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Article Reference Up-regulation of interleukin-8 by novel small cytoplasmic molecules of nontypeable Haemophilus influenzae via p38 and extracellular signal-regulated kinase pathways.
Nontypeable Haemophilus influenzae (NTHI) is an important etiological agent of otitis media (OM) and of exacerbated chronic obstructive pulmonary diseases (COPD). Inflammation is a hallmark of both diseases. Interleukin-8 (IL-8), one of the important inflammatory mediators, is induced by NTHI and may play a significant role in the pathogenesis of these diseases. Our studies demonstrated that a soluble cytoplasmic fraction (SCF) from NTHI induced much greater IL-8 expression by human epithelial cells than did NTHI lipooligosaccharides and envelope proteins. The IL-8-inducing activity was associated with molecules of < or =3 kDa from SCF and was peptidase and lipase sensitive, suggesting that small lipopeptides are responsible for the strong IL-8 induction. Moreover, multiple intracellular signaling pathways were activated in response to cytoplasmic molecules. The results indicated that the p38 mitogen-activated protein kinase (MAPK) and Src-dependent Raf-1-Mek1/2-extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) pathways are required for NTHI-induced IL-8 production. In contrast, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway did not affect IL-8 expression, although this pathway was concomitantly activated upon exposure to NTHI SCF. The PI3K-Akt pathway was also directly activated by IL-8 and significantly inhibited by an antagonist of IL-8 receptors during NTHI stimulation. These results indicated that the PI3K-Akt pathway is activated in response to IL-8 that is induced by NTHI and may lead to other important epithelial cell responses. This work provides insight into essential molecular and cellular events that may impact on the pathogenesis of OM and COPD and identifies rational targets for anti-inflammatory intervention.
Article Reference The role of nuclear factor-kappa B in interleukin-8 expression by human adenoidal fibroblasts.
The production of cytokines by adenoids is known to be associated with inflammation of nasopharynx and the pathogenesis of otitis media with effusion. However, the role of adenoids in producing inflammatory cytokines such as interleukin-8 (IL-8) is not yet clear. In the present study, expression of IL-8 in adenoidal fibroblasts was investigated at the level of transcription factors. Further, the effects of clarithromycin, a 14-member ring macrolide, on IL-8 gene expression and nuclear factor-kappa B (NF-kappa B) activation in adenoidal fibroblasts were evaluated.
Article Reference S-carboxymethylcysteine inhibits the attachment of Streptococcus pneumoniae to human pharyngeal epithelial cells.
Streptococcus pneumoniae causes respiratory and other invasive infections. Increased resistance of this bacterium to antibiotics necessitates new approaches to the treatment of infections. Attachment of bacteria to human pharyngeal epithelial cells is the initial step in the pathogenesis of infection and S-carboxymethylcysteine (S-CMC) can modulate the attachment of Moraxella catarrhalis and nontypable Haemophilus influenzae to epithelial cells. Unlike these two, S. pneumoniae is gram-positive and has a well-defined capsule. Here we examined the effects of S-CMC on the attachment and detachment of S. pneumoniae to human pharyngeal epithelial cells in vitro. Treatment of these cells with S-CMC significantly reduced the number of attached S. pneumoniae. S-CMC also resulted in a significant increase in the detachment of already attached S. pneumoniae to epithelial cells. In addition, treatment of S. pneumoniae with S-CMC significantly reduced their ability to attach to epithelial cells, but not the number of viable bacteria. Our study shows that S-CMC modulates the attachment of S. pneumoniae to human pharyngeal epithelial cells by acting both on cells and bacteria.
Article Reference Respiratory infections caused by non-typeable Haemophilus influenzae.
This review will consider recent developments in the clinical aspects of infections due to non-typeable Haemophilus influenzae. In addition, newer developments in the areas of mechanisms of pathogenesis, host pathogen interaction, immune responses and efforts toward vaccine development will be reviewed briefly.
Article Reference The Haemophilus influenzae HMW1 adhesin is glycosylated in a process that requires HMW1C and phosphoglucomutase, an enzyme involved in lipooligosaccharide biosynthesis.
Non-typeable Haemophilus influenzae is a common respiratory pathogen and an important cause of morbidity in humans. The non-typeable H. influenzae HMW1 and HMW2 adhesins are related proteins that mediate attachment to human epithelial cells, an essential step in the pathogenesis of disease. Secretion of these adhesins requires accessory proteins called HMW1B/HMW2B and HMW1C/HMW2C. In the present study, we investigated the specific function of HMW1C. Examination of mutant constructs demonstrated that HMW1C influences both the size and the secretion of HMW1. Co-immunoprecipitation and yeast two-hybrid assays revealed that HMW1C interacts with HMW1 and forms a complex in the cytoplasm. Additional experiments and homology analysis established that HMW1C is required for glycosylation of HMW1 and may have glycotransferase activity. The glycan structure contains galactose, glucose and mannose and appears to be generated in part by phosphoglucomutase, an enzyme important for lipooligosaccharide biosynthesis. In the absence of glycosylation, HMW1 is partially degraded and is efficiently released from the surface of the organism, resulting in reduced adherence. Based on these results, we conclude that glycosylation is a prerequisite for HMW1 stability. In addition, glycosylation appears to be essential for optimal HMW1 tethering to the bacterial surface, which in turn is required for HMW1-mediated adherence, thus revealing a novel mechanism by which glycosylation influences cell-cell interactions.
Article Reference Exploitation of host epithelial signaling networks by respiratory bacterial pathogens.
Although tremendous effort has been put towards identifying the surface molecules of nontypeable Haemophilus influenzae (NTHi) for vaccine development over the past decades, it is only recently that we have begun to appreciate the intricate host epithelial signaling networks activated by NTHi, an important human pathogen causing respiratory infections. From what has been reported, it is evident that NTHi activates multiple signaling pathways in host epithelial cells that, in turn, inadvertently contribute to the pathogenesis. Among those signaling pathways, activation of NF-kappaB leads to up-regulation of IL-1beta, IL-8 and TNF-alpha, mucin MUC2 and Toll-like receptor 2 (TLR2), whereas activation of p38 MAP kinase mediates not only up-regulation of inflammatory mediators and mucin MUC5AC but also down-regulation of TLR2. Interestingly, NTHi-induced activation of the PI3K-Akt pathway, however, leads to inhibition of p38 mitogen-activated protein (MAP) kinase. Moreover, the TGF-beta-Smad signaling pathway cooperates with NF-kappaB to mediate up-regulation of mucin MUC2. Finally, glucocorticoids synergistically enhance NTHi-induced TLR2 expression via specific up-regulation of the MAP kinase phosphatase-1 that, in turn, leads to inactivation of p38 MAP kinase, the negative regulator for TLR2 expression. These studies may bring new insights into the molecular pathogenesis of NTHi-induced infections and open up novel therapeutic targets for these diseases.
Article Reference Bacterial otitis media: current vaccine development strategies.
Otitis media is the most common reason for children less than 5 years of age to visit a medical practitioner. Whilst the disease rarely results in death, there is significant associated morbidity. The most common complication is loss of hearing at a critical stage of the development of speech, language and cognitive abilities in children. The cause and pathogenesis of otitis media is multifactorial. Among the contributing factors, the single most important are viral and bacterial infections. Infection with respiratory syncytial virus, influenza viruses, para-influenza viruses, enteroviruses and adenovirus are most commonly associated with acute and chronic otitis media. Streptococcus pneumoniae, non-typeable Haemophilus influenzae and Moraxella catarrhalis are the most commonly isolated bacteria from the middle ears of children with otitis media. Treatment of otitis media has largely relied on the administration of antimicrobials and surgical intervention. However, attention has recently focused on the development of a vaccine. For a vaccine to be effective against bacterial otitis media, it must, at the very least, contain antigens that induce a protective immune response in the middle ear against the three most common infecting bacteria. Whilst over the past decade there has been significant progress in the development of vaccines against invasive S. pneumoniae disease, these vaccines are less efficacious for otitis media. The search for candidate vaccine antigens for non-typeable H. influenzae are well advanced whilst less progress has been made for M. catarrhalis. No human studies have been conducted for non-typeable H. influenzae or M. catarrhalis and the concept of a tribacterial vaccine remains to be tested in animal models. Only when vaccine antigens are determined and an understanding of the immune responses induced in the middle ear by infection and immunization is gained will the formulation of a tribacterial vaccine against otitis media be possible.
Article Reference Adaptive immunity to nontypeable Haemophilus influenzae.
Nontypeable Haemophilus influenzae (NTHi) colonizes the upper respiratory tract of most healthy people and is also a major cause of infection in chronic obstructive lung disease. The immune response to this bacterium has not been well characterized. We tested the hypothesis that recurrent airway infection with NTHi may be associated with nonclearing adaptive immunity. Study subjects were healthy control subjects and patients with idiopathic bronchiectasis who had severe chronic infection with H. influenzae. We established that all subjects in both groups had detectable antibody to NTHi, suggesting that most normal people have developed an adaptive immune response. To characterize the nature of the immune response, we measured antigen-specific production of T helper cell cytokines and CD40 ligand by flow cytometry and immunoglobulin subclass levels in peripheral blood. We found that normal control subjects made Th1 response to NTHi with distinct CD40 ligand production. In contrast, subjects with bronchiectasis had predominant production of Th2 cytokines, decreased expression of CD40 ligand, and different immunoglobulin G subclass production. Therefore, chronic infection with NTHi in bronchiectasis is associated with a change in adaptive immunity that may be important in the pathogenesis of bronchial infection.
Article Reference The pathogenesis of disease due to nontypeable Haemophilus influenzae.
To summarize, the pathogenesis of disease due to nontypeable H. influenzae involves multiple steps and the interplay of a number of bacterial and host factors, as shown in Fig. 1. Following entry into the upper respiratory tract, bacteria encounter the mucociliary escalator. The P2 and P5 outer-membrane proteins and probably other factors promote bacterial binding to mucus, and elaboration of LOS causes damage to ciliated cells and impairs mucociliary function. Subsequently, several adhesins, including HMW1 and HMW2, pili, Hia, Hap, and others, mediate direct adherence to nonciliated epithelial cells. Cleavage of IgA1, invasion into cells and the subepithelial space, and phase and antigenic variation facilitate evasion of local immune mechanisms. Binding and uptake of iron and heme allow organisms to persist on the respiratory mucosa despite the relative scarcity of these nutrients. In the setting of a viral infection, allergic disease, or exposure to cigarette smoke, bacteria spread from the nasopharynx to other sites within the respiratory tract and produce symptomatic disease.
Article Reference Transforming growth factor-beta -Smad signaling pathway cooperates with NF-kappa B to mediate nontypeable Haemophilus influenzae-induced MUC2 mucin transcription.
Transforming growth factor-beta (TGF-beta) and related factors are multifunctional cytokines that regulate diverse cellular processes, including proliferation, differentiation, apoptosis, and immune response. The involvement of TGF-beta receptor-mediated signaling in bacteria-induced up-regulation of mucin, a primary innate defensive response for mammalian airways, however, still remains unknown. Here, we report that the bacterium nontypeable Haemophilus influenzae (NTHi), an important human respiratory pathogen, utilizes the TGF-beta-Smad signaling pathway together with the TLR2-MyD88-TAK1-NIK-IKKbeta/gamma-IkappaBalpha pathway to mediate NF-kappaB-dependent MUC2 mucin transcription. The NTHi-induced TGF-beta receptor Type II phosphorylation occurred at as early as 5 min. Pretreatment of NTHi with TGF-beta neutralization antibody reduced up-regulation of MUC2 transcription. Moreover, functional cooperation of NF-kappaB p65/p50 with Smad3/4 appears to positively mediate NF-kappaB-dependent MUC2 transcription. These data are the first to demonstrate the involvement of TGF-beta receptor-mediated signaling in bacteria-induced up-regulation of mucin transcription, bring insights into the novel role of TGF-beta signaling in bacterial pathogenesis, and may lead to new therapeutic intervention of NTHi infections.
Article Reference Identification of the lipooligosaccharide biosynthesis gene lic2B as a putative virulence factor in strains of nontypeable Haemophilus influenzae that cause otitis media.
Nontypeable (NT) strains of Haemophilus influenzae are an important cause of acute otitis media (OM). The pathogenic process by which NT H. influenzae strains cause OM is poorly understood. In order to identify specific virulence factors important for OM pathogenesis, genomic subtraction of the NT H. influenzae middle ear isolate G622 against H. influenzae strain Rd was conducted and the resulting subtraction products were used to screen a panel of H. influenzae isolates. Subtraction identified 36 PCR fragments unique to strain G622, which were used in a preliminary screen of 48 middle ear isolates and 46 nasopharyngeal and throat isolates to identify genes found more frequently among middle ear isolates. These experiments identified a PCR fragment with high homology to the lipooligosaccharide biosynthesis gene lic2B (originally identified in an H. influenzae type b strain) among 52% of the middle ear isolates and 9% of nasopharyngeal and throat isolates. The lic2B gene cloned from NT H. influenzae strain G622 was 99% identical at the amino acid level to that of the H. influenzae type b strain RM7004. The lic2B gene was used to screen a larger panel of H. influenzae isolates including the original 48 middle ear isolates, 40 invasive type b isolates, 90 NT H. influenzae throat isolates from children attending day care, and 32 NT H. influenzae nasopharyngeal clinical isolates. The lic2B gene was found 3.7 times more frequently among middle ear isolates than in throat isolates from children attending day care. These data suggest that a specific NT H. influenzae gene is associated with OM.
Article Reference Vaccine prevention of acute otitis media.
The incidence of acute otitis media (AOM) in infants and young children has increased dramatically in recent years in the United States. AOM often follows upper respiratory tract infections due to pathogens such as respiratory syncytial virus (RSV), influenza virus, and parainfluenza virus (PIV). These viruses cause eustachian tube dysfunction that is critical to the pathogenesis of AOM. Vaccines against these viruses would likely reduce the incidence of AOM. In three previous studies, influenza virus vaccines reduced the incidence of AOM by 30% to 36%. Vaccines to prevent infections with RSV and PIV type 3 are undergoing clinical testing at this time. Streptococcus pneumoniae, nontypeable Haemophilus influenzae (NTHi), and Moraxella catarrhalis are the three most common AOM pathogens. Heptavalent pneumococcal conjugate vaccine is effective in preventing invasive disease and AOM caused by serotypes contained in the vaccine. Vaccine candidates for NTHi and M. catarrhalis are under development.
Article Reference Nontypeable Haemophilus influenzae in the lower respiratory tract of patients with chronic bronchitis.
The frequency of colonization and intracellular localization of nontypeable Haemophilus influenzae (NTHi) in the lower respiratory tract was determined in healthy adults and in clinically stable and acutely ill chronic bronchitis (CB) patients. NTHi was recovered from bronchial wash or bronchial brush specimens in 6 of 23 (26%) stable CB patients and in 1 of 15 (7%) CB patients with a respiratory exacerbation. No NTHi (0 of 26) was recovered from lower tract specimens of healthy adults undergoing anesthesia for elective surgery. Molecular typing of NTHi strains revealed that five of nine patients with stable CB had different strains in upper respiratory tract and bronchial wash/brush specimens collected simultaneously. Four stable patients with CB had different strains recovered on repeat bronchoscopy. These results demonstrate the frequent colonization of the lower airways of stable CB patients with multiple strains of NTHi. Bronchial biopsies also were examined for intracellular NTHi by in situ hybridization and immunofluorescence microscopy. Intracellular NTHi were found in 0 of 7 healthy adults, 8 of 24 patients with clinically stable CB, and 13 of 15 acutely ill CB patients. This observation suggests a role for intracellular infection by NTHi in the pathogenesis of exacerbations of CB.
Article Reference Novel cytoplasmic proteins of nontypeable Haemophilus influenzae up-regulate human MUC5AC mucin transcription via a positive p38 mitogen-activated protein kinase pathway and a negative phosphoinositide 3-kinase-Akt pathway.
Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that causes chronic otitis media with effusion (COME) in children and exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Mucin overproduction, a hallmark of both diseases, has been shown to directly cause conductive hearing loss in COME and airway obstruction in COPD. The molecular mechanisms underlying mucin overproduction in NTHi infections still remain unclear. Here, we show that NTHi strongly up-regulates MUC5AC mucin transcription only after bacterial cell disruption. Maximal up-regulation is induced by heat-stable bacterial cytoplasmic proteins, whereas NTHi surface membrane proteins induce only moderate MUC5AC transcription. These results demonstrate an important role for cytoplasmic molecules from lysed bacteria in the pathogenesis of NTHi infections, and may well explain why many patients still have persistent symptoms such as middle ear effusion in COME after intensive antibiotic treatment. Furthermore, our results indicate that activation of p38 mitogen-activated protein kinase is required for NTHi-induced MUC5AC transcription, whereas activation of phosphoinositide 3-kinase-Akt pathway leads to down-regulation of NTHi-induced MUC5AC transcription via a negative cross-talk with p38 mitogen-activated protein kinase pathway. These studies may bring new insights into molecular pathogenesis of NTHi infections and lead to novel therapeutic intervention for COME and COPD.
Article Reference A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae: a survey of 24 non-typeable H. influenzae strains.
In view of the importance of 5-N-acetyl neuraminic acid in bacterial pathogenesis, a sensitive, reproducible and reliable method for the determination of 5-N-acetyl neuraminic acid levels in lipopolysaccharide (LPS) is described and applied to 24 different non-typeable Haemophilus influenzae (NTHi) strains. The method involves analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of terminal 5-N-acetyl neuraminic acid residues released by neuraminidase treatment of O-deacylated LPS. The procedure is relatively fast and the instrumental effort is moderate. The results of the procedure were compared with data obtained by 1H NMR and electrospray ionisation-mass spectrometry (ESI-MS). The analysis of LPS from 24 NTHi strains showed that 5-N-acetyl neuraminic acid was found to be a common constituent of LPS in NTHi. Only one strain (NTHi 432) did not show any sialylation. Molar ratios (LPS/5-N-acetyl neuraminic acid) ranged between 5/1 and 500/1. Several strains in which no 5-N-acetyl neuraminic acid could be determined by other methods including 1H NMR and ESI-MS were shown to contain 5-N-acetyl neuraminic acid by this HPAEC-PAD procedure. The method was applied to determine levels of terminal 5-N-acetyl neuraminic acid in LPS from NTHi strains grown under different conditions and mutant strains containing inactive LPS biosynthetic genes.
Article Reference Protein D expression promotes the adherence and internalization of non-typeable Haemophilus influenzae into human monocytic cells.
Protein D, having a glycerol-3-phosphodiester phosphodiesterase activity, is found at the surface of all Haemophilus influenzae strains and is a possible virulence factor. In the present study, the involvement of protein D in the entry of NTHi into human monocytic cells is reported. Primary monocytes and the monocytic cell lines U-937 and THP-1 were infected with NTHi strain 772 and the mutant 772 Delta hpd 1 (lacking the gene for protein D). NTHi 772 adhered to and entered monocytic cells up to four-fold more efficiently compared to 772 Delta hpd 1. When an Escherichia coli transformant expressing protein D was incubated with monocytic cells, the number of intracellular bacteria increased 1.6-fold compared to protein D-deficient controls. Any correlation between internalization and phosphorylcholine expression was not detected. In conclusion, our data suggest that surface-expressed protein D promotes the adherence of NTHi to human monocytes leading to a higher number of internalized bacteria.
Article Reference Activation of NF-kappa B by nontypeable Hemophilus influenzae is mediated by toll-like receptor 2-TAK1-dependent NIK-IKK alpha /beta-I kappa B alpha and MKK3/6-p38 MAP kinase signaling pathways in epithelial cells.
Nontypeable Hemophilus influenzae (NTHi) is an important human pathogen in both children and adults. In children, it causes otitis media, the most common childhood infection and the leading cause of conductive hearing loss in the United States. In adults, it causes lower respiratory tract infections in the setting of chronic obstructive pulmonary disease, the fourth leading cause of death in the United States. The molecular mechanisms underlying the pathogenesis of NTHi-induced infections remain undefined, but they may involve activation of NF-kappa B, a transcriptional activator of multiple host defense genes involved in immune and inflammatory responses. Here, we show that NTHi strongly activates NF-kappa B in human epithelial cells via two distinct signaling pathways, NF-kappa B translocation-dependent and -independent pathways. The NF-kappa B translocation-dependent pathway involves activation of NF-kappa B inducing kinase (NIK)&ndash;IKK alpha/beta complex leading to I kappa B alpha phosphorylation and degradation, whereas the NF-kappa B translocation-independent pathway involves activation of MKK3/6&ndash;p38 mitogen-activated protein (MAP) kinase pathway. Bifurcation of NTHi-induced NIK-IKK alpha/beta-I kappa B alpha and MKK3/6&ndash;p38 MAP kinase pathways may occur at transforming growth factor-beta activated kinase 1 (TAK1). Furthermore, we show that toll-like receptor 2 (TLR2) is required for NTHi-induced NF-kappa B activation. In addition, several key inflammatory mediators including IL-1 beta, IL-8, and tumor necrosis factor-alpha are up-regulated by NTHi. Finally, P6, a 16-kDa lipoprotein highly conserved in the outer membrane of all NTHi and H. influenzae type b strains, appears to also activate NF-kappa B via similar signaling pathways. Taken together, our results demonstrate that NTHi activates NF-kappa B via TLR2-TAK1-dependent NIK&ndash;IKK alpha/beta-I kappa B alpha and MKK3/6&ndash;p38 MAP kinase signaling pathways. These studies may bring new insights into molecular pathogenesis of NTHi-induced infections and open up new therapeutic targets for these diseases.
Article Reference Role of interleukin-1beta in a murine model of otitis media with effusion.
To clarify the role of interleukin-1beta (IL-1beta) in the pathogenesis of otitis media with effusion (OME), we developed and investigated a murine model of this disease. Specific pathogen-free male BALB/c mice received intratympanic injections of 20 microg of endotoxin derived from nontypeable Haemophilus influenzae. Three days after injection, middle ear effusions were observed through the eardrum. Similar pathological changes were observed after inoculation with 100 ng of recombinant IL-1beta. Anti-IL-1 receptor antibodies inhibited the pathological changes induced by the endotoxin. In situ hybridization showed expression of IL-1beta messenger RNA in the epithelium of the middle ear mucosa. These results suggest that IL-1beta might be associated with endotoxin-induced inflammation in the middle ear and might play an important role in the induction of otitis media with effusion.
Article Reference Goblet cell density in acute otitis media caused by Moraxella catarrhalis.
Secretory otitis media is associated with a highly increased goblet cell density, confirming the secretory pathogenesis of this disease. Previous studies have shown that the middle ear goblet cell density, and thus the secretory capacity, are massively increased during experimental acute otitis media and at least 6 months thereafter, conceivably predisposing to the subsequent development of secretory otitis media. These studies used middle ear inoculation of either Streptococcus pneumoniae, nontypeable Haemophilus influenzae, or H. influenzae type b. The present study aimed at determining the goblet cell density during and after acute otitis media caused by Moraxella catarrhalis to clarify whether this bacterium induces an equivalently enhanced secretory capacity.
Article Reference Bacterial infection in chronic obstructive pulmonary disease in 2000: a state-of-the-art review.
Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States. The precise role of bacterial infection in the course and pathogenesis of COPD has been a source of controversy for decades. Chronic bacterial colonization of the lower airways contributes to airway inflammation; more research is needed to test the hypothesis that this bacterial colonization accelerates the progressive decline in lung function seen in COPD (the vicious circle hypothesis). The course of COPD is characterized by intermittent exacerbations of the disease. Studies of samples obtained by bronchoscopy with the protected specimen brush, analysis of the human immune response with appropriate immunoassays, and antibiotic trials reveal that approximately half of exacerbations are caused by bacteria. Nontypeable Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae are the most common causes of exacerbations, while Chlamydia pneumoniae causes a small proportion. The role of Haemophilus parainfluenzae and gram-negative bacilli remains to be established. Recent progress in studies of the molecular mechanisms of pathogenesis of infection in the human respiratory tract and in vaccine development guided by such studies promises to lead to novel ways to treat and prevent bacterial infections in COPD.