Pharmacologic Inhibition of COX-1 and COX-2 in Influenza A Viral Infection in Mice

Background

We previously demonstrated that cyclooxygenase (COX)-1 deficiency results in greater morbidity and inflammation, whereas COX-2 deficiency leads to reduced morbidity, inflammation and mortality in influenza infected mice.

Methodology/Principal Findings

We investigated the effects of COX-1 and COX-2 inhibitors in influenza A viral infection. Mice were given a COX-1 inhibitor (SC-560), a COX-2 inhibitor (celecoxib) or no inhibitor beginning 2 weeks prior to influenza A viral infection (200 PFU) and throughout the course of the experiment. Body weight and temperature were measured daily as indicators of morbidity. Animals were sacrificed on days 1 and 4 post-infection and bronchoalveolar lavage (BAL) fluid was collected or daily mortality was recorded up to 2 weeks post-infection. Treatment with SC-560 significantly increased mortality and was associated with profound hypothermia and greater weight loss compared to celecoxib or control groups. On day 4 of infection, BAL fluid cells were modestly elevated in celecoxib treated mice compared to SC-560 or control groups. Viral titres were similar between treatment groups. Levels of TNF-α and G-CSF were significantly attenuated in the SC-560 and celecoxib groups versus control and IL-6 levels were significantly lower in BAL fluid of celecoxib treated mice versus control and versus the SC-560 group. The chemokine KC was significantly lower in SC-560 group versus control.

Conclusions/Significance

Treatment with a COX-1 inhibitor during influenza A viral infection is detrimental to the host whereas inhibition of COX-2 does not significantly modulate disease severity. COX-1 plays a critical role in controlling the thermoregulatory response to influenza A viral infection in mice.     

Avian Influenza A H7N9 Virus Induces Severe Pneumonia in Mice without Prior Adaptation and Responds to a Combination of Zanamivir and COX-2 Inhibitor

Background

Human infection caused by the avian influenza A H7N9 virus has a case-fatality rate of over 30%. Systematic study of the pathogenesis of avian H7N9 isolate and effective therapeutic strategies are needed.

Methods

BALB/c mice were inoculated intranasally with an H7N9 virus isolated from a chicken in a wet market epidemiologically linked to a fatal human case, (A/chicken/Zhejiang/DTID-ZJU01/2013 [CK1]), and with an H7N9 virus isolated from a human (A/Anhui/01/2013 [AH1]). The pulmonary viral loads, cytokine/chemokine profiles and histopathological changes of the infected mice were compared. The therapeutic efficacy of a non-steroidal anti-inflammatory drug (NSAID), celecoxib, was assessed.

Results

Without prior adaptation, intranasal inoculation of 10⁶ plaque forming units (PFUs) of CK1 caused a mortality rate of 82% (14/17) in mice. Viral nucleoprotein and RNA expression were limited to the respiratory system and no viral RNA could be detected from brain, liver and kidney tissues. CK1 caused heavy alveolar inflammatory exudation and pulmonary hemorrhage, associated with high pulmonary levels of proinflammatory cytokines. In the mouse lung cell line LA-4, CK1 also induced high levels of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) mRNA. Administration of the antiviral zanamivir did not significantly improve survival in mice infected with CK1, but co-administration of the non-steroidal anti-inflammatory drug (NSAID) celecoxib in combination with zanamivir improved survival and lung pathology.

Conclusions

Our findings suggested that H7N9 viruses isolated from chicken without preceding trans-species adaptation can cause lethal mammalian pulmonary infection. The severe proinflammatory responses might be a factor contributing to the mortality. Treatment with combination of antiviral and NSAID could ameliorate pulmonary inflammation and may improve survival.     

Cigarette smoke worsens lung inflammation and impairs resolution of influenza infection in mice

Background

Cigarette smoke has both pro-inflammatory and immunosuppressive effects. Both active and passive cigarette smoke exposure are linked to an increased incidence and severity of respiratory virus infections, but underlying mechanisms are not well defined. We hypothesized, based on prior gene expression profiling studies, that upregulation of pro-inflammatory mediators by short term smoke exposure would be protective against a subsequent influenza infection.

Methods

BALB/c mice were subjected to whole body smoke exposure with 9 cigarettes/day for 4 days. Mice were then infected with influenza A (H3N1, Mem71 strain), and analyzed 3 and 10 days later (d3, d10). These time points are the peak and resolution (respectively) of influenza infection.

Results

Inflammatory cell influx into the bronchoalveolar lavage (BALF), inflammatory mediators, proteases, histopathology, viral titres and T lymphocyte profiles were analyzed. Compared to smoke or influenza alone, mice exposed to smoke and then influenza had more macrophages, neutrophils and total lymphocytes in BALF at d3, more macrophages in BALF at d10, lower net gelatinase activity and increased activity of tissue inhibitor of metalloprotease-1 in BALF at d3, altered profiles of key cytokines and CD4+ and CD8+ T lymphocytes, worse lung pathology and more virus-specific, activated CD8+ T lymphocytes in BALF. Mice smoke exposed before influenza infection had close to 10-fold higher lung virus titres at d3 than influenza alone mice, although all mice had cleared virus by d10, regardless of smoke exposure. Smoke exposure caused temporary weight loss and when smoking ceased after viral infection, smoke and influenza mice regained significantly less weight than smoke alone mice.

Conclusion

Smoke induced inflammation does not protect against influenza infection.In most respects, smoke exposure worsened the host response to influenza. This animal model may be useful in studying how smoke worsens respiratory viral infections.     

Preserved ex vivo inflammatory status in decidual cells from women with preterm labor and subclinical intrauterine infection

Objective

To compare the inflammatory response preserved ex vivo by decidual cells isolated from women who experienced preterm labor with and without subclinical intrauterine infection.

Methods

Fetal membranes were obtained after cesarean section from 35 women who delivered before 37 weeks of gestation following spontaneous preterm labor, with no clinical evidence of intrauterine infection. Decidua was microbiologically tested and cultured. Concentrations of anti-inflammatory cytokines (IL-2, IL-4, IL-10), pro-inflammatory cytokines (IL-6, IL-8, IL-1β and TNF-α), and matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9) were measured in the supernatants using Bio-Plex, and prostaglandin E₂ (PGE₂) was measured by enzyme immunoassay.

Results

Subclinical infection was confirmed in 10 women (28.5%). Microorganisms isolated were Ureaplasma urealyticum (4), group B streptococci (3), Gardnerella vaginalis (1), and Escherichia coli (2). We found a significant increase of pro-inflammatory cytokines and a significant decrease of anti-inflammatory cytokines in supernatants from decidual cells obtained from women with preterm labor and subclinical intrauterine infection compared to women without infection. Secretion of MMP-1, MMP-8, MMP-9 and PGE₂ was significantly higher in infected women. Secretion of IL-8 by decidual cells from infected women persisted upon repeated in vitro culture passages.

Conclusions

Almost 30% of idiopathic preterm labor cases were associated with subclinical intrauterine infection, and decidual cells isolated from these cases preserved an ex vivo inflammatory status after in vivo bacterial exposure.     

Use of praziquantel as an adjuvant enhances protection and Tc-17 responses to killed H5N1 virus vaccine in mice

Background

H5N1 is a highly pathogenic influenza A virus, which can cause severe illness or even death in humans. Although the widely used killed vaccines are able to provide some protection against infection via neutralizing antibodies, cytotoxic T-lymphocyte responses that are thought to eradicate viral infections are lacking.

Methodology/Principal Findings

Aiming to promote cytotoxic responses against H5N1 infection, we extended our previous finding that praziquantel (PZQ) can act as an adjuvant to induce IL-17-producing CD8⁺ T cells (Tc17). We found that a single immunization of 57BL/6 mice with killed viral vaccine plus PZQ induced antigen-specific Tc17 cells, some of which also secreted IFN-γ. The induced Tc17 had cytolytic activities. Induction of these cells was impaired in CD8 knockout (KO) or IFN-γ KO mice, and was even lower in IL-17 KO mice. Importantly, the inoculation of killed vaccine with PZQ significantly reduced virus loads in the lung tissues and prolonged survival. Protection against H5N1 virus infection was obtained by adoptively transferring PZQ-primed wild type CD8⁺ T cells and this was more effective than transfer of activated IFN-γ KO or IL-17 KO CD8⁺ T cells.

Conclusions/Significance

Our results demonstrated that adding PZQ to killed H5N1 vaccine could promote broad Tc17-mediated cytotoxic T lymphocyte activity, resulting in improved control of highly pathogenic avian influenza virus infection.     

Cigarette smoke inhibits BAFF expression and mucosal immunoglobulin A responses in the lung during influenza virus infection

Background

It is incompletely understood how cigarette smoke (CS) exposure affects lung mucosal immune responses during viral respiratory infections. B cell activating factor belonging to the tumor necrosis factor family (BAFF) plays an important role in the induction of secretory immunoglobulin A (S-IgA) which is the main effector of the mucosal immune system. We therefore investigated the effects of CS exposure on BAFF expression and S-IgA responses in the lung during influenza virus infection.

Methods

Mice were exposed to CS and/or infected with influenza virus. Bronchoalveolar lavage fluid and lung compartments were analyzed for BAFF expression, influenza-specific S-IgA level and histological changes. Lung B cells were isolated and the activation-induced cytidine deaminase (Aicda) expression was determined. BEAS-2B cells were treated with CS extract (CSE), influenza virus, interferon beta or N-acetylcysteine and BAFF expression was measured.

Results

CS inhibited BAFF expression in the lung, particularly after long-term exposure. BAFF and S-IgA levels were increased during influenza virus infection. Three-month CS exposure prior to influenza virus infection resulted in reduced BAFF and S-IgA levels in the lung as well as augmented pulmonary inflammation on day 7 after infection. Prior CS exposure also caused decreased Aicda expression in lung B cells during infection. Neutralization of BAFF in the lung resulted in reduced S-IgA levels during influenza virus infection. CSE inhibited virus-mediated BAFF induction in a dose-dependent manner in BEAS-2B cells, while this inhibition of BAFF by CSE was prevented by pretreatment with the antioxidant N-acetylcysteine.

Conclusions

Our findings indicate that CS may hinder early mucosal IgA responses in the lung during influenza virus infection through oxidative inhibition of BAFF, which might contribute to the increased incidence and severity of viral infections in smokers.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0201-y) contains supplementary material, which is available to authorized users.     

IL-32 and IL-17 interact and have the potential to aggravate osteoclastogenesis in rheumatoid arthritis

Introduction

Interleukin (IL)-32 and IL-17 play critical roles in pro-inflammatory responses and are highly expressed in the synovium of patients with rheumatoid arthritis (RA). We investigated the relations between these two cytokines (IL-17 and IL-32) for their ability to induce each other and to stimulate osteoclasts in RA fibroblast-like synoviocytes (FLSs) and T cells.

Methods

FLSs were isolated through surgical synovectomy obtained from patients with RA or osteoarthritis (OA). Real-time PCR were performed to evaluate the expression of IL-32, IL-17 and osteoclast-related genes. Immunohistochemical staining and tartrate-resistant acid phosphatase (TRAP) staining were performed to determine the distribution of inflammatory cytokines and the presence of osteoclastogenesis.

Results

IL-17 induced the expression of IL-32 in the FLSs from RA patients, as assessed by microarray. IL-32 production was increased by IL-17. IL-32 in the FLSs from RA patients induced the production of IL-17 in CD4⁺ T cells. IL-32 and IL-17 were colocalized near TRAP-positive areas in joint specimens. IL-17 and IL-32 synergistically induced the differentiation of osteoclasts, as demonstrated by the expression of osteoclast-related genes. IL-32 and IL-17 also could induce resorption by osteoclasts in a RANKL-dependent manner.

Conclusions

IL-17 affected the expression of IL-32 in FLSs of RA patients and IL-32 induced the production of IL-17 in CD4+ T cells. Both IL-17 and IL-32 cytokines can reciprocally influence each other''s production and amplify the function of osteoclastogenesis in the in RA synovium. Separately, IL-17 and IL-32 each stimulated osteoclastogenesis without RANKL. Together, the two cytokines synergistically amplified the differentiation of osteoclasts, independent of RANKL stimulation.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Induction of influenza-specific mucosal immunity by an attenuated recombinant Sendai virus

Background

Many pathogens initiate infection at the mucosal surfaces; therefore, induction of mucosal immune responses is a first level of defense against infection and is the most powerful means of protection. Although intramuscular injection is widely used for vaccination and is effective at inducing circulating antibodies, it is less effective at inducing mucosal antibodies.

Methodology/Principal Findings

Here we report a novel recombinant, attenuated Sendai virus vector (GP42-H1) in which the hemagglutinin (HA) gene of influenza A virus was introduced into the Sendai virus genome as an additional gene. Infection of CV-1 cells by GP42-H1 resulted in cell surface expression of the HA protein. Intranasal immunization of mice with 1,000 plaque forming units (pfu) of GP42-H1 induced HA-specific IgG and IgA antibodies in the blood, brochoalveolar lavage fluid, fecal pellet extracts and saliva. The HA-specific antibody titer induced by GP42-H1 closely resembles the titer induced by sublethal infection by live influenza virus; however, in contrast to infection by influenza virus, immunization with GP42-H1 did not result in disease symptoms or the loss of body weight. In mice that were immunized with GP42-H1 and then challenged with 5LD₅₀ (1250 pfu) of influenza virus, no significant weight loss was observed and other visual signs of morbidity were not detected.

Conclusions

These results demonstrate that the GP42-H1 Sendai virus recombinant is able to confer full protection from lethal infection by influenza virus, supporting the conclusion that it is a safe and effective mucosal vaccine vector.     

A novel genetic locus linked to pro-inflammatory cytokines after virulent H5N1 virus infection in mice

Background

Genetic variation in the human population is a key determinant of influenza disease severity. A single nucleotide polymorphism in the antiviral gene IFITM3 was linked to outcomes during the 2009 H1N1 pandemic. To identify variant host genes associated with increased virus replication and severe disease, we performed a quantitative trait locus analysis on pro-inflammatory cytokine production 48 hours after intranasal infection with highly pathogenic H5N1 influenza virus.

Results

Pro-inflammatory cytokines CCL2, TNFα and IFN-α, were measured by ELISA in lung homogenates of DBA/2J (D2), C57BL/6J (B6) and 44 different BXD recombinant inbred mouse strains. Virus titer was also assessed in a subset of these animals. CCL2 (8-fold), TNFα (24-fold) and IFN-α (8-fold) concentrations varied significantly among the different BXD RI strains. Importantly, cytokine concentration correlated very well (r =0.86-0.96, P <0.0001) with virus titer suggesting that early cytokine production is due to increased virus infection and replication. Linkage analysis of cytokine concentration revealed a significant locus on chromosome 6 associated with differences in TNFα, IFN-α and CCL2 cytokine concentration (LRS =26). This locus accounted for nearly 20% of the observed phenotypic variation in the BXD population studied. Sequence and RNA expression analysis identified several candidate host genes containing missense mutations or deletions; Samd9l, Ica1, and Slc25a13. To study the role of Slc25a13, we obtained Slc25a13 knockout line, but upon challenge with H5N1 influenza virus observed no effect on CCL2 production, or morbidity and mortality.

Conclusion

A novel genetic locus on chromosome 6 modulates early pro-inflammatory cytokine production and virus replication after highly pathogenic influenza virus infection. Candidate genes, Samd9l and Ica1, may be important for the control of influenza virus infection and pathogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1017) contains supplementary material, which is available to authorized users.     

The effect of rosuvastatin in a murine model of influenza A infection

Rationale

HMG-CoA reductase inhibitors such as rosuvastatin may have immunomodulatory and anti-inflammatory effects that may reduce the severity of influenza A infection. We hypothesized that rosuvastatin would decrease viral replication, attenuate lung injury, and improve mortality following influenza A infection in mice.

Methods

C57Bl/6 mice were treated daily with rosuvastatin (10 mg/kg/day) supplemented in chow (or control chow) beginning three days prior to infection with either A//Udorn/72 [H3N2] or A/WSN/33 [H1N1] influenza A virus (1×10⁵ pfu/mouse). Plaque assays were used to examine the effect of rosuvastatin on viral replication in vitro and in the lungs of infected mice. We measured cell count with differential, protein and cytokines in the bronchoalveolar lavage (BAL) fluid, histologic evidence of lung injury, and wet-to-dry ratio on Day 1, 2, 4, and 6. We also recorded daily weights and mortality.

Results

The administration of rosuvastatin had no effect on viral clearance of influenza A after infection. Weight loss, lung inflammation and lung injury severity were similar in the rosuvastatin and control treated mice. In the mice infected with influenza A (A/WSN/33), mortality was unaffected by treatment with rosuvastatin.

Conclusions

Statins did not alter the replication of influenza A in vitro or enhance its clearance from the lung in vivo. Statins neither attenuated the severity of influenza A-induced lung injury nor had an effect on influenza A-related mortality. Our data suggest that the association between HMG CoA reductase inhibitors and improved outcomes in patients with sepsis and pneumonia are not attributable to their effects on influenza A infection.     

The role of IL-27 in susceptibility to post-influenza Staphylococcus aureus pneumonia

Background

Influenza is a common respiratory virus and Staphylococcus aureus frequently causes secondary pneumonia during influenza infection, leading to increased morbidity and mortality. Influenza has been found to attenuate subsequent Type 17 immunity, enhancing susceptibility to secondary bacterial infections. IL-27 is known to inhibit Type 17 immunity, suggesting a potential critical role for IL-27 in viral and bacterial co-infection.

Methods

A murine model of influenza and Staphylococcus aureus infection was used to mimic human viral, bacterial co-infection. C57BL/6 wild-type, IL-27 receptor α knock-out, and IL-10 knock-out mice were infected with Influenza H1N1 (A/PR/8/34) or vehicle for 6 days followed by challenge with Staphylococcus aureus or vehicle for 24 hours. Lung inflammation, bacterial burden, gene expression, and cytokine production were determined.

Results

IL-27 receptor α knock-out mice challenged with influenza A had increased morbidity compared to controls, but no change in viral burden. IL-27 receptor α knock-out mice infected with influenza displayed significantly decreased IL-10 production compared to wild-type. IL-27 receptor α knock-out mice co-infected with influenza and S. aureus had improved bacterial clearance compared to wild-type controls. Importantly, there were significantly increased Type 17 responses and decreased IL-10 production in IL-27 receptor α knock-out mice. Dual infected IL-10−/− mice had significantly less bacterial burden compared to dual infected WT mice.

Conclusions

These data reveal that IL-27 regulates enhanced susceptibility to S. aureus pneumonia following influenza infection, potentially through the induction of IL-10 and suppression of IL-17.     

Microarray analysis identifies IL-1 receptor type 2 as a novel candidate biomarker in patients with acute respiratory distress syndrome

Background

Acute respiratory distress syndrome (ARDS) is a disease associated with a high mortality rate. The initial phase is characterized by induction of inflammatory cytokines and chemokines and influx of circulating inflammatory cells, including macrophages which play a pivotal role in the innate and adaptive immune responses to injury. Growing evidence points to phenotypic heterogeneity and plasticity between various macrophage activation states.

Methods

In this study, gene expression in alveolar macrophages and circulating leukocytes from healthy control subjects and patients with ARDS was assessed by mRNA microarray analysis.

Results

Both alveolar macrophages and circulating leukocytes demonstrated up-regulation of genes encoding chemotactic factors, antimicrobial peptides, chemokine receptors, and matrix metalloproteinases. Two genes, the pro-inflammatory S100A12 and the anti-inflammatory IL-1 decoy receptor IL-1R2 were significantly induced in both cell populations in ARDS patients, which was confirmed by protein quantification. Although S100A12 levels did not correlate with disease severity, there was a significant association between early plasma levels of IL-1R2 and APACHE III scores at presentation. Moreover, higher levels of IL-1R2 in plasma were observed in non-survivors as compared to survivors at later stages of ARDS.

Conclusions

These results suggest a hybrid state of alveolar macrophage activation in ARDS, with features of both alternative activation and immune tolerance/deactivation.. Furthermore, we have identified a novel plasma biomarker candidate in ARDS that correlates with the severity of systemic illness and mortality.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0190-x) contains supplementary material, which is available to authorized users.     

Nrf2 protects human alveolar epithelial cells against injury induced by influenza A virus

Background

Influenza A virus (IAV) infection primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. Recent studies have shown the importance of lung antioxidant defense systems against injury by IAV. Nuclear factor-erythroid 2 related factor 2 (Nrf2) activates the majority of antioxidant genes.

Methods

Alveolar type II (ATII) cells and alveolar macrophages (AM) were isolated from human lungs not suitable for transplantation and donated for medical research. In some studies ATII cells were transdifferentiated to alveolar type I-like (ATI-like) cells. Alveolar epithelial cells were infected with A/PR/8/34 (PR8) virus. We analyzed PR8 virus production, influenza A nucleoprotein levels, ROS generation and expression of antiviral genes. Immunocytofluorescence was used to determine Nrf2 translocation and western blotting to detect Nrf2, HO-1 and caspase 1 and 3 cleavage. We also analyzed ingestion of PR8 virus infected apoptotic ATII cells by AM, cytokine levels by ELISA, glutathione levels, necrosis and apoptosis by TUNEL assay. Moreover, we determined the critical importance of Nrf2 using adenovirus Nrf2 (AdNrf2) or Nrf2 siRNA to overexpress or knockdown Nrf2, respectively.

Results

We found that IAV induced oxidative stress, cytotoxicity and apoptosis in ATI-like and ATII cells. We also found that AM can ingest PR8 virus-induced apoptotic ATII cells (efferocytosis) but not viable cells, whereas ATII cells did not ingest these apoptotic cells. PR8 virus increased ROS production, Nrf2, HO-1, Mx1 and OAS1 expression and Nrf2 translocation to the nucleus. Nrf2 knockdown with siRNA sensitized ATI-like cells and ATII cells to injury induced by IAV and overexpression of Nrf2 with AdNrf2 protected these cells. Furthermore, Nrf2 overexpression followed by infection with PR8 virus decreased virus replication, influenza A nucleoprotein expression, antiviral response and oxidative stress. However, AdNrf2 did not increase IFN-λ1 (IL-29) levels.

Conclusions

Our results indicate that IAV induces alveolar epithelial injury and that Nrf2 protects these cells from the cytopathic effects of IAV likely by increasing the expression of antioxidant genes. Identifying the pathways involved in protecting cells from injury during influenza infection may be particularly important for developing new therapeutic strategies.     

Gamma-irradiated influenza virus uniquely induces IFN-I mediated lymphocyte activation independent of the TLR7/MyD88 pathway

Background

We have shown previously in mice, that infection with live viruses, including influenza/A and Semliki Forest virus (SFV), induces systemic partial activation of lymphocytes, characterized by cell surface expression of CD69 and CD86, but not CD25. This partial lymphocytes activation is mediated by type-I interferons (IFN-I). Importantly, we have shown that γ-irradiated SFV does not induce IFN-I and the associated lymphocyte activation.

Principal Findings

Here we report that, in contrast to SFV, γ-irradiated influenza A virus elicits partial lymphocyte activation in vivo. Furthermore, we show that when using influenza viruses inactivated by a variety of methods (UV, ionising radiation and formalin treatment), as well as commercially available influenza vaccines, only γ-irradiated influenza virus is able to trigger IFN-I-dependent partial lymphocyte activation in the absence of the TLR7/MyD88 signalling pathways.

Conclusions

Our data suggest an important mechanism for the recognition of γ-irradiated influenza vaccine by cytosolic receptors, which correspond with the ability of γ-irradiated influenza virus to induce cross-reactive and cross-protective cytotoxic T cell responses.     

Profiling of Humoral Response to Influenza A(H1N1)pdm09 Infection and Vaccination Measured by a Protein Microarray in Persons with and without History of Seasonal Vaccination

Background

The influence of prior seasonal influenza vaccination on the antibody response produced by natural infection or vaccination is not well understood.

Methods

We compared the profiles of antibody responses of 32 naturally infected subjects and 98 subjects vaccinated with a 2009 influenza A(H1N1) monovalent MF59-adjuvanted vaccine (Focetria®, Novartis), with and without a history of seasonal influenza vaccination. Antibodies were measured by hemagglutination inhibition (HI) assay for influenza A(H1N1)pdm09 and by protein microarray (PA) using the HA1 subunit for seven recent and historic H1, H2 and H3 influenza viruses, and three avian influenza viruses. Serum samples for the infection group were taken at the moment of collection of the diagnostic sample, 10 days and 30 days after onset of influenza symptoms. For the vaccination group, samples were drawn at baseline, 3 weeks after the first vaccination and 5 weeks after the second vaccination.

Results

We showed that subjects with a history of seasonal vaccination generally exhibited higher baseline titers for the various HA1 antigens than subjects without a seasonal vaccination history. Infection and pandemic influenza vaccination responses in persons with a history of seasonal vaccination were skewed towards historic antigens.

Conclusions

Seasonal vaccination is of significant influence on the antibody response to subsequent infection and vaccination, and further research is needed to understand the effect of annual vaccination on protective immunity.     

Virus-Like Particle Vaccine Protects against 2009 H1N1 Pandemic Influenza Virus in Mice

Background

The 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines.

Methodology/Principal Findings

We generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection.

Conclusion/Significance

This study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production.     

Evidence for regulated interleukin-4 expression in chondrocyte-scaffolds under in vitro inflammatory conditions

Objective

To elucidate the anti-inflammatory and anabolic effects of regulated expression of IL-4 in chondrocyte-scaffolds under in vitro inflammatory conditions.

Methods

Mature articular chondrocytes from dogs (n = 3) were conditioned through transient transfection using pcDNA3.1.cIL-4 (constitutive) or pCOX-2.cIL-4 (cytokine-responsive) plasmids. Conditioned cells were seeded in alginate microspheres and rat-tail collagen type I matrix (CaReS®) to generate two types of tissue-engineered 3-dimensional scaffolds. Inflammatory arthritis was simulated in the packed chondrocytes through exogenous addition of recombinant canine (rc) IL-1β (100 ng/ml) plus rcTNFα (50 ng/ml) in culture media for 96 hours. Harvested cells and culture media were analyzed by various assays to monitor the anti-inflammatory and regenerative (anabolic) properties of cIL-4.

Results

cIL-4 was expressed from COX-2 promoter exclusively on the addition of rcIL-1β and rcTNFα while its expression from CMV promoter was constitutive. The expressed cIL-4 downregulated the mRNA expression of IL-1β, TNFα, IL-6, iNOS and COX-2 in the cells and inhibited the production of NO and PGE₂ in culture media. At the same time, it up-regulated the expression of IGF-1, IL-1ra, COL2a1 and aggrecan in conditioned chondrocytes in both scaffolds along with a diminished release of total collagen and sGAG into the culture media. An increased amount of cIL-4 protein was detected both in chondrocyte cell lysate and in concentrated culture media. Neutralizing anti-cIL-4 antibody assay confirmed that the anti-inflammatory and regenerative effects seen are exclusively driven by cIL-4. There was a restricted expression of IL-4 under COX-2 promoter possibly due to negative feedback loop while it was over-expressed under CMV promoter (undesirable). Furthermore, the anti-inflammatory /anabolic outcomes from both scaffolds were reproducible and the therapeutic effects of cIL-4 were both scaffold- and promoter-independent.

Conclusions

Regulated expression of therapeutic candidate gene(s) coupled with suitable scaffold(s) could potentially serve as a useful tissue-engineering tool to devise future treatment strategies for osteoarthritis.     

The role of neutrophils in the upper and lower respiratory tract during influenza virus infection of mice

Background

Neutrophils have been shown to play a role in host defence against highly virulent and mouse-adapted strains of influenza virus, however it is not clear if an effective neutrophil response is an important factor moderating disease severity during infection with other virus strains. In this study, we have examined the role of neutrophils during infection of mice with influenza virus strain HKx31, a virus strain of the H3N2 subtype and of moderate virulence for mice, to determine the role of neutrophils in the early phase of infection and in clearance of influenza virus from the respiratory tract during the later phase of infection.

Methods

The anti-Gr-1 monoclonal antibody (mAb) RB6-8C5 was used to (i) identify neutrophils in the upper (nasal tissues) and lower (lung) respiratory tract of uninfected and influenza virus-infected mice, and (ii) deplete neutrophils prior to and during influenza virus infection of mice.

Results

Neutrophils were rapidly recruited to the upper and lower airways following influenza virus infection. We demonstrated that use of mAb RB6-8C5 to deplete C57BL/6 (B6) mice of neutrophils is complicated by the ability of this mAb to bind directly to virus-specific CD8⁺ T cells. Thus, we investigated the role of neutrophils in both the early and later phases of infection using CD8⁺ T cell-deficient B6.TAP^-/- mice. Infection of B6.TAP^-/- mice with a low dose of influenza virus did not induce clinical disease in control animals, however RB6-8C5 treatment led to profound weight loss, severe clinical disease and enhanced virus replication throughout the respiratory tract.

Conclusion

Neutrophils play a critical role in limiting influenza virus replication during the early and later phases of infection. Furthermore, a virus strain of moderate virulence can induce severe clinical disease in the absence of an effective neutrophil response.     

The Association between Serum Biomarkers and Disease Outcome in Influenza A(H1N1)pdm09 Virus Infection: Results of Two International Observational Cohort Studies

Background

Prospective studies establishing the temporal relationship between the degree of inflammation and human influenza disease progression are scarce. To assess predictors of disease progression among patients with influenza A(H1N1)pdm09 infection, 25 inflammatory biomarkers measured at enrollment were analyzed in two international observational cohort studies.

Methods

Among patients with RT-PCR-confirmed influenza A(H1N1)pdm09 virus infection, odds ratios (ORs) estimated by logistic regression were used to summarize the associations of biomarkers measured at enrollment with worsened disease outcome or death after 14 days of follow-up for those seeking outpatient care (FLU 002) or after 60 days for those hospitalized with influenza complications (FLU 003). Biomarkers that were significantly associated with progression in both studies (p<0.05) or only in one (p<0.002 after Bonferroni correction) were identified.

Results

In FLU 002 28/528 (5.3%) outpatients had influenza A(H1N1)pdm09 virus infection that progressed to a study endpoint of complications, hospitalization or death, whereas in FLU 003 28/170 (16.5%) inpatients enrolled from the general ward and 21/39 (53.8%) inpatients enrolled directly from the ICU experienced disease progression. Higher levels of 12 of the 25 markers were significantly associated with subsequent disease progression. Of these, 7 markers (IL-6, CD163, IL-10, LBP, IL-2, MCP-1, and IP-10), all with ORs for the 3^rd versus 1^st tertile of 2.5 or greater, were significant (p<0.05) in both outpatients and inpatients. In contrast, five markers (sICAM-1, IL-8, TNF-α, D-dimer, and sVCAM-1), all with ORs for the 3^rd versus 1^st tertile greater than 3.2, were significantly (p≤.002) associated with disease progression among hospitalized patients only.

Conclusions

In patients presenting with varying severities of influenza A(H1N1)pdm09 virus infection, a baseline elevation in several biomarkers associated with inflammation, coagulation, or immune function strongly predicted a higher risk of disease progression. It is conceivable that interventions designed to abrogate these baseline elevations might affect disease outcome.