Influenza Infection In Mice Research Articles

Single-cell BCR and transcriptome analysis after influenza infection reveals spatiotemporal dynamics of antigen-specific B cells.

SummaryB cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity.

Physical properties of lactic acid bacteria influence the level of protection against influenza infection in mice.

We evaluated whether the water dispersibility of lactic acid bacteria (Enterococcus faecalis KH2) affects their efficacy. When cultured lactic acid bacteria are washed, heat-killed, and powdered, adhesion occurs between results in aggregation (non-treated lactic acid bacteria, n-LAB). However, dispersed lactic acid bacteria (d-LAB) with a lower number of aggregates can be prepared by treating them with a high-pressure homogenizer and adding an excipient during powdering. Mice were administered n-LAB or d-LAB Peyer's patches in the small intestine were observed. Following n-LAB administration, a high amount of aggregated bacteria drifting in the intestinal mucosa was observed; meanwhile, d-LAB reached the Peyer's patches and was absorbed into them. Evaluation in a mouse influenza virus infection model showed that d-LAB was more effective than n-LAB in the influenza yield of bronchoalveolar lavage fluids on day 3 post-infection and neutralizing antibody titers of sera and influenza virus-specific immunoglobulin A in the feces on day 14 post-infection. Therefore, the physical properties of lactic acid bacteria affect their efficacy; controlling their water dispersibility can improve their effectiveness.

Regulation of Inflammatory Response by Transmembrane Adaptor Protein LST1.

LST1 is a small adaptor protein expressed in leukocytes of myeloid lineage. Due to the binding to protein tyrosine phosphatases SHP1 and SHP2 it was thought to have negative regulatory function in leukocyte signaling. It was also shown to be involved in cytoskeleton regulation and generation of tunneling nanotubes. LST1 gene is located in MHCIII locus close to many immunologically relevant genes. In addition, its expression increases under inflammatory conditions such as viral infection, rheumatoid arthritis and inflammatory bowel disease and its deficiency was shown to result in slightly increased sensitivity to influenza infection in mice. However, little else is known about its role in the immune system homeostasis and immune response. Here we show that similar to humans, LST1 is expressed in mice in the cells of the myeloid lineage. In vivo, its deficiency results in alterations in multiple leukocyte subset abundance in steady state and under inflammatory conditions. Moreover, LST1-deficient mice show significant level of resistance to dextran sodium sulphate (DSS) induced acute colitis, a model of inflammatory bowel disease. These data demonstrate that LST1 regulates leukocyte abundance in lymphoid organs and inflammatory response in the gut.

The study of neuraminidase immunity in protection against secondary bacterial pneumonia induced by <i>S. aureus</i> after influenza infection in mice

Introduction. Pneumonia often occurs secondary to influenza infection and accounts for a large proportion of the morbidity and mortality associated with seasonal and pandemic influenza outbreaks. We previously have shown that vaccination with Virus-like particles (VLPs) containing hemagglutinin (HA) of influenza virus reduces mortality caused by bacterial infections after an influenza infections in mice.The aim of this work is to study whether this protective effect may be potentiated by supplementing the HA preparation with the influenza neuraminidase (NA).Materials and methods. We studied the effect of Gag-VLPs with the influenza HA or NA from А/PR/8/34 alone or in combination, in a lethal BALB/c mouse model of S. aureus infection after vaccine-matched or mismatched influenza virus challenge.Results. A cocktail of HA-Gag and NA-Gag-VLPs fully protected from weight loss, mortality and viral replication and significantly reduced the bacterial burden in the lungs of А/PR/8/34 infected animals. Immunization with this cocktail HA-Gag-VLPs 100 ng + NA-Gag-VLPs 20 ng also protected 60% of animals from mortality associated with secondary bacterial S. aureus infection following a heterologous H1N1 influenza virus challenge, and led to the significant protection from weight loss and pulmonary pathogen replication even in the absence of HA-inhibition and NA-inhibition antibodies.Conclusion. Our results indicate that influenza vaccination may improve the outcome of a secondary bacterial pneumonia induced by S. aureus after influenza even when the virus is antigenically different from the vaccine strain. At the same time, in our model, the significance of the immunity to influenza virus HA was prevalent.

Topical exposure to triclosan inhibits Th1 immune responses and reduces T cells responding to influenza infection in mice.

Healthcare workers concurrently may be at a higher risk of developing respiratory infections and allergic disease, such as asthma, than the general public. Increased incidence of allergic diseases is thought to be caused, in part, due to occupational exposure to chemicals that induce or augment Th2 immune responses. However, whether exposure to these chemical antimicrobials can influence immune responses to respiratory pathogens is unknown. Here, we use a BALB/c murine model to test if the Th2-promoting antimicrobial chemical triclosan influences immune responses to influenza A virus. Mice were dermally exposed to 2% triclosan for 7 days prior to infection with a sub-lethal dose of mouse adapted PR8 A(H1N1) virus (50 pfu); triclosan exposure continued until 10 days post infection (dpi). Infected mice exposed to triclosan did not show an increase in morbidity or mortality, and viral titers were unchanged. Assessment of T cell responses at 10 dpi showed a decrease in the number of total and activated (CD44hi) CD4+ and CD8+ T cells at the site of infection (BAL and lung) in triclosan exposed mice compared to controls. Influenza-specific CD4+ and CD8+ T cells were assessed using MHCI and MHCII tetramers, with reduced populations, although not reaching statistical significance at these sites following triclosan exposure. Reductions in the Th1 transcription factor T-bet were seen in both activated and tetramer+ CD4+ and CD8+ T cells in the lungs of triclosan exposed infected mice, indicating reduced Th1 polarization and providing a potential mechanism for numerical reduction in T cells. Overall, these results indicate that the immune environment induced by triclosan exposure has the potential to influence the developing immune response to a respiratory viral infection and may have implications for healthcare workers who may be at an increased risk for developing infectious diseases.

Orally administered recombinant baculovirus vaccine elicits partial protection against avian influenza virus infection in mice

Avian influenza outbreaks have placed a tremendous economic burden on the poultry industry, necessitating the need for an effective vaccine. Although multiple vaccine candidates are available, its development is hindered by several drawbacks associated with the vaccine platforms and as such, more improvements to the vaccines are needed. Therefore, in this study, the vaccine efficacy in the murine models was assessed prior to evaluation in chickens. An oral recombinant baculovirus (rBV) vaccine expressing influenza hemagglutinin (HA) (A/H5N1) was generated and its efficacy was investigated against homologous avian influenza infection in mice. Our results confirmed that oral administration of rBVs enhanced the level of virus-specific antibodies in the sera following boost immunization. Upon challenge infection with a lethal dose of highly pathogenic avian influenza virus (HPAI, H5N1) virus, a marked increase in mucosal IgG and IgA were observed. Drastically increased antibody secretory cell responses from the bone marrow cells and splenocytes of vaccinated mice were observed, in addition to the strongly elicited germinal center responses in the lungs and the spleens. Vaccinated mice showed significantly reduced lung pro-inflammatory cytokine responses, lung viral loads, body weight loss, and mortality. Though mice were only partially protected upon challenge infection, these results highlight the potential of orally administered rBVs expressing the HA as a vaccine candidate for controlling avian influenza outbreaks.

Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice.

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.

Mouse-adapted influenza B virus for in vitro and in vivo assessment of therapeutic and preventive efficacy of antiviral drugs

Objective: to develop a new antigenic relevance influenza B virus suitable for modeling influenza infection in mice to assess of in vivo and in vitro therapeutic and preventive efficacy of antiviral drugs. Materials and methods : was carried out an adaptation of influenza B virus in BALB/c mice. Was performed, comparative assessment of in vivo and in vitro pathogenicity of the parenta! virus and. adapted, influenza B virus. Was assessed, inhibition of neuraminidase with antiviral drugs (oseltamivir ethoxyacrylate and. Tamiflu) in relation to the adapted, influenza B virus. Results: adapted influenza B virus (B/Novosibirsk/40/2017-MA strain) models non-lethal influenza infection with pronounced, clinical signs of the disease in experimental animals. Were described the destructive changes in lungs and. brain that increases during infection. Analysis of internal organs (lungs, brain, liver, heart, kidneys, spleen) were revealed viral load only in the lungs. Were evaluated, in vivo and in vitro efficacy of antiviral drugs (oseltamivir ethoxysuccinate and Tamiflu®) on the model of influenza infection. Were proved, the high, efficiency of the innovative drug — oseltamivir ethoxysuccinate. Conclusion: the antigen-relevant adapted, influenza B virus (B/Novosibirsk/40/2017-MA strain) can be used, to assess the drug effectiveness against influenza, as well as an additional tool for predicting the effectiveness of the vaccine against drifting strains.

Early production of IL-17A by γδ T cells in the trachea promotes viral clearance during influenza infection in mice.

The innate immune response generated against influenza infection is critical for the inhibition of viral dissemination. The trachea contains different types of innate immune cells that protect the respiratory tract from pathogen invasion. Among them, γδ T cells have the ability to rapidly generate large amounts of pro‐inflammatory cytokines to preserve mucosal barrier homeostasis during infection. However, little is known about their role during the early phase of influenza infection in the airways. In this study, we found that, early after infection, γδ T cells are recruited and activated in the trachea and outnumber αβ T cells during the course of the influenza infection that follows. We also showed that the majority of the recruited γδ T cells express the Vγ4 TCR chain and infiltrate in a process that involves the chemokine receptor CXCR3. In addition, we demonstrated that γδ T cells promote the recruitment of protective neutrophils and NK cells to the tracheal mucosa. Altogether, our results highlight the importance of the immune responses mediated by γδ T cells.

Combination Therapy Targeting Platelet Activation and Virus Replication Protects Mice against Lethal Influenza Pneumonia.

Excessive neutrophils recruited during influenza pneumonia contribute to severe lung pathology through induction of neutrophil extracellular traps (NETs) and release of extracellular histones. We have recently shown that activation of platelets during influenza enhances pulmonary microvascular thrombosis, leading to vascular injury and hemorrhage. Emerging evidence indicates that activated platelets also interact with neutrophils, forming neutrophil-platelet aggregates (NPAs) that contribute to tissue injury. Here, we examined neutrophil-platelet interactions and evaluated the formation of NPAs during influenza pneumonia. We also evaluated the efficacy of clopidogrel (CLP), an antagonist of the ADP-P2Y12 platelet receptor, alone or in combination with an antiviral agent (oseltamivir) against influenza infection in mice. Our studies demonstrated increased platelet activation and induction of NPAs in influenza-infected lungs, and that these NPAs led to NET release both in vitro and in vivo. Furthermore, neutrophil integrin Mac-1 (macrophage-1 antigen)-mediated platelet binding was critical for NPA formation and NET release. Administration of CLP reduced platelet activation and NPA formation but did not protect the mice against lethal influenza challenge. However, administration of CLP together with oseltamivir improved survival rates in mice compared with oseltamivir alone. The combination treatment reduced lung pathology, neutrophil influx, NPAs, NET release, and inflammatory cytokine release in infected lungs. Taken together, these results provide the first evidence that NPAs formed during influenza contribute to acute lung injury. Targeting both platelet activation and virus replication could represent an effective therapeutic option for severe influenza pneumonia.

IgM, IgG, and IgA Influenza-Specific Plasma Cells Express Divergent Transcriptomes.

Ab-secreting cells (ASC) or plasma cells are essential components of the humoral immune system. Although Abs of different isotypes have distinct functions, it is not known if the ASC that secrete each isotype are also distinct. ASC downregulate their surface BCR upon differentiation, hindering analyses that couple BCR information to other molecular characteristics. In this study, we developed a methodology using fixation, permeabilization, and intracellular staining coupled with cell sorting and reversal of the cross-links to allow RNA sequencing of isolated cell subsets. Using hemagglutinin and nucleoprotein Ag-specific B cell tetramers and intracellular staining for IgM, IgG, and IgA isotypes, we were able to derive and compare the gene expression programs of ASC subsets that were responding to the same Ags following influenza infection in mice. Intriguingly, whereas a shared ASC signature was identified, each ASC isotype-specific population expressed distinct transcriptional programs controlling cellular homing, metabolism, and potential effector functions. Additionally, we extracted and compared BCR clonotypes and found that each ASC isotype contained a unique, clonally related CDR3 repertoire. In summary, these data reveal specific complexities in the transcriptional programming of Ag-specific ASC populations.

Immunomodulatory and prophylactic effects of Bifidobacterium bifidum probiotic strain on influenza infection in mice.

The limited efficacy of available influenza vaccines against rapidly emerging new viral strains stresses the need for the development of new antigen-independent prophylactic treatment for enhancing immunity against influenza infection. Recent studies suggest that probiotics possess immunomodulatory properties and can reduce the severity of respiratory infections. Here, we investigated the potential of prophylactic Bifidobacterium bifidum in improving anti-influenza immune responses in an experimental lethal mouse-adapted influenza A (H1N1) infection in a BALB/c mouse model. One week after viral challenge, splenocyte proliferation assay (MTT), IFN-gamma, IL-12, and IL-4 in spleen and IL-6 in the lung homogenates were conducted using ELISA assays. Sera samples were collected to measure IgG1 and IgG2a levels. Furthermore, the mice challenged with lethal influenza virus were assessed for survival rate. The findings demonstrated a strong induction of both humoral and cellular immunities, as well as decreased level of IL-6 production in the lung and an increase in survival rate in the mice receiving Bifidobacterium than those of the control group were observed. Taken together, the results indicate a robust potential for Bifidobacterium to modulate humoral and cellular immune responses and induce balanced Th1/Th2 immune responses against influenza infection.

CD5+ B-1 cell responses to infection are facilitated by toll-like receptor stimulation

Abstract B-1 lymphocytes are neonatally-derived, self-reactive, innate-like B cells that produce natural IgM at steady state. B-1 cells also actively respond to infections with induced local IgM production. These two distinct functions of B-1 cells have been linked to the expression CD5, an inhibitor of BCR signaling, with CD5+ cells thought to contribute to steady state IgM, and CD5− cells to local IgM production during infection. The mechanisms underlying such “division of labor” are poorly understood. We aim to test our hypothesis that CD5+ B-1 cells are stimulated by innate signals via TLR, during infection, resulting in their activation and differentiation into CD5− IgM antibody-secreting cells. For that we first cultured CD5+ B-1 cells with TLR9 agonist, ODN-CpG. The cells proliferated, secreted IgM and showed increased Nur77 expression, Akt phosphorylation and interaction of Syk with CD79a, signs of BCR signaling. They lost expression of the BCR-inhibitor CD5 and upregulated the plasma cell marker CD138+. Following influenza infection of mice, CD5+ B-1 cells migrated to the mediastinal lymph nodes (medLN) where they lost CD5 and became IgM secreting cells. Both, global TLR−/− mice and chimeric mice lacking TLR only on B-1 cells had reduced numbers of B-1 derived plasma cells and antibody secreting cells in the medLN compared to controls. Our findings show that TLR-mediated activation of CD5+ B-1 cells results in the rapid reorganization of the BCR-complex, followed by loss of CD5 and that TLR stimulation of CD5+ B-1 is required for differentiation of B-1 cells to infection in vivo. Overall our data suggest that CD5+ and CD5− B-1 cells are not distinct subsets, but rather that CD5 expression indicates the activation state of B-1 cells.

Differential effect of GITR on lung effector T cell subpopulations during influenza infection

Abstract Influenza remains an important global threat. Tissue resident memory T cells (Trm) are critical in protection against influenza, and it is important to identify key mechanisms regulating their formation and persistence. GITR, an NF-κB activating TNFR family member, is required on CD8 T cells for maximal responses against influenza. Our recent work (Mucosal Immunology, doi:10.1038/s41385-018-0105-5) provided evidence that GITRL on monocyte-derived inflammatory APCs provides crucial signals through GITR on T cells in the lung tissue (termed Signal 4), allowing effector T cell accumulation and optimal Trm formation during influenza infection in mice. During influenza infection, responding lung T cells are heterogeneous, some will give rise to terminally differentiated effector cells, while others will become Trm. It remains unknown how GITR affects these subpopulations. To address this issue, we characterized these CD8 T cell subsets using markers Ly6C, KLRG1, T-bet, and the chemokine receptor CX3CR1. By staining with Ly6C and CX3CR1, we identified three different CD8 T cell subsets with differential expression of Ly6C and CX3CR1 (Ly6ChiCX3CR1 hi, Ly6ChiCX3CR1 lo, Ly6CloCX3CR1 lo). Ly6ChiCX3CR1 hi CD8 T cell subset likely represents the most terminally differentiated effector cells as they have the highest expression of KLRG1 and T-bet. Using the adoptive transfer of transgenic OT-I cells, we discovered that there is a larger loss of the Ly6CloCX3CR1 loCD8 T cell in the absence of GITR when compared to the other two subsets. We are currently investigating which of the three subsets give rise to the lung Trm population after influenza infection and the timing and nature of the GITR-dependent signals involved. Funded by CIHR:FDN143250

Neutrophils Induce a Novel Chemokine Receptors Repertoire During Influenza Pneumonia.

Exaggerated host innate immune responses have been implicated in severe influenza pneumonia. We have previously demonstrated that excessive neutrophils recruited during influenza infection drive pulmonary pathology through induction of neutrophil extracellular traps (NETs) and release of extracellular histones. Chemokine receptors (CRs) are essential in the recruitment and activation of leukocytes. Although neutrophils have been implicated in influenza pathogenesis, little is known about their phenotypic changes, including expression of CRs occurring in the infected -lung microenvironment. Here, we examined CC and CXC CRs detection in circulating as well as lung-recruited neutrophils during influenza infection in mice using flow cytometry analyses. Our studies revealed that lung-recruited neutrophils displayed induction of CRs, including CCR1, CCR2, CCR3, CCR5, CXCR1, CXCR3, and CXCR4, all of which were marginally induced in circulating neutrophils. CXCR2 was the most predominant CR observed in both circulating and lung-infiltrated neutrophils after infection. The stimulation of these induced CRs modulated neutrophil phagocytic activity, ligand-specific neutrophil migration, bacterial killing, and NETs induction ex vivo. These findings indicate that neutrophils induce a novel CR repertoire in the infectious lung microenvironment, which alters their functionality during influenza pneumonia.

Inducible Bronchus-Associated Lymphoid Tissues (iBALT) Serve as Sites of B Cell Selection and Maturation Following Influenza Infection in Mice.

Seasonally recurrent influenza virus infections are a significant cause of global morbidity and mortality. In murine models, primary influenza infection in the respiratory tract elicits potent humoral responses concentrated in the draining mediastinal lymph node and the spleen. In addition to immunity within secondary lymphoid organs (SLO), pulmonary infection is also associated with formation of ectopic inducible bronchus-associated tissues (iBALT) in the lung. These structures display a lymphoid organization, but their function and protective benefits remain unclear. Here we examined the phenotype, transcriptional profile and antigen specificity of B cell populations forming iBALT in influenza infected mice. We show that the cellular composition of iBALT was comparable to SLO, containing populations of follicular dendritic cells (FDC), T-follicular helper (Tfh) cells, and germinal center (GC)-like B cells with classical dark- and light-zone polarization. Transcriptional profiles of GC B cells in iBALT and SLO were conserved regardless of anatomical localization. The architecture of iBALT was pleiomorphic and less structurally defined than SLO. Nevertheless, we show that GC-like structures within iBALT serve as a distinct niche that independently support the maturation and selection of B cells primarily targeted against the influenza virus nucleoprotein. Our findings suggest that iBALT, which are positioned at the frontline of the lung mucosa, drive long-lived, and unique GC reactions that contribute to the diversity of the humoral response targeting influenza.

GITRL on inflammatory antigen presenting cells in the lung parenchyma provides signal 4 for T-cell accumulation and tissue-resident memory T-cell formation.

T-cell responses in the lung are critical for protection against respiratory pathogens. TNFR superfamily members play important roles in providing survival signals to T cells during respiratory infections. However, whether these signals take place mainly during priming in the secondary lymphoid organs and/or in the peripheral tissues remains unknown. Here we show that under conditions of competition, GITR provides a T-cell intrinsic advantage to both CD4 and CD8 effector T cells in the lung tissue, as well as for the formation of CD4 and CD8 tissue-resident memory T cells during respiratory influenza infection in mice. In contrast, under non-competitive conditions, GITR has a preferential effect on CD8 over CD4 T cells. The nucleoprotein-specific CD8 T-cell response partially compensated for GITR deficiency by expansion of higher affinity T cells; whereas, the polymerase-specific response was less flexible and more GITR dependent. Following influenza infection, GITR is expressed on lung T cells and GITRL is preferentially expressed on lung monocyte-derived inflammatory antigen presenting cells. Accordingly, we show that GITR+/+ T cells in the lung parenchyma express more phosphorylated-ribosomal protein S6 than their GITR-/- counterparts. Thus, GITR signaling within the lung tissue critically regulates effector and tissue-resident memory T-cell accumulation.

Innate Immunity Induces the Accumulation of Lung Mast Cells During Influenza Infection.

Mast cells release disease-causing mediators and accumulate in the lung of asthmatics. The most common cause of exacerbations of asthma is respiratory virus infections such as influenza. Recently, we demonstrated that influenza infection in mice triggers the recruitment of mast cell progenitors to the lung. This process starts early after infection and leads to the accumulation of mast cells. Previous studies showed that an adaptive immune response was required to trigger the recruitment of mast cell progenitors to the lung in a mouse model of allergic lung inflammation. Therefore, we set out to determine whether an adaptive immune response against the virus is needed to cause the influenza-induced recruitment of mast cell progenitors to the lung. We found that influenza-induced recruitment of mast cell progenitors to the lung was intact in Rag2−/− mice and mice depleted of CD4+ cells, implicating the involvement of innate immune signals in this process. Seven weeks after the primary infection, the influenza-exposed mice harbored more lung mast cells than unexposed mice. As innate immunity was implicated in stimulating the recruitment process, several compounds known to trigger innate immune responses were administrated intranasally to test their ability to cause an increase in lung mast cell progenitors. Poly I:C, a synthetic analog of viral dsRNA, induced a TLR3-dependent increase in lung mast cell progenitors. In addition, IL-33 induced an ST2-dependent increase in lung mast cell progenitors. In contrast, the influenza-induced recruitment of mast cell progenitors to the lung occurred independently of either TLR3 or ST2, as demonstrated using Tlr3−/− or Il1rl1−/− mice. Furthermore, neutralization of IL-33 in Tlr3−/− mice could not abrogate the influenza-induced influx of mast cell progenitors to the lung. These results suggest that other innate receptor(s) contribute to mount the influx of mast cell progenitors to the lung upon influenza infection. Our study establishes that mast cell progenitors can be rapidly recruited to the lung by innate immune signals. This indicates that during life various innate stimuli of the respiratory tract trigger increases in the mast cell population within the lung. The expanded mast cell population may contribute to the exacerbations of symptoms which occurs when asthmatics are exposed to respiratory infections.

GM-CSF overexpression after influenza a virus infection prevents mortality and moderates M1-like airway monocyte/macrophage polarization

BackgroundInfluenza A viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. Application of high GM-CSF levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established.MethodsMice were infected intranasally with influenza A virus (PR8 strain). Supra-physiologic levels of GM-CSF were induced in the airways using the double transgenic GM-CSF (DTGM) or littermate control mice starting on 3 days post-infection (dpi). Assessment of respiratory mechanical parameters was performed using the flexiVent rodent ventilator. RNA sequence analysis was performed on FACS-sorted airway macrophage subsets at 8 dpi.ResultsSupra-physiologic levels of GM-CSF conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (BAL) fluid to near baseline levels. Transcriptome analysis, and subsequent validation ELISA assays, revealed that excess GM-CSF re-directs macrophages from an “M1-like” to a more “M2-like” activation state as revealed by alterations in the ratios of CXCL9 and CCL17 in BAL fluid, respectively. Ingenuity pathway analysis predicted that GM-CSF surplus during IAV infection elicits expression of anti-inflammatory mediators and moderates M1 macrophage pro-inflammatory signaling by Type II interferon (IFN-γ).ConclusionsOur data indicate that application of high levels of GM-CSF in the lung after influenza A virus infection alters pathogenic “M1-like” macrophage inflammation. These results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury.

Effect of triazavirine on the outcome of a lethal influenza infection and secondary bacterial pneumonia following influenza in mice

ABSTRACT Pneumonia often occurs as secondary infection post influenza disease and accounts for a large proportion of the morbidity and mortality associated with seasonal and pandemic influenza outbreaks. The antiviral drug triazavirine is licensed in Russia for the treatment and prophylaxis of acute respiratory infections, including influenza A and B viruses. In the present study, we investigated the efficacy of triazavirine in a mouse model of secondary Staphylococcus aureus pneumonia following A/California/04/2009 (H1N1)pdm09 influenza virus infection. We also performed a study of the efficacy of triazavirine against the A/California/04/2009 (H1N1)pdm09 lethal influenza infection in mice. In this model, triazavirine at the dose of 25 mg/kg/day significantly enhanced the survival of animals (60% compared to 20%) and the mean survival time to death, prevented weight loss, and reduced viral titer in the lungs of mice infected with influenza virus. At doses of 50 and 100 mg/kg/day, triazavirine was highly effective in the treatment of the secondary bacterial pneumonia following influenza infection in mice. At these doses, triazavirine protected 67-75% of animals against death, increased the mean survival time to death by twofold, and reduced the virus titer by 2.2-3.0 log10TCID50/ml compared to the mice in the control group. These findings suggest the possible benefit of triazavirine treatment in reducing post influenza pneumonia incidence in humans.