Influenza Virus-induced Acute Lung Injury Research Articles

From bioinformatics to anti-inflammation and immune regulation

Background: ACT001 is a potent anti-inflammatory small-molecule drug. However, the single cell and spatial molecular basis of pyroptosis and whether ACT001 exerts a therapeutic effect by preventing pyroptosis on acute lung injury (ALI) remains unclear. Methods: The bioinformatics approach was employed to identify single cell and spatial landscape of nucleotide-binding domains and leucine-rich repeat protein 3 (NLRP3)-dependent pyroptosis in lipopolysaccharide (LPS) and influenza virus-induced ALI. Molecular docking was performed to elucidate the relationship between ACT001 and NLRP3. LPS-induced ALI mice model was established. Histopathological analysis and bronchoalveolar lavage fluid collection were conducted to investigate the anti-inflammatory and protective effects. In vitro experiments were also performed on bone marrow–derived macrophages to explore the effect of ACT001 on the balance of mitochondrial fusion and fission protein. Results: Single cell transcriptomic and spatial transcriptomic analysis predicted that NLRP3-dependent pyroptosis significantly correlated with the development of ALI both in single cell and spatial distribution. Molecular docking provided a stable and reliable docking between ACT001 and NLRP3. ACT001 improved the 7-day survival of mice by approximately 50% over the loading dose of LPS-induced ALI. ACT001 (5 uM) attenuated the disruption of mitochondrial integrity and reactive oxygen species. Further, ACT001 reduced the overexpression of the mitochondrial fission protein DRP1 without affecting fusion protein Mitofusin2 levels. Moreover, ACT001 exerted a similar protective effect of suppressing pyroptosis as the DRP1-inhibitor Mdivi-1. Conclusions: Our study revealed that pyroptosis genes were highly expressed in single-cell and spatial mapping along the first week of ALI occurrence. ACT001 attenuates ALI by reducing the NLRP3-dependent pyroptosis and balancing mitochondrial fission and fusion.

From bioinformatics to anti-inflammation and immune regulation: ACT001 in lipopolysaccharide-induced lung injury.

ACT001 is a potent anti-inflammatory small-molecule drug. However, the single cell and spatial molecular basis of pyroptosis and whether ACT001 exerts a therapeutic effect by preventing pyroptosis on acute lung injury (ALI) remains unclear. The bioinformatics approach was employed to identify single cell and spatial landscape of nucleotide-binding domains and leucine-rich repeat protein 3 (NLRP3)-dependent pyroptosis in lipopolysaccharide (LPS) and influenza virus-induced ALI. Molecular docking was performed to elucidate the relationship between ACT001 and NLRP3. LPS-induced ALI mice model was established. Histopathological analysis and bronchoalveolar lavage fluid collection were conducted to investigate the anti-inflammatory and protective effects. In vitro experiments were also performed on bone marrow-derived macrophages to explore the effect of ACT001 on the balance of mitochondrial fusion and fission protein. Single cell transcriptomic and spatial transcriptomic analysis predicted that NLRP3-dependent pyroptosis significantly correlated with the development of ALI both in single cell and spatial distribution. Molecular docking provided a stable and reliable docking between ACT001 and NLRP3. ACT001 improved the 7-day survival of mice by approximately 50% over the loading dose of LPS-induced ALI. ACT001 (5 uM) attenuated the disruption of mitochondrial integrity and reactive oxygen species. Further, ACT001 reduced the overexpression of the mitochondrial fission protein DRP1 without affecting fusion protein Mitofusin2 levels. Moreover, ACT001 exerted a similar protective effect of suppressing pyroptosis as the DRP1-inhibitor Mdivi-1. Our study revealed that pyroptosis genes were highly expressed in single-cell and spatial mapping along the first week of ALI occurrence. ACT001 attenuates ALI by reducing the NLRP3-dependent pyroptosis and balancing mitochondrial fission and fusion.

Extracellular vesicles: A promising therapy against SARS-CoV-2 infection

Extracellular vesicles: A promising therapy against SARS-CoV-2 infection

Diosmetin alleviates benzo[a]pyrene-exacerbated H1N1 influenza virus-induced acute lung injury and dysregulation of inflammation through modulation of the PPAR-γ-NF-κB/P38 MAPK signaling axis.

The severity of a viral respiratory illness was greatly exacerbated after exposure to a contaminant containing benzo[a]pyrene (B[a]P). Flavonoid-rich fruit intake has gained intense interest due to their health-promoting benefits for viral respiratory diseases, including influenza viruses. In our study, diosmetin (3',5,7-trihydroxy-4'-methoxyflavone), a naturally occurring hydroxylated methoxyflavone that is abundant in Citrus fruits, was explored for its effects on B[a]P-exacerbated H1N1 influenza virus-mediated inflammation and lung injury. Initially, in vivo results demonstrated that diosmetin protected against H1N1 virus-elicited acute lung injury. Simultaneously, H1N1 virus or B[a]P-stimulated A549 cells treated with diosmetin inhibited NF-κB and P-P38 activation, resulting in suppression of pro-inflammatory cytokines and apoptosis. Interestingly, diosmetin obviously promoted the expression of PPAR-γ as well as nuclear translocation of PPAR-γ, whereas, PPAR-γ inhibition by GW9662 weakened the inhibitory effects of diosmetin on H1N1 virus or B[a]P-mediated activation of NF-κB and P-P38, elevated expression of pro-inflammatory mediators as well as apoptosis. Furthermore, it was surprising to discover that mice exposed to both B[a]P and H1N1 viruses contributed to exacerbated acute lung injury, which were significantly ameliorated by diosmetin administration. In vitro studies showed that A549 cells with the combination of B[a]P and H1N1 virus augmented NF-κB and P-P38 activation, accompanied by higher levels of pro-inflammatory mediators and apoptosis, all of which were also significantly reduced by diosmetin treatment. Repressing PPAR-γ abrogated the inhibitory effects of diosmetin on B[a]P-exacerbated H1N1 virus-mediated NF-κB and P-P38 activation, inflammation, and apoptosis in A549 cells. Our findings suggest that diosmetin protected against B[a]P-exacerbated H1N1 virus-mediated lung injury by suppressing the exacerbation of NF-κB and P38 kinase activation in a PPAR-γ-dependent manner, suggesting potential benefits for B[a]P-exacerbated influenza-related illness therapeutics.

The Potential of Medicinal Plants and Bioactive Compounds in the Fight Against COVID-19

The Potential of Medicinal Plants and Bioactive Compounds in the Fight Against COVID-19

Effect of paeoniflorin on acute lung injury induced by influenza A virus in mice. Evidences of its mechanism of action

BackgroundInfluenza often leads to acute lung injury (ALI). Few therapeutics options such as vaccines and other antiviral drugs are available. Paeoniflorin is a monoterpene glucoside isolated from the roots of Paeonia lactiflora Pall. that has showed good anti-inflammatory and anti-fibrotic effects. However, it is not known whether paeoniflorin has an effect on influenza virus-induced ALI. PurposeTo investigative the protective effect and potential mechanism of paeoniflorin on ALI induced by influenza A virus (IAV). Study design and methodsThe anti-influenza activity of paeoniflorin in vitro was investigated. Influenza virus A/FM/1/47 was intranasally infected in mice to induce ALI, and paeoniflorin (50 and 100 mg/kg) was given orally to mice during 5 days, beginning 2 h after infection. On day 6 post-infection, body and lung weights, histology and survival were observed, and the lungs were examined for viral load, cytokine and cellular pathway protein expression. ResultsResults showed that paeoniflorin (50 and 100 mg/kg) reduced IAV-induced ALI. It reduces pulmonary oedema and improves histopathological changes in the lung, and also diminishes the accumulation of inflammatory cells in the lung. It was shown that paeoniflorin (50 and 100 mg/kg) alleviated IAV-induced ALI, as evidenced by improved survival in infected mice (40% and 50%, respectively), reduced viral titer in lung tissue, improved histological changes, and reduced lung inflammation. Paeoniflorin also improves pulmonary fibrosis by reducing the levels of pulmonary fibrotic markers (collagen type IV, alpha-smooth muscle actin, hyaluronic acid, laminin, and procollagen type III) and downregulating the expression levels of type I collagen (Col I) and type III collagen (Col III) in the lung tissues. Additionally, paeoniflorin inhibits the expression of αvβ3, TGF-β1, Smad2, NF-κB, and p38MAPK in the lung tissues. ConclusionThe results showed that paeoniflorin (50 and 100 mg/kg) protected against IAV-induced ALI, and the underlying mechanism may be related to the reduction of pro-inflammatory cytokine production and lung collagen deposition through down-regulation of activation of αvβ3/TGF-β1 pathway in lung tissue.

The active GLP-1 analogue liraglutide alleviates H9N2 influenza virus-induced acute lung injury in mice

Influenza virus is responsible for significant morbidity and mortality worldwide. Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is the major cause of death in influenza virus infected patients. Recent studies indicated that active glucagon like peptide-1 (GLP-1) encoded by glucagon (GCG) gene exerts anti-inflammatory functions. The aim of this study was to determine the potential role of active GLP-1 in H9N2 influenza virus-induced ALI/ARDS in mice. First, we uncovered that GCG mRNA expression levels and GCG precursor protein levels were significantly increased, but total GLP-1 and active GLP-1 levels were decreased in the lungs of H9N2-infected mice. Next, liraglutide, an active GLP-1 analogue, was used to treat infected mice and to observe its effects on H9N2 virus-induced ALI. Liraglutide treatment ameliorated the declined body weight, decreased food intake and mortality observed in infected mice. It also alleviated the severity of lung injury, including lowering lung index, decreasing inflammatory cell infiltration and lowing total protein levels in bronchoalveolar lavage fluid (BALF). In addition, liraglutide did not influence viral titers in infected lungs, but decreased the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-α in BALF. These results indicated that liraglutide alleviated H9N2 virus-induced ALI in mice most likely due to lower levels of pro-inflammatory cytokines.

β-sitosterol ameliorates influenza A virus-induced proinflammatory response and acute lung injury in mice by disrupting the cross-talk between RIG-I and IFN/STAT signaling.

β-Sitosterol (24-ethyl-5-cholestene-3-ol) is a common phytosterol Chinese medical plants that has been shown to possess antioxidant and anti-inflammatory activity. In this study we investigated the effects of β-sitosterol on influenza virus-induced inflammation and acute lung injury and the molecular mechanisms. We demonstrate that β-sitosterol (150–450 μg/mL) dose-dependently suppresses inflammatory response through NF-κB and p38 mitogen-activated protein kinase (MAPK) signaling in influenza A virus (IAV)-infected cells, which was accompanied by decreased induction of interferons (IFNs) (including Type I and III IFN). Furthermore, we revealed that the anti-inflammatory effect of β-sitosterol resulted from its inhibitory effect on retinoic acid-inducible gene I (RIG-I) signaling, led to decreased STAT1 signaling, thus affecting the transcriptional activity of ISGF3 (interferon-stimulated gene factor 3) complexes and resulting in abrogation of the IAV-induced proinflammatory amplification effect in IFN-sensitized cells. Moreover, β-sitosterol treatment attenuated RIG-I-mediated apoptotic injury of alveolar epithelial cells (AEC) via downregulation of pro-apoptotic factors. In a mouse model of influenza, pre-administration of β-sitosterol (50, 200 mg·kg−1·d−1, i.g., for 2 days) dose-dependently ameliorated IAV-mediated recruitment of pathogenic cytotoxic T cells and immune dysregulation. In addition, pre-administration of β-sitosterol protected mice from lethal IAV infection. Our data suggest that β-sitosterol blocks the immune response mediated by RIG-I signaling and deleterious IFN production, providing a potential benefit for the treatment of influenza.

Expression pattern of the interferon regulatory factor family members in influenza virus induced local and systemic inflammatory responses

Type I interferon is considered to be a key cytokine in influenza virus-induced acute lung injury (ALI), in which IRF3 and IRF7 play particularly important roles. However, whether all nine members of IRF family are involved in influenza virus-induced immune response is currently unknown. In this study, we found that all members of IRF family responded to influenza virus. The IRF family expression profile seems to be related to the pathogenicity of the particular influenza virus strain. The influenza virus mainly relies on endosomal TLR signals and the positive feedback loop of IFN-I to cause either direct or indirect different expression of all IRF family members locally or systemically. Interestingly, IRF6 was somewhat different from other IRF family members during influenza virus infection. Overall, the expression profile of the IRF family may be a valuable reference for the prevention and treatment of influenza complications, which encourage further, more in-depth research.

C5a receptor1 inhibition alleviates influenza virus-induced acute lung injury

Influenza A virus is an important human pathogen that causes 3 to 5 million severe cases of influenza worldwide each year. An aberrant innate immune response, particularly hypercytokinemia, is thought to play an important role in the disease, although the pathogenesis of severe influenza virus infection remains unclear and no specific and efficacious immunotherapy is available. This study reports dysregulated complement activation in mice after infection with A/Puerto Rico/8/34 (PR8). C5aR1-deficient mice and mice treated with an anti-C5aR1 antibody were used as models to study the C5a-C5aR1 axis during acute lung injury (ALI) induced by influenza virus infection. The results showed that blocking the C5a-C5aR1 axis alleviated ALI by inhibiting endothelial cell activation and dampening the host immune response (i.e., reduced TNF-α, IL-1β, IL-6, IP-10, MCP-1, IL-12p70, and IFN-γ concentrations in plasma), particularly CTL-mediated immunopathology. Furthermore, blockade of the C5a-C5aR1 axis inhibited viral replication in lung tissue. Taken together, the results indicate that the C5a-C5aR1 axis plays an important role in the outcome of ALI induced by influenza virus infection and that regulation of complement activation, particularly the C5aR1 inhibition, is a promising intervention and adjunctive treatment.

Mesenchymal stem cell-derived extracellular vesicles attenuate influenza virus-induced acute lung injury in a pig model

BackgroundMesenchymal stem (stromal) cells (MSCs) mediate their immunoregulatory and tissue repair functions by secreting paracrine factors, including extracellular vesicles (EVs). In several animal models of human diseases, MSC-EVs mimic the beneficial effects of MSCs. Influenza viruses cause annual outbreaks of acute respiratory illness resulting in significant mortality and morbidity. Influenza viruses constantly evolve, thus generating drug-resistant strains and rendering current vaccines less effective against the newly generated strains. Therefore, new therapies that can control virus replication and the inflammatory response of the host are needed. The objective of this study was to examine if MSC-EV treatment can attenuate influenza virus-induced acute lung injury in a preclinical model.MethodsWe isolated EVs from swine bone marrow-derived MSCs. Morphology of MSC-EVs was determined by electron microscopy and expression of mesenchymal markers was examined by flow cytometry. Next, we examined the anti-influenza activity of MSC-EVs in vitro in lung epithelial cells and anti-viral and immunomodulatory properties in vivo in a pig model of influenza virus.ResultsMSC-EVs were isolated from MSC-conditioned medium by ultracentrifugation. MSC-EVs were round-shaped and, similarly to MSCs, expressed mesenchymal markers and lacked the expression of swine leukocyte antigens I and II. Incubation of PKH-26-labeled EVs with lung epithelial cells revealed that MSC-EVs incorporated into the epithelial cells. Next, we examined the anti-influenza and anti-inflammatory properties of MSC-EVs. MSC-EVs inhibited the hemagglutination activity of avian, swine, and human influenza viruses at concentrations of 1.25–5 μg/ml. MSC-EVs inhibited influenza virus replication and virus-induced apoptosis in lung epithelial cells. The anti-influenza activity of MSC-EVs was due to transfer of RNAs from EVs to epithelial cells since pre-incubation of MSC-EVs with RNase enzyme abrogated the anti-influenza activity of MSC-EVs. In a pig model of influenza virus, intratracheal administration of MSC-EVs 12 h after influenza virus infection significantly reduced virus shedding in the nasal swabs, influenza virus replication in the lungs, and virus-induced production of proinflammatory cytokines in the lungs of influenza-infected pigs. The histopathological findings revealed that MSC-EVs alleviated influenza virus-induced lung lesions in pigs.ConclusionsOur data demonstrated in a relevant preclinical large animal model of influenza virus that MSC-EVs possessed anti-influenza and anti-inflammatory properties and that EVs may be used as cell-free therapy for influenza in humans.

Kaempferol ameliorates H9N2 swine influenza virus-induced acute lung injury by inactivation of TLR4/MyD88-mediated NF-κB and MAPK signaling pathways.

Kaempferol, a very common type of dietary flavonoids, has been found to exert antioxidative and anti-inflammatory properties. The purpose of our investigation was designed to reveal the effect of kaempferol on H9N2 influenza virus-induced inflammation in vivo and in vitro. In vivo, BALB/C mice were infected intranasally with H9N2 influenza virus with or without kaempferol treatment to induce acute lung injury (ALI) model. In vitro, MH-S cells were infected with H9N2 influenza virus with or without kaempferol treatment. In vivo, kaempferol treatment attenuated pulmonary edema, the W/D mass ratio, pulmonary capillary permeability, myeloperoxidase (MPO) activity, and the numbers of inflammatory cells. Kaempferol reduced ROS and Malondialdehyde (MDA) production, and increased the superoxide dismutase (SOD) activity. Kaempferol also reduced overproduction of TNF-α, IL-1β and IL-6. In addition, kaempferol decreased the H9N2 viral titre. In vitro, ROS, MDA, TNF-α, IL-1β and IL-6 was also reduced by kaempferol. Moreover, our data showed that kaempferol significantly inhibited the upregulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphorylation level of IκBα and nuclear factor-κB (NF-κB) p65, NF-κB p65 DNA binding activity, and phosphorylation level of MAPKs, both in vivo and in vitro. These results suggest that kaempferol exhibits a protective effect on H9N2 virus-induced inflammation via suppression of TLR4/MyD88-mediated NF-κB and MAPKs pathways, and kaempferol may be considered as an effective drug for the potential treatment of influenza virus-induced ALI.

Mesenchymal stromal cell treatment prevents H9N2 avian influenza virus-induced acute lung injury in mice.

BackgroundThe avian influenza virus (AIV) can cross species barriers and expand its host range from birds to mammals, even humans. Avian influenza is characterized by pronounced activation of the proinflammatory cytokine cascade, which perpetuates the inflammatory response, leading to persistent systemic inflammatory response syndrome and pulmonary infection in animals and humans. There are currently no specific treatment strategies for avian influenza.MethodsWe hypothesized that mesenchymal stromal cells (MSCs) would have beneficial effects in the treatment of H9N2 AIV-induced acute lung injury in mice. Six- to 8-week-old C57BL/6 mice were infected intranasally with 1 × 104 MID50 of A/HONG KONG/2108/2003 [H9N2 (HK)] H9N2 virus to induce acute lung injury. After 30 min, syngeneic MSCs were delivered through the caudal vein. Three days after infection, we measured the survival rate, lung weight, arterial blood gas, and cytokines in both bronchoalveolar lavage fluid (BALF) and serum, and assessed pathological changes to the lungs.ResultsMSC administration significantly palliated H9N2 AIV-induced pulmonary inflammation by reducing chemokines and proinflammatory cytokines levels, as well as reducing inflammatory cell recruit into the lungs. Thus, H9N2 AIV-induced lung injury was markedly alleviated in mice treated with MSCs. Lung histopathology and arterial blood gas analysis were improved in mice with H9N2 AIV-induced lung injury following MSC treatment.ConclusionsMSC treatment significantly reduces H9N2 AIV-induced acute lung injury in mice and is associated with reduced pulmonary inflammation. These results indicate a potential role for MSC therapy in the treatment of clinical avian influenza.

Carnosine markedly ameliorates H9N2 swine influenza virus-induced acute lung injury

Oxidative stress injury is an important pathogenesis of influenza virus in critically ill patients. The present study investigated the efficacy of carnosine, an antioxidant and free radical scavenger, on a model of acute lung injury (ALI) induced by H9N2 swine influenza virus. Female specific-pathogen-free BALB/c mice were randomized into four groups and treated as follows: (1) H9N2 group, (2) mock control group, (3) H9N2+carnosine group and (4) carnosine control group. The H9N2 group mice were inoculated intranasally with A/Swine/Hebei/012/2008/ (H9N2) virus (100 μl) in allantoic fluid (AF), whilst mock-infected animals were intranasally inoculated with non-infectious AF. Carnosine [10 mg (kg body mass)- 1] was administered orally (100 μl) for 7 days consecutively. The survival rate, lung water content, TNF-α and IL-1β levels, lung histopathology, myeloperoxidase (MPO) activity, and Toll-like receptor (TLR)-4 levels were determined at 2, 4, 6, 8 and 14 days after inoculation. Carnosine treatment effectively decreased the mortality (43 versus 75 %, P < 0.05), significantly ameliorated pathological lesions in lungs and decreased the lung wet/dry mass ratio (P < 0.05). It also inhibited MPO activity, suppressed TNF-α and IL-1β release, decreased the H9N2 viral titre, and markedly inhibited levels of TLR-4 mRNA and protein in the lungs of infected mice (P < 0.05), which supported the use of carnosine for managing severe influenza cases.

Activation of A1-adenosine receptors promotes leukocyte recruitment to the lung and attenuates acute lung injury in mice infected with influenza A/WSN/33 (H1N1) virus.

We have shown that bronchoalveolar epithelial A1-adenosine receptors (A1-AdoR) are activated in influenza A virus-infected mice. Alveolar macrophages and neutrophils also express A1-AdoRs, and we hypothesized that activation of A1-AdoRs on these cells will promote macrophage and neutrophil chemotaxis and activation and thereby play a role in the pathogenesis of influenza virus-induced acute lung injury. Wild-type (WT) C57BL/6 mice, congenic A1-AdoR knockout (A1-KO) mice, and mice that had undergone reciprocal bone marrow transfer were inoculated intranasally with 10,000 PFU/mouse influenza A/WSN/33 (H1N1) virus. Alternatively, WT mice underwent daily treatment with the A1-AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) from 1 day prior to inoculation. Infection increased bronchoalveolar lining fluid (BALF) adenosine comparably in WT and A1-KO mice. Infection of WT mice resulted in reduced carotid arterial O2 saturation (hypoxemia), lung pathology, pulmonary edema, reduced lung compliance, increased basal airway resistance, and hyperresponsiveness to methacholine. These effects were absent or significantly attenuated in A1-KO mice. Levels of BALF leukocytes, gamma interferon (IFN-γ), and interleukin 10 (IL-10) were significantly reduced in infected A1-KO mice, but levels of KC, IP-10, and MCP-1 were increased. Reciprocal bone marrow transfer resulted in WT-like lung injury severity, but BALF leukocyte levels increased only in WT and A1-KO mice with WT bone barrow. Hypoxemia, pulmonary edema, and levels of BALF alveolar macrophages, neutrophils, IFN-γ, and IL-10 were reduced in DPCPX-treated WT mice. Levels of viral replication did not differ between mouse strains or treatment groups. These findings indicate that adenosine activation of leukocyte A1-AdoRs plays a significant role in their recruitment to the infected lung and contributes to influenza pathogenesis. A1-AdoR inhibitor therapy may therefore be beneficial in patients with influenza virus-induced lung injury. Because antiviral drugs are of limited efficacy in patients hospitalized for influenza virus-induced respiratory failure, there is an urgent need for new therapeutics that can limit the progression of lung injury and reduce influenza death rates. We show that influenza A virus infection results in increased production of the nucleoside adenosine in the mouse lung and that activation of A1-subtype adenosine receptors by adenosine contributes significantly to both recruitment of innate immune cells to the lung and development of acute lung injury following influenza virus infection. We also show that treatment with an A1-adenosine receptor antagonist reduces the severity of lung injury in influenza virus-infected mice. Our findings indicate that adenosine plays an important and previously unrecognized role in the innate immune response to influenza virus infection and suggest that drugs which can inhibit either generation of adenosine or activation of A1-adenosine receptors may be beneficial in treating influenza patients hospitalized for respiratory failure.

N-acetyl-l-cystine (NAC) protects against H9N2 swine influenza virus-induced acute lung injury.

The antioxidant N-acetyl-l-cysteine (NAC) had been shown to inhibit replication of seasonal human influenza A viruses. Here, the effects of NAC on H9N2 swine influenza virus-induced acute lung injury (ALI) were investigated in mice. BALB/c mice were inoculated intranasally with 107 50% tissue culture infective doses (TCID50) of A/swine/HeBei/012/2008/(H9N2) viruses with or without NAC treatments to induce ALI model. The result showed that pulmonary inflammation, pulmonary edema, MPO activity, total cells, neutrophils, macrophages, TNF-α, IL-6, IL-1β and CXCL-10 in BALF were attenuated by NAC. Moreover, our data showed that NAC significantly inhibited the levels of TLR4 protein and TLR4 mRNA in the lungs. Pharmacological inhibitors of TLR4 (E5564) exerted similar effects like those determined for NAC in H9N2 swine influenza virus-infected mice. These results suggest that antioxidants like NAC represent a potential additional treatment option that could be considered in the case of an influenza A virus pandemic.

Roles of p38 MAPK in the regulation of the inflammatory response to swine influenza virus-induced acute lung injury in mice.

Swine influenza virus (SIV), one of the most important zoonotic agents, is associated with major public health concerns. The current study was conducted to investigate the role of p38 mitogen-activated protein kinase (p38 MAPK) in the regulation of the inflammatory response to acute lung injury (ALI) induced by SIV of H9N2 subtype (H9N2-SIV) in mice. For this purpose, BALB/c mice were intranasally infected with 20 LD(50) of H9N2-SIV (infected group), while non-infected mice served as control (control group). To assess the effect of p38 MAPK, its specific inhibitor SB203580 was employed followed by SIV infection (SB group). At various times after infection, mouse lungs were subjected to pathological and histological observations and detection of inflammatory cytokines tumor necrosis factor α (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 and phosphorylated p38 MAPK. The obtained results showed obvious inflammatory responses, injury and raised levels of inflammatory cytokines and phosphorylated p38 MAPK in the lungs of virus-infected mice. In the mice inoculated with the virus alone, the level of phosphorylated p38 MAPK increased from day 2 and peaked at day 6 post infection (p.i.). However, SB203580 caused lower increases in inflammatory cytokines and phosphorylated p38 MAPK and a milder lung injury. These findings indicate that the activation of p38 MAPK upregulated the inflammatory responses to H9N2-SIV-induced ALI, increased its severity and promoted the production of inflammatory cytokines.

Type I interferon limits influenza virus-induced acute lung injury by regulation of excessive inflammation in mice

Antiviral immune responses play as a double edged sword in resolution of infection and pathogenesis of acute lung injury caused by infection with highly pathogenic influenza A viruses. Here we show that type I interferons (IFNs) are important in protection against acute influenza A virus infection not only via their antiviral activity but also via their anti-inflammatory activity. IFN α receptor (IFNAR) knock-out (KO) mice exhibited increased mortality and morbidity with higher viral load after infection with influenza virus A/FM/1/47 (H1N1, a mouse-adapted strain) compared with wild-type (WT) mice, though the viruses were finally eliminated in both groups. The levels of proinflammatory cytokines in the lungs were significantly higher, while the level of IL-10 in the lungs was significantly lower in IFNAR KO mice than in WT mice during the course of infection. Restoration of IL-10 during an ongoing virus infection significantly reduced the levels of proinflammatory cytokines and improved mortality of IFNAR KO mice. These results suggest that type I IFNs are responsible not only for direct resolution of viral load but also for suppression of immunopathology caused by influenza A virus through IL-10 production.