IAV Infection Research Articles

Profiling Cullin4-E3 Ligases Interactomes and Their Rewiring in Influenza a Virus Infection

Understanding the integrated regulation of cellular processes during viral infection is crucial for developing host-targeted approaches. We have previously reported that an optimal in vitro infection by influenza A (IAV) requires three components of Cullin 4-RING E3 ubiquitin ligases (CRL4) complexes, namely the DDB1 adaptor and two Substrate Recognition Factors (SRF), DCAF11 and DCAF12L1, which mediate non-degradative poly-ubiquitination of the PB2 subunit of the viral polymerase. However, the impact of IAV infection on the CRL4 interactome remains elusive. Here, using Affinity Purification coupled with Mass Spectrometry (AP-MS) approaches, we identified cellular proteins interacting with these CRL4 components in IAV-infected and non-infected contexts. IAV infection induces significant modulations in protein interactions, resulting in a global loss of DDB1 and DCAF11 interactions, and an increase in DCAF12L1-associated proteins. The distinct rewiring of CRL4's associations upon infection impacted cellular proteins involved in protein folding, ubiquitination, translation, splicing, and stress responses. Using a split-nanoluciferase-based assay, we identified direct partners of CRL4 components and via siRNA-mediated silencing validated their role in IAV infection, representing potential substrates or regulators of CRL4 complexes. Our findings unravel the dynamic remodeling of the proteomic landscape of CRL4's E3 ubiquitin ligases during IAV infection, likely involved in shaping a cellular environment conducive to viral replication and offer potential for the exploration of future host-targeted antiviral therapeutic strategies.

Matrix metalloproteinases mediate influenza A-associated shedding of the alveolar epithelial glycocalyx.

The alveolar epithelium is protected by a heparan sulfate-rich, glycosaminoglycan layer called the epithelial glycocalyx. It is cleaved in patients with acute respiratory distress syndrome (ARDS) and in murine models of influenza A (IAV) infection, shedding fragments into the airspace from the cell surface. Glycocalyx shedding results in increased permeability of the alveolar-capillary barrier, amplifying acute lung injury. The mechanisms underlying alveolar epithelial glycocalyx shedding in IAV infection are unknown. We hypothesized that induction of host sheddases such as matrix metalloproteinases (MMPs) during IAV infection results in glycocalyx shedding and increased lung injury. We measured glycocalyx shedding and lung injury during IAV infection with and without treatment with the pan-MMP inhibitor Ilomastat (ILO) and in an MMP-7 knock out (MMP-7KO) mouse. C57BL/6 or MMP-7KO male and female mice were given IAV A/PR/8/34 (H1N1) at 30,000 PFU/mouse or PBS intratracheally. For some experiments, C56BL/6 mice were infected in the presence of ILO (100mg/kg) or vehicle given daily by IP injection. Bronchoalveolar lavage (BAL) and lung tissue were collected on day 1, 3, and 7 for analysis of glycocalyx shedding (BAL Syndecan-1) and lung injury (histology, BAL protein, BAL cytokines, BAL immune cell infiltrates, BAL RAGE). Expression and localization of the sheddase MMP-7 and its inhibitor TIMP-1 was examined by RNAScope. For in vitro experiments, MLE-12 mouse lung epithelial cells were cultured and treated with active or heat-inactivated heparinase (2.5 U/mL) prior to infection with IAV (MOI 1) and viral load and MMP-7 and TIMP-1 expression analyzed. IAV infection caused shedding of the epithelial glycocalyx into the BAL. Inhibition of MMPs with ILO reduced glycocalyx shedding by 36% (p = 0.0051) and reduced lung epithelial injury by 40% (p = 0.0404). ILO also reduced viral load by 68% (p = 0.027), despite having no significant effect on lung cytokine production. Both MMP-7 and its inhibitor TIMP-1 were upregulated in IAV infected mice: MMP-7 colocalized with IAV, while TIMP-1 was limited to cells adjacent to infection. However, MMP-7KO mice had similar glycocalyx shedding, epithelial injury, and viral load compared to WT littermates, suggesting redundancy in MMP sheddase function in the lung. In vitro, heparinase treatment before infection led to a 52% increase in viral load (p = 0.0038) without altering MMP-7 or TIMP-1 protein levels. Glycocalyx shedding and MMPs play key roles in IAV-induced epithelial injury, with significant impact on IAV viral load. Further studies are needed to understand which specific MMPs regulate lung epithelial glycocalyx shedding.

Cooperation of immune regulators Tollip and surfactant protein A inhibits influenza A virus infection in mice

BackgroundInfluenza A virus (IAV) infection is a significant risk factor for respiratory diseases, but the host defense mechanisms against IAV remain to be defined. Immune regulators such as surfactant protein A (SP-A) and Toll-interacting protein (Tollip) have been shown to be involved in IAV infection, but whether SP-A and Tollip cooperate in more effective host defense against IAV infection has not been investigated.MethodsWild-type (WT), Tollip knockout (KO), SP-A KO, and Tollip/SP-A double KO (dKO) mice were infected with IAV for four days. Lung macrophages were isolated for bulk RNA sequencing. Precision-cut lung slices (PCLS) from WT and dKO mice were pre-treated with SP-A and then infected with IAV for 48 h.ResultsViral load was significantly increased in bronchoalveolar lavage (BAL) fluid of dKO mice compared to all other strains of mice. dKO mice had significantly less recruitment of neutrophils into the lung compared to Tollip KO mice. SP-A treatment of PCLS enhanced expression of TNF and reduced viral load in dKO mouse lung tissue. Pathway analysis of bulk RNA sequencing data suggests that macrophages from IAV-infected dKO mice reduced expression of genes involved in neutrophil recruitment, IL-17 signaling, and Toll-like receptor signaling.ConclusionsOur data suggests that both Tollip and SP-A are essential for the lung to exert more effective innate defense against IAV infection.

The Role of Invariant Natural Killer T (iNKT) Cells in Early Responses to Influenza A Virus (IAV)

Abstract iNKT cells are a CD1d-restricted innate-like lineage of ⍺ßT cells that are prevalent in mucosal tissues, where they influence early immune responses. iNKT cells differentiate in the thymus into—iNKT1, iNKT2, and iNKT17 analogous to Th1, Th2, and Th17 cells. We have shown in ET-2;CD4cre mice, that sustained E protein activity results in changes in iNKT subset composition, with a shift from iNKT1s towards iNKT2/NKT17 cells and other subsets. To understand the physiological relevance of these changes, we tested the response of ET2 mice to IAV. ET2 mice showed less weight loss, reduction in lung-infected areas, decreased neutrophil infiltration, lower activation markers in inflammatory monocytes, reduced levels of CCL2 and interferon-responsive genes in ET2. We observed increased levels of IFNλ in infected ET2 mice vs. elevated IFNα in WT mice 24 hours post-infection. scRNA-seq analysis of sorted iNKT from WT and ET2 mice showed that ET2 profiles are closer to conventional naïve T cells, and that, after infection, their response is marked by overexpression of Ccr7, -Lselectin, IL1R1. WT response is predominantly type 1. Gene Ontology pathway analysis of infected WT show enrichment of cytokine production and immune cell activation pathways, while pathways enriched in infected ET-2 are associated with adhesion and repair. These experiments suggest that early type 1 responses driven by NKT cells during IAV infection can be deleterious, intensifying inflammation, and neutrophilia.

Melatonin improves influenza virus infection-induced acute exacerbation of COPD by suppressing macrophage M1 polarization and apoptosis

BackgroundInfluenza A viruses (IAV) are extremely common respiratory viruses for the acute exacerbation of chronic obstructive pulmonary disease (AECOPD), in which IAV infection may further evoke abnormal macrophage polarization, amplify cytokine storms. Melatonin exerts potential effects of anti-inflammation and anti-IAV infection, while its effects on IAV infection-induced AECOPD are poorly understood.MethodsCOPD mice models were established through cigarette smoke exposure for consecutive 24 weeks, evaluated by the detection of lung function. AECOPD mice models were established through the intratracheal atomization of influenza A/H3N2 stocks in COPD mice, and were injected intraperitoneally with melatonin (Mel). Then, The polarization of alveolar macrophages (AMs) was assayed by flow cytometry of bronchoalveolar lavage (BAL) cells. In vitro, the effects of melatonin on macrophage polarization were analyzed in IAV-infected Cigarette smoking extract (CSE)-stimulated Raw264.7 macrophages. Moreover, the roles of the melatonin receptors (MTs) in regulating macrophage polarization and apoptosis were determined using MTs antagonist luzindole.ResultsThe present results demonstrated that IAV/H3N2 infection deteriorated lung function (reduced FEV20,50/FVC), exacerbated lung damages in COPD mice with higher dual polarization of AMs. Melatonin therapy improved airflow limitation and lung damages of AECOPD mice by decreasing IAV nucleoprotein (IAV-NP) protein levels and the M1 polarization of pulmonary macrophages. Furthermore, in CSE-stimulated Raw264.7 cells, IAV infection further promoted the dual polarization of macrophages accompanied with decreased MT1 expression. Melatonin decreased STAT1 phosphorylation, the levels of M1 markers and IAV-NP via MTs reflected by the addition of luzindole. Recombinant IL-1β attenuated the inhibitory effects of melatonin on IAV infection and STAT1-driven M1 polarization, while its converting enzyme inhibitor VX765 potentiated the inhibitory effects of melatonin on them. Moreover, melatonin inhibited IAV infection-induced apoptosis by suppressing IL-1β/STAT1 signaling via MTs.ConclusionsThese findings suggested that melatonin inhibited IAV infection, improved lung function and lung damages of AECOPD via suppressing IL-1β/STAT1-driven macrophage M1 polarization and apoptosis in a MTs-dependent manner. Melatonin may be considered as a potential therapeutic agent for influenza virus infection-induced AECOPD.Graphical Schematic mechanisms underlying the regulatory effects of melatonin on macrophage polarization and apoptosis in IAV infection plus cigarette stimulation-induced AECOPD model.

Xiaoer niuhuang qingxin powder alleviates influenza a virus infection by inhibiting the activation of the TLR4/MyD88/NF-κB signaling pathway

Ethnopharmacological relevanceXiaoer Niuhuang Qingxin Powder (XNQP) is a classic traditional Chinese medicine formula with significant clinical efficacy for treating febrile convulsions and influenza. Aim of the studyThis study aims to explore the potential mechanisms of XNQP in combating combating the influenza A virus, providing a theoretical basis for its clinical application. Materials and methodsThe present investigation employed network pharmacology and bioinformatics analysis to determine the TLR4/MyD88/NF-κB signaling pathway as a viable target for XNQP intervention in IAV infection.Subsequently, a mouse model of influenza A virus infection was established, and different doses of XNQP were used for intervention. The protein expression levels of TLR4/MyD88/NF-κB were detected using HE staining, Elisa, immunohistochemistry, immunofluorescence, and western blot. ResultsThe results showed that treatment with XNQP after IAV infection reduced the mortality and prolonged the survival time of infected mice. It reduced the release of TNF-α and IFN-γ in the serum and alleviated pathological damage in the lung tissue following infection. Additionally, the levels of TLR4, MyD88, NF-κB, and p–NF–κB P65 proteins were significantly reduced in lung tissue by XNQP. The inhibitory effect of XNQP on the expression of MyD88 and NF-κB was antagonized when TLR4 signaling was overexpressed. Consequently, the expression levels of MyD88, NF-κB, and p–NF–κB P65 were increased in lung tissue. Conversely, the expression levels of the proteins MyD88, NF-κB, and p–NF–κB P65 were downregulated when TLR4 signaling was inhibited. ConclusionsXNQP alleviated lung pathological changes, reduced serum levels of inflammatory factors, reduced mortality, and prolonged survival time in mice by inhibiting the overexpression of the TLR4/MyD88/NF-κB signaling pathway in lung tissues after IAV infection.

Prior infection with unrelated neurotropic virus exacerbates influenza disease and impairs lung T cell responses

Immunity to infectious diseases is predominantly studied by measuring immune responses towards a single pathogen, although co-infections are common. In-depth mechanisms on how co-infections impact anti-viral immunity are lacking, but are highly relevant to treatment and prevention. We established a mouse model of co-infection with unrelated viruses, influenza A (IAV) and Semliki Forest virus (SFV), causing disease in different organ systems. SFV infection eight days before IAV infection results in prolonged IAV replication, elevated cytokine/chemokine levels and exacerbated lung pathology. This is associated with impaired lung IAV-specific CD8+ T cell responses, stemming from suboptimal CD8+ T cell activation and proliferation in draining lymph nodes, and dendritic cell paralysis. Prior SFV infection leads to increased blood brain barrier permeability and presence of IAV RNA in brain, associated with increased trafficking of IAV-specific CD8+ T cells and establishment of long-term tissue-resident memory. Relative to lung IAV-specific CD8+ T cells, brain memory IAV-specific CD8+ T cells have increased TCR repertoire diversity within immunodominant DbNP366+CD8+ and DbPA224+CD8+ responses, featuring suboptimal TCR clonotypes. Overall, our study demonstrates that infection with an unrelated neurotropic virus perturbs IAV-specific immune responses and exacerbates IAV disease. Our work provides key insights into therapy and vaccine regimens directed against unrelated pathogens.

Impaired influenza A virus replication by the host restriction factor SAMHD1 which inhibited by PA-mediated dephosphorylation of the host transcription factor IRF3

BackgroundInfluenza A virus (IAV) can cause severe and life-threatening illness in humans and animals. Therefore, it is important to search for host antiviral proteins and elucidate their antiviral mechanisms for the development of potential treatments. As a part of human innate immunity, host restriction factors can inhibit the replication of viruses, among which SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) can restrict the replication of viruses, such as HIV and enterovirus EV71. Viruses also developed countermeasures in the arms race with their hosts. There are few reports about whether SAMHD1 has a restriction effect on IAV.MethodsTo investigate the impact of IAV infection on SAMHD1 expression in A549 cells, we infected A549 cells with a varying multiplicity of infection (MOI) of IAV and collected cell samples at different time points for WB and RT-qPCR analysis to detect viral protein and SAMHD1 levels. The virus replication level in the cell culture supernatant was determined using TCID50 assay. Luciferase assay was used to reveal that H5N1 virus polymerase acidic protein (PA) affected the activity of the SAMHD1 promoter. To assess the antiviral capacity of SAMHD1, we generated a knockdown and overexpressed cell line for detecting H5N1 replication.ResultsIn this study, we observed that SAMHD1 can restrict the intracellular replication of H5N1 and that the H5N1 viral protein PA can downregulate the expression of SAMHD1 by affecting SAMHD1 transcriptional promoter activity. We also found that SAMHD1's ability to restrict H5N1 is related to phosphorylation at 592-tyrosine.ConclusionsIn conclusion, we found that SAMHD1 may affect the replication of IAVs as a host restriction factor and be countered by PA. Furthermore, SAMHD1 may be a potential target for developing antiviral drugs.

Influenza Virus-Induced Paracrine Cellular Senescence of the Lung Contributes to Enhanced Viral Load.

Aging is a major risk factor associated with increased morbidity and mortality rates observed during respiratory infections. In this study, we investigated the role of influenza virus infections in the establishment of premature cellular senescence and paracrine macrophage-activated inflammation. We observed in our murine model a premature aging by the appearance of senescent cells in the lungs after 21 d of influenza A virus infection. By using murine ex vivo lung models, the influence of TNF-α on the establishment of cellular senescence was detectable. Our findings were proven by using conditioned media of infected human monocyte-derived macrophages on primary lung fibroblasts. Here, a distinct expression of senescence-associated parameters could be confirmed. Furthermore, senescent cells in the lungs strongly influenced subsequent viral infections. Our data demonstrated a higher viral load in senescent primary lung fibroblasts, indicating an intracellular effect on viral replication. Transcriptomic data revealed an increased regulation of JAK/STAT signaling in senescent IAV-infected cells accompanied with increased TRAIL expression. Additionally, senescent cells indicating low pH values, accelerating viral replication. Our study provides new insights into pathomechanisms of virus-induced cellular senescence. Hence, IAV infection induces premature senescence and subsequent infections in senescent cells lead to an increased viral replication.

Hoveniae Semen Seu Fructus water extract inhibits influenza A virus infection

Hoveniae Semen Seu Fructus (HSF) is a fruit and seed of Hovenia dulcis Thunb. Here, we first report HSF water extract contains a potent anti-influenza A virus (IAV) effect via inhibition of neuraminidaseand hemagglutinin and a direct virus-killing effect. We examined the effect of WHSF on IAV infection using GFP-tagged Influenza A/PR/8/34. FACS analysis and fluorescent microscopy showed a dose-dependent inhibitory effect of WHSF against IAV infection. WHSF also suppressed the cytopathic effects of H1N1 and H3N2 IAV. Immunofluorescence and qPCR analyses confirmed WHSF significantly decreases IAV protein and RNA expressions. Time of addition and plaque assays revealed WHSF affects viral replication in the early stage via modulatinghemagglutinin and inducing virucidal effects. Furthermore, WHSF suppressed the neuraminidase activity of H1N1 and H3N2 IAV. Quercetin, not taxifolin in WHSF inhibited IAV infection. Our results suggest WHSF could be developed as a natural antiviral agent to prevent IAV infection.

Systemic perturbations in amino acids/amino acid derivatives and tryptophan pathway metabolites associated with murine influenza A virus infection

BackgroundInfluenza A virus (IAV) is the only influenza virus causing flu pandemics (i.e., global epidemics of flu disease). Influenza (the flu) is a highly contagious disease that can be deadly, especially in high-risk groups. Worldwide, these annual epidemics are estimated to result in about 3 to 5 million cases of severe illness and in about 290,000 to 650,000 respiratory deaths. We intend to reveal the effect of IAV infection on the host′s metabolism, immune response, and neurotoxicity by using a mouse IAV infection model.Methods51 metabolites of murine blood plasma (33 amino acids/amino acid derivatives (AADs) and 18 metabolites of the tryptophan pathway) were analyzed by using Ultra-High-Performance Liquid Chromatography-Mass Spectrometry with Electrospray Ionization at the acute (7 days post-infection (dpi)), resolution (14 dpi), and recovery (21 dpi) stages of the virus infection in comparison with controls.ResultsAmong the 33 biogenic amino acids/AADs, the levels of five amino acids/AADs (1-methylhistidine, 5-oxoproline, α-aminobutyric acid, glutamine, and taurine) increased by 7 dpi, whereas the levels of ten amino acids/AADs (4-hydroxyproline, alanine, arginine, asparagine, cysteine, citrulline, glycine, methionine, proline, and tyrosine) decreased. By 14 dpi, the levels of one AAD (3-methylhistidine) increased, whereas the levels of five amino acids/AADs (α-aminobutyric acid, aminoadipic acid, methionine, threonine, valine) decreased. Among the 18 metabolites from the tryptophan pathway, the levels of kynurenine, quinolinic acid, hydroxykynurenine increased by 7 dpi, whereas the levels of indole-3-acetic acid and nicotinamide riboside decreased.ConclusionsOur data may facilitate understanding the molecular mechanisms of host responses to IAV infection and provide a basis for discovering potential new mechanistic, diagnostic, and prognostic biomarkers and therapeutic targets for IAV infection.

Inhibition of influenza a virus infection by natural stilbene piceatannol targeting virus hemagglutinin.

Given the magnitude of influenza pandemics as a threat to the global population, it is crucial to have as many prevention and treatment options as possible. Piceatannol (PIC) is a tetrahydroxylated stilbenoid (trans-3,4,3',5'-tetrahydroxystilbene), also known as 3'- hydroxy resveratrol, which has demonstrated many different biological activities such as anti-inflammatory and antiviral activities. In this study, the anti-influenza A virus (IAV) activities and mechanisms of PIC in vitro and in vivo were investigated in order to provide reference for the development of novel plant-derived anti-IAV drugs. The viral plaque assay, RT-PCR and western blot assay were used to evaluate the anti-IAV effects of PIC in vitro. The anti-IAV mechanism of PIC was determined by HA syncytium assay, DARTS assay and Surface Plasmon Resonance assay. The mouse pneumonia model combined with HE staining were used to study the anti-IAV effects of PIC in vivo. PIC shows inhibition on the multiplication of both H1N1 and H3N2 viruses, and blocks the infection of H5N1 pseudovirus with low toxicity. PIC may directly act on the envelope of IAV to induce the rupture and inactivation of IAV particles. PIC can also block membrane fusion via binding to HA2 rather than HA1 and cleavage site of HA0. PIC may interact with the two residues (HA2-T68 and HA2-I75) of HA2 to block the conformational change of HA so as to inhibit membrane fusion. Importantly, oral therapy of PIC also markedly improved survival and reduced viral titers in IAV-infected mice. PIC possesses significant anti-IAV effects both in vitro and in vivo and may block IAV infection mainly through interaction with HA to block membrane fusion. Thus, PIC has the potential to be developed into a new broad-spectrum anti-influenza drug for the prevention and treatment of influenza.

Prevention of severe lung immunopathology associated with influenza infection through adeno-associated virus vector administration

BackgroundInfluenza A viruses (IAVs) have long posed a threat to humans, occasionally causing significant morbidity and mortality. The initial immune response is triggered by infected epithelial cells, alveolar macrophages and dendritic cells. However, an exaggerated innate immune response can result in severe lung injury and even host mortality. One notable pathology observed in hosts succumbing to severe influenza is the excessive influx of neutrophils and monocytes into the lung. In this study, we investigated a strategy for controlling lung immunopathology following severe influenza infection.ResultsTo evaluate the impact of innate immunity on influenza-associated lung injury, we employed CB17.SCID and NOD.SCID mice. NOD.SCID mice exhibited slower weight loss and longer survival than CB17.SCID mice following influenza infection. Lung inflammation was reduced in NOD.SCID mice compared to CB17.SCID mice. Bulk RNA sequencing analysis of lung tissue showed significant downregulation of 827 genes, and differentially expressed gene analysis indicated that the cytokine-cytokine receptor interaction pathway was predominantly downregulated in NOD.SCID mice. Interestingly, the expression of the Cxcl14 gene was higher in the lungs of influenza-infected NOD.SCID mice than in CB17.SCID mice. Therefore, we induced overexpression of the Cxcl14 gene in the lung using the adeno-associated virus 9 (AAV9)-vector system for target gene delivery. However, when we administered the AAV9 vector carrying the Cxcl14 gene or a control AAV9 vector to BALB/c mice from both groups, the morbidity and mortality rates remained similar. Both groups exhibited lower morbidity and mortality than the naive group that did not receive the AAV9 vector prior to IAV infection, suggesting that the pre-administration of the AAV9 vector conferred protection against lethal influenza infection, irrespective of Cxcl14 overexpression. Furthermore, we found that pre-inoculation of BALB/c mice with AAV9 attenuated the infiltration of trans-macrophages, neutrophils and monocytes in the lungs following IAV infection. Although there was no difference in lung viral titers between the naive group and the AAV9 pre-inoculated group, pre-inoculation with AAV9 conferred lung injury protection against lethal influenza infection in mice.ConclusionsOur study demonstrated that pre-inoculation with AAV9 prior to IAV infection protected mouse lungs from immunopathology by reducing the recruitment of inflammatory cells.

Influenza enhances host susceptibility to non-pulmonary invasive Streptococcus pyogenes infections

ABSTRACT Streptococcus pyogenes (group A streptococcus; GAS) causes a variety of invasive diseases (iGAS) such as bacteremia, toxic shock syndrome, and pneumonia, which are associated with high mortality despite the susceptibility of the bacteria to penicillin ex vivo. Epidemiologic studies indicate that respiratory influenza virus infection is associated with an increase in the frequency of iGAS diseases, including those not directly involving the lung. We modified a murine model of influenza A (IAV)-GAS superinfection to determine if viral pneumonia increased the susceptibility of mice subsequently infected with GAS in the peritoneum. The results showed that respiratory IAV infection increased the morbidity (weight loss) of mice infected intraperitoneally (i.p.) with GAS 3, 5, and 10 d after the initial viral infection. Mortality was also significantly increased when mice were infected with GAS 3 and 5 d after pulmonary IAV infection. Increased mortality among mice infected with virus 5 d prior to bacterial infection correlated with increased dissemination of GAS from the peritoneum to the blood, spleen, and lungs. The interval was also associated with a significant increase in the pro-inflammatory cytokines IFN-γ, IL-12, TNF-α, MCP-1 and IL-27 in sera. We conclude, using a murine model, that respiratory influenza virus infection increases the likelihood and severity of systemic iGAS disease, even when GAS infection does not originate in the respiratory tract.

Metformin reduces the risk of developing influenza A virus related cardiovascular disease

This study investigated the drug repositioning potential of metformin for cardiovascular risk due to influenza A virus infection. Statistical analysis was performed to analyze factors related to the risk of death after IAV infection in diabetic patients. Through in vitro and in vivo experiments, the effect of metformin on influenza A virus infection in non-diabetic conditions was analyzed. In logistic regression analysis, influenza vaccination (OR = 0.378, p-value = 0.007, 0.186 < 95% C·I < 0.768) and metformin treatment (OR = 0.380, p-value = 0.016, 0.173 < 95% C·I < 0.835) were associated with a decreased influenza-related mortality in diabetic patients. In vitro and in vivo studies showed that viral replication and influenza A virus-induced cytokine expression were inhibited by metformin. In particular, MCP-1 and IP-10, cytokines related to cell infiltration and cardiovascular disease development, were significantly reduced by metformin under influenza A virus infection condition. As a result, the acute exacerbation of atherosclerosis caused by influenza A virus in mouse aorta was inhibited by metformin. In addition, we found that regulation of AKT/MAPK signaling plays an important role in the mechanism of metformin. In conclusion, we demonstrated the potential and mechanism of metformin as a treatment for acute exacerbation of atherosclerosis caused by influenza A virus infection.

The antiviral activity of Thuja orientalis folium against Influenza A virus

Thuja orientalis Folium (TOF) has been prescribed traditionally as an expectorant for inflammatory airway disease. In this study, we evaluated the anti-influenza A virus (IAV) activity of TOF by detecting GFP expressed by influenza A virus (A/PR/8/34-GFP) infection. The fluorescence microscopy and fluorescence-activated cell sorting analysis showed that TOF potently inhibited IAV infection, dose-dependently. Consistently, immunofluorescence and Q-PCR analysis results confirmed TOF significantly represses IAV protein and RNA expression. TOF inhibited IAV infection at the binding and entry step upon viral infection and interferes with HA protein. Further, TOF exhibited a virucidal effect and inhibited the neuraminidase activity of IAV. Additionally, TOF prevented the cytopathic effect caused by H1N1 and H3N2 IAV infection. Amentoflavone among the constituents in TOF exerted the strongest anti-IAV effect. Myricetin, quercetin, and quercitrin also inhibited IAV infection. However, the potent anti-IAV effect of TOF may be related to the synergistic effect of constituents, not by a single specific compound. Our results suggest TOF exhibits a significant inhibitory effect against IAV infection at multi-stages via the blockage of viral attachment and entry, inhibition of neuraminidase, and induction of virucidal effects.

Stimulation of lipopolysaccharide from Pseudomonas aeruginosa following H9N2 IAV infection exacerbates inflammatory responses of alveolar macrophages and decreases virus replication

H9N2 IAV infection contributed to P. aeruginosa coinfection, causing severe hemorrhagic pneumonia in mink. In this study, the in vitro alveolar macrophage models were developed to investigate the innate immune responses to P. aeruginosa LPS stimulation following H9N2 IAV infection, using MH-S cells. The cytokine levels, apoptosis levels and the viral nucleic acid levels were detected and analyzed. As a result, the levels of IFN-α, IL-1β, TNF-α, and IL-10 in MH-S cells with P. aeruginosa LPS stimulation following H9N2 IAV infection were significantly higher than those in MH-S cells with single H9N2 IAV infection and single LPS stimulation (P < 0.05), exacerbating inflammatory responses. LPS stimulation aggravated the apoptosis of MH-S cells with H9N2 IAV infection. Interestingly, LPS stimulation influences H9N2 IAV replication and indirectly reduced H9N2 IAV replications in in vitro AMs. It implied that LPS should play an important role in the pathogenesis of H9N2 IAV and P. aeruginosa coinfection.

Interferon-lambda modulates dendritic cell IL-10 production and CD8 T cell immunity during influenza A virus infection

Abstract Influenza A virus (IAV) infection remains a major global health burden, requiring development of novel strategies to limit infection and drive durable protective immunity. Type III interferon (IFN), IFN-lambda, plays an important role in regulating IAV infection and immunity, but it’s contribution to regulating adaptive immunity remains less understood. Our previous research found mice lacking the IFN-lambda receptor (Ifnlr1−/−) are more susceptible to heterosubtypic rechallenge with influenza A virus. Ifnlr1−/−mice also have reduced effector and memory IAV-specific CD8 T cells compared to WT, highlighting a unique role for IFN-lambda in regulating protective CD8 T cell immunity against IAV. However, this defect in the CD8 T cell response was not T cell-intrinsic, but rather due to aberrant dendritic cell (DC) function in the absence of IFN-lambda signaling. RNA-Seq analysis of DC from the draining lymph nodes of mice following IAV infection revealed enhancement of an immunosuppressive network containing IL-10 in Ifnlr1−/−not present in WT. We hypothesize that IFN-lambda suppresses IL-10, playing a crucial role in regulation of protective CD8 T cell responses against IAV. Our results demonstrate IL-10 production is increased in bone marrow-derived DC (BMDC) in vitro and CD103 +DC in vivo following IAV infection in Ifnlr1−/−compared to WT. Ongoing experiments will unravel the contribution of IFN-lambda regulated IL-10 in regulation of CD8 T cell responses during IAV infection in vivo. Overall, understanding the mechanisms by which IFN-lambda signaling regulates DC function and protective CD8 T cell immunity will inform therapeutic potential of this cytokine during respiratory virus infection. Supported by NIH grant K22 AI146141

Abstract 156: Role of Platelets in Maintaining Hemostasis and Coagulation Activation in Viral-induced Acute Respiratory Distress Syndrome (ards) in Mice

Platelets are crucial players during viral infections, as bridge hemostasis, inflammation, and immune response. RNA viruses like IAV are associated with pathologic platelet activation and thrombosis. We are currently investigating how platelet depletion before or post initial IAV infection can affect disease outcome. WT mice were infected with IAV (0.02HAU, intranasally) for 7 days to induce viral ARDS. Mice were injected with platelet depletion antibody R300 or control IgG at day 0 (prior) and day 3 (after infection). Mice treated with R300/control IgG were subjected to dsRNA mimetic poly IC intranasally for 3 days to induce virus-like ARDS. Global lung function was recorded, plasma and BALF were collected 7 or 3 dpi or final poly IC treatment, respectively. BALF cellularity and blood cellular composition was analyzed. Markers of lung injury and cytokines were measured. We observed that IAV infection leads to local lung inflammation, coagulation activation and ARDS-like lung dysfunction. Mice treated with R300 showed severe thrombocytopenia in blood. We observed more impaired alveolar hemostasis and increased hemorrhages into the airspace when platelet depletion was initiated 3 dpi compared to mice where platelets were depleted prior to infection. In addition, in dsRNA-induced ARDS model, platelet depletion resulted in increased signs of lung dysfunction, increased lung injury and local inflammation compared to mice with normal platelet levels. In conclusion, depletion of platelets after IAV infection resulted in increased alveolar hemorrhage and was associated with more pronounced lung inflammation, injury, and lung dysfunction. Our study suggests that interfering with platelet before viral infection can be beneficial as it limits the role of platelets as potential multiplicator of an initial pathologic immune reaction subsequently reducing collateral damage and organ dysfunction/failure. However, at later stages of viral infection, platelet activation is beneficial to facilitate tissue hemostasis as their absence later can result in increased hemorrhagic risk and more pronounced lung dysfunction. We aim to address the mechanism by which platelet contribute later to pathologic immune processes in dsRNA-induced or viral ARDS

Abstract 419: Liberation Of Soluble Urokinase Plasminogen Receptor (supar) Occurs In Response To Coronavirus Infection But Not To Influenza Virus Infection

Objectives: Urokinase (uPA), and its receptor, uPAR, mediate fibrinolysis and matrix remodeling. Liberation of monocyte/macrophage (MO/Mϕ) bound uPAR to yield soluble uPAR (suPAR) occurs after infection with coronaviruses (CoVs) such as SARS-CoV-2, and suPAR levels correlate with systemic inflammation and degree of end organ injury. How suPAR liberation from MO/Mϕs occurs after CoV infection, and whether suPAR induction occurs after pneumotropic viral infections such as influenza A (IAV) is unclear. Methods: Murine bone marrow derived Mϕs (BMDMs) and immortalized Mϕs (RAW264.7) were infected with CoV [MHVA59 (murine model for SARS-CoV-2 infection) or MHV1] or with IAV (A/PR/8/34) and levels of mRNA and protein were measured using qRT-PCR and ELISA, respectively. uPA activity of cell supernatants was quantitated using a commercially available assay. uPA knockdown of Mϕs was performed using siRNA transfection. C57BL6/J mice underwent intranasal inoculation of either 2x10 5 pfu of MHVA59, 25 pfu of IAV, or with PBS (sham). Plasma levels of suPAR and splenic Mϕ (surrogate for circulating MOs) levels of mRNA and protein were analyzed at post-infection day 3. Results: Though both CoV or IAV infection resulted in induction of mRNA and protein levels of cellular uPAR and uPA, and uPA activity, only CoV infection yielded liberation of suPAR from Mϕs (Fig. 1A-C). Indicating a functional role in suPAR liberation, infection of uPA-silenced Mϕs yielded a blunted induction of suPAR release despite not affecting mRNA levels of uPAR (Fig 1D). Finally, inoculation of mice with MHVA59, but not with IAV, resulted in elevated plasma levels of suPAR, despite inducing a robust induction of uPAR in splenic Mϕs (Fig. 1E). Conclusions: suPAR induction results after CoV infection, but not IAV infection, and occurs through a uPA-dependent pathway. Antagonism of co-regulators of uPAR and uPA may prove to be a therapeutic strategy to combat post-CoV tissue injury mediated by Mϕs.