The therapeutic potential of thiocyanate and hypothiocyanous acid against pulmonary infections.

Hypothiocyanous acid (HOSCN) is an endogenous oxidant produced by peroxidase oxidation of thiocyanate (SCN-), an ubiquitous sulfur-containing pseudohalide synthesized from cyanide. HOSCN serves as a potent microbicidal agent against pathogenic bacteria, viruses, and fungi, functioning through thiol-targeting mechanisms, independent of currently approved antimicrobials. Additionally, SCN- reacts with hypochlorous acid (HOCl), a highly reactive oxidant produced by myeloperoxidase (MPO) at sites of inflammation, also producing HOSCN. This imparts both antioxidant and antimicrobial potential to SCN-. In this review, we discuss roles of HOSCN/SCN- in immunity and potential therapeutic implications for combating infections.

Bone marrow stromal cell-derived hepcidin has antimicrobial and immunomodulatory activities.

Bone marrow stromal cells (BMSCs) have immunomodulatory activities in numerous species and have been used in clinical trials. BMSCs also make antibacterial agents. Since hepcidin is known to have antimicrobial effects in fish, we wondered if it might also be used as an antimicrobial agent by mammalian BMSCs. In the present study, we show hepcidin expression in both mouse (mBMSC) and human BMSCs (hBMSC). We observed a hBMSC hepcidin-dependent degradation of ferroportin in HEK-293 reporter cells in vitro. In human and mouse bone marrows (BM) we detected hepcidin-positive BMSCs in close proximity to hematopoietic progenitors. The conditioned culture medium of hBMSCs significantly reduced bacterial proliferation that was partially blocked by a hepcidin-neutralizing antibody. Similarly, medium in which hepcidin-deficient (Hamp-/-) mouse BMSCs had been grown was significantly less effective in reducing bacterial counts than the medium of wild-type cells. In a zymosan-induced peritonitis mouse model we found that mBMSC-derived hepcidin reduced the number of invading polymorphonuclear (PMN) cells in the peritoneal cavity. Our results show that BMSC-derived hepcidin has antimicrobial properties in vitro and also reduces inflammation in vivo. We conclude that hepcidin should be added to the expanding arsenal of agents available to BMSCs to fight infections and inflammation.

Variant-dependent oxidative and cytokine responses of human neutrophils to SARS-CoV-2 spike protein and anti-spike IgG1 antibodies.

Introduction: Severe forms of COVID-19, the disease caused by SARS-CoV-2, are characterized by acute respiratory distress syndrome, robust lung inflammation and death in some patients. Strong evidence has been accumulating that polymorphonuclear neutrophilic granulocytes (PMN) play an important role in the pathophysiology of severe COVID-19. SARS-CoV-2 directly induces in vitro PMN activation, mainly the release of neutrophil extracellular traps (NETs). However, the viral components inducing this PMN response remain unclear. Methods: In this work human PMN responses were assessed in vitro in response to the spike (S) protein of two different SARS-CoV-2 variants, anti-S IgG1 antibodies or immune complexes formed by them. Production of reactive oxygen species (ROS) was measured by Diogenes-based chemiluminescence. Release of myeloperoxidase (MPO) was assessed by ELISA while secretion of a list of cytokines and growth factors was determined by high-performance multiplex cytokine assay. Results and discussion: We show that the SARS-CoV-2 Omicron variant S protein and anti-spike IgG1, either alone or together, stimulate ROS production in human PMNs. We also observed that the SARS-CoV-2 Wuhan S protein and anti-S IgG1 antibody together trigger MPO release from PMNs. Based on the relevance of SARS-CoV-2 and influenza co-infections, we have also investigated the impact of influenza virus infection on the previous PMN responses to S proteins or anti-S antibodies. We did not detect any significant effect of influenza co-infection on ROS generation in PMNs. Our data also show that PMN stimulation by S proteins induced the release of different chemokines, growth factors, regulatory and proinflammatory cytokines. Overall, our findings show that the SARS-CoV-2 S protein, an anti-spike IgG1 antibody or their immune complex, promote oxidative responses of PMNs in a variant-dependent manner, contributing to a better understanding of the role of PMN responses during SARS-CoV-2 infection.

Sputum from People with Cystic Fibrosis Reduces the Killing of Methicillin-Resistant Staphylococcus aureus by Neutrophils and Diminishes Phagosomal Production of Reactive Oxygen Species.

Cystic fibrosis (CF) airway disease is characterized by chronic polymicrobial infections and an infiltration of neutrophils (PMNs). Staphylococcus aureus has been the most prevalent respiratory pathogen in CF. In particular, methicillin-resistant S. aureus (MRSA) represents a huge clinical burden in CF due to its association with lung disease and increased resistance to antibiotics. In CF, PMNs are unable to kill and clear MRSA. The reason for this remains largely unknown. Our study found that CF PMNs are as equally capable of killing MRSA as healthy PMNs. We show that the CF sputum, however, significantly impairs the ability of human PMNs to kill CF MRSA isolates. In the absence of CF sputum, PMNs kill MRSA via intracellular mechanisms mediated by phagocytosis, rather than extracellular mechanisms via NET formation. CF sputum does not affect the phagocytosis of MRSA via healthy or CF PMNs. Our results demonstrate that CF sputum exposure impairs phagosomal levels of reactive oxygen species (ROS) in MRSA-phagocytosing PMNs. While phagosomal co-localizations of MRSA with primary granule markers, myeloperoxidase and cathepsin D, were significantly reduced upon CF sputum exposure, that of a third azurophilic granule marker, neutrophil elastase, remained unaffected. This suggests that CF sputum does not compromise the fusion of primary granules with phagosomes but diminishes phagosomal ROS levels via another, likely more specific, mechanism. Overall, we identified the airway environment as an important factor that restricts neutrophils' oxidative microbicidal activities in CF against MRSA. These results deliver new details of the complex host-pathogen interactions present in the CF lung.

Cystic fibrosis autoantibody signatures associate with Staphylococcus aureus lung infection or cystic fibrosis-related diabetes.

Introduction: While cystic fibrosis (CF) lung disease is characterized by persistent inflammation and infections and chronic inflammatory diseases are often accompanied by autoimmunity, autoimmune reactivity in CF has not been studied in depth. Methods: In this work we undertook an unbiased approach to explore the systemic autoantibody repertoire in CF using autoantibody microarrays. Results and discussion: Our results show higher levels of several new autoantibodies in the blood of people with CF (PwCF) compared to control subjects. Some of these are IgA autoantibodies targeting neutrophil components or autoantigens linked to neutrophil-mediated tissue damage in CF. We also found that people with CF with higher systemic IgM autoantibody levels have lower prevalence of S. aureus infection. On the other hand, IgM autoantibody levels in S. aureus-infected PwCF correlate with lung disease severity. Diabetic PwCF have significantly higher levels of IgA autoantibodies in their circulation compared to nondiabetic PwCF and several of their IgM autoantibodies associate with worse lung disease. In contrast, in nondiabetic PwCF blood levels of IgA autoantibodies correlate with lung disease. We have also identified other autoantibodies in CF that associate with P. aeruginosa airway infection. In summary, we have identified several new autoantibodies and associations of autoantibody signatures with specific clinical features in CF.

Short chain fatty acids reduce the respiratory burst of human neutrophils in response to cystic fibrosis isolates of Staphylococcus aureus.

Short chain fatty acids (SCFA) are produced by anaerobic bacteria. The most common SCFAs are acetate, propionate and butyrate. SCFAs have been implicated in several inflammatory diseases including cystic fibrosis (CF) where they are present in the airways at millimolar concentrations. Staphylococcus aureus is one of the main respiratory pathogens in CF. Polymorphonuclear neutrophil granulocytes (PMN) represent the most important immune defense the host uses against S. aureus. However, the reason why PMNs are unable to clear S. aureus in CF remains largely unclear. We hypothesized that SCFAs impair effector functions of PMNs in response to S. aureus. To test this, human PMNs were exposed to CF clinical isolates of S. aureus in vitro in the presence or absence of SCFAs and effector functions of PMNs were assessed. Our data show that SCFAs do not affect the viability of PMNs and do not stimulate the release of neutrophil extracellular traps (NET) from human PMNs. Production of reactive oxygen species (ROS), another important antimicrobial function of PMNs, on the other hand, was significantly inhibited by SCFAs in response to the bacterium. SCFAs did not compromise the ability of PMNs to kill CF isolates of S. aureus in vitro. Overall, our results provide new knowledge into the interactions between SCFAs and the immune system, and indicate that SCFAs produced by anaerobic bacteria in the CF lung could interfere with reactive oxidant production of PMNs in response to S. aureus, one of the prominent respiratory pathogens in this disease.

Dual oxidase 1 is dispensable during Mycobacterium tuberculosis infection in mice.

Introduction: Mycobacterium tuberculosis (Mtb) is the primary cause of human tuberculosis (TB) and is currently the second most common cause of death due to a singleinfectious agent. The first line of defense against airborne pathogens, including Mtb, is the respiratory epithelium. One of the innate defenses used by respiratory epithelial cells to prevent microbial infection is an oxidative antimicrobial system consisting of the proteins, lactoperoxidase (LPO) and Dual oxidase 1 (Duox1), the thiocyanate anion (SCN-) and hydrogen peroxide (H2O2), which together lead to the generation of antimicrobial hypothiocyanite (OSCN-) in the airway lumen. OSCN- kills bacteria and viruses in vitro, but the role of this Duox1-based system in bacterial infections in vivo remains largely unknown. The goal of this study was to assess whether Duox1 contributes to the immune response against the unique respiratory pathogen, Mtb. Methods: Duox1-deficient (Duox1 KO) and wild-type (WT) mice were infected with Mtb aerosols and bacterial titers, lung pathology, cytokines and immune cell recruitment were assessed. Results and discussion: Mtb titers in the lung, spleen and liver were not different 30 days after infection between WT and Duox1 KO mice. Duox1 did not affect lung histology assessed at days 0, 30, and 90 post-Mtb infection. Mtb-infected Duox1 KO animals exhibited enhanced production of certain cytokines and chemokines in the airway; however, this response was not associated with significantly higher numbers of macrophages or neutrophils in the lung. B cell numbers were lower, while apoptosis was higher in the pulmonary lesions of Mtb-infected Duox1 KO mice compared to infected WT animals. Taken together, these data demonstrate that while Duox1 might influence leukocyte recruitment to inflammatory cell aggregates, Duox1 is dispensable for the overall clinical course of Mtb lung infection in a mouse model.

The Hypothiocyanite and Amantadine Combination Treatment Prevents Lethal Influenza A Virus Infection in Mice.

The influenza virus has a large clinical burden and is associated with significant mortality and morbidity. The development of effective drugs for the treatment or prevention of influenza is important in order to reduce its impact. Adamantanes and neuraminidase inhibitors are two classes of anti-influenza drugs in which resistance has developed; thus, there is an urgent need to explore new therapeutic options. Boosting antiviral innate immune mechanisms in the airways represents an attractive approach. Hypothiocyanite (OSCN-) is produced by the airway epithelium and is effective in reducing the replication of several influenza A virus strains in vitro. It remains, however, largely unexplored whether OSCN- has such an antiviral effect in vivo. Here we determined the therapeutic potential of OSCN-, alone or in combination with amantadine (AMT), in preventing lethal influenza A virus replication in mice and in vitro. Mice intranasally infected with a lethal dose of A/Puerto Rico/8/1934 (H1N1) or A/Hong Kong/8/1968 (H3N2) were cured by the combination treatment of OSCN- and AMT. Monotherapy with OSCN- or AMT alone did not substantially improve survival outcomes. However, AMT+OSCN- treatment significantly inhibited viral replication, and in vitro treatment inhibited viral entry and nuclear transport of different influenza A virus strains (H1N1 and H3N2) including the AMT-resistant strain A/WSN/33 (H1N1). A triple combination treatment consisting of AMT, oseltamivir, and OSCN- was also tested and further inhibited in vitro viral replication of the AMT-resistant A/WSN/33 strain. These results suggest that OSCN- is a promising anti-influenza treatment option when combined with other antiviral drugs.

Human Intelectin-1 Promotes Cellular Attachment and Neutrophil Killing of Streptococcus pneumoniae in a Serotype-Dependent Manner.

Human intelectin-1 (hIntL-1) is a secreted glycoprotein capable of binding exocyclic 1,2-diols within surface glycans of human pathogens such as Streptococcus pneumoniae, Vibrio cholerae, and Helicobacter pylori. For the latter, lectin binding was shown to cause bacterial agglutination and increased phagocytosis, suggesting a role for hIntL-1 in pathogen surveillance. In this study, we investigated the interactions between hIntL-1 and S. pneumoniae, the leading cause of bacterial pneumonia. We show that hIntL-1 also agglutinates S. pneumoniae serotype 43, which displays an exocyclic 1,2-diol moiety in its capsular polysaccharide but is unable to kill in a complement-dependent manner or to promote bacterial killing by peripheral blood mononuclear cells. In contrast, hIntL-1 not only significantly increases serotype-specific S. pneumoniae killing by neutrophils but also enhances the attachment of these bacteria to A549 lung epithelial cells. Taken together, our results suggest that hIntL-1 participates in host surveillance through microbe sequestration and enhanced targeting to neutrophils.

Serum anti-PAD4 autoantibodies are present in cystic fibrosis children and increase with age and lung disease severity.

Cystic fibrosis (CF) lung disease begins early in childhood and is characterized by neutrophilic inflammation of the airways. Neutrophil extracellular traps (NETs) represent one mechanism by which neutrophils contribute to lung damage. The enzyme peptidylarginine deiminase 4 (PAD4) is required for NET formation. Our overall concept is that NET formation delivers PAD4 outside the neutrophil resulting in autoantibody generation, and this autoimmunity may be a novel mechanism contributing to CF lung disease progression. The aim of this study was to investigate clinical predictors of serum anti-PAD4 autoantibody (PAD4 Ab) levels in CF subjects with a wide range of ages from early childhood through middle age. We measured PAD4 Ab levels in sera from 104 CF subjects. PAD4 Abs were detectable among CF children as young as one year of age and elevated compared to paediatric healthy controls. PAD4 Ab levels increased significantly with age (r = 0.584, p <.001) and correlated with lower lung function (r = -0.481, n = 99, p <.001). PAD4 Abs were elevated in subjects with chronic Pseudomonas aeruginosa airways infection (p <.001), but not with other key clinical CF co-variates including sex, CFTR genotype, sweat chloride, pancreatic enzyme use, nutritional status, recent pulmonary exacerbations, Staphylococcus aureus, or CF-related diabetes. PAD4 Ab levels were also correlated with serum anti-double-stranded DNA IgA autoantibodies, which have similarly been shown to be elevated in CF subjects and associated with lung damage. In multivariable analysis, age and lung function remained correlated with PAD4 Ab levels. In summary, we describe novel findings of anti-PAD4 autoantibodies in CF that are present early in childhood, increase over time with age, and correlate with lung disease severity. Autoimmunity to antigens extruded by NETs appears to be an early event in CF lung disease, and airway autoimmunity related to NET formation is a potential mechanism of lung disease progression in CF.HighlightsSerum anti-PAD4 autoantibodies are detected in paediatric CF serum and are elevated compared to healthy paediatric controlsAnti-PAD4 autoantibodies increase with ageAnti-PAD4 autoantibodies correlate with lower lung function, Pseudomonas aeruginosa airway infection and anti-dsDNA IgA autoantibodies, but not with other key clinical CF co-variatesAge and lung function remain correlated with anti-PAD4 autoantibodies in multivariable analysis.

RGS10 Reduces Lethal Influenza Infection and Associated Lung Inflammation in Mice.

Seasonal influenza epidemics represent a significant global health threat. The exacerbated immune response triggered by respiratory influenza virus infection causes severe pulmonary damage and contributes to substantial morbidity and mortality. Regulator of G-protein signaling 10 (RGS10) belongs to the RGS protein family that act as GTPase activating proteins for heterotrimeric G proteins to terminate signaling pathways downstream of G protein-coupled receptors. While RGS10 is highly expressed in immune cells, in particular monocytes and macrophages, where it has strong anti-inflammatory effects, its physiological role in the respiratory immune system has not been explored yet. Here, we show that Rgs10 negatively modulates lung immune and inflammatory responses associated with severe influenza H1N1 virus respiratory infection in a mouse model. In response to influenza A virus challenge, mice lacking RGS10 experience enhanced weight loss and lung viral titers, higher mortality and significantly faster disease onset. Deficiency of Rgs10 upregulates the levels of several proinflammatory cytokines and chemokines and increases myeloid leukocyte accumulation in the infected lung, markedly neutrophils, monocytes, and inflammatory monocytes, which is associated with more pronounced lung damage. Consistent with this, influenza-infected Rgs10-deficent lungs contain more neutrophil extracellular traps and exhibit higher neutrophil elastase activities than wild-type lungs. Overall, these findings propose a novel, in vivo role for RGS10 in the respiratory immune system controlling myeloid leukocyte infiltration, viral clearance and associated clinical symptoms following lethal influenza challenge. RGS10 also holds promise as a new, potential therapeutic target for respiratory infections.

Microbicidal Activity of Hypothiocyanite against Pneumococcus.

Infections caused by Streptococcus pneumoniae (pneumococcus, Spn) manifest in several forms such as pneumonia, meningitis, sinusitis or otitis media and are associated with severe morbidity and mortality worldwide. While current vaccines and antibiotics are available to treat Spn infections, the rise of antibiotic resistance and limitations of the vaccines to only certain Spn serotypes urge the development of novel treatments against Spn. Hypothiocyanite (OSCN-) is a natural antimicrobial product produced by the body's own innate immune system to fight a variety of pathogens. We recently showed that OSCN- is also capable of killing Spn in vitro. OSCN- is an oxidative agent attacking microbes in a nonspecific manner, is safe for the host and also has anti-inflammatory effects that make it an ideal candidate to treat a variety of infections in humans. However, OSCN- has a short life span that makes its use, dosage and administration more problematic. This minireview discusses the antimicrobial mechanism of action of OSCN- against Spn and elaborates on the potential therapeutic use of OSCN- against Spn and other infectious agents, either alone or in combination with other therapeutic approaches.

Myeloperoxidase and Other Markers of Neutrophil Activation Associate With Malaria and Malaria/HIV Coinfection in the Human Placenta.

Introduction: Placental malaria (PM) is characterized by accumulation of inflammatory leukocytes in the placenta, leading to poor pregnancy outcomes. Understanding of the underlying mechanisms remains incomplete. Neutrophils respond to malaria parasites by phagocytosis, generation of oxidants, and externalization of Neutrophil Extracellular Traps (NETs). NETs drive inflammation in malaria but evidence of NETosis in PM has not been reported. Neutrophil activity in the placenta has not been directly investigated in the context of PM and PM/HIV-co-infection. Methods: Using peripheral and placental plasma samples and placental tissue collected from Kenyan women at risk for malaria and HIV infections, we assessed granulocyte levels across all gravidities and markers of neutrophil activation, including NET formation, in primi- and secundigravid women, by ELISA, western blot, immunohistochemistry and immunofluorescence. Results: Reduced peripheral blood granulocyte numbers are observed with PM and PM/HIV co-infection in association with increasing parasite density and placental leukocyte hemozoin accumulation. In contrast, placental granulocyte levels are unchanged across infection groups, resulting in enhanced placental: peripheral count ratios with PM. Within individuals, PM- women have reduced granulocyte counts in placental relative to peripheral blood; in contrast, PM stabilizes these relative counts, with HIV coinfection tending to elevate placental counts relative to the periphery. In placental blood, indicators of neutrophil activation, myeloperoxidase (MPO) and proteinase 3 (PRTN3), are significantly elevated with PM and, more profoundly, with PM/HIV co-infection, in association with placental parasite density and hemozoin-bearing leukocyte accumulation. Another neutrophil marker, matrix metalloproteinase (MMP9), together with MPO and PRTN3, is elevated with self-reported fever. None of these factors, including the neutrophil chemoattractant, CXCL8, differs in relation to infant birth weight or gestational age. CXCL8 and MPO levels in the peripheral blood do not differ with infection status nor associate with birth outcomes. Indicators of NETosis in the placental plasma do not vary with infection, and while structures consistent with NETs are observed in placental tissue, the results do not support an association with PM. Conclusions: Granulocyte levels are differentially regulated in the peripheral and placental blood in the presence and absence of PM. PM, both with and without pre-existing HIV infection, enhances neutrophil activation in the placenta. The impact of local neutrophil activation on placental function and maternal and fetal health remains unclear. Additional investigations exploring how neutrophil activation and NETosis participate in the pathogenesis of malaria in pregnant women are needed.

Granulocytic Myeloid-Derived Suppressor Cells in Cystic Fibrosis.

Cystic Fibrosis (CF) is a genetic disease that causes chronic and severe lung inflammation and infection associated with high rates of mortality. In CF, disrupted ion exchange in the epithelium results in excessive mucus production and reduced mucociliary clearance, leading to immune system exacerbation and chronic infections with pathogens such as P. aeruginosa and S. aureus. Constant immune stimulation leads to altered immune responses including T cell impairment and neutrophil dysfunction. Specifically, CF is considered a Th17-mediated disease, and it has been proposed that both P. aeruginosa and a subset of neutrophils known as granulocytic myeloid suppressor cells (gMDSCs) play a role in T cell suppression. The exact mechanisms behind these interactions are yet to be determined, but recent works demonstrate a role for arginase-1. It is also believed that P. aeruginosa drives gMDSC function as a means of immune evasion, leading to chronic infection. Herein, we review the current literature regarding immune suppression in CF by gMDSCs with an emphasis on T cell impairment and the role of P. aeruginosa in this dynamic interaction.

PI3K/ NF-κB-dependent TNF-α and HDAC activities facilitate LPS-induced RGS10 suppression in pulmonary macrophages.

Regulator of G-protein signaling 10 (RGS10) is a member of the superfamily of RGS proteins that canonically act as GTPase activating proteins (GAPs). RGS proteins accelerate GTP hydrolysis on the G-protein α subunits and result in termination of signaling pathways downstream of G protein-coupled receptors. Beyond its GAP function, RGS10 has emerged as an anti-inflammatory protein by inhibiting LPS-mediated NF-κB activation and expression of inflammatory cytokines, in particular TNF-α. Although RGS10 is abundantly expressed in resting macrophages, previous studies have shown that RGS10 expression is suppressed in macrophages following Toll-like receptor 4 (TLR4) activation by LPS. However, the molecular mechanism by which LPS induces Rgs10 silencing has not been clearly defined. The goal of the current study was to determine whether LPS silences Rgs10 expression through an NF-κB-mediated proinflammatory mechanism in pulmonary macrophages, a unique type of innate immune cells. We demonstrate that Rgs10 transcript and RGS10 protein levels are suppressed upon LPS treatment in the murine MH-S alveolar macrophage cell line. We show that pharmacological inhibition of PI3K/ NF-κB/p300 (NF-κB co-activator)/TNF-α signaling cascade and the activities of HDAC (1-3) enzymes block LPS-induced silencing of Rgs10 in MH-S cells as well as microglial BV2 cells and BMDMs. Further, loss of RGS10 generated by using CRISPR/Cas9 amplifies NF-κB phosphorylation and inflammatory gene expression following LPS treatment in MH-S cells. Together, our findings strongly provide critical insight into the molecular mechanism underlying RGS10 suppression by LPS in pulmonary macrophages.

Cystic Fibrosis Sputum Impairs the Ability of Neutrophils to Kill Staphylococcus aureus.

Cystic fibrosis (CF) airway disease is characterized by chronic microbial infections and infiltration of inflammatory polymorphonuclear (PMN) granulocytes. Staphylococcus aureus (S. aureus) is a major lung pathogen in CF that persists despite the presence of PMNs and has been associated with CF lung function decline. While PMNs represent the main mechanism of the immune system to kill S. aureus, it remains largely unknown why PMNs fail to eliminate S. aureus in CF. The goal of this study was to observe how the CF airway environment affects S. aureus killing by PMNs. PMNs were isolated from the blood of healthy volunteers and CF patients. Clinical isolates of S. aureus were obtained from the airways of CF patients. The results show that PMNs from healthy volunteers were able to kill all CF isolates and laboratory strains of S. aureus tested in vitro. The extent of killing varied among strains. When PMNs were pretreated with supernatants of CF sputum, S. aureus killing was significantly inhibited suggesting that the CF airway environment compromises PMN antibacterial functions. CF blood PMNs were capable of killing S. aureus. Although bacterial killing was inhibited with CF sputum, PMN binding and phagocytosis of S. aureus was not diminished. The S. aureus-induced respiratory burst and neutrophil extracellular trap release from PMNs also remained uninhibited by CF sputum. In summary, our data demonstrate that the CF airway environment limits killing of S. aureus by PMNs and provides a new in vitro experimental model to study this phenomenon and its mechanism.

Modeling Immune Evasion and Vaccine Limitations by Targeted Nasopharyngeal Bordetella pertussis Inoculation in Mice.

Conventional pertussis animal models deliver hundreds of thousands of Bordetella pertussis bacteria deep into the lungs, rapidly inducing severe pneumonic pathology and a robust immune response. However, human infections usually begin with colonization and growth in the upper respiratory tract. We inoculated only the nasopharynx of mice to explore the course of infection in a more natural exposure model. Nasopharyngeal colonization resulted in robust growth in the upper respiratory tract but elicited little immune response, enabling prolonged and persistent infection. Immunization with human acellular pertussis vaccine, which prevents severe lung infections in the conventional pneumonic infection model, had little effect on nasopharyngeal colonization. Our infection model revealed that B. pertussis can efficiently colonize the mouse nasopharynx, grow and spread within and between respiratory organs, evade robust host immunity, and persist for months. This experimental approach can measure aspects of the infection processes not observed in the conventional pneumonic infection model.

Dual oxidase 1 promotes antiviral innate immunity.

Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre-ssed in respiratory epithelial cells and produces H2O2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN-) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1-/- mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1ß, IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell recruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN- is generated by LPO using DUOX1-derived H2O2 and inactivates several influenza strains in vitro. We also show that OSCN- diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H2O2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN- in an H2O2-dependent manner in vitro. OSCN- does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN-, and proposes that boosting this mechanism in vivo can have therapeutic potential in treating viral infections.

DUOX1 in mammalian disease pathophysiology.

Dual oxidase 1 (DUOX1) is a member of the protein family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. DUOX1 has several normal physiological, immunological, and biochemical functions in different parts of the body. Dysregulated oxidative metabolism interferes with various disease pathologies and numerous therapeutic options are based on targeting cellular redox pathways. DUOX1 forms an important enzymatic source of biological oxidants, and DUOX1 expression is frequently dysregulated in various diseases. While this review shortly addresses the biochemical and cellular properties and proposed physiological roles of DUOX1, its main purpose is to summarize the current knowledge with respect to the potential role of DUOX1 enzyme in disease pathology, especially in mammalian organisms. Although DUOX1 is normally prominently expressed in epithelial lineages, it is frequently silenced in epithelial-derived cancers by epigenetic mechanisms. While an abundance of information is available on DUOX1 transcription in different diseases, an increasing number of mechanistic studies indicate a causative relationship between DUOX1 function and disease pathophysiology. Additionally, specific functions of the DUOX1 maturation factor, DUOXA1, will also be addressed. Lastly, urgent and outstanding questions on the field of DUOX1 will be discussed that could provide valuable new diagnostic tools and novel therapeutic options.