IAV-induced Lung Injury Research Articles

Gut microbiota-derived acetate attenuates lung injury induced by influenza infection via protecting airway tight junctions

BackgroundGut microbiota (GM) have been implicated as important regulators of gastrointestinal symptom which is commonly occurred along with respiratory influenza A virus (IAV) infection, suggesting the involvement of the gut-to-lung axis in a host’s response to IAV. IAV primarily destroys airway epithelium tight junctions (TJs) and consequently causes acute respiratory disease syndrome. It is known that GM and their metabolism produce an anti-influenza effect, but their role in IAV-induced airway epithelial integrity remains unknown.MethodsA mouse model of IAV infection was established. GM were analyzed using 16S rRNA gene sequencing, and short-chain fatty acids (SCFAs) levels were measured. GM depletion and fecal microbiota transplantation (FMT) were conducted to validate the role of GM in IAV infection. A pair-feeding experiment was conducted to reveal whether IAV-induced GM dysbiosis is attributed to impaired food intake. Furthermore, human bronchial epithelial (HBE) cells were cocultured with IAV in the presence or absence of acetate. TJs function was analyzed by paracellular permeability and transepithelial electronic resistance (TEER). The mechanism of how acetate affects TJs integrity was evaluated in HBE cells transfected with G protein-coupled receptor 43 (GPR43) short hairpin RNA (shRNA).ResultsIAV-infected mice exhibited lower relative abundance of acetate-producing bacteria (Bacteroides, Bifidobacterium, and Akkermansia) and decreased acetate levels in gut and serum. These changes were partly caused by a decrease in food consumption (due to anorexia). GM depletion exacerbated and FMT restored IAV-induced lung inflammatory injury. IAV infection suppressed expressions of TJs (occludin, ZO-1) leading to disrupted airway epithelial barrier function as evidenced by decreased TEER and increased permeability. Acetate pretreatment activated GPR43, partially restored IAV-induced airway epithelial barrier function, and reduced inflammatory cytokines levels (TNF-α, IL-6, and IL-1β). Such protective effects of acetate were absent in HBE cells transfected with GPR43 shRNA. Acetate and GPR43 improved TJs in an AMP-activated protein kinase (AMPK)-dependent manner.ConclusionCollectively, our results demonstrated that GM protected airway TJs by modulating GPR43-AMPK signaling in IAV-induced lung injury. Therefore, improving GM dysbiosis may be a potential therapeutic target for patients with IAV infection.

The pathogenesis of influenza in intact alveoli: virion endocytosis and its effects on the lung's air-blood barrier.

Lung infection by influenza A virus (IAV) is a major cause of global mortality from lung injury, a disease defined by widespread dysfunction of the lung's air-blood barrier. Endocytosis of IAV virions by the alveolar epithelium - the cells that determine barrier function - is central to barrier loss mechanisms. Here, we address the current understanding of the mechanistic steps that lead to endocytosis in the alveolar epithelium, with an eye to how the unique structure of lung alveoli shapes endocytic mechanisms. We highlight where future studies of alveolar interactions with IAV virions may lead to new therapeutic approaches for IAV-induced lung injury.

Activation of TREK-1 (K2P2.1) potassium channels protects against influenza A-induced lung injury.

Influenza-A virus (IAV) infects yearly an estimated one billion people worldwide, resulting in 300,000-650,000 deaths. Preventive vaccination programs and antiviral medications represent the mainstay of therapy, but with unacceptably high morbidity and mortality rates, new targeted therapeutic approaches are urgently needed. Since inflammatory processes are commonly associated with measurable changes in the cell membrane potential (Em), we investigated whether Em hyperpolarization via TREK-1 (K2P2.1) K+ channel activation can protect against influenza-A virus (IAV)-induced pneumonia. We infected mice with IAV, which after 5 days caused 10-15% weight loss and a decrease in spontaneous activity, representing a clinically relevant infection. We then started a 3-day intratracheal treatment course with the novel TREK-1 activating compounds BL1249 or ML335. We confirmed TREK-1 activation with both compounds in untreated and IAV-infected primary human alveolar epithelial cells (HAECs) using high-throughput fluorescent imaging plate reader (FLIPR) assays. In mice, TREK-1 activation with BL1249 and ML335 counteracted IAV-induced histological lung injury and decrease in lung compliance and improved BAL fluid total protein levels, cell counts, and inflammatory IL-6, IP-10/CXCL-10, MIP-1α, and TNF-α levels. To determine whether these anti-inflammatory effects were mediated by activation of alveolar epithelial TREK-1 channels, we studied the effects of BL1249 and ML335 in IAV-infected HAEC, and found that TREK-1 activation decreased IAV-induced inflammatory IL-6, IP-10/CXCL10, and CCL-2 secretion. Dissection of TREK-1 downstream signaling pathways and construction of protein-protein interaction (PPI) networks revealed NF-κB1 and retinoic acid-inducible gene-1 (RIG-1) cascades as the most likely targets for TREK-1 protection. Therefore, TREK-1 activation may represent a novel therapeutic approach against IAV-induced lung injury.

Lignans from Mosla scabra Ameliorated Influenza A Virus-Induced Pneumonia via Inhibiting Macrophage Activation.

The lower respiratory tract infection, induced by influenza virus, coronaviruses, and respiratory syncytial virus, remains a serious threat to human health that can cause a global pandemic. Thus, finding effective chemicals and therapeutic measures to advance the functional restoration of the respiratory tract after infection has been the emphasis of the studies on the subjects. Mosla scabra is a natural medicinal plant used for treating various lung and gastrointestinal diseases, including viral infection, cough, chronic obstructive pulmonary disease, acute gastroenteritis, and diarrhoea. In this study, the antiviral and anti-inflammatory effects of total lignans (MSTL) extracted from the plant were investigated in influenza A virus (IAV)-infected mice and RAW 264.7 macrophages. MSTL could not only protect the macrophages against IAV-induced pyroptosis but also could lighten the lung inflammation induced by IAV in vivo and in vitro. The network pharmacology analysis revealed that differentially expressed genes, mainly involving in EGFR tyrosine kinase inhibitor resistance, endocrine resistance, HIF-1 signaling pathway, C-type lectin receptor signaling pathway, and FOXO signaling pathway, contributed to the IAV-induced alveolar macrophage dysfunction. It indicated that MSTL enhanced the function of alveolar macrophages and improved IAV-induced lung injury in mice.

CircRNA expression profiling and bioinformatics analysis indicate the potential biological role and clinical significance of circRNA in influenza A virus-induced lung injury

Circular RNA (circRNA) plays an important role in the regulation of multiple biological processes. However, circRNA profiling and the potential biological role of circRNA in influenza A virus (IAV)-induced lung injury have not been investigated. In the present study, circRNA expression profiles in lung tissues from mice with and without IAV-induced lung injury were analyzed using high-throughput sequencing, and differentially expressed circRNAs were verified by quantitative PCR. The gene homology of candidate circRNAs was investigated and the expression of plasma circRNAs from patients with IAV-induced acute respiratory distress syndrome (ARDS) was detected. The target microRNAs (miRNAs) of circRNAs were predicted. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. In total, 781 circRNAs were differentially expressed between ARDS mice and control (467 were up-regulated and 314 were down-regulated). Moreover, the candidate circRNAs (Slco3a1, Nfatc2, Wdr33, and Dmd) expression showed the same trend with the sequencing results. The isoforms of circRNA Slco3a1 and Wdr33 were highly conserved between humans and mice. Plasma circRNA Slco3a1 and Wdr33 presented differential expression in patients with IAV-induced ARDS compared to control. The circRNA-miRNA interaction network and GO and KEGG analyses indicated the potential biological role of circRNAs in the development of IAV-induced lung injury. Taken together, a large number of differentially expressed circRNAs were identified in our study. CircRNA Slco3a1 and Wdr33 had significantly different expression in specimens from mice and humans, and showed a potential biological role in IAV-induced lung injury by bioinformatics analysis.Electronic supplementary materialThe online version contains supplementary material available at 10.1007/s12038-021-00152-8.

STIM1 mediates IAV-induced inflammation of lung epithelial cells by regulating NLRP3 and inflammasome activation via targeting miR-223

AimsInfluenza A virus (IAV) infection accelerates the inflammatory injury of lung epithelial cells that contributes to pulmonary lesion. Recently, stromal interaction molecule 1 (STIM1) was found to mediate cellular immune response and participated in lung tumorigenesis. Our study aimed to illustrate the function and mechanism of STIM1 in IAV-induced inflammation injury and oxidative stress of lung epithelial cells. Main methodsWe evaluated the levels of STIM1 in IAV-infected patients' serum and BEAS-2B cells using RT-qPCR, Elisa and western blotting methods. MTT and Elisa were performed to measure cell viability and cytokine contents. Besides, ROS intensity, SOD contents and cell apoptosis were detected based on DCFH-DA probe, colorimetry and cell death kits. A luciferase assay and Pearson's correlation analysis evaluated the associations between target genes. Key findingsSTIM1 was dramatically up-regulated in IAV-infected patients' serum and BEAS-2B cells. Silencing STIM1 in vitro inhibited oxidative stress and inflammatory responses induced by IAV, and reversed cell viability and suppressed apoptosis. Moreover, miR-223 and NLRP3 were negatively and positively correlated with STIM1. STIM1 was found to regulate NLRP3 expression by binding the AACUGAC motif in miR-223. STIM1/miR-223/NLRP3 axis modulated IAV-induced inflammation injury of lung epithelial cells. SignificanceOur evidence indicated that silencing STIM1 alleviated IAV-induced inflammation injury of lung epithelial cells by inactivating NLRP3 and inflammasome via promoting miR-223 expression. These findings may contribute to understand the mechanism of IAV-induced lung injury and help for therapy of IAV infection.

Pharmacological inhibition of poly (ADP-ribose) polymerase by olaparib ameliorates influenza-virus-induced pneumonia in mice

Treatments against influenza A viruses (IAV) have to be updated regularly due to antigenic drift and drug resistance. Poly (ADP-ribose) polymerases (PARPs) are considered effective therapeutic targets of acute lung inflammatory injury. This study aimed to explore the effects of PARP-1 inhibitor olaparib on IAV-induced lung injury and the underlying mechanisms. Male wild-type C57BL/6 mice were intranasally infected with IAV strain H1N1 to mimic pneumonia experimentally. Olaparib at different doses was intraperitoneally injected 2 days before and 5 consecutive days after virus stimulation. On day 6 post-infection, lung tissues as well as bronchoalveolar lavage fluid (BALF) were sampled for histological and biochemical analyses. Olaparib increased the survival rate of IAV mice dose-dependently. Olaparib remarkably reduced IAV mRNA expression, myeloperoxidase (MPO) level, and inflammatory cell infiltration in IAV lungs. Moreover, olaparib significantly reduced the level of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6, and IL-4 and increased IL-10 in IAV lungs. Also, olaparib efficiently reduced IL-6, monocyte chemotactic protein (MCP)-1, granulocyte colony-stimulating factor (G-CSF), TNF-α, chemokine (C–X–C motif) ligand (CXCL)1, CXCL10, chemokine (C–C motif) ligand (CCL)3, and regulated on activation, normal T cell expressed and secreted (RANTES) release in IAV BALF. Olaparib decreased PARylated protein content and p65, IκBα phosphorylation in IAV lung tissues. This study successfully constructed the pneumonia murine model using IAV. Olaparib decreased IAV-induced mortality in mice, lung injury, and cytokine production possibly via modulation of PARP-1/NF-κB axis.Electronic supplementary materialThe online version of this article (10.1007/s10096-020-04020-5) contains supplementary material, which is available to authorized users.

EMAPII Monoclonal Antibody Ameliorates Influenza A Virus-Induced Lung Injury.

Influenza A virus (IAV) remains a major worldwide health threat, especially to high-risk populations, including the young and elderly. There is an unmet clinical need for therapy that will protect the lungs from damage caused by lower respiratory infection. Here, we analyzed the role of EMAPII, a stress- and virus-induced pro-inflammatory and pro-apoptotic factor, in IAV-induced lung injury. First, we demonstrated that IAV induces EMAPII surface translocation, release, and apoptosis in cultured endothelial and epithelial cells. Next, we showed that IAV induces EMAPII surface translocation and release to bronchoalveolar lavage fluid (BALF) in mouse lungs, concomitant with increases in caspase 3 activity. Injection of monoclonal antibody (mAb) against EMAPII attenuated IAV-induced EMAPII levels, weight loss, reduction of blood oxygenation, lung edema, and increase of the pro-inflammatory cytokine TNF alpha. In accordance with the pro-apoptotic properties of EMAPII, levels of caspase 3 activity in BALF were also decreased by mAb treatment. Moreover, we detected EMAPII mAb-induced increase in lung levels of M2-like macrophage markers YM1 and CD206. All together, these data strongly suggest that EMAPII mAb ameliorates IAV-induced lung injury by limiting lung cell apoptosis and shifting the host inflammatory setting toward resolution of inflammation.

Impact of Monocyte Age on Influenza A Virus‐induced Lung Injury

RationaleInfluenza A virus (IAV) causes more life‐threatening infections in healthy children than in healthy adults. In our mouse model of juvenile IAV infection, juvenile mice clear the virus at the same rate as adult mice, but develop more severe lung injury and have increased mortality compared to adult mice. We previously found that the monocytes recruited to the lungs during IAV infection exhibited a more inflammatory phenotype in juvenile mice than in adult mice. These juvenile monocytes exacerbated IAV‐induced lung injury in juvenile mice, and inhibition of their recruitment protected juvenile mice from IAV‐induced lung injury without impacting viral clearance. Based on these findings, we hypothesized that juvenile monocytes contribute to excessive inflammation and lung injury during IAV infection, and that adult monocytes could protect juvenile mice from IAV‐induced lung injury and death.MethodsPregnant mice were treated with busulfan (15 mg/kg i.p. day E18 and E19) to cause myelosuppression in fetal pups. Following birth, additional busulfan was given to juvenile pups (10 mg/kg i.p. day of life 1). Bone marrow derived stem cells were harvested from adult (10 week old) donor mice bearing the CD45.1 marker and injected into the myelosuppressed recipient pups (CD45.2) on day 2 of life (7.5 × 106 cells intravenously). Four weeks following transplantation, juvenile recipient mice were then infected with IAV (strain WSN, 25 PFU, intratracheally) and monitored for the development of lung injury and survival.ResultsJuvenile mice expressing juvenile CD45.2 cells had a median survival of 9 dpi. In contrast, 90% of the juvenile mice expressing adult CD45.1 were alive at 9 dpi and the median survival was >62%. Histological examination of the lungs showed decreased lung injury in IAV‐infected juvenile mice expressing adult CD45.1, as compared to control mice. Juvenile mice that recruited adult monocytes to the lungs during IAV infection had decreased lung injury compared to juvenile mice that recruited juvenile monocytes to the lungs during IAV infection.ConclusionsJuvenile mice are more susceptible to IAV‐induced lung injury and death than adult mice. Recruitment of juvenile monocytes to the lungs during IAV infection exacerbates inflammation and worsens outcomes. Transplantation of adult monocytes into juvenile mice lessens lung injury and improves survival. This suggests that juvenile monocytes are particularly injurious in juvenile IAV infection and may have age‐related cell‐intrinsic properties that exacerbate lung injury.Support or Funding InformationPediatric Critical Care and Trauma Scientist Development Program (NICHD K12)American Thoracic Society/American Lung AssociationNHLBIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protective effects of diketopiperazines from Moslae Herba against influenza A virus-induced pulmonary inflammation via inhibition of viral replication and platelets aggregation

Ethnopharmacological relevanceMoslae Herba (MH) is broadly used as an antiviral, antipyretic and anticoagulant drug which effectively treats respiratory diseases including cough, asthma, throat, cold and flu. Aim of this studyThe excessive inflammation of the lungs is the hallmark of severe influenza A virus (IAV) infection, while platelet aggregation and its subsequent microvascular thrombosis can exacerbate IAV-induced lung injury. Thus, inhibition of platelet aggregation can be a potential target for IAV treatment. Previous studies focus on the flavonoids from MH and their anti-inflammatory activities, but the anticoagulant compounds and potential molecular mechanism of MH remains unclear. This study was to isolate and characterize diketopiperazines (DKPs) from MH and to explore the underlying anticoagulant mechanism on IAV infection models. Materials and methodsEtOAc sub-extract separated from MH ethanolic extract was subjected to fractionation through column chromatography. The chemical structures of pure compounds were characterized by the spectral analysis. Antiviral activities of DKPs were assayed in IAV-infected Madin-Darby canine kidney (MDCK) cells and mice. Anticoagulant effects of DKPs were investigated on adenosine 5′-diphosphate (ADP)-induced acute pulmonary embolism and IAV-induced lung injury in vivo, as well as the inhibition on platelet activating factor (PAF), arachidonic acid (AA) and ADP-induced platelet aggregation in vitro. The serum levels of thromboxane B2 (TXB2) and 6-keto-PGF1α were detected by ELISA. The expressions of key proteins in CD41-mediated PI3K/AKT pathways were determined by western blotting analysis. ResultsSix DKPs were, for the first time, isolated from MH and identified as cyclo(Tyr-Leu) (1), cyclo(Phe-Phe) (2), cyclo(Phe-Tyr) (3), cyclo(Ala-Ile) (4), cyclo(Ala-Leu) (5) and Bz-Phe-Phe-OMe (6). Among these DKPs, cyclo(Ala-Ile) and Bz-Phe-Phe-OMe possessed low cytotoxicities and significant inhibition against cytopathic effects induced by IAV (H1N1 and H3N2) replication in MDCK cells. Furthermore, cyclo(Ala-Ile) and Bz-Phe-Phe-OMe significantly alleviated IAV-induced platelet activation and lung inflammation in mice. They could reduce the expression of CD41 and the phosphorylation of PI3K and AKT in PLTs of IAV-infected mice. ConclusionThese results suggested that cyclo(Ala-Ile) and Bz-Phe-Phe-OMe isolated from MH have antiviral and anticoagulant effects against IAV-induced PLT aggregation and lung inflammation via regulating CD41/PI3K/AKT pathway, and could be used as the potential agents for IAV treatment.

MiR-194 Inhibits Innate Antiviral Immunity by Targeting FGF2 in Influenza H1N1 Virus Infection.

Fibroblast growth factor 2 (FGF2 or basic FGF) regulates a wide range of cell biological functions including proliferation, angiogenesis, migration, differentiation, and injury repair. However, the roles of FGF2 and the underlying mechanisms of action in influenza A virus (IAV)-induced lung injury remain largely unexplored. In this study, we report that microRNA-194-5p (miR-194) expression is significantly decreased in A549 alveolar epithelial cells (AECs) following infection with IAV/Beijing/501/2009 (BJ501). We found that miR-194 can directly target FGF2, a novel antiviral regulator, to suppress FGF2 expression at the mRNA and protein levels. Overexpression of miR-194 facilitated IAV replication by negatively regulating type I interferon (IFN) production, whereas reintroduction of FGF2 abrogated the miR-194-induced effects on IAV replication. Conversely, inhibition of miR-194 alleviated IAV-induced lung injury by promoting type I IFN antiviral activities in vivo. Importantly, FGF2 activated the retinoic acid-inducible gene I signaling pathway, whereas miR-194 suppressed the phosphorylation of tank binding kinase 1 and IFN regulatory factor 3. Our findings suggest that the miR-194-FGF2 axis plays a vital role in IAV-induced lung injury, and miR-194 antagonism might be a potential therapeutic target during IAV infection.

Basic fibroblast growth factor protects against influenza A virus-induced acute lung injury by recruiting neutrophils.

Influenza virus (IAV) infection is a major cause of severe respiratory illness that affects almost every country in the world. IAV infections result in respiratory illness and even acute lung injury and death, but the underlying mechanisms responsible for IAV pathogenesis have not yet been fully elucidated. In this study, the basic fibroblast growth factor 2 (FGF2) level was markedly increased in H1N1 virus-infected humans and mice. FGF2, which is predominately derived from epithelial cells, recruits and activates neutrophils via the FGFR2-PI3K-AKT-NFκB signaling pathway. FGF2 depletion or knockout exacerbated influenza-associated disease by impairing neutrophil recruitment and activation. More importantly, administration of the recombinant FGF2 protein significantly alleviated the severity of IAV-induced lung injury and promoted the survival of IAV-infected mice. Based on the results from experiments in which neutrophils were depleted and adoptively transferred, FGF2 protected mice against IAV infection by recruiting neutrophils. Thus, FGF2 plays a critical role in preventing IAV-induced lung injury, and FGF2 is a promising potential therapeutic target during IAV infection.

39 - Using electrophilic fatty acid derivatives to tame host-induced lung injury during influenza infection

In the past century, there have been four major Influenza A Virus (IAV) pandemics including the H1N1 strain that killed an estimated 50 million people in 1918. Seasonal strains still claim over 250,000 deaths globally each year and circulating strains in migratory birds and poultry that could potentially mutate and transmit in humans, are known to be highly pathogenic with reported mortality rates of over 50%. In cases of severe infection, an exuberant host-response induces lung injury by generating reactive oxygen and nitrogen species, recruiting immune cells, and damaging the epithelial-endothelial barrier. Vaccines and antiviral treatments do not focus on host-mediated lung injury; therefore, novel therapeutic strategies that target the host to minimize lung damage are required. Electrophilic fatty acid derivatives (EFADs) can dampen inflammation through nuclear factor κB (NF-ĸB) inhibition as well as activate antioxidant responses via nuclear factor erythroid 2-related factor 2 (Nrf2) by alkylating key cysteine residues involved in redox signaling. We hypothesized that EFADs will mitigate IAV-induced lung injury by dampening inflammation (NF-ĸB) and activating antioxidant responses (Nrf2). Our group successfully used nitro-oleic acid (NO2-OA, an EFAD) to mitigate lung injury observed 70% survival of NO2-OA treated mice versus 10% survival in those treated with the vehicle. A significant decrease in both CD4 and CD8 T cell recruitment was observed on day 6 post infection and correlated with maximal plasma levels of NO2-OA. Furthermore, pilot study data showed decreased Nrf2 nuclear translocation during infection and subsequent restoration upon treatment with NO2-OA. Together, these data suggest EFADs can rescue mice infected with a lethal dose of IAV and both NF-ĸB and Nrf2 are possible mechanisms of action since they are implicated in immune cell infiltration through their ability to alter the expression of genes associated with cytokine/chemokine release, reaction oxygen and nitrogen species generation, and antiviral signaling. These studies will shed light on how EFADs can “tame” the host response to dampen the collateral damage associated with severe infection and how both Nrf2 and NF-ĸB are regulated during influenza infection.

Hesperidin attenuates influenza A virus (H1N1) induced lung injury in rats through its anti-inflammatory effect.

Influenza A viruses (IAV) can cause pandemics and are big threats to human health. Inflammation is the main pathological process in the lungs after IAV infection. We aimed to investigate whether hesperidin, a well-known anti-inflammatory compound, could be effective in improving IAV-induced lung injury. We generated a rat model using H1N1 virus infection, and intraperitoneally injected different doses of hesperidin for 5 days. Pulmonary function was analysed. Local inflammatory state was profiled by immune cells and cytokines. Pulmonary microvascular endothelial cells were isolated from rats and used to test the effects of hesperidin in vitro. Hesperidin showed efficacy in improving H1N1-induced impairment of pulmonary function in a dose-dependent manner. Local numbers of immune cells and concentrations of cytokines were significantly limited by hesperidin. However, we found that hesperidin neither inhibited virus replication, nor rescued infected pulmonary microvascular endothelial cells. Rather, we observed that hesperidin reduced pro-inflammatory cytokine production by suppressing mitogen-activated protein kinase (MAPK) signalling pathways. Hesperidin could alleviate H1N1-induced impairment of pulmonary function by inhibiting cytokine production in pulmonary microvascular endothelial cells through MAPK signalling pathways.

AIM2 Inflammasome Is Critical for Influenza-Induced Lung Injury and Mortality.

The absent in melanoma 2 (AIM2) inflammasome plays an important role in many viral and bacterial infections, but very little is known about its role in RNA virus infection, including influenza A virus (IAV). In this study, we have designed in vivo and in vitro studies to determine the role of AIM2 in infections with lethal doses of IAVs A/PR8/34 and A/California/07/09. In wild-type mice, IAV infection enhanced AIM2 expression, induced dsDNA release, and stimulated caspase-1 activation and release of cleaved IL-1β in the lung, which was significantly reduced in AIM2-deficient mice. Interestingly, AIM2 deficiency did not affect the transcription of caspase-1 and IL-1β. In addition, AIM2-deficient mice exhibited attenuated lung injury and significantly improved survival against IAV challenges, but did not alter viral burden in the lung. However, AIM2 deficiency did not seem to affect adaptive immune response against IAV infections. Furthermore, experiments with AIM2-specific small interfering RNA-treated and AIM2-deficient human and mouse lung alveolar macrophages and type II cells indicated a macrophage-specific function of AIM2 in regulation of IAV-stimulated proinflammatory response. Collectively, our results demonstrate that influenza infection activates the AIM2 inflammasome, which plays a critical role in IAV-induced lung injury and mortality. AIM2 might serve as a therapeutic target for combating influenza-associated morbidity and mortality without compromising the host antiviral responses.

Antiviral activities of atractylon from Atractylodis Rhizoma.

Atractylodis Rhizoma is a traditional medicinal herb, which has antibacterial, antiviral, anti-inflammatory and anti-allergic, anticancer, gastroprotective and neuroprotective activities. It is widely used for treating fever, cold, phlegm, edema and arthralgia syndrome in South-East Asian nations. In this study, 6 chemical compositions of Atractylodis Rhizoma were characterized by spectral analysis and their antiviral activities were evaluated in vitro and in vivo. Among them, atractylon showed most significant antiviral activities. Atractylon treatment at doses of 10–40 mg/kg for 5 days attenuated influenza A virus (IAV)-induced pulmonary injury and decreased the serum levels of interleukin (IL)-6, tumor necrosis factor-α and IL-1β, but increased interferon-β (IFN-β) levels. Atractylon treatment upregulated the expression of Toll-like receptor 7 (TLR7), MyD88, tumor necrosis factor receptor-associated factor 6 and IFN-β mRNA but downregulated nuclear factor-κB p65 protein expression in the lung tissues of IAV-infected mice. These results demonstrated that atractylon significantly alleviated IAV-induced lung injury via regulating the TLR7 signaling pathway, and may warrant further evaluation as a possible agent for IAV treatment.

Macrophage-epithelial paracrine crosstalk inhibits lung edema clearance during influenza infection.

Influenza A viruses (IAV) can cause lung injury and acute respiratory distress syndrome (ARDS), which is characterized by accumulation of excessive fluid (edema) in the alveolar airspaces and leads to hypoxemia and death if not corrected. Clearance of excess edema fluid is driven mostly by the alveolar epithelial Na,K-ATPase and is crucial for survival of patients with ARDS. We therefore investigated whether IAV infection alters Na,K-ATPase expression and function in alveolar epithelial cells (AECs) and the ability of the lung to clear edema. IAV infection reduced Na,K-ATPase in the plasma membrane of human and murine AECs and in distal lung epithelium of infected mice. Moreover, induced Na,K-ATPase improved alveolar fluid clearance (AFC) in IAV-infected mice. We identified a paracrine cell communication network between infected and noninfected AECs and alveolar macrophages that leads to decreased alveolar epithelial Na,K-ATPase function and plasma membrane abundance and inhibition of AFC. We determined that the IAV-induced reduction of Na,K-ATPase is mediated by a host signaling pathway that involves epithelial type I IFN and an IFN-dependent elevation of macrophage TNF-related apoptosis-inducing ligand (TRAIL). Our data reveal that interruption of this cellular crosstalk improves edema resolution, which is of biologic and clinical importance to patients with IAV-induced lung injury.

ID: 159: IFN-dependent macrophage-epithelial crosstalk induces and sustains lung injury in viral pneumonia

Influenza A viruses (IAV) may cause lung injury and acute respiratory distress syndrome (ARDS) characterized by apoptotic injury to the alveolar epithelium and accumulation of excessive fluid (edema) in the alveolar airspaces resulting in loss of barrier function, respiratory failure, and death. We recently demonstrated that inflammatory macrophages recruited from the circulation into the alveoli substantially contribute to influenza-induced lung epithelial injury by IFN-dependent expression of pro-apoptotic factors such as TNF-related apoptosis-inducing ligand (TRAIL). Following severe injury, clearance of excess edema fluid, driven by the alveolar epithelial Na, K-ATPase, is crucial for survival of patients with ARDS. We demonstrate that, apart from causing direct injury to the delicate alveolar barrier, macrophage mediators significantly impair resolution of inflammation in terms of edema clearance from the infected lungs. Using ex vivo and in vivo infection models including transgenic mice and adoptive cell transfer strategies, we identify a paracrine cell communication network between alveolar epithelial cells and alveolar macrophages leading to decreased alveolar epithelial Na, K-ATPase plasma membrane abundance and inhibition of alveolar fluid clearance. Our data furthermore reveal that therapeutic blockade of this cellular cross-talk improves edema resolution which is of clinical significance to patients with IAV-induced lung injury.

Delivery of GM-CSF to Protect against Influenza Pneumonia.

BackgroundSince adaptive immunity is thought to be central to immunity against influenza A virus (IAV) pneumonias, preventive strategies have focused primarily on vaccines. However, vaccine efficacy has been variable, in part because of antigenic shift and drift in circulating influenza viruses. Recent studies have highlighted the importance of innate immunity in protecting against influenza.MethodsGranulocyte-macrophage colony stimulating factor (GM-CSF) contributes to maturation of mononuclear phagocytes, enhancing their capacity for phagocytosis and cytokine production.ResultsOverexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) in the lung of transgenic mice provides remarkable protection against IAV, which depends on alveolar macrophages (AM). In this study, we report that pulmonary delivery of GM-CSF to wild type young and aged mice abrogated mortality from IAV.ConclusionWe also demonstrate that protection is species specific and human GM-CSF do not protect the mice nor stimulates mouse immunity. We also show that IAV-induced lung injury is the culprit for side-effects of GM-CSF in treating mice after IAV infection, and introduce a novel strategy to deliver the GM-CSF to and retain it in the alveolar space even after IAV infection.

Abstract 17261: Inhibition of Fibrinolysis Attenuates Influenza A-induced Lung Injury

Rationale: Influenza A virus (IAV) is the leading cause of death from an infectious cause and ranks 8th in the list of attributable annual mortality in the United States. Currently available antiviral treatments are limited and may become ineffective as resistant strains emerge. The coexistence of hemostatic alterations with inflammatory responses to viral infections support the idea that common molecular mechanisms contribute to the regulation of these systems. Previous studies demonstrated that IAV induces the activation of both coagulation and fibrinolytic pathways, and that the impairment of the latter is mainly attributable to high levels of plasminogen activator inhibitor type I (PAI-1), the major inhibitor of tissue-type plasminogen activator (t-PA). Interferon-α (IFN-α) plays a key role in the control of viral replication and overall shaping of the immune response. We sought to determine whether PAI-1, plays a role in the immune response and morbidity and mortality associated with IAV infection. Methods: Wild-type (C57BL/6J), PAI-1-/-, t-PA-/- and PAI-1 stab (transgenic mice expressing an active form of human PAI-1) mice were treated intratracheally with IAV (A/WSN/33 [H1N1] 500 pfu/mouse in 50μl PBS). We assessed the levels of IFN-α in bronchoalveolar lavage fluid (BALF) on day 2 and 4, and influenza A-induced morbidity (weight loss) and mortality. Results: Compared with wild-type mice, PAI-1-/- mice had increased IAV-induced morbidity and mortality. In contrast, both PAI-1 stab and t-PA-/- mice had improved survival. In wild-type mice, IAV induced a significant elevation IFN-α levels in BALF. PAI-1 stab mice had a 2-fold increase, while PAI-1-/-mice had a 69% reduction in IFN-α levels in BALF compared to wild-type mice. Conclusions: Inhibition of fibrinolysis by means of genetic overexpression of PAI-1 or deletion of t-PA augments the antiviral response and attenuates the morbidity and mortality associated with IAV infection. These results suggest an important functional role for PAI-1 and fibrinolysis in the pathogenesis of influenza A infection. Modulation of the fibrinolytic pathway or PAI-1 may potentially be useful as a target for novel therapeutics in the management of IAV-induced lung injury.