Nontypeable Haemophilus Influenzae Infection Research Articles

Invasive Nontypeable Haemophilus influenzae Infection Among Adults With HIV in Metropolitan Atlanta, Georgia, 2008-2018

Invasive nontypeable Haemophilus influenzae (NTHi) infection among adults is typically associated with bacteremic pneumonia. Nontypeable H influenzae is genetically diverse and clusters of infection are uncommon. To evaluate an increase in invasive NTHi infection from 2017-2018 among HIV-infected men who have sex with men in metropolitan Atlanta, Georgia. A population-based surveillance study with a cohort substudy and descriptive epidemiological analysis identified adults aged 18 years or older with invasive NTHi infection (isolation of NTHi from a normally sterile site) between January 1, 2008, and December 31, 2018 (final date of follow-up). Time period, HIV status, and genetic relatedness (ie, cluster status) of available NTHi isolates. The primary outcome was incidence of invasive NTHi infection (from 2008-2016 and 2017-2018) among persons with HIV and compared with NTHi infection from 2008-2018 among those without HIV. The secondary outcomes were assessed among those aged 18 to 55 years with invasive NTHi infection and included epidemiological, clinical, and geographic comparisons by cluster status. Among 553 adults with invasive NTHi infection (median age, 66 years [Q1-Q3, 48-78 years]; 52% male; and 38% black), 60 cases occurred among persons with HIV. Incidence of invasive NTHi infection from 2017-2018 among persons with HIV (41.7 cases per 100 000) was significantly greater than from 2008-2016 among those with HIV (9.6 per 100 000; P < .001) and from 2008-2018 among those without HIV (1.1 per 100 000; P < .001). Among adults aged 18 to 55 years with invasive NTHi infections from 2017-2018 (n = 179), persons with HIV (n = 31) were significantly more likely than those from 2008-2018 without HIV (n = 124) to be male (94% vs 49%, respectively; P < .001), black (100% vs 53%; P < .001), and have septic arthritis (35% vs 1%; P < .001). Persons with HIV who had invasive NTHi infection from 2017-2018 (n = 31) were more likely than persons with HIV who had invasive NTHi infection from 2008-2016 (n = 24) to have septic arthritis (35% vs 4%, respectively; P = .01). Pulsed-field gel electrophoresis of 174 of 179 NTHi isolates from 18- to 55-year-olds identified 2 genetically distinct clonal groups: cluster 1 (C1; n = 24) and cluster 2 (C2; n = 23). Whole-genome sequencing confirmed 2 clonal lineages of NTHi infection and revealed all C1 isolates (but none of the C2 isolates) carried IS1016 (an insertion sequence associated with H influenzae capsule genes). Persons with HIV were significantly more likely to have C1 or C2 invasive NTHi infection from 2017-2018 (28/31 [90%]) compared with from 2008-2016 among persons with HIV (10/24 [42%]; P < .001) and compared with from 2008-2018 among those without HIV (9/119 [8%]; P < .001). Among persons with C1 or C2 invasive NTHi infection who had HIV (n = 38) (median age, 34.5 years; 100% male; 100% black; 82% men who have sex with men), 32 (84%) lived in 2 urban counties and an area of significant spatial aggregation was identified compared with those without C1 or C2 invasive NTHi infection. Among persons with HIV in Atlanta, the incidence of invasive nontypeable H influenzae infection increased significantly from 2017-2018 compared with 2008-2016. Two unique but genetically related clonal strains were identified and were associated with septic arthritis among black men who have sex with men and who lived in geographic proximity.

Preclinical Evaluation of the Antimicrobial-Immunomodulatory Dual Action of Xenohormetic Molecules against Haemophilus influenzae Respiratory Infection.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammation and impaired airway immunity, providing an opportunistic platform for nontypeable Haemophilus influenzae (NTHi) infection. In this context, therapies targeting not only overactive inflammation without significant adverse effects, but also infection are of interest. Increasing evidence suggests that polyphenols, plant secondary metabolites with anti-inflammatory and antimicrobial properties, may be protective. Here, a Cistus salviifolius plant extract containing quercetin, myricetin, and punicalagin was shown to reduce NTHi viability. Analysis of these polyphenols revealed that quercetin has a bactericidal effect on NTHi, does not display synergies, and that bacteria do not seem to develop resistance. Moreover, quercetin lowered NTHi airway epithelial invasion through a mechanism likely involving inhibition of Akt phosphorylation, and reduced the expression of bacterially-induced proinflammatory markers il-8, cxcl-1, il-6, pde4b, and tnfα. We further tested quercetin’s effect on NTHi murine pulmonary infection, showing a moderate reduction in bacterial counts and significantly reduced expression of proinflammatory genes, compared to untreated mice. Quercetin administration during NTHi infection on a zebrafish septicemia infection model system showed a bacterial clearing effect without signs of host toxicity. In conclusion, this study highlights the therapeutic potential of the xenohormetic molecule quercetin against NTHi infection.

Cellular Immune Response against Nontypeable Haemophilus influenzae Infecting the Preinflamed Middle Ear of the Junbo Mouse.

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing acute otitis media (AOM). The pathology of AOM increases during long-term infection in the middle ear (ME), but the host cellular immune response to bacterial infection in this inflamed environment is poorly understood. Using the Junbo mouse, a characterized NTHi infection model, we analyzed the cellular response to NTHi infection in the Junbo mouse middle ear fluid (MEF). NTHi infection increased the total cell number and significantly decreased the proportion of live cells in the MEF at day 1, and this further decreased gradually on each day up to day 7. Flow cytometry analysis showed that neutrophils were the dominant immune cell population in the MEF and that NTHi infection significantly increased their proportion whereas it decreased the monocyte, macrophage, and dendritic cell proportions. Neutrophil and macrophage numbers increased in blood and spleen after NTHi infection. The T-cell population was dominated by T-helper (Th) cells in noninoculated MEF, and the effector Th (CD44+) cell population increased at day 2 of NTHi infection with an increase in IL-12p40 levels. Sustained NTHi infection up to 3 days increased the transforming growth factor β levels, decreasing the effector cell population and increasing the T-regulatory (T-reg) cell population. In the preinflamed ME environment of the Junbo mouse, neutrophils are the first responder to NTHi infection followed by T-reg immune suppressive cells. These data indicate that sustained NTHi infection in the ME induces the immune suppressive response by inducing the T-reg cell population and reducing immune cell infiltration, thus promoting longer-term infection.

Vaccine Prevention of Paediatric Chronic Lung Disease: Targeting Haemophilus

Abstract Background Chronic Suppurative Lung Disease (CSLD) is an emerging term encompassing a spectrum of chronic childhood respiratory disease. Characterised by early and severe infections and recurrent exacerbations, it is associated with progressive deterioration in lung function and quality of life. CSLD is highly unequally distributed and largely preventable, and is a major contributor to the global burden of chronic paediatric respiratory disease. Relatively few pathogens comprise the main culprit organisms identified in the aetiology of CSLD, over which Non-Typeable Haemophilus influenzae (NTHi) predominates. Methods We review developments that establish the role of NTHi in disease progression, focusing on high-risk population subgroups with an established CSLD burden. We consider current studies examining the role of prenatal NTHi and pneumococcal vaccination in preventing CSLD through reducing infections in high-risk populations, and discuss current directions in future research, including the need for precise identification of the pulmonary microbiome in CSLD. Results Early, repeated NTHi infections are clearly implicated in the aetiology of CLSD. Early results of current studies indicate NTHi vaccination may reduce the frequency of causal infections in high-risk groups. Clinical and immunological data show vaccination also reduces frequency of exacerbations and antibiotic usage in CSLD. Conclusions We provide the first clinical and immunological data for H. influenzae vaccination in children with CSLD. Targeted vaccination strategies may prevent CSLD establishment, slow progression, and potentially reduce morbidity, healthcare presentations and antibiotic consumption. This implicates vaccination as a potential aid in both antimicrobial stewardship and prevention of chronic disease. In the face of threats posed to health systems by antimicrobial resistance and the growing burden of chronic disease, vaccination is again emerging as a uniquely powerful public health tool. Key messages Hi vaccination can reduce infective exacerbations and antibiotic use in children with CSL. Vaccines may have a role in preventing chronic disease progression and reducing antibiotic reliance.

Non-typeable Haemophilus influenzae-associated early pregnancy loss: an emerging neonatal and maternal pathogen

ObjectivesThere is increasing evidence indicating an association between invasive non-typeable Haemophilus influenzae (NTHi) infection in pregnancy and early pregnancy loss. As the diagnosis relies on microbiological investigation of post-mortem placental and foetal samples, a significant proportion of NTHi-related pregnancy loss remains unrecognised. To better characterise NTHi in septic abortion, we report NTHi cases associated with early pregnancy loss.MethodsWe reviewed all post-mortems at <24 weeks gestation with histologically proven acute chorioamnionitis on placental histology and enrolled cases with at least one matched foetal and placental sample culture positive for NTHi. The study was approved by the NHS Lothian Caldicott Guardian.ResultsIn our cohort, invasive NTHi has accounted for 20% of infections associated with early pregnancy loss prior to 24 weeks gestation. All patients were young and healthy pregnant women at < 20 weeks' gestation who presented with abdominal pain, PV bleed /discharge and were septic at the time of presentation. One patient with previous history of miscarriage who presented with cervical incompetence had more severe pathology suggestive of early intrauterine pneumonia.ConclusionThe burden of invasive NTHi disease in early pregnancy loss is likely to be much larger than currently recognised. NTHi should be considered in pregnant women presenting with abdominal pain and PV bleed/discharge in whom clinical signs of sepsis are present. Active surveillance should be considered in this patient group including septic abortion to capture the true prevalence of this emerging pathogen to inform preventative and therapeutic approaches.

Combined treatment with SB203580 and dexamethasone suppresses non-typeable Haemophilus influenzae-induced Th17 inflammation response in murine allergic asthma

Accumulating evidence suggests that non-typeable Haemophilus influenzae (NTHi) infection drives the development of steroid-resistant allergic airway disease (SRAAD), exacerbates clinical symptoms, worsens quality of life, and accounts for most of the related healthcare burden. The poor understanding of the pathogenesis of SRAAD deters the development of more effective therapeutic strategies. Here, we established a murine model of NTHi infection-induced exacerbation of allergic airway disease. We showed that NTHi infection drove Th 17-mediated pulmonary neutrophilic inflammation, aggravated airway hyper-responsiveness, and upset the balance of MUC5AC and MUC5B expression. Dexamethasone treatment effectively inhibited the features of allergic airway disease but failed to reduce NTHi-induced exacerbation, which was associated with the hyper-phosphorylation of p38 mitogen-activated protein kinase (MAPK). Interestingly, inhibition of p38 using a specific inhibitor (SB203580) only partly suppressed the airway hyper-responsiveness and mucus hyper-secretion but failed to abrogate the infection-induced neutrophilic inflammatory response in SRAAD. However, SB203580 and dexamethasone co-treatment substantially suppressed all the features of NTHi-induced SRAAD. Our findings highlight the importance of p38 MAPK in the pathogenesis of NTHi-induced steroid resistance, and this combined treatment approach may be a novel strategy against steroid-resistant asthma.

Non-typeable Haemophilus influenzae protein vaccine in adults with COPD: A phase 2 clinical trial

Loss of airway microbial diversity is associated with non-typeable Haemophilus influenzae (NTHi) infection and increased risk of exacerbation in chronic obstructive pulmonary disease (COPD). We assessed the safety and immunogenicity of an investigational vaccine containing NTHi antigens, recombinant protein D (PD) and combined protein E and Pilin A (PE-PilA), and AS01 adjuvant in adults with moderate/severe COPD and prior exacerbations.In this phase 2, observer-blind, controlled trial (NCT02075541), 145 COPD patients aged 40–80 years randomly (1:1) received two doses of NTHi vaccine or placebo 60 days apart, on top of standard care.Reactogenicity in the 7-day post-vaccination period was higher following NTHi vaccine than placebo. Most solicited adverse events (AEs) were mild/moderate. At least one unsolicited AE was reported during the 30-day post-vaccination period by 54.8% of NTHi vaccine and 51.4% of placebo recipients. One serious AE (placebo group) was assessed by the investigator as vaccine-related. Anti-PD, anti-PE and anti-PilA geometric mean antibody concentrations increased up to 30 days after each NTHi vaccine dose, waned thereafter, but remained higher than baseline (non-overlapping confidence intervals) up to 13 months post-dose 2. The frequency of specific CD4+ T cells increased following two doses of NTHi vaccine and remained higher than baseline. Exploratory analysis showed a statistically non-significant lower yearly rate of moderate/severe exacerbations in the NTHi vaccine group than following placebo (1.49 versus 1.73) in the one-year period post-dose 2, with estimated vaccine efficacy of 13.3% (95% confidence interval −24.2 to 39.5; p = 0.44).The NTHi vaccine had an acceptable safety and reactogenicity profile and good immunogenicity in adults with COPD.

Pellino-1 Regulates Immune Responses to Haemophilus influenzae in Models of Inflammatory Lung Disease.

Non-typeable Haemophilus influenzae (NTHi) is a frequent cause of lower respiratory tract infection in people with chronic obstructive pulmonary disease (COPD). Pellino proteins are a family of E3 ubiquitin ligases that are critical regulators of TLR signaling and inflammation. The aim of this study was to identify a role for Pellino-1 in airway defense against NTHi in the context of COPD. Pellino-1 is rapidly upregulated by LPS and NTHi in monocyte-derived macrophages (MDMs) isolated from individuals with COPD and healthy control subjects, in a TLR4 dependent manner. C57BL/6 Peli1−/− and wild-type (WT) mice were subjected to acute (single LPS challenge) or chronic (repeated LPS and elastase challenge) airway inflammation followed by NTHi infection. Both WT and Peli1−/− mice develop airway inflammation in acute and chronic airway inflammation models. Peli1−/− animals recruit significantly more neutrophils to the airway following NTHi infection which is associated with an increase in the neutrophil chemokine, KC, in bronchoalveolar lavage fluid as well as enhanced clearance of NTHi from the lung. These data suggest that therapeutic inhibition of Pellino-1 may augment immune responses in the airway and enhance bacterial clearance in individuals with COPD.

Antibiotic modulation of mucins in otitis media; should this change our approach to watchful waiting?

BackgroundGel-forming mucins (GFMs) play important roles in otitis media (OM) pathogenesis. Increased mucin expression is activated by pathogens and proinflammatory cytokines. Bacterial biofilms influence inflammation and resolution of OM and may contribute to prolonged mucin production. The influence of specific pathogens on mucin expression and development of chronic OM with effusion (OME) remains an area of significant knowledge deficit. ObjectivesTo assess the relationship between GFM expression, specific pathogens, middle ear mucosal (MEM) changes, biofilm formation, and antibiotic utilization. MethodsMixed gender chinchillas were inoculated with nontypeable Haemophilus influenzae (NTHi) strain 86028NP or Streptococcus pneumoniae (SP) strain TIGR4 via transbulla injection. Antibiotic was administered on day 3–5 post inoculation. GFM expression was measured by quantitative PCR. Biofilm formation was identified and middle ear histologic changes were measured. ResultsSP infection resulted in higher incidence of biofilm and ME effusion compared with NTHi infection. However, NTHi persisted in the ME longer than SP with no substantive bacterial clearance detected on day 10 compared with complete bacterial clearance on day 10 for 50–60% of the SP-infected chinchillas. Both infections increased MEM inflammatory cell infiltration and thickening. NTHi upregulated the Muc5AC, Muc5B and Muc19 expression on day 10 (p = 0.0004, 0.003, and 0.002 respectively). SP-induced GFM upregulations were trended toward significant. In both NTHi and SP infections, the degree of GFM upregulation had a direct relationship to increased MEM hypertrophy, inflammatory cell infiltration and biofilm formation. Antibiotic treatment reduced the incidence of ME effusion and biofilm, limited the MEM changes and reversed the GFM upregulation. In NTHi infection, the rate of returning to baseline level of GFMs in treated chinchillas was quicker than those without treatment. ConclusionsIn an animal model of OM, GFM genes are upregulated in conjunction with MEM hypertrophy and biofilm formation. This upregulation is less robust and more quickly ameliorated to a significant degree in the NTHi infection with appropriate antibiotic therapy. These findings contribute to the understanding of pathogen specific influences on mucin expression during OM pathogenesis and provide new data which may have implications in clinical approach for OM treatment.

Type I interferon induced by DNA of nontypeable Haemophilus influenza modulates inflammatory cytokine profile to promote susceptibility to this bacterium

Type I interferon (IFN) is indispensable for antiviral immunity, but its role in bacterial infections is controversial and not fully described. Nontypeable Haemophilus influenzae (NTHi) is one of the most common bacterial pathogens in patients with chronic obstructive pulmonary disease (COPD). NTHi-DNA activates type I IFN production in macrophages, but the function of type I IFN in host-pathogen interactions, in the context of NTHi infection, is still unclear. Here, we showed that type I IFN, induced by NTHi-DNA, restrained bacterial killing in vitro and promoted COPD development in vivo in response to NTHi. Mice deficient for type I IFN receptor (IFNAR) exhibited improved resistance to NTHi infection. Moreover, similar to exogenous IFN-β, NTHi-DNA-induced type I IFN increased the production of IL-6, IL-1β, IL-12 and CXCL10 via p38 MAPK activation. Our findings demonstrated that NTHi-DNA-induced type I IFN signaling played a negative role in host defense against NTHi infection and identified potential targets for future therapeutic management of COPD.

Uptake of Sialic Acid by Nontypeable Haemophilus influenzae Increases Complement Resistance through Decreasing IgM-Dependent Complement Activation.

Although nontypeable Haemophilus influenzae (NTHi) is a human-specific nasopharyngeal commensal bacterium, it also causes upper respiratory tract infections in children and lower respiratory tract infections in the elderly, resulting in frequent antibiotic use. The transition from symbiotic colonizing bacterium to opportunistic pathogen is not completely understood. Incorporation of sialic acids into lipooligosaccharides is thought to play an important role in bacterial virulence. It has been known for more than 25 years that sialic acids increase resistance to complement-mediated killing; however, the mechanism of action has not been elucidated thus far. Here, we provide evidence that growth of NTHi in the presence of sialic acids Neu5Ac and Neu5Gc decreases complement-mediated killing through abrogating the classical pathway of complement activation by preventing mainly IgM antibody binding to the bacterial surface. Therefore, strategies that interfere with uptake or incorporation of sialic acids into the lipooligosaccharide, such as novel antibiotics and vaccines, might be worth exploring to prevent or treat NTHi infections.

The Interplay Between Immune Response and Bacterial Infection in COPD: Focus Upon Non-typeable Haemophilus influenzae.

Chronic obstructive pulmonary disease (COPD) is a debilitating respiratory disease and one of the leading causes of morbidity and mortality worldwide. It is characterized by persistent respiratory symptoms and airflow limitation due to abnormalities in the lower airway following consistent exposure to noxious particles or gases. Acute exacerbations of COPD (AECOPD) are characterized by increased cough, purulent sputum production, and dyspnea. The AECOPD is mostly associated with infection caused by common cold viruses or bacteria, or co-infections. Chronic and persistent infection by non-typeable Haemophilus influenzae (NTHi), a Gram-negative coccobacillus, contributes to almost half of the infective exacerbations caused by bacteria. This is supported by reports that NTHi is commonly isolated in the sputum from COPD patients during exacerbations. Persistent colonization of NTHi in the lower airway requires a plethora of phenotypic adaptation and virulent mechanisms that are developed over time to cope with changing environmental pressures in the airway such as host immuno-inflammatory response. Chronic inhalation of noxious irritants in COPD causes a changed balance in the lung microbiome, abnormal inflammatory response, and an impaired airway immune system. These conditions significantly provide an opportunistic platform for NTHi colonization and infection resulting in a “vicious circle.” Episodes of large inflammation as the consequences of multiple interactions between airway immune cells and NTHi, accumulatively contribute to COPD exacerbations and may result in worsening of the clinical status. In this review, we discuss in detail the interplay and crosstalk between airway immune residents and NTHi, and their effect in AECOPD for better understanding of NTHi pathogenesis in COPD patients.

Airway infection with Nontypeable Haemophilus influenzae is more rapidly eradicated in vitamin D deficient mice

Chronic obstructive pulmonary disease (COPD), which is characterized by an excessive inflammatory response of the airways, is often complicated by exacerbations. Vitamin D deficiency has been associated with an increased risk for COPD and may predispose COPD patients to a higher exacerbation rate, particularly during smoking. In the current study, we investigated the effect of vitamin D deficiency and cigarette smoke (CS)-exposure on lung inflammation and bacterial clearance after an acute infection with Nontypeable Haemophilus influenzae (NTHi). Vitamin D deficient or sufficient mice were exposed to nose-only CS or ambient air for 6 weeks and oropharyngeally instilled with 106 NTHi. Residual viable NTHi were measured at different time points post-infection. Mechanisms of bacterial clearance (e.g. phagocytosis, pattern recognition receptors, antimicrobial peptides, surfactant proteins and mucin) and lung remodeling (e.g. metalloproteinases, MMP’s) were assessed. Although smoking resulted in reduced phagocytosis capacity of macrophages and neutrophils, bacterial clearance was similar to control mice. By contrast and independent of smoking, bacterial clearance was significantly accelerated in vitamin D deficient mice already from 24 h post-infection (p = 0.0087). This faster and complete eradication was associated with a more rapid resolution of cytokines and neutrophils 72 h post-infection and dominated by an upregulation of cathelicidin-related antimicrobial peptide (CRAMP) mRNA during infection (p = 0.026). However, vitamin D deficiency also resulted in more MMP12 protein in broncho-alveolar lavage and a shift in mRNA expression of MMP12/TIMP1 (p = 0.038) and MMP9/TIMP1 (p = 0.024) ratio towards more protease activity. Overall, vitamin D deficient mice resolved NTHi infection faster with a faster resolution of local lung inflammation, possibly through upregulation of CRAMP. This was associated with a disruption of the protease/anti-protease balance, which may potentially scale towards a higher extracellular matrix breakdown.

Cellular content plays a crucial role in Non-typeable Haemophilus influenzae infection of preinflamed Junbo mouse middle ear.

Non‐typeable Haemophilus influenzae (NTHi) is a major pathogen causing acute otitis media (AOM). The relationship between the cellular content of the middle ear fluid (MEF) during AOM and infection of NTHi is poorly understood. Using the Junbo mouse, a characterised NTHi infection model, we analysed the cellular content of MEF and correlated the data with NTHi titres. The MEF of the Junbo mouse was heterogeneous between ears and was graded from 1 to 5; 1 being highly serous/clear and 5 being heavily viscous/opaque. At seven‐day post‐intranasal inoculation, NTHi was not found in grade‐1 or 2 fluids, and the proportion of MEF that supported NTHi increased with the grade. Analyses by flow cytometry indicated that the cellular content was highest in grade‐4 and 5 fluids, with a greater proportion of necrotic cells and a low‐live cell count. NTHi infection of the middle ear increased the cell count and led to infiltration of immune cells and changes in the cytokine and chemokine levels. Following NTHi inoculation, high‐grade infected MEFs had greater neutrophil infiltration whereas monocyte infiltration was significantly higher in serous noninfected low‐grade fluids. These data underline a role for immune cells, specifically monocytes and neutrophils, and cell necrosis in NTHi infection of the Junbo mouse middle ear.

Epigenetic Regulation Alters Biofilm Architecture and Composition in Multiple Clinical Isolates of Nontypeable Haemophilus influenzae.

Biofilms play a critical role in the colonization, persistence, and pathogenesis of many human pathogens. Multiple mucosa-associated pathogens have evolved a mechanism of rapid adaptation, termed the phasevarion, which facilitates a coordinated regulation of numerous genes throughout the bacterial genome. This epigenetic regulation occurs via phase variation of a DNA methyltransferase, Mod. The phasevarion of nontypeable Haemophilus influenzae (NTHI) significantly affects the severity of experimental otitis media and regulates several disease-related processes. However, the role of the NTHI phasevarion in biofilm formation is unclear. The present study shows that the phasevarions of multiple NTHI clinical isolates regulate in vitro biofilm formation under disease-specific microenvironmental conditions. The impact of phasevarion regulation was greatest under alkaline conditions that mimic those known to occur in the middle ear during disease. Under alkaline conditions, NTHI strains that express the ModA2 methyltransferase formed biofilms with significantly greater biomass and less distinct architecture than those formed by a ModA2-deficient population. The biofilms formed by NTHI strains that express ModA2 also contained less extracellular DNA (eDNA) and significantly less extracellular HU, a DNABII DNA-binding protein critical for biofilm structural stability. Stable biofilm structure is critical for bacterial pathogenesis and persistence in multiple experimental models of disease. These results identify a role for the phasevarion in regulation of biofilm formation, a process integral to the chronic nature of many infections. Understanding the role of the phasevarion in biofilm formation is critical to the development of prevention and treatment strategies for these chronic diseases.IMPORTANCE Upper respiratory tract infections are the number one reason for a child to visit the emergency department, and otitis media (middle ear infection) ranks third overall. Biofilms contribute significantly to the chronic nature of bacterial respiratory tract infections, including otitis media, and make these diseases particularly difficult to treat. Several mucosa-associated human pathogens utilize a mechanism of rapid adaptation termed the phasevarion, or phasevariable regulon, to resist environmental and host immune pressures. In this study, we assessed the role of the phasevarion in regulation of biofilm formation by nontypeable Haemophilus influenzae (NTHI), which causes numerous respiratory tract diseases. We found that the NTHI phasevarion regulates biofilm structure and critical biofilm matrix components under disease-specific conditions. The findings of this work could be significant in the design of improved strategies against NTHI infections, as well as diseases due to other pathogens that utilize a phasevarion.

The Pulmonary Extracellular Matrix Is a Bactericidal Barrier Against Haemophilus influenzae in Chronic Obstructive Pulmonary Disease (COPD): Implications for an in vivo Innate Host Defense Function of Collagen VI.

Non-typeable Haemophilus influenzae (NTHi) is a Gram-negative human commensal commonly residing in the nasopharynx of preschool children. It occasionally causes upper respiratory tract infection such as acute otitis media, but can also spread to the lower respiratory tract causing bronchitis and pneumonia. There is increasing recognition that NTHi has an important role in chronic lower respiratory tract inflammation, particularly in persistent infection in patients suffering from chronic obstructive pulmonary disease (COPD). Here, we set out to assess the innate protective effects of collagen VI, a ubiquitous extracellular matrix component, against NTHi infection in vivo. In vitro, collagen VI rapidly kills bacteria through pore formation and membrane rupture, followed by exudation of intracellular content. This effect is mediated by specific binding of the von Willebrand A (VWA) domains of collagen VI to the NTHi surface adhesins protein E (PE) and Haemophilus autotransporter protein (Hap). Similar observations were made in vivo specimens from murine airways and COPD patient biopsies. NTHi bacteria adhered to collagen fibrils in the airway mucosa and were rapidly killed by membrane destabilization. The significance in host-pathogen interplay of one of these molecules, PE, was highlighted by the observation that it confers partial protection from bacterial killing. Bacteria lacking PE were more prone to antimicrobial activity than NTHi expressing PE. Altogether the data shed new light on the carefully orchestrated molecular events of the host-pathogen interplay in COPD and emphasize the importance of the extracellular matrix as a novel branch of innate host defense.

Recognition of conserved antigens by Th17 cells provides broad protection against pulmonary Haemophilus influenzae infection

Nontypeable Haemophilus influenzae (NTHi) is a major cause of community acquired pneumonia and exacerbation of chronic obstructive pulmonary disease. A current effort in NTHi vaccine development has focused on generating humoral responses and has been greatly impeded by antigenic variation among the numerous circulating NTHi strains. In this study, we showed that pulmonary immunization of mice with killed NTHi generated broad protection against lung infection by different strains. While passive transfer of immune antibodies protected only against the homologous strain, transfer of immune T cells conferred protection against both homologous and heterologous strains. Further characterization revealed a strong Th17 response that was cross-reactive with different NTHi strains. Responding Th17 cells recognized both cytosolic and membrane-associated antigens, while immune antibodies preferentially responded to surface antigens and were highly strain specific. We further identified several conserved proteins recognized by lung Th17 cells during NTHi infection. Two proteins yielding the strongest responses were tested as vaccine candidates by immunization of mice with purified proteins plus an adjuvant. Immunization induced antigen-specific Th17 cells that recognized different strains and, upon adoptive transfer, conferred protection. Furthermore, immunized mice were protected against challenge with not only NTHi strains but also a fully virulent, encapsulated strain. Together, these results show that the immune mechanism of cross-protection against pneumonia involves Th17 cells, which respond to a broad spectrum of antigens, including those that are highly conserved among NTHi strains. These mechanistic insights suggest that inclusion of Th17 antigens in subunit vaccines offers the advantage of inducing broad protection and complements the current antibody-based approaches.

Toll-like receptor 4 plays an important role to enhance bacterial clearance from the nose in synergy with triggering receptor expressed on myeloid cells (TREM)-1 expression on polymorphonuclear neutrophils

ObjectiveAcute rhinosinusitis (ARS) is among the most common infectious diseases. Neutrophils play a major role in innate host defenses against pathogenic microorganisms such as fungi and bacteria. Recently, in neutrophils, ligation of the triggering receptor expressed on myeloid cells (TREM)-1 was found to activate the full spectrum of neutrophil effector mechanisms, including the release of inflammatory mediators, degranulation, phagocytosis, and oxidative burst in synergy with Toll-like receptors (TLRs). In this study, we investigated the effect of TREM-1 on the functions of neutrophils in relation to TLR4 in a nasal and nasopharyngeal inflammation mouse model via nontypeable Haemophilus influenzae (NTHi) intranasal inoculation. MethodsWe used C3H/HeJ (TLR4-deficient) mice, which arose spontaneously and have non-functional TLR4 protein, and normal wild-type (WT) C3H/HeN mice. Mice were inoculated intranasally with NTHi (107 cfu/mouse) to investigate the effects of TLR4 on the function of Neutrophils. We examined the kinetics of bacterial clearance and inflammatory cell infiltration in nasal washes at 6, 12, 24, and 72 h after inoculation. The expression of TREM-1 on neutrophils, and TREM-1 mRNA expression in neutrophils in the nasal washes were examined by flow cytometric analysis and RT-PCR. ResultsBacterial counts of NTHi from nasal washes were significantly lower in WT mice than in TLR4-mutant mice after inoculation. The numbers of inflammatory cells in nasal washes were significantly higher in WT mice at 6 h, 12 h, and 24 h after inoculation than in TLR4-deficient mice. The expression of TREM-1 protein on neutrophils and the mRNA levels were greater in WT mice than in TLR4-mutant mice. The concentrations of soluble TREM-1 in WT nasal washes were also significantly higher than in those of TLR4-deficient mice. ConclusionTREM-1 may play an important role together with TLR4 in the nasopharyngeal clearance of NTHi by neutrophils. Further studies will need to clarify the innate immune responses of neutrophils via TLR4 to prevent NTHi infection.

Secondhand Smoke Induces Inflammation and Impairs Immunity to Respiratory Infections.

Despite advocacy to reduce smoking-related diseases, >1 billion people worldwide continue to smoke. Smoking is immunosuppressive and an important etiological factor in the development of several human disorders including respiratory diseases like chronic obstructive pulmonary disease. However, there is a critical gap in the knowledge of the role of secondhand smoke (SHS) in inflammation and immunity. We therefore studied the influence of SHS on pulmonary inflammation and immune responses to respiratory infection by nontypeable Haemophilus influenzae (NTHI) recurrently found in chronic obstructive pulmonary disease patients. Chronic SHS-exposed mice were chronically infected with NTHI and pulmonary inflammation was evaluated by histology. Immune cell numbers and cytokines were measured by flow cytometry and ELISA, respectively. Chronic SHS exposure impaired NTHI P6 Ag-specific B and T cell responses following chronic NTHI infection as measured by ELISPOT assays, reduced the production of Abs in serum and bronchoalveolar lavage, and enhanced albumin leak into the bronchoalveolar lavage as determined by ELISA. Histopathological examination of lungs revealed lymphocytic accumulation surrounding airways and bronchovasculature following chronic SHS exposure and chronic infection. Chronic SHS exposure enhanced the levels of inflammatory cytokines IL-17A, IL-6, IL-1β, and TNF-α in the lungs, and impaired the generation of adaptive immunity following either chronic infection or P6 vaccination. Chronic SHS exposure diminished bacterial clearance from the lungs after acute NTHI challenge, whereas P6 vaccination improved clearance equivalent to the level seen in air-exposed, non-vaccinated mice. Our study provides unequivocal evidence that SHS exposure has long-term detrimental effects on the pulmonary inflammatory microenvironment and immunity to infection and vaccination.

Nontypeable Haemophilus influenzae DNA stimulates type I interferon expression via STING signaling pathway

Nontypeable Haemophilus influenzae (NTHI) is one of the leading causes of acute exacerbations of COPD (AECOPD). Although the immunoregulation function of NTHI outer member protein and endotoxin were confirmed, the role of NTHI DNA in activating immune responses remains to be elucidated. In this study, we found expression of IFN-β and IFN stimulated gene CXCL10 in host cells was forcefully elevated after treating with NTHI and NTHI DNA. Interestingly, we tested increased level of STING in NTHI infected mice lung. Meanwhile, STING expression in lung of mimic COPD murine model was higher than healthy mice after NTHI infection. Importantly, knockout of STING or overexpression of STING, TBK1 and IRF3 respectively impaired or enhanced IFN-β and CXCL10 expression during treating with NTHI and NTHI DNA. NTHI and NTHI DNA-induced I-IFN response appeared to be mediated by cGAS. Collectively, we suggested that NTHI DNA as a PAMP triggered I-IFN response, which was STING/TBK1/IRF3 dependent. SummaryNTHI is the leading cause of acute exacerbations of COPD (AECOPD). Since AECOPD is an immune event, it is meaningful to elucidate the mechanism under NTHI induced immune response. It has been revealed that lipooligosaccharides and protein of NTHI could induce host immune response, but the function of NTHI nuclide acid during infection is unclear. In this research, we demonstrate NTHI DNA is a trigger for I-IFN expression, and the STING/TBK1/IRF3 pathway plays an integral role in sensing NTHI DNA to induce I-IFN expression. Moreover, by long-term intrabronchial infection of LPS, we constructed a mimic COPD murine model, in which the STING expression in lung tissues were higher than healthy mice after NTHI infection, which led us to surmise that NTHI cause AECOPD by inducing I-IFN production via STING signal pathway.