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Mucin overproduction is a hallmark of otitis media (OM). Streptococcus pneumoniae is one of the most common bacterial pathogens causing OM. Mucin MUC5AC plays an important role in mucociliary clearance of bacterial pathogens. However, if uncontrolled, excessive mucus contributes significantly to conductive hearing loss. Currently, there is a lack of effective therapeutic agents that suppress mucus overproduction. In this study, we show that a currently existing antistroke drug, vinpocetine, a derivative of the alkaloid vincamine, inhibited S. pneumoniae-induced mucin MUC5AC upregulation in cultured middle ear epithelial cells and in the middle ear of mice. Moreover, vinpocetine inhibited MUC5AC upregulation by inhibiting the MAPK ERK pathway in an MKP-1-dependent manner. Importantly, ototopical administration of vinpocetine postinfection inhibited MUC5AC expression and middle ear inflammation induced by S. pneumoniae and reduced hearing loss and pneumococcal loads in a well-established mouse model of OM. Thus, these studies identified vinpocetine as a potential therapeutic agent for inhibiting mucus production in the pathogenesis of OM., (Copyright © 2015 by The American Association of Immunologists, Inc.)

There is considerable evidence that influenza A virus (IAV) promotes adherence, colonization, and superinfection by S. pneumoniae (Spn) and contributes to the pathogenesis of otitis media (OM). The complement system is a critical innate immune defense against both pathogens. To assess the role of the complement system in the host defense and the pathogenesis of acute pneumococcal OM following IAV infection, we employed a well-established transtympanically-induced mouse model of acute pneumococcal OM. We found that antecedent IAV infection enhanced the severity of acute pneumococcal OM. Mice deficient in complement C1qa (C1qa-/-) or factor B (Bf -/-) exhibited delayed viral and bacterial clearance from the middle ear and developed significant mucosal damage in the eustachian tube and middle ear. This indicates that both the classical and alternative complement pathways are critical for the oto-immune defense against acute pneumococcal OM following influenza infection. We also found that Spn increased complement activation following IAV infection. This was characterized by sustained increased levels of anaphylatoxins C3a and C5a in serum and middle ear lavage samples. In contrast, mice deficient in the complement C5a receptor (C5aR) demonstrated enhanced bacterial clearance and reduced severity of OM. Our data support the concept that C5a-C5aR interactions play a significant role in the pathogenesis of acute pneumococcal OM following IAV infection. It is possible that targeting the C5a-C5aR axis might prove useful in attenuating acute pneumococcal OM in patients with influenza infection.

Streptococcus pneumoniae (Spn) is the predominant causative organism of acute otitis media in children. To better understand the genes that are regulated at the onset of AOM caused by Spn infection in the middle ear, the transcriptome profile of peripheral blood mononuclear cells isolated from children prior to and during an AOM event was evaluated by microarray. We found that 1903 (6.2%) of 29,187 genes were differentially regulated greater than 2-fold at the onset of AOM compared to the pre-infection stage of the same children. The ontology of differentially regulated genes was dominated by those involved with the immune response. At onset of infection, genes associated with bacterial defenses were significantly up-regulated, including beta-defensin123, S100 protein A12, Toll-like receptor 5, IL-10, and those involved in the classical and alternative complement pathways. Genes associated with inhibition of bacterial entry through clathrin-dependent endocytosis were also up-regulated. In contrast, genes associated with cell-mediated immune responses were broadly down-regulated. The results provide the first human transcriptome data identifying genes differentially regulated at the onset of AOM in children., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

To define the roles of specific complement activation pathways in host defense against Streptococcus pneumoniae in acute otitis media (AOM), we investigated the susceptibility to AOM in mice deficient in complement factor B and C2 (Bf/C2(-/)(-)), C1qa (C1qa(-/)(-)), and factor B (Bf(-)(/)(-)). Bacterial titers of both S. pneumoniae serotype 6A and 14 in the middle ear lavage fluid samples from Bf/C2(-/)(-), Bf(-)(/)(-), and C1qa(-/)(-) mice were significantly higher than in samples from wild-type mice 24 h after transtympanical infection (P < 0.05) and remained persistently higher in samples from Bf/C2(-/)(-) mice than in samples from wild-type mice. Bacteremia occurred in Bf/C2(-/)(-), Bf(-)(/)(-), and C1qa(-/)(-) mice infected with both strains, but not in wild-type mice. Recruitment of inflammatory cells was paralleled by enhanced production of inflammatory mediators in the middle ear lavage samples from Bf/C2(-/)(-) mice. C3b deposition on both strains was greatest for sera obtained from wild-type mice, followed by C1qa(-)(/)(-) and Bf(-)(/)(-) mice, and least for Bf/C2(-)(/)(-) mice. Opsonophagocytosis and whole-blood killing capacity of both strains were significantly decreased in the presence of sera or whole blood from complement-deficient mice compared to wild-type mice. These findings indicate that both the classical and alternative complement pathways are critical for middle ear immune defense against S. pneumoniae. The reduced capacity of complement-mediated opsonization and phagocytosis in the complement-deficient mice appears to be responsible for the impaired clearance of S. pneumoniae from the middle ear and dissemination to the bloodstream during AOM.

Background: Lysozyme is an antimicrobial innate immune molecule degrading peptidoglycan of the bacterial cell wall. Lysozyme shows the ubiquitous expression in wide varieties of species and tissues including the tubotympanum of mammals. We aim to investigate the effects of lysozyme depletion on pneumococcal clearance from the middle ear cavity., Methods: Immunohistochemistry was performed to localize lysozyme in the Eustachian tube. Lysozyme expression was compared between the wild type and the lysozyme M-/- mice using real time quantitative RT-PCR and western blotting. Muramidase activity and bactericidal activity of lysozyme was measured using a lysoplate radial diffusion assay and a liquid broth assay, respectively. To determine if depletion of lysozyme M increases a susceptibility to pneumococal otitis media, 50 CFU of S. pneumoniae 6B were transtympanically inoculated to the middle ear and viable bacteria were counted at day 3 and 7 with clinical grading of middle ear inflammation., Results: Immunolabeling revealed that localization of lysozyme M and lysozyme P is specific to some/particular cell types of the Eustachian tube. Lysozyme P of lysozyme M-/- mice was mainly expressed in the submucosal gland but not in the tubal epithelium. Although lysozyme M-/- mice showed compensatory up-regulation of lysozyme P, lysozyme M depletion resulted in a decrease in both muramidase and antimicrobial activities. Deficiency in lysozyme M led to an increased susceptibility to middle ear infection with S. pneumoniae 6B and resulted in severe middle ear inflammation, compared to wild type mice., Conclusion: The results suggest that lysozyme M plays an important role in protecting the middle ear from invading pathogens, particularly in the early phase. We suggest a possibility of the exogenous lysozyme as an adjuvant therapeutic agent for otitis media, but further studies are necessary.

Objective: To define the gene expression patterns during the early phases of a bacterial middle ear infection in the rat model., Method: Using cDNA gene array technology, we profiled the mRNA expression of 1176 genes in a rat model of acute otitis media. We identified changes in gene expression two-fold or greater 12 and 48 h after bilateral ME inoculation with either tryptic soy broth (TSB) or Streptococcus pneumoniae in TSB., Results: Transcripts of cytokines and cell adhesion molecules were up-regulated by 12 h, but returned to placebo transcription levels by 48 h. Three of six stress-response genes, including inducible nitric oxide synthase, GADD45 and heat shock protein 27 (HSP27) were up-regulated by 12 h, with HSP27 transcription levels continuing to rise through 48 h. All assayed transcription factors were up-regulated by 12 h, but only c-fos and c-jun up-regulation persisted to the 48-h time point. Up-regulation of apoptosis-related genes, except for bcl-x, was not evident until 48 h. These gene expression patterns reflected an early proinflammatory response consisting of cytokines, cell adhesion and stress-response molecules at 12 h followed by an up-regulation of apoptosis-related genes at 48 h., Conclusion: Downstream targets of several transcription factors, up-regulated transiently at 12 h, control secondary effects of S. pneumoniae infection, including apoptosis of neutrophils and mucosal epithelial cells, bone proliferation and promotion of leukocyte differentiation. These observations lead to a greater understanding of the early events in the pathogenesis of an AOM episode and highlight therapeutic targets, which may play a roll in the sequelae of AOM.

Phase variation in the colonial opacity phenotype of Streptococcus pneumoniae has been implicated as a factor in bacterial adherence, colonization, and invasion in the pathogenesis of pneumococcal otitis media (OM). The purpose of this study was to determine whether S. pneumoniae opacity variants influence the induction of gene expression for proinflammatory mediators in vivo using the rat model of OM. Both the opaque and transparent phenotype variants induced a significant up-regulation in gene expression for interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-10, tumor necrosis factor alpha, and inducible nitric oxide synthase (iNOS) compared to saline sham-inoculated controls at both 4 and 24 h postinoculation (P < 0.05 in all cases). Furthermore, whereas a significant difference in gene expression was evident for only IL-6 (greater following challenge with the opaque variant) and IL-1beta (greater following challenge with the transparent variant) at 4 h, by 24 h the opaque variant cohort demonstrated a significant increase in gene expression for IL-1alpha, IL-1beta, IL-6, IL-10, and iNOS relative to animals inoculated with the transparent phenotype variant (P < 0.05 in all cases). Enzyme-linked immunosorbent assay results confirmed the gene expression data as determined by real-time PCR. Moreover, the concentrations of the opaque variant in the middle ear lavage fluid were a full log higher than those of the transparent variant. The aforementioned results indicate that the opaque phenotype variant is more efficient at survival and multiplication within the middle ear space, resulting in the accumulation of more inflammatory cells and the enhanced expression and production of inflammatory mediators. However, when the data were normalized to account for differences in middle ear bacterial titers, it became apparent that the transparent variant of S. pneumoniae is a more potent inducer of inflammation, triggering the accumulation of more inflammatory cells and substantially greater fold increases in the expression and production of inflammatory mediators. Data from this study indicate that S. pneumoniae opacity variants influence the temporal mRNA expression of inflammatory mediators within the middle ear.

Objective: Recently, we reported that gene transcripts encoding 3 Na+ transport proteins (pump, channel and exchanger) in the middle ear mucosa (MEM) were simultaneously suppressed at 12 and 48 h after Streptococcus pneumoniae (SP) challenge of rat middle ears., Material and Methods: From cDNA microarray screening of those specimens, several gene clusters, including Nos2 and the transcription factors Fos, Fosl1, Jun and Nfkb1, were identified as possible upstream regulators of Na+ transport protein expression. The altered expression of those genes in MEM was validated and quantified using real-time polymerase chain reaction and MEM protein expression for Atp1a1, Nos2 and Nfkb1 was studied using Western blot and/or immunohistochemistry assays., Results: At both time-points. Atp1a1 mRNA and protein were decreased and Nos2 mRNA and protein were increased in MEM. While Nfkb1 protein was decreased at those times. the corresponding mRNA was increased at 12 h but decreased at 48 h. Gene expression for Fos was suppressed at both times, while that for Fosl1 and Jun was augmented at 12 h and suppressed at 48 h. Immunohistochemical study of specimens challenged with SP showed a swollen MEM with infiltration of inflammatory cells that stained positive for Nos2., Conclusion: Given the known activities of Nos2, these results can be interpreted as evidencing a transcriptional suppression of Na+ transport protein synthesis secondary to upregulated Nos2 expression during SP infection of the rat MEM. This proposed signaling pathway does not require the continuous upregulation of Nfkb1 or the other assayed transcription factors as early as 12 h after middle ear infection.

Streptococcus pneumoniae is leading cause of bacterial otitis media in young children. Increasing rates of antibiotic resistance and the changing epidemiology of pneumococcal strains dictate the need to develop new methods to study, control, and prevent these important infections. Investigation into the molecular epidemiology of these bacteria will provide important insights into disease; progress in this field is described below.

Objective: Streptococcus pneumoniae is the most common pathogen in otitis media. Infection of the middle ear with S. pneumoniae potentiates development of thick effusion in the middle ear which frequently causes hearing loss and communication disorders in children. What has changed immediately in the middle ear cleft following pneumococcal infection is extensively studied and characterized but what has changed ever after remains elusive. The purpose of this study is to explore the cellular and molecular basis that remains on a longer time after acute pneucmococcal middle ear infection and potentiates development of thick effusion in the middle ear., Methods: 12 rats were intrabullarly inoculated with pneumococcus at 2.5x10(6) CFU/ear and profiles of gene expression in the middle ear were examined by cDNA microarrays in combination with reverse transcription-polymer chain reaction (RT-PCR) 6 weeks after infection while the morphologic changes in middle ear were simultaneously characterized by histopathologic techniques. Twelve rats receiving phosphate-buffered saline (PBS) served as controls., Results: it demonstrated that pneumococcus infected ears had the expression of the following genes at a high level compared to the controls: mitogenic signaling proteins (mitogen-activated protein kinase [MEK1 and MEK2], helix-loop-helix transcriptional regulators (Id3 and Id1), ion channels (sodium channel beta 1 and sodium channel 2), and mucin glycoproteins (Muc2 and Muc5). The morphology demonstrated a thickened mucosa and submucosa with increased expression of macroglycoconjugates compared to the controls., Conclusion: the expression of several genes remains high even after the acute episode of pneumococcal otitis media has been resolved. The up-regulated expression of these genes may serve as the basis for the development of thick effusion and mucous cell metaplasia/hyperplasia once it is complicated with other factors such as dysfunction of the Eustachian tube.

Until recently, it was not feasible to conduct genome-wide screening for gene transcript variations that play key roles in the pathogenesis of otitis media. In this study microarray technology was used to profile differential gene expression patterns from rat middle ear mucosa at 12 and 48 h after Streptococcus pneumoniae challenge. Real-time polymerase chain reaction was performed for independent verification of the microarray results. Three ion transport mRNAs were simultaneously suppressed more than 4-fold at 12 h in bacteria-challenged ears, including Na,K-ATPase alpha I subunit (SPATPa1), sodium channel beta 2 subunit (SCNB2) and sodium-hydrogen exchange protein isoform 2 subunit (NHE2). At 48 h after infection, the mRNA levels of SCNB2 and NHE2 had decreased 7- and 10-fold, respectively, whereas the relatively abundant SPATPa1 transcript showed recovery. The downregulation of Na(+)-transporting transcripts suggests a reduced number of epithelial cells and transporting proteins and/or the dysfunction of sodium transporters secondary to the bacterial infection. These changes can disrupt the coupling of the apical Na + entry and basolateral Na + extrusion, deplete the electrochemical Na+ transmembrane gradient, disrupt the intracellular osmotic equilibrium and lead to intracellular acidification and the accumulation of excess sodium, water and other organic and inorganic molecules in the middle ear cavity. Any or all of these changes may contribute to the initiation and persistence of middle ear mucosa inflammation and effusion during an episode of bacterial acute otitis media.

Acute otitis media (AOM) elicits potent inflammatory responses from the cells of the middle ear mucosa as well as from infiltrating leukocytes. To explore host responses during experimental AOM induced by Streptococcus pneumoniae type 3 and nontypeable Haemophilus influenzae (NTHi), otomicroscopy findings and expression of cytokine genes in the middle ear were monitored up to 1 month postinoculation. The mucosa and infiltrating cells responded rapidly to the bacterial challenge. Otomicroscopically, AOM appeared 1 day after NTHi inoculation and 3 days after pneumococcus inoculation. Pneumococcal AOM was more severe than NTHi otitis, but in general, lower transcript levels were detected in pneumococcus-infected than in NTHi-infected animals. Interleukin-6 (IL-6) mRNA levels peaked at 3 to 6 h for both pneumococcus-infected and NTHi-infected animals. IL-1alpha, tumor necrosis factor alpha, and IL-10 mRNA levels peaked at 6 h for NTHi otitis and 1 to 3 days for pneumococcal otitis. Comparing otomicroscopy with expression profiles, it would appear that the majority of cytokine mRNAs had passed their peak before the AOM diagnosis could be made clinically. Only transforming growth factor beta mRNA followed a slower time course, peaking very late and continuing expression even after the AOM was otomicroscopically resolved. IL-2 and IL-4 mRNAs were not detected in any animal at any time. Most of the investigated cytokines are very early markers for AOM and may be involved in initiation of inflammation, but they would be poor targets for pharmacological manipulation since their levels decline before clinical signs appear.

A multiply resistant serotype 23F pneumococcus was cultured from a 3-year-old child with otitis media and five carriers at the child's day-care centre in South Africa. Attempts to eradicate carriage with mupirocin failed. The strains were identical to the Spanish clone of multiply resistant pneumococci by serotype, antimicrobial susceptibility pattern, restriction analysis of the penicillin-binding protein 1A, 2B and 2X genes and by multilocus enzyme electrophoresis. The data suggest that this clone has acquired erythromycin resistance. A possible link between the index case and residence of a family member in Spain was found.