Intestinal Interleukin-17 Receptor Signaling Mediates Reciprocal Control of the Gut Microbiota and Autoimmune Inflammation.

Interleukin-17 (IL-17) and IL-17 receptor (IL-17R) signaling are essential for regulating mucosal host defense against many invading pathogens. Commensal bacteria, especially segmented filamentous bacteria (SFB), are a crucial factor that drives T helper 17 (Th17) cell development in the gastrointestinal tract. In this study, we demonstrate that Th17 cells controlled SFB burden. Disruption of IL-17R signaling in the enteric epithelium resulted in SFB dysbiosis due to reduced expression of α-defensins, Pigr, and Nox1. When subjected to experimental autoimmune encephalomyelitis, IL-17R-signaling-deficient mice demonstrated earlier disease onset and worsened severity that was associated with increased intestinal Csf2 expression and elevated systemic GM-CSF cytokine concentrations. Conditional deletion of IL-17R in the enteric epithelium demonstrated that there was a reciprocal relationship between the gut microbiota and enteric IL-17R signaling that controlled dysbiosis, constrained Th17 cell development, and regulated the susceptibility to autoimmune inflammation.

Viral PB1-F2 and host IFN-γ guide ILC2 and T cell activity during influenza virus infection.

Functional plasticity of innate lymphoid cells (ILCs) and T cells is regulated by host environmental cues, but the influence of pathogen-derived virulence factors has not been described. We now report the interplay between host interferon (IFN)-γ and viral PB1-F2 virulence protein in regulating the functions of ILC2s and T cells that lead to recovery from influenza virus infection of mice. In the absence of IFN-γ, lung ILC2s from mice challenged with the A/California/04/2009 (CA04) H1N1 virus, containing nonfunctional viral PB1-F2, initiated a robust IL-5 response, which also led to improved tissue integrity and increased survival. Conversely, challenge with Puerto Rico/8/1934 (PR8) H1N1 virus expressing fully functional PB1-F2, suppressed IL-5+ ILC2 responses, and induced a dominant IL-13+ CD8 T cell response, regardless of host IFN-γ expression. IFN-γ-deficient mice had increased survival and improved tissue integrity following challenge with lethal doses of CA04, but not PR8 virus, and increased resistance was dependent on the presence of IFN-γR+ ILC2s. Reverse-engineered influenza viruses differing in functional PB1-F2 activity induced ILC2 and T cell phenotypes similar to the PB1-F2 donor strains, demonstrating the potent role of viral PB1-F2 in host resistance. These results show the ability of a pathogen virulence factor together with host IFN-γ to regulate protective pulmonary immunity during influenza infection.

Sequential targeting of interferon pathways for increased host resistance to bacterial superinfection during influenza.

Bacterial co-infections represent a major clinical complication of influenza. Host-derived interferon (IFN) increases susceptibility to bacterial infections following influenza, but the relative roles of type-I versus type-II IFN remain poorly understood. We have used novel mouse models of co-infection in which colonizing pneumococci were inoculated into the upper respiratory tract; subsequent sublethal influenza virus infection caused the bacteria to enter the lungs and mediate lethal disease. Compared to wild-type mice or mice deficient in only one pathway, mice lacking both IFN pathways demonstrated the least amount of lung tissue damage and mortality following pneumococcal-influenza virus superinfection. Therapeutic neutralization of both type-I and type-II IFN pathways similarly provided optimal protection to co-infected wild-type mice. The most effective treatment regimen was staggered neutralization of the type-I IFN pathway early during co-infection combined with later neutralization of type-II IFN, which was consistent with the expression and reported activities of these IFNs during superinfection. These results are the first to directly compare the activities of type-I and type-II IFN during superinfection and provide new insights into potential host-directed targets for treatment of secondary bacterial infections during influenza.

IFN-γ Drives TNF-α Hyperproduction and Lethal Lung Inflammation during Antibiotic Treatment of Postinfluenza Staphylococcus aureus Pneumonia.

Inflammatory cytokine storm is a known cause for acute respiratory distress syndrome. In this study, we have investigated the role of IFN-γ in lethal lung inflammation using a mouse model of postinfluenza methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. To mimic the clinical scenario, animals were treated with antibiotics for effective bacterial control following MRSA superinfection. However, antibiotic therapy alone is not sufficient to improve survival of wild-type animals in this lethal acute respiratory distress syndrome model. In contrast, antibiotics induce effective protection in mice deficient in IFN-γ response. Mechanistically, we show that rather than inhibiting bacterial clearance, IFN-γ promotes proinflammatory cytokine response to cause lethal lung damage. Neutralization of IFN-γ after influenza prevents hyperproduction of TNF-α, and thereby protects against inflammatory lung damage and animal mortality. Taken together, the current study demonstrates that influenza-induced IFN-γ drives a stepwise propagation of inflammatory cytokine response, which ultimately results in fatal lung damage during secondary MRSA pneumonia, despite of antibiotic therapy.

The Interleukin-33-Group 2 Innate Lymphoid Cell Axis Represents a Potential Adjuvant Target To Increase the Cross-Protective Efficacy of Influenza Vaccine.

Interleukin-33 (IL-33) is a multifunctional cytokine that mediates type 2-dominated immune responses. In contrast, the role of IL-33 during viral vaccination, which often aims to induce type 1 immunity, has not been fully investigated. Here, we examined the effects of IL-33 on influenza vaccine responses. We found that intranasal coadministration of IL-33 with an inactivated influenza virus vaccine increases vaccine efficacy against influenza virus infection, not only with the homologous strain but also with heterologous strains, including the 2009 H1N1 influenza virus pandemic strain. Cross-protection was dependent on group 2 innate lymphoid cells (ILC2s), as the beneficial effect of IL-33 on vaccine efficacy was abrogated in ILC2-deficient C57BL/6 Il7rCre/+ Rorafl/fl mice. Furthermore, mechanistic studies revealed that IL-33-activated ILC2s potentiate vaccine efficacy by enhancing mucosal humoral immunity, particularly IgA responses, potentially in a Th2 cytokine-dependent manner. Our results demonstrate that IL-33-mediated activation of ILC2s is a critical early event that is important for the induction of mucosal humoral immunity, which in turn is responsible for cross-strain protection against influenza. Thus, we reveal a previously unrecognized role for the IL-33-ILC2 axis in establishing broadly protective and long-lasting humoral mucosal immunity against influenza, knowledge that may help in the development of a universal influenza vaccine. IMPORTANCE Current influenza vaccines, although capable of protecting against predicted viruses/strains included in the vaccine, are inept at providing cross-protection against emerging/novel strains. Thus, we are in critical need of a universal vaccine that can protect against a wide range of influenza viruses. Our novel findings show that a mucosal vaccination strategy involving the activation of lung ILC2s is highly effective in eliciting cross-protective humoral immunity in the lungs. This suggests that the biology of lung ILC2s can be exploited to increase the cross-reactivity of commercially available influenza subunit vaccines.

Bacterial Second Messenger Cyclic di-AMP Modulates the Competence State in Streptococcus pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is a naturally competent organism that causes diseases such as pneumonia, otitis media, and bacteremia. The essential bacterial second messenger cyclic di-AMP (c-di-AMP) is an emerging player in the stress responses of many pathogens. In S. pneumoniae, c-di-AMP is produced by a diadenylate cyclase, CdaA, and cleaved by phosphodiesterases Pde1 and Pde2. c-di-AMP binds a transporter of K+ (Trk) family protein, CabP, which subsequently halts K+ uptake via the transporter TrkH. Recently, it was reported that Pde1 and Pde2 are essential for pneumococcal virulence in mouse models of disease. To elucidate c-di-AMP-mediated transcription that may lead to changes in pathogenesis, we compared the transcriptomes of wild-type (WT) and Δpde1 Δpde2 strains by transcriptome sequencing (RNA-Seq) analysis. Notably, we found that many competence-associated genes are significantly upregulated in the Δpde1 Δpde2 strain compared to the WT. These genes play a role in DNA uptake, recombination, and autolysis. Competence is induced by a quorum-sensing mechanism initiated by the secreted factor competence-stimulating peptide (CSP). Surprisingly, the Δpde1 Δpde2 strain exhibited reduced transformation efficiency compared to WT bacteria, which was c-di-AMP dependent. Transformation efficiency was also directly related to the [K+] in the medium, suggesting a link between c-di-AMP function and the pneumococcal competence state. We found that a strain that possesses a V76G variation in CdaA produced less c-di-AMP and was highly susceptible to CSP. Deletion of cabP and trkH restored the growth of these bacteria in medium with CSP. Overall, our study demonstrates a novel role for c-di-AMP in the competence program of S. pneumoniaeIMPORTANCE Genetic competence in bacteria leads to horizontal gene transfer, which can ultimately affect antibiotic resistance, adaptation to stress conditions, and virulence. While the mechanisms of pneumococcal competence signaling cascades have been well characterized, the molecular mechanism behind competence regulation is not fully understood. The bacterial second messenger c-di-AMP has previously been shown to play a role in bacterial physiology and pathogenesis. In this study, we provide compelling evidence for the interplay between c-di-AMP and the pneumococcal competence state. These findings not only attribute a new biological function to this dinucleotide as a regulator of competence, transformation, and survival under stress conditions in pneumococci but also provide new insights into how pneumococcal competence is modulated.

Effects of Influenza on Alveolar Macrophage Viability Are Dependent on Mouse Genetic Strain.

Secondary bacterial coinfections following influenza virus pose a serious threat to human health. Therefore, it is of significant clinical relevance to understand the immunological causes of this increased susceptibility. Influenza-induced alterations in alveolar macrophages (AMs) have been shown to be a major underlying cause of the increased susceptibility to bacterial superinfection. However, the mechanisms responsible for this remain under debate, specifically in terms of whether AMs are depleted in response to influenza infection or are maintained postinfection, but with disrupted phagocytic activity. The data presented in this article resolves this issue by showing that either mechanism can differentially occur in individual mouse strains. BALB/c mice exhibited a dramatic IFN-γ-dependent reduction in levels of AMs following infection with influenza A, whereas AM levels in C57BL/6 mice were maintained throughout the course of influenza infection, although the cells displayed an altered phenotype, namely an upregulation in CD11b expression. These strain differences were observed regardless of whether infection was performed with low or high doses of influenza virus. Furthermore, infection with either the H1N1 A/California/04/2009 (CA04) or H1N1 A/PR8/1934 (PR8) virus strain yielded similar results. Regardless of AM viability, both BALB/c and C57BL/6 mice showed a high level of susceptibility to postinfluenza bacterial infection. These findings resolve the apparent inconsistencies in the literature, identify mouse strain-dependent differences in the AM response to influenza infection, and ultimately may facilitate translation of the mouse model to clinical application.

Characterization of the local wound environment following treatment of chronic leg ulcers with SIS wound matrix.

BACKGROUND: The SIS Wound Matrix (SISWM) has been shown to improve healing of chronic ulcers over standard of care. In this study, we tested the hypothesis that chronic venous ulcers responsive to treatment with SISWM would more closely mimic an acute wound state as opposed to unresponsive ulcers. METHODS: Serum and wound exudate were collected at baseline and then weekly for up to 12 weeks from 12 patients receiving multiple applications of the SISWM. Levels of matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9, and MMP-12), pro-inflammatory cytokines (IL-1ß, TNF-α, IL-8), and transforming growth factor beta (TGF-ß1) were evaluated. A variety of Th1/Th2 cytokines were also assayed, as were systemic anti-SIS and anti-α-gal antibody titers. RESULTS: Seven of the 12 patients eventually healed their wounds. Results showed significant decreases in MMP-1, MMP-2, MMP-3, MMP-9, TNF-α and IL-8, and significant increases in TGF-ß1 in wounds responding to treatment with the SISWM versus wounds that did not respond to treatment. None of the 12 patients formed a measurable serum antibody response to the SISWM. CONCLUSIONS: These data show that SISWM does not lead to immune system recognition or sensitization to the matrix and that wounds that went on to heal following treatment were characterized by a more acute wound state. The study confirms that the wound environment is important to healing and that turning a wound toward an acute biochemical state is key to the healing process.

Topical application of nebulized human IgG, IgA and IgAM in the lungs of rats and non-human primates.

BACKGROUND: Recurrent and persistent infections are known to affect airways of patients with Primary Immunodeficiency despite appropriate replacement immunoglobulin serum levels. Interestingly, patients with Chronic Obstructive Pulmonary Disease or with non-CF bronchiectasis also show similar susceptibility to such infections. This may be due to the limited availability of immunoglobulins from the systemic circulation in the conductive airways, resulting in local immunodeficiency. Topical application of nebulized plasma-derived immunoglobulins may represent a means to address this deficiency. In this study, we assessed the feasibility of nebulizing plasma-derived immunoglobulins and delivering them into the airways of rats and non-human primates. METHODS: Distinct human plasma-derived immunoglobulin isotype preparations were nebulized with an investigational eFlow® nebulizer and analyzed in vitro or deposited into animals. Biochemical and immunohistological analysis of nebulized immunoglobulins were then performed. Lastly, efficacy of topically applied human plasma-derived immunoglobulins was assessed in an acute Streptococcus pneumoniae respiratory infection in mice. RESULTS: Characteristics of the resulting aerosols were comparable between preparations, even when using solutions with elevated viscosity. Neither the structural integrity nor the biological function of nebulized immunoglobulins were compromised by the nebulization process. In animal studies, immunoglobulins levels were assessed in plasma, broncho-alveolar lavages (BAL) and on lung sections of rats and non-human primates in samples collected up to 72 h following application. Nebulized immunoglobulins were detectable over 48 h in the BAL samples and up to 72 h on lung sections. Immunoglobulins recovered from BAL fluid up to 24 h after inhalation remained structurally and functionally intact. Importantly, topical application of human plasma-derived immunoglobulin G into the airways of mice offered significant protection against acute pneumococcal pneumonia. CONCLUSION: Taken together our data demonstrate the feasibility of topically applying plasma-derived immunoglobulins into the lungs using a nebulized liquid formulation. Moreover, topically administered human plasma-derived immunoglobulins prevented acute respiratory infection.

Live Vaccination Generates Both Disease Tolerance and Host Resistance During Chronic Pulmonary Infection With Highly Virulent Francisella tularensis SchuS4.

Disease tolerance can preserve host homeostasis and limit the negative impact of infections. We report that vaccinated mice survived pulmonary challenge with the extremely virulent SchuS4 strain of Francisella tularensis for at least 100 days, despite the persistence of large numbers (~104) of organisms. Transfer of 100 of these resident bacteria to naive animals caused 100% lethality, demonstrating that virulence was maintained. Tissue damage in the lung was limited over the course of infection and was associated with increased levels of amphiregulin. Mice depleted of CD4+ cells had reduced amphiregulin and succumbed to infection. In addition, neutralization of interferon-γ or depletion of CD8+ cells resulted in increased pathogen loads, bacteremia, and death of the host. Conversely, depletion of Ly6G+ neutrophils had no effect on survival and actually resulted in reduced bacterial levels. Understanding the interplay between host resistance and disease tolerance will provide new insights into the understanding of chronic infectious diseases.

Stress Suppressor Screening Leads to Detection of Regulation of Cyclic di-AMP Homeostasis by a Trk Family Effector Protein in Streptococcus pneumoniae.

Cyclic di-AMP (c-di-AMP) is a newly discovered bacterial second messenger. However, regulation of c-di-AMP homeostasis is poorly understood. In Streptococcus pneumoniae, a sole diadenylate cyclase, CdaA, produces c-di-AMP and two phosphodiesterases, Pde1 and Pde2, cleave the signaling dinucleotide. To expand our knowledge of the pneumococcal c-di-AMP signaling network, we performed whole-genome sequencing of Δpde1 Δpde2 heat shock suppressors. In addition to their effects on surviving heat shock, these suppressor mutations restored general stress resistance and improved growth in rich medium. Mutations in CdaA or in the potassium transporter TrkH paired with an insertion leading to a frameshift at the C terminus of CdaA significantly reduced c-di-AMP levels. These observations indicate that the elevated c-di-AMP levels in the Δpde1 Δpde2 mutant enhance susceptibility of S. pneumoniae to the stress conditions. Interestingly, we have previously shown that TrkH complexes with a Trk family c-di-AMP-binding protein, CabP, to mediate potassium uptake. In this study, we found that deletion of cabP significantly reduced pneumococcal c-di-AMP levels. This is the first observation that a c-di-AMP effector protein modulates bacterial c-di-AMP homeostasis.IMPORTANCE Second messengers, including c-di-AMP, are prevalent among bacterial species. In S. pneumoniae, c-di-AMP phosphodiesterase-encoding gene null mutants are attenuated during mouse models of infection, but the role of c-di-AMP signaling in pneumococcal pathogenesis is enigmatic. In this work, we found that heat shock suppressor mutations converge on undermining c-di-AMP toxicity by changing intracellular c-di-AMP concentrations. These mutations improve the growth and restore the stress response generally in c-di-AMP phosphodiesterase-deficient pneumococci, thereby demonstrating the essentiality for tight regulation of c-di-AMP homeostasis in order to respond to stress. Likewise, this work demonstrates that a c-di-AMP effector protein, CabP, affects c-di-AMP homeostasis, which provides new perception into c-di-AMP regulation. This study has implications for c-di-AMP-producing bacteria since many species contain CabP homologs.

Detrimental Influence of Alveolar Macrophages on Protective Humoral Immunity during Francisella tularensis SchuS4 Pulmonary Infection.

Opsonizing antibody is a critical component of the host protective immune response against many respiratory pathogens. However, the role of antibodies in protection against pulmonary infection with highly virulent Francisella tularensis strain SchuS4 is unclear, and the mechanism that allows F. tularensis to evade antibody-mediated bacterial clearance is not fully understood. We have now found that depletion of alveolar macrophages reveals an otherwise cryptic protective effect of opsonizing antibody. While antibody opsonization alone failed to confer any survival benefit against SchuS4 lung infection, significant protection was observed when mice were depleted of alveolar macrophages prior to infection. Blood immune signature analyses and bacterial burden measurements indicated that the treatment regimen blocked establishment of productive, systemic infection. In addition, protection was found to be dependent upon neutrophils. The results show for the first time a protective effect of opsonizing antibodies against highly virulent F. tularensis SchuS4 pulmonary infection through depletion of alveolar macrophages, the primary bacterial reservoir, and prevention of systemic dissemination. These findings have important implications for the potential use of therapeutic antibodies against intracellular pathogens that may escape clearance by residing within mucosal macrophages.

Protective Role for Macrophages in Respiratory Francisella tularensis Infection.

Francisella tularensis causes lethal pneumonia following infection of the lungs by targeting macrophages for intracellular replication; however, macrophages stimulated with interferon gamma (IFN-γ) can resist infection in vitro We therefore hypothesized that the protective effect of IFN-γ against F. tularensisin vivo requires macrophages receptive to stimulation. We found that the lethality of pulmonary F. tularensis LVS infection was exacerbated under conditions of alveolar macrophage depletion and in mice with a macrophage-specific defect in IFN-γ signaling (termed mice with macrophages insensitive to IFN-γ [MIIG mice]). We previously found that treatment with exogenous interleukin 12 (IL-12) protects against F. tularensis infection; this protection was lost in MIIG mice. MIIG mice also exhibited reduced neutrophil recruitment to the lungs following infection. Systemic neutrophil depletion was found to render wild-type mice highly sensitive to respiratory F. tularensis infection, and depletion beginning at 3 days postinfection led to more pronounced sensitivity than depletion beginning prior to infection. Furthermore, IL-12-mediated protection required NADPH oxidase activity. These results indicate that lung macrophages serve a critical protective role in respiratory F. tularensis LVS infection. Macrophages require IFN-γ signaling to mediate protection, which ultimately results in recruitment of neutrophils to further aid in survival from infection.

Prevention of Influenza Virus-Induced Immunopathology by TGF-ß Produced during Allergic Asthma.

Asthma is believed to be a risk factor for influenza infection, however little experimental evidence exists to directly demonstrate the impact of asthma on susceptibility to influenza infection. Using a mouse model, we now report that asthmatic mice are actually significantly more resistant to a lethal influenza virus challenge. Notably, the observed increased resistance was not attributable to enhanced viral clearance, but instead, was due to reduced lung inflammation. Asthmatic mice exhibited a significantly reduced cytokine storm, as well as reduced total protein levels and cytotoxicity in the airways, indicators of decreased tissue injury. Further, asthmatic mice had significantly increased levels of TGF-ß1 and the heightened resistance of asthmatic mice was abrogated in the absence of TGF-ß receptor II. We conclude that a transient increase in TGF-ß expression following acute asthma can induce protection against influenza-induced immunopathology.

Border Patrol Gone Awry: Lung NKT Cell Activation by Francisella tularensis Exacerbates Tularemia-Like Disease.

The respiratory mucosa is a major site for pathogen invasion and, hence, a site requiring constant immune surveillance. The type I, semi-invariant natural killer T (NKT) cells are enriched within the lung vasculature. Despite optimal positioning, the role of NKT cells in respiratory infectious diseases remains poorly understood. Hence, we assessed their function in a murine model of pulmonary tularemia--because tularemia is a sepsis-like proinflammatory disease and NKT cells are known to control the cellular and humoral responses underlying sepsis. Here we show for the first time that respiratory infection with Francisella tularensis live vaccine strain resulted in rapid accumulation of NKT cells within the lung interstitium. Activated NKT cells produced interferon-γ and promoted both local and systemic proinflammatory responses. Consistent with these results, NKT cell-deficient mice showed reduced inflammatory cytokine and chemokine response yet they survived the infection better than their wild type counterparts. Strikingly, NKT cell-deficient mice had increased lymphocytic infiltration in the lungs that organized into tertiary lymphoid structures resembling induced bronchus-associated lymphoid tissue (iBALT) at the peak of infection. Thus, NKT cell activation by F. tularensis infection hampers iBALT formation and promotes a systemic proinflammatory response, which exacerbates severe pulmonary tularemia-like disease in mice.

Expression of suppressor of cytokine signaling 1 (SOCS1) impairs viral clearance and exacerbates lung injury during influenza infection.

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of cytokine signaling. SOCS1-/- mice die within three weeks postnatally due to IFN-γ-induced hyperinflammation. Since it is well established that IFN-γ is dispensable for protection against influenza infection, we generated SOCS1-/-IFN-γ-/- mice to determine whether SOCS1 regulates antiviral immunity in vivo. Here we show that SOCS1-/-IFN-γ-/- mice exhibited significantly enhanced resistance to influenza infection, as evidenced by improved viral clearance, attenuated acute lung damage, and consequently increased survival rates compared to either IFN-γ-/- or WT animals. Enhanced viral clearance in SOCS1-/-IFN-γ-/- mice coincided with a rapid onset of adaptive immune responses during acute infection, while their reduced lung injury was associated with decreased inflammatory cell infiltration at the resolution phase of infection. We further determined the contribution of SOCS1-deficient T cells to antiviral immunity. Anti-CD4 antibody treatment of SOCS1-/-IFN-γ-/- mice had no significant effect on their enhanced resistance to influenza infection, while CD8+ splenocytes from SOCS1-/-IFN-γ-/- mice were sufficient to rescue RAG1-/- animals from an otherwise lethal infection. Surprisingly, despite their markedly reduced viral burdens, RAG1-/- mice reconstituted with SOCS1-/-IFN-γ-/- adaptive immune cells failed to ameliorate influenza-induced lung injury. In conclusion, in the absence of IFN-γ, the cytoplasmic protein SOCS1 not only inhibits adaptive antiviral immune responses but also exacerbates inflammatory lung damage. Importantly, these detrimental effects of SOCS1 are conveyed through discrete cell populations. Specifically, while SOCS1 expression in adaptive immune cells is sufficient to inhibit antiviral immunity, SOCS1 in innate/stromal cells is responsible for aggravated lung injury.

Influenza infection suppresses NADPH oxidase-dependent phagocytic bacterial clearance and enhances susceptibility to secondary methicillin-resistant Staphylococcus aureus infection.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a leading contributor to mortality during recent influenza pandemics. The mechanism for this influenza-induced susceptibility to secondary S. aureus infection is poorly understood. In this study, we show that innate antibacterial immunity was significantly suppressed during the recovery stage of influenza infection, even though MRSA superinfection had no significant effect on viral burdens. Compared with mice infected with bacteria alone, postinfluenza MRSA-infected mice exhibited impaired bacterial clearance, which was not due to defective phagocyte recruitment, but rather coincided with reduced intracellular reactive oxygen species levels in alveolar macrophages and neutrophils. NADPH oxidase is responsible for reactive oxygen species production during phagocytic bacterial killing, a process also known as oxidative burst. We found that gp91(phox)-containing NADPH oxidase activity in macrophages and neutrophils was essential for optimal bacterial clearance during respiratory MRSA infections. In contrast to wild-type animals, gp91(phox-/-) mice exhibited similar defects in MRSA clearance before and after influenza infection. Using gp91(phox+/-) mosaic mice, we further demonstrate that influenza infection inhibits a cell-intrinsic contribution of NADPH oxidase to phagocyte bactericidal activity. Taken together, our results establish that influenza infection suppresses NADPH oxidase-dependent bacterial clearance and leads to susceptibility to secondary MRSA infection.

Limited Efficacy of Antibacterial Vaccination Against Secondary Serotype 3 Pneumococcal Pneumonia Following Influenza Infection.

BACKGROUND: Secondary bacterial infections following influenza represent a major cause of mortality in the human population, which, in turn, has led to a call for stockpiling of bacterial vaccines for pandemic preparedness. METHODS: To investigate the efficacy of bacterial vaccination for protection against secondary pneumococcal infection, mice were immunized with pneumococcal capsular polysaccharide conjugate vaccine, and then sequentially coinfected 5 weeks later with PR8 influenza virus and A66.1 Streptococcus pneumoniae. RESULTS: In the absence of influenza virus exposure, vaccination with polysaccharide conjugate vaccine was highly effective, as indicated by 100% survival from lethal pneumococcal pneumonia and 10 000-fold greater efficiency in clearance of bacteria from the lung compared to unvaccinated mice. Enhanced clearance after vaccination was dependent upon Fc receptor (FcR) expression. However, following influenza, <40% of vaccinated mice survived bacterial coinfection and FcR-dependent clearance of antibody-opsonized bacteria reduced bacterial levels in the lungs only 5-10 fold. No differences in lung myeloid cell numbers or in FcR cell surface expression were observed following influenza. CONCLUSIONS: The results show that induction of antibacterial humoral immunity is only partially effective in protection against secondary bacterial infections that occur following influenza, and suggest that additional therapeutic strategies to overcome defective antibacterial immunity should be explored.

Allergic Lung Inflammation Reduces Tissue Invasion and Enhances Survival from Pulmonary Pneumococcal Infection in Mice, Which Correlates with Increased Expression of Transforming Growth Factor ß1 and SiglecF(low) Alveolar Macrophages.

Asthma is generally thought to confer an increased risk for invasive pneumococcal disease (IPD) in humans. However, recent reports suggest that mortality rates from IPD are unaffected in patients with asthma and that chronic obstructive pulmonary disease (COPD), a condition similar to asthma, protects against the development of complicated pneumonia. To clarify the effects of asthma on the subsequent susceptibility to pneumococcal infection, ovalbumin (OVA)-induced allergic lung inflammation (ALI) was induced in mice followed by intranasal infection with A66.1 serotype 3 Streptococcus pneumoniae. Surprisingly, mice with ALI were significantly more resistant to lethal infection than non-ALI mice. The heightened resistance observed following ALI correlated with enhanced early clearance of pneumococci from the lung, decreased bacterial invasion from the airway into the lung tissue, a blunted inflammatory cytokine and neutrophil response to infection, and enhanced expression of transforming growth factor ß1 (TGF-ß1). Neutrophil depletion prior to infection had no effect on enhanced early bacterial clearance or resistance to IPD in mice with ALI. Although eosinophils recruited into the lung during ALI appeared to be capable of phagocytizing bacteria, neutralization of interleukin-5 (IL-5) to inhibit eosinophil recruitment likewise had no effect on early clearance or survival following infection. However, enhanced resistance was associated with an increase in levels of clodronate-sensitive, phagocytic SiglecF(low) alveolar macrophages within the airways following ALI. These findings suggest that, while the risk of developing IPD may actually be decreased in patients with acute asthma, additional clinical data are needed to better understand the risk of IPD in patients with different asthma phenotypes.

Role of Interleukin-12 in Protection against Pulmonary Infection with Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common pathogen associated with nosocomial pneumonia and is an increasing threat for severe community-acquired pneumonia. We have now investigated the role of interleukin-12 (IL-12) in protective immunity against lung infection with MRSA. The importance of IL-12 in protection from pulmonary MRSA infection was demonstrated by the finding that IL-12p35-deficient mice had a lower survival rate, higher bacterial burdens in lung and spleen, and decreased expression of interferon gamma (IFN-γ) in the lung compared to wild-type mice. These effects were completely reversed by replacement intranasal therapy with recombinant IL-12. Furthermore, exogenous IL-12 treatment of wild-type mice 24 h before pulmonary challenge with a lethal dose of MRSA significantly improved bacterial clearance and resulted in protection from death. The IL-12-treated mice had increased numbers of lung natural killer (NK) cells and neutrophils and higher levels of IFN-γ in the lung and serum compared to untreated mice. The major source of IL-12-driven IFN-γ expression in the lung was the NK cell, and the direct target of pulmonary IFN-γ was the lung macrophage, as shown using mice with a macrophage-specific defect in interferon gamma (IFN-γ) signaling (MIIG mice). Importantly, combination therapy with linezolid and IL-12 following intranasal MRSA infection significantly increased survival compared to that of mice receiving linezolid or IL-12 alone. These results indicate that IL-12-based immunotherapy may hold promise for treatment of MRSA pneumonia.