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Cigarette smoke (CS) promotes the development of chronic pulmonary disease and has been associated with increased risk for influenza-related illness. Here, we directly addressed the impact of CS disordered microbiota on the severity of influenza A virus (IAV) infection. Specific and opportunistic pathogen-free (SOPF) C57BL/6J mice were exposed to CS or room air (RA) for 5.5 months. Each exposed mouse was then cohoused with a group of recipient germ-free (GF) mice for 1 month for microbial transfer. Colonized GF mice were then infected intranasally with IAV and disease development was monitored. Upper and lower airway and fecal microbiota were longitudinally investigated by 16S rRNA gene sequencing and bacterial cultures in donor and recipient mice. The bacterial family Streptococcaceae accounted for the largest difference between CS- and RA-exposed microbiota in the oropharynx. Analysis of the oropharynx and fecal microbiota indicated an efficient transfer to coprophagic recipient mice, which replicated the differences in microbiota composition observed in donor mice. Subsequent IAV infection revealed significantly higher weight loss for CS microbiota recipient mice at 8-10 days post infection (dpi) compared to control recipient mice. In addition, H1N1 infection inflicted substantial changes in the microbiota composition, especially at days 4 and 8 after infection. In conclusion, mice with a CS-associated microbiota suffer from higher disease severity upon IAV infection compared to mice colonized with a normal SOPF microbiota. Our data suggest that independently of CS exposure and concomitant structural lung damage, microbial distortion due to CS exposure may impact the severity of IAV disease course.IMPORTANCEIt has been reported that chronic exposure to CS is associated with a disordered microbiota composition. In this study, we colonized germ-free (GF) mice with the microbiota from SOPF mice which were chronically exposed to CS or RA. This allowed disentangling the effect of the disordered microbiota from the immune-modulating effects of actual CS exposure. We observed a successful transfer of the microbiotas after cohousing including specific microbiota differences induced by CS exposure in formerly GF mice, which were never exposed to CS. We then investigated the effects of IAV infection on the disease course and microbiotas of formerly GF mice. We found that mice with CS-associated microbiota reveal worse disease course compared to the control group. We hypothesize that CS-induced disordering of the microbiota may, indeed, impact the severity of influenza A disease., Competing Interests: The authors declare no conflict of interest.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

In this study, whether H9N2 influenza A virus (IAV) infection contributed to secondary Klebsiella pneumoniae infection was investigated. From post-infection onwards, clinical symptoms were monitored, examined and recorded daily for 11 days. As a result, no clinical signs were observed in the mice infected with single H9N2 IAV, implying that H9N2 IAV was less pathogenic to mice. Compared to single K. pneumonia infection, K. pneumoniae infection following H9N2 IAV infection exacerbates lung histopathological lesions and apoptosis, resulting in more severe diseases. Lung index of the mice with H9N2 IAV and K. pneumoniae co-infection was significantly higher than those in the other groups. Bacterial loads in the tissues in H9N2 IAV and K. pneumoniae co-infection group were significantly higher than those in the single K. pneumoniae infection group at 7 dpi. It demonstrated that prior H9N2 IAV infection contributed to K. pneumonia proliferation and delayed bacterial clearance in mice. Secondary K. pneumoniae infection influences seroconversion of anti-H9N2 antibody titers and the cytokine profiles. The findings demonstrated that H9N2 IAV infection facilitated secondary K. pneumonia infection, causing severe the diseases in mice., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Influenza is a respiratory virus that alone or in combination with secondary bacterial pathogens can contribute to the development of acute pneumonia in persons >65 years of age. Host innate immune antiviral signaling early in response to influenza is essential to inhibit early viral replication and guide the initiation of adaptive immune responses. Using young adult (3 months) and aged adult mice infected with mouse adapted H1N1 or H3N2, the results of our study illustrate dysregulated and/or diminished activation of key signaling pathways in aged lung contribute to increased lung inflammation and morbidity. Specifically, within the first seven days of infection, there were significant changes in genes associated with TLR and RIG-I signaling detected in aged murine lung in response to H1N1 or H3N2. Taken together, the results of our study expand our current understanding of age-associated changes in antiviral signaling in the lung.

Transient receptor potential (TRP) channels, neuronal stimulations widely known to be associated with thermal responses, pain induction, and osmoregulation, have been shown in recent studies to have underlying mechanisms associated with inflammatory responses. The role of TRP channels on inflammatory milieu during bacterial infections has been widely demonstrated. It may vary among types of channels/pathogens, however, and it is not known how TRP channels function during pneumococcal infections. Streptococcus pneumoniae can cause severe infections such as pneumonia, bacteremia, and meningitis, with systemic inflammatory responses. This study examines the role of TRP channels (TRPV1 and TRPV4) for pneumococcal nasal colonization and subsequent development of invasive pneumococcal disease in a mouse model. Both TRPV1 and TRPV4 channels were shown to be related to regulation of the development of pneumococcal diseases. In particular, the influx of neutrophils (polymorphonuclear cells) in the nasal cavity and the bactericidal activity were significantly suppressed among TRPV4 knockout mice. This may lead to severe pneumococcal pneumonia, resulting in dissemination of the bacteria to various organs and causing high mortality during influenza virus coinfection. Regulating host immune responses by TRP channels could be a novel strategy against pathogenic microorganisms causing strong local/systemic inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kono, Nanushaj, Sakatani, Murakami, Hijiya, Kinoshita, Shiga, Kaneko, Enomoto, Sugita, Miyajima, Okada, Saika and Hotomi.)

Influenza A virus (IAV) infection predisposes the host to secondary bacterial pneumonia, known as a major cause of morbidity and mortality during influenza virus epidemics. Analysis of interactions between IAV-infected human epithelial cells and Streptococcus pneumoniae revealed that infected cells ectopically exhibited the endoplasmic reticulum chaperone glycoprotein 96 (GP96) on the surface. Importantly, efficient pneumococcal adherence to epithelial cells was imparted by interactions with extracellular GP96 and integrin α V , with the surface expression mediated by GP96 chaperone activity. Furthermore, abrogation of adherence was gained by chemical inhibition or genetic knockout of GP96 as well as addition of RGD peptide, an inhibitor of integrin-ligand interactions. Direct binding of extracellular GP96 and pneumococci was shown to be mediated by pneumococcal oligopeptide permease components. Additionally, IAV infection induced activation of calpains and Snail1, which are responsible for degradation and transcriptional repression of junctional proteins in the host, respectively, indicating increased bacterial translocation across the epithelial barrier. Notably, treatment of IAV-infected mice with the GP96 inhibitor enhanced pneumococcal clearance from lung tissues and ameliorated lung pathology. Taken together, the present findings indicate a viral-bacterial synergy in relation to disease progression and suggest a paradigm for developing novel therapeutic strategies tailored to inhibit pneumococcal colonization in an IAV-infected respiratory tract. IMPORTANCE Secondary bacterial pneumonia following an influenza A virus (IAV) infection is a major cause of morbidity and mortality. Although it is generally accepted that preceding IAV infection leads to increased susceptibility to secondary bacterial infection, details regarding the pathogenic mechanism during the early stage of superinfection remain elusive. Here, we focused on the interaction of IAV-infected cells and Streptococcus pneumoniae, which revealed that human epithelial cells infected with IAV exhibit a cell surface display of GP96, an endoplasmic reticulum chaperon. Notably, extracellular GP96 was shown to impart efficient adherence for secondary infection by S. pneumoniae, and GP96 inhibition ameliorated lung pathology of superinfected mice, indicating it to be a useful target for development of therapeutic strategies for patients with superinfection.

Experimental experience suggests that microbial agents including probiotics and prebiotics (representative microbial agents) play a critical role in defending against respiratory virus infection. We aim to systematically examine these agents' effect on respiratory viral infection and encourage research into clinical applications. An electronic literature search was conducted from published data with a combination of a microbial agents search component containing synonyms for microbial agents-related terms and a customized search component for respiratory virus infection. Hazard ratio (HR), risk ratio (RR) and standard deviation (SD) were employed as effect estimates. In 45 preclinical studies, the mortality rates decreased in the respiratory viral infection models that included prebiotics or prebiotics as interventions (HR: 0.70; 95% confidence interval (CI): 0.56-0.87; P=0.002). There was a significant decrease in viral load due to improved gut microbiota (SD: -1.22; 95% CI: -1.50 to -0.94; P<0.001). Concentrations of interferon (IFN)-α (SD: 1.05; 95% CI: 0.33-1.77; P=0.004), IFN-γ (SD: 0.83; 95% CI: 0.01-1.65; P=0.05) and interleukin (IL)-12 (SD: 2.42; 95% CI: 0.32-4.52; P=0.02), IL-1β (SD: 0.01; 95% CI: -0.37 to 0.40; P=0.94) increased, whereas those of TNF-α (SD: -0.58; 95% CI: -1.59 to 0.43; P=0.26) and IL-6 (SD: -0.59; 95% CI: -1.24 to 0.07; P=0.08) decreased. Six clinical studies had lower symptom scores (SD: -0.09; 95% CI: -0.44 to 0.26; P=0.61) and less incidence of infection (RR: 0.80; 95% CI: 0.64-1.01; P=0.06). Our research indicates that probiotics and prebiotics pose a defensive possibility on respiratory viral infection and may encourage the clinical application., (© 2021 The Author(s).)

Influenza A virus (IAV)-related mortality is often due to secondary bacterial infections, primarily by pneumococci. Here, we study how IAV-modulated changes in the lungs affect bacterial replication in the lower respiratory tract (LRT). Bronchoalveolar lavages (BALs) from coinfected mice showed rapid bacterial proliferation 4 to 6 h after pneumococcal challenge. Metabolomic and quantitative proteomic analyses demonstrated capillary leakage with efflux of nutrients and antioxidants into the alveolar space. Pneumococcal adaptation to IAV-induced inflammation and redox imbalance increased the expression of the pneumococcal chaperone/protease HtrA. Presence of HtrA resulted in bacterial growth advantage in the IAV-infected LRT and protection from complement-mediated opsonophagocytosis due to capsular production. Absence of HtrA led to growth arrest in vitro that was partially restored by antioxidants. Pneumococcal ability to grow in the IAV-infected LRT depends on the nutrient-rich milieu with increased levels of antioxidants such as ascorbic acid and its ability to adapt to and cope with oxidative damage and immune clearance., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Pneumococcal conjugate vaccination (PCV) may prevent influenza-related pneumonia, including Streptococcus pneumoniae pneumonia. To investigate PCV efficacy against secondary pneumococcal pneumonia following influenza, PCV was administered intramuscularly 2 and 5 weeks before S. pneumoniae serotype-3 colonization of murine nasopharynges followed by intranasal challenge with a sublethal dose of influenza A virus. Bacterial and viral loads, including innate immune responses were compared across conditions. PCV vaccination improved the survival of mice with secondary pneumococcal pneumonia and significantly reduced the pulmonary bacterial burden. Increased monocyte/macrophage influx into the lungs, alleviated loss of alveolar macrophages and decreased neutrophil influx into the lungs occurred in PCV-treated mice irrespective of pneumococcal colonization. Higher monocyte chemoattractant protein 1 levels and lower levels of CXCL1, interferon-γ, interleukin-17A, and IL-10, were detected in PCV-treated mice. Additionally, PCV treatment activated the macrophage intracellular killing of S. pneumoniae. Collectively, PCV potentially modulates the host's innate immunity and specific antibodies induction. Macrophage-related innate immunity should be further explored to elucidate the efficacy and mechanisms of PCV versus influenza-related life-threatening diseases., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Infection with influenza can be aggravated by bacterial co-infections, which often results in disease exacerbation. The effects of influenza infection on the upper respiratory tract (URT) microbiome are largely unknown. Here, we report a longitudinal study to assess the temporal dynamics of the URT microbiomes of uninfected and influenza virus-infected humans and ferrets. Uninfected human patients and ferret URT microbiomes have stable healthy ecostate communities both within and between individuals. In contrast, infected patients and ferrets exhibit large changes in bacterial community composition over time and between individuals. The unhealthy ecostates of infected individuals progress towards the healthy ecostate, coinciding with viral clearance and recovery. Pseudomonadales associate statistically with the disturbed microbiomes of infected individuals. The dynamic and resilient microbiome during influenza virus infection in multiple hosts provides a compelling rationale for the maintenance of the microbiome homeostasis as a potential therapeutic target to prevent IAV associated bacterial co-infections.

Background: Influenza is a severe respiratory illness that continually threatens global health. It has been widely known that gut microbiota modulates the host response to protect against influenza infection, but mechanistic details remain largely unknown. Here, we took advantage of the phenomenon of lethal dose 50 (LD 50 ) and metagenomic sequencing analysis to identify specific anti-influenza gut microbes and analyze the underlying mechanism., Results: Transferring fecal microbes from mice that survive virulent influenza H7N9 infection into antibiotic-treated mice confers resistance to infection. Some gut microbes exhibit differential features to lethal influenza infection depending on the infection outcome. Bifidobacterium pseudolongum and Bifidobacterium animalis levels are significantly elevated in surviving mice when compared to dead or mock-infected mice. Oral administration of B. animalis alone or the combination of both significantly reduces the severity of H7N9 infection in both antibiotic-treated and germ-free mice. Functional metagenomic analysis suggests that B. animalis mediates the anti-influenza effect via several specific metabolic molecules. In vivo tests confirm valine and coenzyme A produce an anti-influenza effect., Conclusions: These findings show that the severity of influenza infection is closely related to the heterogeneous responses of the gut microbiota. We demonstrate the anti-influenza effect of B. animalis, and also find that the gut population of endogenous B. animalis can expand to enhance host influenza resistance when lethal influenza infection occurs, representing a novel interaction between host and gut microbiota. Further, our data suggest the potential utility of Bifidobacterium in the prevention and as a prognostic predictor of influenza.

Influenza A virus (IAV) and bacteria co-infection can influence the host clinical conditions. Both H9N2 IAV and Pseudomonas aeruginosa (P. aeruginosa) are potential pathogens of respiratory diseases in mink. In this study, to clarify the effects of H9N2 IAV and P. aeruginosa co-infections on hemorrhagic pneumonia in mink, we carried out to establish the mink models of the two-pathogen co-infections in different orders. Compared with the single infections with H9N2 IAV or P. aeruginosa, the mink co-infected with H9N2 IAV and P. aeruginosa showed severe respiratory diseases, and exacerbated histopathological lesions and more obvious apoptosis in the lung tissues. H9N2 IAV shedding and viral loads in the lungs of the mink co-infected with H9N2 IAV and P. aeruginosa were higher than those in the mink with single H9N2 IAV infection. Furthermore, the clearance of P. aeruginosa in the co-infected mink lungs was delayed. In addition, the anti-H9N2 antibody titers in mink with P. aeruginosa co-infection following H9N2 IAV infection were significantly higher than those of the other groups. This implied that H9N2 IAV and P. aeruginosa co-infection contributed to the development of hemorrhagic pneumonia in mink, and that P. aeruginosa should play a major role in the disease. The exact interaction mechanism among H9N2 IAV, P. aeruginosa and the host needs to be further investigated., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Influenza has frequently been epidemic in recent years. However, the mechanisms of severe pneumonia with postinfluenza Streptococcus pneumoniae (SP) secondary infection have not been fully understood. In this study, we explored the mechanisms of pneumonia in postinfluenza A virus (IAV) infection via a mouse model. Mice were intranasally inoculated with SP three days after IAV inoculation. We then collected samples at three time points to dynamically observe the pathological progression. In IAV infection alone, lymphocyte infiltration and widened alveolar intervals were observed. In the blood, levels of the CD19+, CD19+CD21+ and CD19+CD79β+B lymphocyte subpopulations were reduced, and IFN-γ and IL-10 were elevated. Slight atrophy was seen in the spleen, which was due to splenic B lymphocyteinitiated apoptosis through the mitochondrial pathway. When SP infection occurred after IAV infection, the pulmonary inflammation was significantly aggravated; a fair number of lymphocytes and neutrophils infiltrated simultaneously with exfoliated bronchial epithelial cells, vascular endothelial cells, widened alveolar septum and hemorrhaging. Increasing edema fluid and bacteria accumulated in the alveolar cavity. Decreased CD19+, CD19+CD21+ and CD19+CD79β+B lymphocyte subpopulations and increased interferon gamma (IFN-γ) or interleukin 10 (IL-10) were more prominent compared to those with viral infection alone. Spleen atrophy resulting from coinfection was more obvious because of massive splenic B lymphocyte apoptosis through the mitochondrial pathway compared to viral infection alone. This study shows that although inflammation caused by SP infection alone was temporary, preceding IAV infection provided favorable conditions for SP colonization and multiplication by destroying lung structure and suppressing humoral immunity. Synergistic IAV-SP coinfection is likely to facilitate more SP colonization and promote B lymphocyte-suppression and reduction. Eventually, the pneumonia worsened., (Copyright 2019 Biolife Sas. www.biolifesas.org.)

Streptococcus suis is an important zoonotic pathogen which can infect humans and pigs worldwide, posing a potential risk to global public health. Suilysin, a pore-forming cholesterol-dependent cytolysin, is considered to play an important role in the pathogenesis of S. suis infections. It is known that infection with influenza A viruses may favor susceptibility to secondary bacterial infection, resulting in more severe disease and increased mortality. However, the molecular mechanisms underlying these coinfections are incompletely understood. Applying highly differentiated primary porcine respiratory epithelial cells grown under air-liquid interface (ALI) conditions, we analyzed the contribution of swine influenza viruses (SIV) to the virulence of S. suis , with a special focus on its cytolytic toxin, suilysin. We found that during secondary bacterial infection, suilysin of S. suis contributed to the damage of well-differentiated respiratory epithelial cells in the early stage of infection, whereas the cytotoxic effects induced by SIV became prominent at later stages of infection. Prior infection by SIV enhanced the adherence to and colonization of porcine airway epithelial cells by a wild-type (wt) S. suis strain and a suilysin-negative S. suis mutant in a sialic acid-dependent manner. A striking difference was observed with respect to bacterial invasion. After bacterial monoinfection, only the wt S. suis strain showed an invasive phenotype, whereas the mutant remained adherent. When the epithelial cells were preinfected with SIV, the suilysin-negative mutant also showed an invasion capacity. Therefore, we propose that coinfection with SIV may compensate for the lack of suilysin in the adherence and invasion process of suilysin-negative S. suis ., (Copyright © 2019 American Society for Microbiology.)

We previously reported that the Toll-like receptor 4 (TLR4) antagonist Eritoran blocks acute lung injury (ALI) therapeutically in mouse and cotton rat models of influenza. However, secondary (2°) bacterial infection following influenza virus infection is associated with excess morbidity and mortality. Wild-type (WT) mice infected with mouse-adapted influenza A/Puerto Rico/8/34 virus (PR8) and, 7 days later, with Streptococcus pneumoniae serotype 3 ( Sp3 ) exhibited significantly enhanced lung pathology and lethality that was reversed by Eritoran therapy after PR8 infection but before Sp3 infection. Cotton rats infected with nonadapted pH1N1 influenza virus and then superinfected with methicillin-resistant Staphylococcus aureus also exhibited increased lung pathology and serum high-mobility-group box 1 (HMGB1) levels, both of which were blunted by Eritoran therapy. In mice, PR8 infection suppressed Sp3 -induced CXCL1 and CXCL2 mRNA, reducing neutrophil infiltration and increasing the bacterial burden, all of which were reversed by Eritoran treatment. While beta interferon (IFN-β)-deficient (IFN-β -/- ) mice are highly susceptible to PR8, they exhibited delayed death upon Sp3 superinfection, indicating that while IFN-β was protective against influenza, it negatively impacted the host response to Sp3 IFN-β-treated WT macrophages selectively suppressed Sp3 -induced CXCL1/CXCL2 transcriptionally, as evidenced by reduced recruitment of RNA polymerase II to the CXCL1 promoter. Thus, influenza establishes a "trained" state of immunosuppression toward 2° bacterial infection, in part through the potent induction of IFN-β and its downstream transcriptional regulation of chemokines, an effect reversed by Eritoran. IMPORTANCE Enhanced susceptibility to 2° bacterial infections following infection with influenza virus is a global health concern that accounts for many hospitalizations and deaths, particularly during pandemics. The complexity of the impaired host immune response during 2° bacterial infection has been widely studied. Both type I IFN and neutrophil dysfunction through decreased chemokine production have been implicated as mechanisms underlying enhanced susceptibility to 2° bacterial infections. Our findings support the conclusion that selective suppression of CXCL1/CXCL2 represents an IFN-β-mediated "training" of the macrophage transcriptional response to TLR2 agonists and that blocking of TLR4 therapeutically with Eritoran after influenza virus infection reverses this suppression by blunting influenza-induced IFN-β., (Copyright © 2019 Shirey et al.)

Bovine respiratory disease (BRD) is economically significant, and influenza D virus (IDV) is commonly identified in cattle with BRD. Mannheimia haemolytica (MHA) is an opportunistic bacterial contributor to BRD; surveillance data suggest that MHA and IDV co-infection occurs in cattle. The objective of this study was to evaluate the synergistic pathogenesis in cattle co-infected with IDV and MHA. Sixteen dairy calves were randomly assigned to four groups of four calves. The IDV + MHA + group received D/bovine/C00046 N/Mississippi/2014 (D/46 N) intranasally at 0 days post-inoculation (DPI) and Mannheimia haemolytica D153 (MHA D153) intratracheally at 5 DPI. The IDV + MHA- group received only D/46 N at 0 DPI; the IDV-MHA + group received only MHA D153 at 5 DPI; and the IDV-MHA- group received neither agent. Clinical scores were calculated twice daily. At 10 DPI, IDV + MHA+, IDV-MHA+, and IDV-MHA- calves were euthanized and evaluated for pathologic lesions. The IDV + groups seroconverted to IDV by 10 DPI. Clinical scores were higher in IDV + groups than IDV- groups on 2-5 DPI (p =  0.001). After MHA challenge on 5 DPI, clinical scores (6-10 DPI) were slightly lower in IDV+MHA+ group than IDV-MHA+ group (p <  0.05) but not significantly different between MHA+ groups and MHA- groups. The average gross pathology score was higher for IDV-MHA+ group than groups IDV-MHA- and IDV+MHA+; however, no significant differences were identified among groups. Under the conditions of this study, infection with IDV before MHA enhance neither clinical disease nor lung pathology, relative to calves infected with MHA alone., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The transcription factor, T-bet, regulates type 1 inflammatory responses against a range of infections. Here, we demonstrate a previously unaddressed role of T-bet, to influenza virus and bacterial superinfection. Interestingly, we found that T-bet deficiency did not adversely affect the efficacy of viral clearance or recovery compared to wild-type hosts. Instead, increased infiltration of neutrophils and production of Th17 cytokines (IL-17 and IL-22), in lungs of influenza virus-infected T-bet -/- mice, were correlated with survival advantage against subsequent infection by Streptococcus pneumoniae Neutralization of IL-17, but not IL-22, in T-bet -/- mice increased pulmonary bacterial load, concomitant with decreased neutrophil infiltration and reduced survival of T-bet -/- mice. IL-17 production by CD8 + , CD4 + and γδ T cell types was identified to contribute to this protection against bacterial superinfection. We further showed that neutrophil depletion in T-bet -/- lungs increased pulmonary bacterial burden. These results thus indicate that despite the loss of T-bet, immune defences required for influenza viral clearance are fully functional, which in turn enhances protective type 17 immune responses against lethal bacterial superinfections., (© 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Influenza virus infections particularly when followed by bacterial superinfections (BSI) result in significant morbidities and mortalities especially during influenza pandemics. Type I interferons (IFNs) regulate both anti-influenza immunity and host susceptibility to subsequent BSIs. These type I IFNs consisting of, among others, 14 IFN-α's and a single IFN-β, are recognized by and signal through the heterodimeric type I IFN receptor (IFNAR) comprised of IFNAR1 and IFNAR2. However, the individual receptor subunits can bind IFN-β or IFN-α's independently of each other and induce distinct signaling. The role of type I IFN signaling in regulating host susceptibility to both viral infections and BSI has been only examined with respect to IFNAR1 deficiency. Here, we demonstrate that despite some redundancies, IFNAR1 and IFNAR2 have distinct roles in regulating both anti-influenza A virus (IAV) immunity and in shaping host susceptibility to subsequent BSI caused by S. aureus . We found IFNAR2 to be critical for anti-viral immunity. In contrast to Ifnar1 -/- mice, IAV-infected Ifnar2 -/- mice displayed both increased and accelerated morbidity and mortality compared to WT mice. Furthermore, unlike IFNAR1, IFNAR2 was sufficient to generate protection from lethal IAV infection when stimulated with IFN-β. With regards to BSI, unlike what we found previously in Ifnar1 -/- mice, Ifnar2 -/- mice were not susceptible to BSI induced on day 3 post-IAV, even though absence of IFNAR2 resulted in increased viral burden and an increased inflammatory environment. The Ifnar2 -/- mice similar to what we previously found in Ifnar1 -/- mice were less susceptible than WT mice to BSI induced on day 7 post-IAV, indicating that signaling through a complete receptor increases BSI susceptibility late during clinical IAV infection. Thus, our results support a role for IFNAR2 in induction of anti-IAV immune responses that are involved in altering host susceptibility to BSI and are essential for decreasing the morbidity and mortality associated with IAV infection. These results begin to elucidate some of the mechanisms involved in how the individual IFNAR subunits shape the anti-viral immune response. Moreover, our results highlight the importance of examining the contributions of entire receptors, as individual subunits can induce distinct outcomes as shown here.

Background: Microbiota integrity is essential for a growing number of physiological processes. Consequently, disruption of microbiota homeostasis correlates with a variety of pathological states. Importantly, commensal microbiota provide a shield against invading bacterial pathogens, probably by direct competition. The impact of viral infections on host microbiota composition and dynamics is poorly understood. Influenza A viruses (IAV) are common respiratory pathogens causing acute infections. Here, we show dynamic changes in respiratory and intestinal microbiota over the course of a sublethal IAV infection in a mouse model., Results: Using a combination of 16S rRNA gene-specific next generation sequencing and qPCR as well as culturing of bacterial organ content, we found body site-specific and transient microbiota responses. In the lower respiratory tract, we observed only minor qualitative changes in microbiota composition. No quantitative impact on bacterial colonization after IAV infection was detectable, despite a robust antimicrobial host response and increased sensitivity to bacterial super infection. In contrast, in the intestine, IAV induced robust depletion of bacterial content, disruption of mucus layer integrity, and higher levels of antimicrobial peptides in Paneth cells. As a functional consequence of IAV-mediated microbiota depletion, we demonstrated that the small intestine is rendered more susceptible to bacterial pathogen invasion, in a Salmonella typhimurium super infection model., Conclusion: We show for the first time the consequences of IAV infection for lower respiratory tract and intestinal microbiobiota in a qualitative and quantitative fashion. The discrepancy of relative 16S rRNA gene next-generation sequencing (NGS) and normalized 16S rRNA gene-specific qPCR stresses the importance of combining qualitative and quantitative approaches to correctly analyze composition of organ associated microbial communities. The transiently induced dysbiosis underlines the overall stability of microbial communities to effects of acute infection. However, during a short-time window, specific ecological niches might lose their microbiota shield and remain vulnerable to bacterial invasion.

Investigation of pathogen-host interactions on a molecular level requires sophisticated in vitro infection procedures, especially in the presence of different pathogens.Super-infections of influenza viruses (IV) and bacteria, with increasing incidence of Staphylococcus aureus (S. aureus) cases, are a long-known phenomenon and represent a major complication in IV-infected patients. Although several in vivo studies have improved our knowledge about pathogenesis and immune responses of super-infections that result in increased morbidity and mortality, the consequences of the direct interplay of viruses and bacteria on a molecular level in affected cells that may contribute to the deadly synergism of these pathogens are so far poorly characterized. Here we describe different infection schemes to study IV and S. aureus coinfections of distinct cell populations in vitro. Depending on the focus of interest, regulation of cell responses such as signalling mechanisms or pro- and anti-inflammatory cytokine expression, or consequences for the viral or bacterial life cycle, can be analyzed. The described infection procedures could be used as guidelines and adapted to super-infection settings of other viral and bacterial pathogens.

The lung is constantly exposed to both environmental and microbial challenge. As a "contained" organ, it also constitutes an excellent "self-contained" tissue to examine inflammatory responses and cellular infiltration into a diseased organ. Influenza A virus (IAV) causes both mild and severe inflammation that is strain specific following infection of the lung epithelium that spreads to other cells of the lung environment. Here, we describe a method of intranasal inoculation of the lung with IAV that can be used as a preclinical model of infection. Mice can be monitored for clinical signs of infection and tissue and lung fluid collected for further analysis to dissect the immunological consequences of IAV infection. Importantly, this method can be modified to introduce other pathogens, therapies and environmental stimuli to examine immune responses in the lung.

Pneumocystis carinii f. sp. suis (PCS) nucleic acid and antibody profiles on two Austrian-farrow-to-finish farms were investigated. Furthermore, associations with other respiratory pathogens were evaluated. Respiratory specimen and sera from pigs of five age classes between the 1st week and the 3rd month of life as well as samples from sows were analyzed. On Farm A, PCS infection occurred early in life. The suckling piglets were already infected in the 1st week of life and the pigs remained positive until the 3rd month of life. On Farm B, pigs were infected later, between 3 and 4 months of age. The maximum PCS nucleic acid load on Farm A was 8.3 log10 genome copies/mL BALF, whereas on Farm B the PCS burden was significantly lower, with 4.0 log10 genome copies/mL BALF. Anti-PCS antibodies were detected in sows, as maternal antibodies in suckling piglets and as an immunological reaction to infection. On both farms, PCS infection was accompanied by several co-infections. On Farm A, there were concurrent infections with PRRSV, a virulent strain of Haemophilus parasuis, and Mycoplasma hyopneumoniae. On Farm B, PCS was accompanied by infections with swine influenza virus, Mycoplasma hyopneumoniae, and a non-virulent strain of Haemophilus parasuis. The results clearly show that the PCS profiles can vary between farms. Younger pigs may be more susceptible as they had higher PCS burdens. It is possible that PCS may contribute to a respiratory disease in pigs and further investigation of its potential role is warranted.

Respiratory tract infections such as flu cause severe morbidity and mortality and are among the leading causes of death in children and adults worldwide. Commensal microbiota is critical for orchestrating tissue homeostasis and immunity in the intestine. Probiotics represent an interesting source of immune modulators and several clinical studies have addressed the potential beneficial effects of probiotics against respiratory infections. Therefore, we have investigated the mechanisms of protection conferred by L. paracasei CNCM I-1518 strain in a mouse model of influenza infection. Notably, local myeloid cells accumulation is generated in the lungs after seven days feeding with L. paracasei prior to viral infection. L. paracasei-fed mice showed reduced susceptibility to the influenza infection, associated with less accumulation of inflammatory cells in the lungs, faster viral clearance and general health improvement. Interestingly, Allobaculum was significantly increased in L. paracasei-fed mice 7 days after influenza infection, even if the gut microbiota composition was not altered overall. L. paracasei-purified peptidoglycan partially recapitulated the protective phenotype observed with the entire bacteria. Collectively, our results demonstrate that oral consumption of L. paracasei CNCM I-1518 modulates lung immunity was associated with an improved control of influenza infection. These results further extend the beneficial role for certain lactobacilli to alleviate the burden of respiratory tract infections.

Obesity is a risk factor for developing severe disease following influenza virus infection; however, the comorbidity of obesity and secondary bacterial infection, a serious complication of influenza virus infections, is unknown. To fill this gap in knowledge, lean and obese C57BL/6 mice were infected with a nonlethal dose of influenza virus followed by a nonlethal dose of Streptococcus pneumoniae Strikingly, not only did significantly enhanced death occur in obese coinfected mice compared to lean controls, but also high mortality was seen irrespective of influenza virus strain, bacterial strain, or timing of coinfection. This result was unexpected, given that most influenza virus strains, especially seasonal human A and B viruses, are nonlethal in this model. Both viral and bacterial titers were increased in the upper respiratory tract and lungs of obese animals as early as days 1 and 2 post-bacterial infection, leading to a significant decrease in lung function. This increased bacterial load correlated with extensive cellular damage and upregulation of platelet-activating factor receptor, a host receptor central to pneumococcal invasion. Importantly, while vaccination of obese mice against either influenza virus or pneumococcus failed to confer protection, antibiotic treatment was able to resolve secondary bacterial infection-associated mortality. Overall, secondary bacterial pneumonia could be a widespread, unaddressed public health problem in an increasingly obese population. IMPORTANCE Worldwide obesity rates have continued to increase. Obesity is associated with increased severity of influenza virus infection; however, very little is known about respiratory coinfections in this expanding, high-risk population. Our studies utilized a coinfection model to show that obesity increases mortality from secondary bacterial infection following influenza virus challenge through a "perfect storm" of host factors that lead to excessive viral and bacterial outgrowth. In addition, we found that vaccination of obese mice against either virus or bacteria failed to confer protection against coinfection, but antibiotic treatment did alleviate mortality. Combined, these results represent an understudied and imminent public health concern in a weighty portion of the global population., (Copyright © 2017 Karlsson et al.)

Influenza virus infection ("flu") constitutes an important public health issue and is associated with a significant socio-economic burden. The discovery of new antivirals and a better understanding of antiviral immunity might offer new options for preventing or controlling flu infections. A better understanding of the underlying pathomechanism might also prompt the discovery of key molecules whose activity could be modulated in a clinical setting. Lastly, the development of new immunomodulators and immunostimulants might compensate for impaired host defense mechanisms and improve resistance to secondary bacterial infections, since the latter significantly worsen the clinical status of influenza-infected patients., (© 2017 médecine/sciences – Inserm.)

Secondary bacterial infections (SBIs) exacerbate influenza-associated disease and mortality. Antimicrobial agents can reduce the severity of SBIs, but many have limited efficacy or cause adverse effects. Thus, new treatment strategies are needed. Kinetic models describing the infection process can help determine optimal therapeutic targets, the time scale on which a drug will be most effective, and how infection dynamics will change under therapy. To understand how different therapies perturb the dynamics of influenza infection and bacterial coinfection and to quantify the benefit of increasing a drug's efficacy or targeting a different infection process, I analyzed data from mice treated with an antiviral, an antibiotic, or an immune modulatory agent with kinetic models. The results suggest that antivirals targeting the viral life cycle are most efficacious in the first 2 days of infection, potentially because of an improved immune response, and that increasing the clearance of infected cells is important for treatment later in the infection. For a coinfection, immunotherapy could control low bacterial loads with as little as 20 % efficacy, but more effective drugs would be necessary for high bacterial loads. Antibiotics targeting bacterial replication and administered 10 h after infection would require 100 % efficacy, which could be reduced to 40 % with prophylaxis. Combining immunotherapy with antibiotics could substantially increase treatment success. Taken together, the results suggest when and why some therapies fail, determine the efficacy needed for successful treatment, identify potential immune effects, and show how the regulation of underlying mechanisms can be used to design new therapeutic strategies.

Preceding or concurrent viral respiratory tract infection can predispose to secondary bacterial co-infection throughout the airway. The mechanisms by which viruses promote these superinfections are diverse and replete. Whereas we understand much as to how viruses damage the airway and dysregulate both innate and acquired immune responses which, in turn, supports bacterial growth, adherence and invasion into normally sterile sites within the respiratory tract, new information regarding these co-infections is being gained from recent advances in microbiome research and our enhanced appreciation of the contribution of bacterial biofilms, among others. The advanced understanding obtained by continued research efforts in all aspects of viral-bacterial co-infections of the respiratory tract will allow us to devise novel approaches for disease prevention as well as to develop more effective therapeutics., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Herein, we studied a virulent isolate of the leading bacterial pathogen Streptococcus pneumoniae in an infant mouse model of colonization, disease and transmission, both with and without influenza A (IAV) co-infection. To identify vulnerable points in the multiple steps involved in pneumococcal pathogenesis, this model was utilized for a comprehensive analysis of population bottlenecks. Our findings reveal that in the setting of IAV co-infection the organism must pass through single cell bottlenecks during bloodstream invasion from the nasopharynx within the host and in transmission between hosts. Passage through these bottlenecks was not associated with genetic adaptation by the pathogen. The bottleneck in transmission occurred between bacterial exit from one host and establishment in another explaining why the number of shed organisms in secretions is critical to overcoming it. These observations demonstrate how viral infection, and TLR-dependent innate immune responses it stimulates and that are required to control it, drive bacterial contagion., Competing Interests: The authors have declared that no competing interests exist.

In a closed farrow-to-finish piglet producing farm 80% of 7-week-old piglets displayed respiratory disease with a 5% mortality rate. In addition to purulent bronchopneumonia in combination with interstitial pneumonia predominantly in the apical and middle lobes, fibrinous serositis was present in the thoracic and abdominal cavities. Further investigations succeeded in confirming the non-pandemic strain of porcine influenza A virus (FLUAVsw) subtype H1avN1. The molecular genetic studies on Mycoplasma (M.) hyopneumoniae and porcine reproductive and respiratory syndrome virus were negative, whereas M. hyorhinis and Haemophilus parasuis were isolated from serous membranes. The possible importance of the underrated M. hyorhinis as a cofactor for viral infections should be emphasized and we demonstrated that the cause of apical lobe pneumonia is not restricted to M. hyopneumoniae. Mother pigs had been vaccinated with an influenza vaccine covering the subtype H1avN1. Only 33% of the examined piglets had maternal antibodies in the 7th week of life. The difficulty of prophylaxis of infections by FLUAVsw in weaners due to lack of vaccine authorization for piglets before their 56th day is reflected by this observation.

The incidence of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia in otherwise healthy individuals is increasing. To investigate the mechanism underlying the epidemiological success of predominant community-associated (CA)-MRSA strains, we examined their fitness traits during the initial interaction between bacteria and the host occurring in the lower airway. Using a mouse respiratory infection model, we show that clinical isolates often responsible for CA infections are highly resistant to clearance from healthy airways, whereas S. aureus strains not as prevalent or traditionally associated with hospital-associated infections are relatively susceptible. Mechanistically, the competitive fitness of S. aureus is a result of both agr-dependent and -independent resistance to innate bacterial killing. Furthermore, we show that rather than evasion from neutrophil-dependent bactericidal process, the observed S. aureus fitness in the lower airways is due to its intrinsic resistance to resident alveolar macrophage-mediated intracellular killing. Importantly, we demonstrate that the virulence determinants responsible for bacterial persistence in immune-competent mice are dispensable in mice with predisposing conditions such as influenza infection. Taken together, these novel findings of the improved competence of predominant CA-MRSA strains to survive innate killing in healthy hosts, particularly at the very beginning stage of infection, provide a unique insight into their epidemiological success., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Infectious salmon anemia virus (ISAV) is an orthomyxovirus, which may cause multisystemic disease and high mortality of Atlantic salmon (Salmo salar L). This suggests that ISAV encodes proteins that antagonize the type I interferon (IFN-I) system, which is of crucial importance in innate antiviral immunity. To find out how ISAV might inhibit IFN-I synthesis, we have here studied whether the two ISAV proteins s7ORF1 and s8ORF2 might interfere with activation of the IFNa1 promoter mediated by overexpression of interferon regulatory factors (IRFs) or by the IFN promoter activation protein IPS-1. The IRF tested were IRF1, IRF3, IRF7A and IRF7B. Promoter activation was measured using a luciferase reporter assay where Atlantic salmon TO cells were co-transfected with the IFNa1 promoter reporter plasmid together with an IRF plasmid and the s7ORF1 or the s8ORF2 construct or a control plasmid. The results showed that s7ORF1 significantly inhibited IRF3 and IRF7B induced IFN promoter activity, while s8ORF2 significantly inhibited IRF1 and IRF3 induced promoter activity. Neither s7ORF1 nor s8ORF2 inhibited IPS-1 mediated promoter activation. Immunoprecipitation data suggest that both s7ORF1 and s8ORF2 can bind to all four IRFs. Taken together, this study thus shows that the ISAV proteins s7ORF1 and s8ORF2 antagonizes IFN-I transcription activation mediated by the IRFs. As such this work provides further insight into the pathogenic properties of ISAV., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Postinfluenza bacterial pneumonia is associated with significant mortality and morbidity. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression posttranscriptionally. miR-155 has recently emerged as a crucial regulator of innate immunity and inflammatory responses and is induced in macrophages during infection. We hypothesized upregulation of miR-155 inhibits IL-17 and increases susceptibility to secondary bacterial pneumonia. Mice were challenged with 100 plaque-forming units H1N1 intranasally and were infected with 10(7) colony-forming units of MRSA intratracheally at day 5 postviral challenge. Lungs were harvested 24 h later, and expression of miR-155, IL-17, and IL-23 was measured by real-time RT-PCR. Induction of miR-155 was 3.6-fold higher in dual-infected lungs compared with single infection. miR-155(-/-) mice were protected with significantly lower (4-fold) bacterial burden and no differences in viral load, associated with robust induction of IL-23 and IL-17 (2.2- and 4.8-fold, respectively) postsequential challenge with virus and bacteria, compared with WT mice. Treatment with miR-155 antagomir improved lung bacterial clearance by 4.2-fold compared with control antagomir postsequential infection with virus and bacteria. Moreover, lung macrophages collected from patients with postviral bacterial pneumonia also had upregulation of miR-155 expression compared with healthy controls, consistent with observations in our murine model. This is the first demonstration that cellular miRNAs regulate postinfluenza immune response to subsequent bacterial challenge by suppressing the IL-17 pathway in the lung. Our findings suggest that antagonizing certain microRNA might serve as a potential therapeutic strategy against secondary bacterial infection., (Copyright © 2016 the American Physiological Society.)

To study bacterial co-infection following 1918 H1N1 influenza virus infection, mice were inoculated with the 1918 influenza virus, followed by Streptococcus pneumoniae (SP) 72 h later. Co-infected mice exhibited markedly more severe disease, shortened survival time and more severe lung pathology, including widespread thrombi. Transcriptional profiling revealed activation of coagulation only in co-infected mice, consistent with the extensive thrombogenesis observed. Immunohistochemistry showed extensive expression of tissue factor (F3) and prominent deposition of neutrophil elastase on endothelial and epithelial cells in co-infected mice. Lung sections of SP-positive 1918 autopsy cases showed extensive thrombi and prominent staining for F3 in alveolar macrophages, monocytes, neutrophils, endothelial and epithelial cells, in contrast to co-infection-positive 2009 pandemic H1N1 autopsy cases. This study reveals that a distinctive feature of 1918 influenza virus and SP co-infection in mice and humans is extensive expression of tissue factor and activation of the extrinsic coagulation pathway leading to widespread pulmonary thrombosis., (Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Phosphorylcholine (ChoP) decoration of lipopolysaccharides is an important virulence strategy adopted by Haemophilus influenzae to establish a niche on the mucosal surface and to promote adherence to the host cells. The incorporation of ChoP on the LPS surface involves the lic1 operon, which consists of the licA, licB, licC, and licD genes. Among which, licB is a choline transporter gene required for acquisition of choline from environmental sources. In this study, we investigated the pathogenesis of the licB gene in an aged mice infection model. Due to immediate clearance of H. influenzae upon infection in mice, we employed influenza A virus and H. influenzae co-infection model. Our data showed that in the co-infection model, the secondary bacterial infection with a very low H. influenzae concentration of 100 colony forming unit is lethal to the aged mice. Although we did not observe any differences in weight loss between parent and licB mutant strains during the course of infection, a significant reduction of lung tissue damage was observed in the licB mutant infected aged mice. These results suggest that the licB gene is a virulence factor during H. influenzae infection in the lung in aged mice, possibly due to the increased binding to the host cell receptor via ChoP expression on the bacterial surface. In addition, when aged mice and mature mice were compared in the challenge experiments, we did not observe any protective immunity in the co-infection model suggesting the detrimental effects of the secondary bacterial infection on the aged mice in contrast to obvious immune-protections observed in the mature mice. The results of our experiments also implied that the co-infection model with influenza A virus and H. influenzae may be employed as a model system to study H. influenzae pathogenesis in vivo in aged mice., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Suppression of type 17 immunity by type I interferon (IFN) during influenza A infection has been shown to enhance susceptibility to secondary bacterial pneumonia. Although this mechanism has been described in coinfection with gram-positive bacteria, it is unclear whether similar mechanisms may impair lung defense against gram-negative infections. Furthermore, precise delineation of the duration of type I IFN-associated susceptibility to bacterial infection remains underexplored. Therefore, we investigated the effects of preceding influenza A virus infection on subsequent challenge with the gram-negative bacteria Escherichia coli or Pseudomonas aeruginosa and the temporal association between IFN expression with susceptibility to Staphylococcus aureus challenge in a mouse model of influenza and bacterial coinfection. Here we demonstrate that preceding influenza A virus led to increased lung E. coli and P. aeruginosa bacterial burden, which was associated with suppression of type 17 immunity and attenuation of antimicrobial peptide expression. Enhanced susceptibility to S. aureus coinfection ceased at day 14 of influenza infection, when influenza-associated type I IFN levels had returned to baseline levels, further suggesting a key role for type I IFN in coinfection pathogenesis. These findings further implicate type I IFN-associated suppression of type 17 immunity and antimicrobial peptide production as a conserved mechanism for enhanced susceptibility to both gram-positive and gram-negative bacterial coinfection during influenza infection., (Copyright © 2015 the American Physiological Society.)

Infection with influenza virus has been a significant cause of morbidity and mortality for more than a hundred years. Severe disease and increased mortality often results from bacterial super-infection of patients with influenza virus infection. Preceding influenza infection alters the host's innate and adaptive immune responses, allowing increased susceptibility to secondary bacterial pneumonia. Recent advances in the field have helped to define how influenza alters the immune response to bacteria through the dysregulation of phagocytes, antimicrobial peptides, and lymphocytes. Viral-induced interferons play a key role in altering the phenotype of the immune response. Potential genetic modifiers of disease will help to define additional immunologic mechanisms that predispose to viral, bacterial super-infection with the overarching goal of developing effective therapeutic strategies to prevent and treat disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Seasonal and especially pandemic influenza predispose patients to secondary bacterial pneumonias, which are a major cause of deaths and morbidity. Staphylococcus aureus is a particularly common and deadly form of post-influenza pneumonia, and increasing staphylococcal drug resistance makes the development of new therapies urgent. We explored an innate immune-mediated model of the lung to define novel mechanisms by which the host can be protected against secondary staphylococcal pneumonia after sub-lethal influenza infection. We found that stimulating the innate immunity in the lung by overexpression of GM-CSF will result in resistance to S. aureus pneumonia after sublethal influenza infection. Resistance was mediated by alveolar macrophages and neutrophils, and was associated with increased production of reactive oxygen species (ROS) by alveolar macrophages. Resistance was abrogated by treatment with agents that scavenged ROS. We conclude that stimulating innate immunity in the lung markedly reduces susceptibility to post-influenza staphylococcal pneumonia and that this may represent a novel immunomodulatory strategy for prevention and treatment of secondary bacterial pneumonia after influenza.

Influenza A represents a significant cause of morbidity and mortality worldwide. Bacterial complications of influenza A confer the greatest risk to patients. TH17 pathway inhibition has been implicated as a mechanism by which influenza A alters bacterial host defense. Here we show that preceding influenza causes persistent Staphylococcus aureus infection and suppression of TH17 pathway activation in mice. Influenza does not inhibit S. aureus binding and uptake by phagocytic cells but instead attenuates S. aureus induced TH17 related antimicrobial peptides necessary for bacterial clearance in the lung. Importantly, exogenous lipocalin 2 rescued viral exacerbation of S. aureus infection and decreased free iron levels in the bronchoalveolar lavage from mice coinfected with S. aureus and influenza. These findings indicate a novel mechanism by which influenza A inhibits TH17 immunity and increases susceptibility to secondary bacterial pneumonia. Identification of new mechanisms in the pathogenesis of bacterial pneumonia could lead to future therapeutic targets.

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

Bacterial coinfection represents a major cause of morbidity and mortality in epidemics of influenza A virus (IAV). The bacterium Haemophilus influenzae typically colonizes the human upper respiratory tract without causing disease, and yet in individuals infected with IAV, it can cause debilitating or lethal secondary pneumonia. Studies in murine models have detected immune components involved in susceptibility and pathology, and yet few studies have examined bacterial factors contributing to coinfection. We conducted genome-wide profiling of the H. influenzae genes that promote its fitness in a murine model of coinfection with IAV. Application of direct, high-throughput sequencing of transposon insertion sites revealed fitness phenotypes of a bank of H. influenzae mutants in viral coinfection in comparison with bacterial infection alone. One set of virulence genes was required in nonvirally infected mice but not in coinfection, consistent with a defect in anti-bacterial defenses during coinfection. Nevertheless, a core set of genes required in both in vivo conditions indicated that many bacterial countermeasures against host defenses remain critical for coinfection. The results also revealed a subset of genes required in coinfection but not in bacterial infection alone, including the iron-sulfur cluster regulator gene, iscR, which was required for oxidative stress resistance. Overexpression of the antioxidant protein Dps in the iscR mutant restored oxidative stress resistance and ability to colonize in coinfection. The results identify bacterial stress and metabolic adaptations required in an IAV coinfection model, revealing potential targets for treatment or prevention of secondary bacterial pneumonia after viral infection.

Bacterial superinfection and associated lung immunopathology are major contributors to hospitalizations and mortality after influenza. However, the underlying mechanisms and effective intervention strategies remain poorly defined. By using a model of influenza and pneumococcal superinfection, we found that dual-infected animals experienced rapid weight loss and succumbed to infection. Bacterial outgrowth, dysregulated cytokines, including keratinocyte-derived chemokine and macrophage inflammatory protein 2, and severe lung neutrophilia and immunopathology were linked to the poor clinical outcome. In vivo neutralization of highly induced macrophage inflammatory protein 2 did not affect clinical outcome, bacterial loads, or lung immunopathology. On the other hand, in vivo neutrophil depletion did not alter the clinical outcome and bacterial burden, although it moderately improved lung immunopathology. Treatment with a bacteriostatic antibiotic, azithromycin, alone significantly improved clinical outcome and bacterial clearance, but failed to reduce lung immunopathology. In comparison, treatment with a global inflammation inhibitor, dexamethasone, alone failed to alter clinical outcome, bacterial infection, and immunopathology, despite its moderate reducing effects on neutrophilic and cytokine responses. In contrast, combined treatment with both azithromycin and dexamethasone best improved clinical outcome, bacterial clearance, lung cellular and cytokine responses, and immunopathology. Our study suggests that marked improvement of clinical outcome and lung immunopathology caused by bacterial superinfection requires the control of both bacterial infection and aberrant host immune responses. Our findings hold implications in clinical management for influenza-associated bacterial superinfections., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Individuals infected with mycobacteria are likely to experience episodes of concurrent infections with unrelated respiratory pathogens, including the seasonal or pandemic circulating influenza A virus strains. We analyzed the impact of influenza A virus and mycobacterial respiratory coinfection on the development of CD8 T cell responses to each pathogen. Coinfected mice exhibited reduced frequency and numbers of CD8 T cells specific to Mycobacterium bovis bacille Calmette-Guérin (BCG) in the lungs, and the IFN-γ CD8 T cell response to BCG-encoded OVA was decreased in the lungs of coinfected mice, when compared with mice infected with BCG alone. Moreover, after 2 wk of infection, mice coinfected with both pathogens showed a significant increase in the number of mycobacteria present in the lung compared with mice infected with BCG only. Following adoptive transfer into coinfected mice, transgenic CD8 T cells specific for OVA(257-264) failed to proliferate as extensively in the mediastinal lymph nodes as in mice infected only with BCG-OVA. Also noted was a reduction in the proliferation of BCG-specific CD4 transgenic T cells in mice coinfected with influenza compared with mice infected with BCG alone. Furthermore, phenotypic analysis of CD11c(+) dendritic cells from mediastinal lymph nodes of the infected mice showed that coinfection was associated with decreased surface expression of MHC class II and class I. Thus, concurrent pulmonary infection with influenza A virus is associated with decreased MHC expression on dendritic cells, reduced activation of BCG-specific CD4 and CD8 T cells, and impaired clearance of mycobacteria.

Influenza A virus (IAV) predisposes individuals to secondary infections with the bacterium Streptococcus pneumoniae (the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis, or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV-induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes such as OM. Here, we used an infant mouse model, human middle ear epithelial cells, and a series of reverse-engineered influenza viruses to investigate how IAV promotes bacterial OM. Our data suggest that the influenza virus HA facilitates disease by inducing a proinflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings suggest that it is the inflammatory response to IAV infection that mediates pneumococcal replication. This study thus provides the first evidence that inflammation drives pneumococcal replication in the middle ear cavity, which may have important implications for the treatment of pneumococcal OM.

Background: Swine influenza (SI) is an acute respiratory disease caused by swine influenza virus (SIV). Swine influenza is generally characterized by acute onset of fever and respiratory symptoms. The most frequent complications of influenza are secondary bacterial pneumonia. The objective of this work was to study the acute phase proteins (APP) responses after coinfection of piglets with H1N1 swine influenza virus (SwH1N1) and Pasteurella multocida (Pm) in order to identify whether the individual APP response correlate with disease severity and whether APP could be used as markers of the health status of coinfected pigs., Results: In all coinfected pigs clinical sings, including fever, coughing and dyspnea, were seen. Viral shedding was observed from 2 to 7 dpi. The mean level of antibodies against Pm dermonecrotoxin in infected piglets increase significantly from 7 dpi. Anti-SwH1N1 antibodies in the serum were detected from 7 dpi. The concentration of C-reactive protein (CRP) increased significantly at 1 dpi as compared to control pigs, and remained significantly higher to 3 dpi. Level of serum amyloid A (SAA) was significantly higher from 2 to 3 dpi. Haptoglobin (Hp) was significantly elevated from 3 dpi to the end of study, while pig major acute phase protein (Pig-MAP) from 3 to 7 dpi. The concentrations of CRP, Hp and SAA significantly increased before specific antibodies were detected. Positive correlations were found between serum concentration of Hp and SAA and lung scores, and between clinical score and concentrations of Pig-MAP and SAA., Conclusions: The results of current study confirmed that monitoring of APP may revealed ongoing infection, and in this way may be useful in selecting clinically healthy pigs (i.e. before integration into an uninfected herd). Present results corroborated our previous findings that SAA could be a potentially useful indicator in experimental infection studies (e.g. vaccine efficiency investigations) or as a marker for disease severity, because of correlation observed between its concentration in serum and disease severity (lung scores, clinical scores).

While the presence of airway bacteria is known to be associated with improved immunity against influenza virus, the mechanism by which endogenous microbiota influence antiviral immunity remains unclear. Here we show that specific pathogen-free mice are more sensitive to influenza-mediated death than mice living in a natural environment. Priming with Toll-like receptor 2-ligand(+) Staphylococcus aureus, which commonly colonizes the upper respiratory mucosa, significantly attenuates influenza-mediated lung immune injury. Toll-like receptor 2 deficiency or alveolar macrophage depletion abolishes this protection. S. aureus priming recruits peripheral CCR2(+)CD11b(+) monocytes into the alveoli that polarize to M2 alveolar macrophages in an environment created by Toll-like receptor 2 signalling. M2 alveolar macrophages inhibit influenza-mediated lethal inflammation via anti-inflammatory cytokines and inhibitory ligands. Our results suggest a previously undescribed mechanism by which the airway microbiota may protect against influenza-mediated lethal inflammation.

The control of infectious diseases such as swine influenza viruses (SwIV) plays an important role in food production both from the animal health and from the public health point of view. Probiotic microorganisms and other health improving food supplements have been given increasing attention in recent years, but, no information on the effects of probiotics on swine influenza virus is available. Here we address this question by assessing the inhibitory potential of the probiotic Enterococcus faecium NCIMB 10415 (E. faecium) on the replication of two porcine strains of influenza virus (H1N1 and H3N2 strain) in a continuous porcine macrophage cell line (3D4/21) and in MDBK cells. Cell cultures were treated with E. faecium at the non-toxic concentration of 1×10(6) CFU/ml in growth medium for 60 to 90 min before, during and after SwIV infection. After further incubation of cultures in probiotic-free growth medium, cell viability and virus propagation were determined at 48 h or 96 h post infection. The results obtained reveal an almost complete recovery of viability of SwIV infected cells and an inhibition of virus multiplication by up to four log units in the E. faecium treated cells. In both 3D4/21- and MDBK-cells a 60 min treatment with E. faecium stimulated nitric oxide (NO) release which is in line with published evidence for an antiviral function of NO. Furthermore, E. faecium caused a modified cellular expression of selected mediators of defence in 3D4-cells: while the expression of TNF-α, TLR-3 and IL-6 were decreased in the SwIV-infected and probiotic treated cells, IL-10 was found to be increased. Since we obtained experimental evidence for the direct adsorptive trapping of SwIV through E. faecium, this probiotic microorganism inhibits influenza viruses by at least two mechanisms, direct physical interaction and strengthening of innate defence at the cellular level.

Influenza causes >250,000 deaths annually in the industrialized world, and bacterial infections frequently cause secondary illnesses during influenza outbreaks, including pneumonia, bronchitis, sinusitis, and otitis media. In this study, we demonstrate that cross-reactive immunity to mismatched influenza strains can reduce susceptibility to secondary bacterial infections, even though this fails to prevent influenza infection. Specifically, infecting mice with H3N2 influenza before challenging with mismatched H1N1 influenza reduces susceptibility to either Gram-positive Streptococcus pneumoniae or Gram-negative Klebsiella pneumoniae. Vaccinating mice with the highly conserved nucleoprotein of influenza also reduces H1N1-induced susceptibility to lethal bacterial infections. Both T cells and Abs contribute to defense against influenza-induced bacterial diseases; influenza cross-reactive T cells reduce viral titers, whereas Abs to nucleoprotein suppress induction of inflammation in the lung. These findings suggest that nonneutralizing influenza vaccines that fail to prevent influenza infection may nevertheless protect the public from secondary bacterial diseases when neutralizing vaccines are not available.

Swine influenza virus (SIV) and Mycoplasma hyopneumoniae (Mhp) are widespread in farms and are major pathogens involved in the porcine respiratory disease complex (PRDC). The aim of this experiment was to compare the pathogenicity of European avian-like swine H1N1 and European human-like reassortant swine H1N2 viruses in naïve pigs and in pigs previously infected with Mhp. Six groups of SPF pigs were inoculated intra-tracheally with either Mhp, or H1N1, or H1N2 or Mhp+H1N1 or Mhp+H1N2, both pathogens being inoculated at 21 days intervals in these two last groups. A mock-infected group was included. Although both SIV strains induced clinical signs when singly inoculated, results indicated that the H1N2 SIV was more pathogenic than the H1N1 virus, with an earlier shedding and a greater spread in lungs. Initial infection with Mhp before SIV inoculation increased flu clinical signs and pathogenesis (hyperthermia, loss of appetite, pneumonia lesions) due to the H1N1 virus but did not modify significantly outcomes of H1N2 infection. Thus, Mhp and SIV H1N1 appeared to act synergistically, whereas Mhp and SIV H1N2 would compete, as H1N2 infection led to the elimination of Mhp in lung diaphragmatic lobes. In conclusion, SIV would be a risk factor for the severity of respiratory disorders when associated with Mhp, depending on the viral subtype involved. This experimental model of coinfection with Mhp and avian-like swine H1N1 is a relevant tool for studying the pathogenesis of SIV-associated PRDC and testing intervention strategies for the control of the disease., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: To evaluate effects on the innate immune system after exposure to, a consumable low-molecular weight fraction (CLMWF) of immunoglobulin-depleted bovine colostrum whey., Methodology: Cell-based immune assays were performed in vitro, and host resistance towards bacterial and viral infection was evaluated in two mouse studies., Results: In vitro data showed a multimodal effect, as CLMWF treatment resulted in a rapid increase in phagocytosis. CLMWF increased chemotaxis of polymorphonuclear cells towards the bacterial peptide f-MLP. CLMWF treatment of natural killer cells increased expression of the CD69 activation marker. Mononuclear phagocytes showed decreased numbers of CD14(bright) and increased number of CD14(dim) cells. The remaining CD14(bright) cells showed reduced expression of CD80 and CD86, whereas CD14(dim) cells showed increased expression of CD80 and CD86, suggesting dendritic cell maturation. Mouse models were applied to evaluate the immune-modulating capacity of CLMWF when consumed acutely during bacterial (Streptococcus) and viral (Influenza) infections in vivo. Reduced bacterial and viral loads were observed in lungs within 24h. Viral load was also reduced when CLMWF was introduced intranasally., Conclusion: The data suggest that the support of antimicrobial immune defense mechanisms and maturation of antigen-presenting cells in vitro translates to protection in vivo when product is introduced across mucosal membranes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Lactic acid bacteria (LAB) are known to have effects on immune function. From 203 strains of LAB isolated from fermented foods, we selected a beneficial strain, Lactobacillus plantarum strain YU (LpYU), which has high interleukin (IL)-12-inducing activity in mouse peritoneal macrophages. This activity of LpYU was partially mediated by Toll-like receptor (TLR) 2, but not TLR4 or TLR9. Oral administration of LpYU to ovalbumin (OVA)-immunized mice caused suppression of serum OVA-specific immunoglobulin E (IgE) levels, enhancing interferon (IFN)-γ production from spleen cells in response to OVA. Furthermore, LpYU enhanced natural killer cell activity in spleen cells and the production of IgA from Peyer's patch cells. Because activation of Th1 immune responses and IgA production induce antiviral effects, we evaluated the inhibitory effects of LpYU against the influenza A virus (A/NWS/33, H1N1) (IFV). Oral administration of LpYU suppressed viral proliferation in the lungs and in bronchoalveolar lavage fluids (BALFs). Both levels of IFV-specific secretory IgA in BALF and feces and titers of IFV-specific neutralizing antibody in BALFs and sera were increased. These results indicate that LpYU has a protective effect against IFV replication. We conclude that this strain has a beneficial effect in activating Th1 immune responses and preventing viral infection., (Copyright © 2011 Elsevier B.V. All rights reserved.)