production of cytokines TNF-α and IL-6, pulmonary transvascular protein permeability, and lung water content in response to LPS. We demonstrated that endothelial p120 modulates lung innate immune function by interfering with the association of TLR4 with its adaptor MyD88 to block TLR4 signaling and NF-κB activation in endothelial cells. In conclusion, these studies have uncovered a novel innate immune function of endothelial p120 in downregulating the lung inflammatory response to endotoxin through the suppression of TLR4 signaling.     

Surfactant protein D enhances clearance of influenza A virus from the lung in vivo.

Mice lacking surfactant protein surfactant protein D (SP-D(-/-)) and wild-type mice (SP-D(+/+)) were infected with influenza A virus (IAV) by intranasal instillation. IAV infection increased the endogenous SP-D concentration in wild-type mice. SP-D-deficient mice showed decreased viral clearance of the Phil/82 strain of IAV and increased production of inflammatory cytokines in response to viral challenge. However, the less glycosylated strain of IAV, Mem/71, which is relatively resistant to SP-D in vitro, was cleared efficiently from the lungs of SP-D(-/-) mice. Viral clearance of the Phil/82 strain of IAV and the cytokine response were both normalized by the coadministration of recombinant SP-D. Since the airway is the usual portal of entry for influenza A virus and other respiratory pathogens, SP-D is likely to play an important role in innate defense responses to IAV.     

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20^AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20^AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20^AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20^AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20^AEC-KO mice during later stages of infection. When A20^AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20^AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.     

Moraxella catarrhalis activates murine macrophages through multiple toll like receptors and has reduced clearance in lungs from TLR4 mutant mice

Moraxella catarrhalis is a gram negative bacterium and a leading causative agent of otitis media (OM) in children. Several recent reports have provided strong evidence for an association between toll like receptors and OM. It has been found that both Streptococcus pneumoniae and nontypeable Haemophilus influenzae activate host protective immune responses through toll like receptors (TLRs), however, the precise mechanism by which Moraxella catarrhalis initiates the host immune response is currently unknown. In this report, using murine macrophages generated from a series of knock-out mice, we have demonstrated that M. catarrhalis lipooligosaccharide (LOS) and either heat killed or live bacteria are recognized by one or more TLRs. LOS activates the host immune response through a membrane bound CD14-TLR4 complex, while both heat killed and live M.cat require recognition by multiple toll like receptors such as TLR2, TLR4 and TLR9 without the requirement of CD14. We have also shown that M.cat stimuli are capable of triggering the host innate immune response by both MyD88- and TRIF- dependent signaling pathways. We further showed that M.cat induced activation of mitogen activated protein kinase (MAPK) is essential in order to achieve optimal secretion of pro-inflammatory cytokine TNF-α. We finally showed that TLR4 mutant C3H/HeJ mice produce significantly lower levels of pro-inflammatory cytokines TNF-α and IL-6 in vivo, An increased bacterial loads at 12 and 24 hours (P<0.001) in their lungs upon challenge with live M.cat in an aerosol chamber compared to wild-type (WT) control mice. These data suggest that TLRs are crucial for an effective innate immune response induced by M.cat. The results of these studies contribute to an increased understanding of molecular mechanism and possible novel treatment strategies for diseases caused by M.cat by specifically targeting TLRs and their signaling pathways.