Acute Pneumococcal Otitis Media Research Articles

Deletion of the complement C5a receptor alleviates the severity of acute pneumococcal otitis media following influenza A virus infection in mice.

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.

Role of the alternative and classical complement activation pathway in complement mediated killing against Streptococcus pneumoniae colony opacity variants during acute pneumococcal otitis media in mice

There is considerable evidence that phase variation among transparent and opaque colony phenotypes of Streptococcus pneumoniae (Spn) plays an important role in the pneumococcal adherence and invasion. The current study was designed to investigate the interactions of the opacity phenotype variants of Spn with specific complement pathway activation in a mouse model of acute otitis media (AOM). Although the opaque colony phenotype was expected to be more resistant to complement mediated killing compared to the transparent Spn variant, we discovered that C3b deposition on the transparent Spn is, in large part, dependent on the alternative pathway activation. There were no significant differences in resistance to complement mediated opsonophagocytosis between the two variants in factor B deficient mice. In addition, an in vitro study demonstrated that significantly more C4b-binding protein (C4BP) (the classical pathway inhibitor) and factor H (FH) (the alternative pathway inhibitor) bound to the transparent strain compared with the opaque one. Our data suggest that the difference in the relative virulence of Spn opacity phenotypes is associated with its ability to evade complement-mediated opsonophagocytosis in a mouse model of pneumococcal AOM.

Essential role of factor B of the alternative complement pathway in complement activation and opsonophagocytosis during acute pneumococcal otitis media in mice

Essential role of factor B of the alternative complement pathway in complement activation and opsonophagocytosis during acute pneumococcal otitis media in mice

Essential Role of Factor B of the Alternative Complement Pathway in Complement Activation and Opsonophagocytosis during Acute Pneumococcal Otitis Media in Mice

We recently reported that the complement system plays a pivotal role in innate immune defense against Streptococcus pneumoniae during acute otitis media (OM) in mice. The current study was designed to determine which of the complement pathways are activated during acute pneumococcal OM and whether components of complement are expressed in the middle ear epithelium. Gene expression was determined by quantitative PCR, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining. We found that S. pneumoniae induced increased gene expression of factor B of the alternative complement pathway and C3 in mouse middle ear epithelium. Activation of factor B and C3 in the middle ear lavage fluids was significantly greater than in simultaneously obtained serum samples as determined by Western blotting. Using mice deficient in complement C1qa, factor B, and factor B/C2, we found that complement C3 activation and opsonophagocytosis of S. pneumoniae were greatly attenuated in factor B- and factor B/C2-deficient mice. These findings support the concept that local complement activation is an important host innate immune response and that activation of the alternative complement pathway represents one of the innate immune defense mechanisms against pneumococcal infection during the early stage of acute OM.

Pattern changes of mucin gene expression with pneumococcal otitis media

Objective: mucins, known to be important components of the mucociliary transport system in the middle ear and Eustachian tube, are subject to changes under inflammatory conditions. Which mucin genes are up-regulated or activated during an inflammatory reaction of the middle ear and Eustachian tube, however, is poorly understood. The purpose of this study was to characterize mucin gene expression in middle ears and Eustachian tubes with pneumococcal ear infection. Methods: sixteen rats received intrabullar inoculation of Streptococcus pneumoniae type 6A at 2.5×10 6 colony forming units (CFU). Four animals were sacrificed on days 1, 3, 7, and 14, respectively. The profile of mucin gene expression in the middle ear and Eustachian tube was examined by reverse transcription polymerase chain reaction (RT-PCR) at the above time points. Sixteen rats that received intrabullar inoculation of phosphate-buffered saline (PBS) served as controls. Results: the Muc2 mucin gene was expressed in middle ear mucosa of the control rats. Following pneumococcal inoculation, Muc1–Muc5 mucin genes were expressed in the middle ear mucosa in a time-dependent manner. In the Eustachian tube, the Muc2, Muc4 and Muc5 mucin genes were expressed in both control and pneumococcal inoculation groups. Conclusion: Muc1, Muc3, Muc4, and Muc5 mucin genes were activated in the middle ear mucosa by pneumococci, which may contribute to hyper-production of mucin in acute pneumococcal otitis media.

Clinical Outcome of Acute Pneumococcal Otitis Media and Serum Antibody Responses to Pneumococcal Pneumolysin and Polysaccharides in Children

Serum antibody responses to pneumococcal antigens and their relationship to the clinical outcome were determined in a prospective study of 121 children with acute otitis media (AOM). Pneumococcus positive children with a pneumolysin response more often had a recurrence and middle ear effusion (MEE) after 1 month than did the non-responders ( p =0.005 and p =0.04, respectively). All the children who responded to pneumolysin also had clinically strong symptoms and signs of AOM. Children who responded to pneumococcal polysaccharides developed otitis media with effusion within a 6-month follow-up period more often than did the non-responders ( p =0.005). The results of this study suggest that children with pneumococcal AOM and an antibody response to the intracellular pneumococcal protein pneumolysin behave clinically differently from children with an antibody response to polysaccharides.

Effect of Penicillin on Experimental Acute Otitis Media: A histopathological study of goblet cell density, bone modelling dynamics, polyp and adhesion formation

Clinical studies have shown that the effect of antibiotic treatment on acute otitis media is modest. Experimental acute otitis media induces a number of histopathological changes in the middle ear mucosa. Among these are increased goblet cell density, polyp and adhesion formation, as well as massive osteoneogenesis. To investigate the effect of penicillin administration on these histopathological features, we employed a rat model of acute pneumococcal otitis media. Five of 25 rats were sacrificed on days 4, 8, 16, 90 and 180 post-inoculation, preceded by oral administration of penicillin V 100 mg/kg/day, initiated on day 2 and lasting 5 days. Using a light microscope, qualitative and quantitative histopathology of middle ear goblet cell density, bone-modelling dynamics, polyp and adhesion formation was registered and compared with previous studies of untreated animals. Increase in goblet cell density and new bone formation was reduced significantly by treatment, whereas polyp and adhesion formation was unaffected by penicillin administration. It is concluded that penicillin reduces middle ear secretory capacity and new bone formation during and following acute otitis media, conceivably preventing subsequent development of secretory otitis media, leaving polyp and adhesion formation unchanged.

Role of the bacterial cell wall in middle ear inflammation caused by Streptococcus pneumoniae.

The pathogenesis of middle ear inflammation caused by Streptococcus pneumoniae was explored in the chinchilla model with different pneumococcal cell wall (CW) preparations, including isolated native CW, M1 muramidase CW (M1-CW) digest, amidase CW digest, and M1 peptidoglycan (M1-PG) digest. Inflammatory cell and lysozyme concentrations in middle ear fluid (MEF) were measured between 6 and 72 h after the middle ears were inoculated with one of the preparations or sterile saline. Middle ear histopathology was measured quantitatively at 72 h. Native CW, M1-CW digest, and amidase-CW digest caused significantly more inflammatory cell influx and lysozyme accumulation in MEF than saline did. M1-PG digest also caused more inflammatory cell influx and lysozyme accumulation in MEF than saline did but caused less inflammation than native CW or either CW digest. Epithelial metaplasia was significantly greater in ears inoculated with native CW than in ears inoculated with the CW or PG digest or with saline. Pneumococcal CW is, therefore, the principal factor that initiates middle ear inflammation in acute pneumococcal otitis media, and CW teichoication seems to be important in initiating this response.

Middle Ear Fluid Lysozyme Source in Experimental Pneumococcal Otitis Media

Middle ear infection with Streptococcus pneumoniae is important in the pathogenesis of acute and chronic otitis media, and lysozyme in middle ear fluid (MEF) is an important inflammatory mediator in this disease. To determine the source of lysozyme during the early period of acute pneumococcal otitis media, chinchillas were irradiated to induce neutropenia, and their middle ears were inoculated with heat-killed, encapsulated pneumococci. The number of inflammatory cells and concentration of lysozyme were measured in MEF between 6 and 72 hours after inoculation. In pneumococcus-inoculated ears, the mean number of inflammatory cells but not lysozyme was significantly lower in MEF from irradiated animals than that from nonirradiated animals at 6 hours. Since lysozyme accumulated in MEF before the influx of inflammatory cells in irradiated animals, the initial release of this inflammatory mediator is most likely not from inflammatory cells; and mucosal epithelial cells, the only other known source of lysozyme in the middle ear environment, were the probable source induced by the direct stimulation of pneumococci. Inflammatory cells may contribute lysozyme later in the inflammatory response, since cellular and lysozyme concentrations in irradiated and nonirradiated animals were similar between 24 and 72 hours. These results suggest that future therapeutic interventions to limit middle ear inflammation in acute otitis media may need to recognize the direct action of pneumococcal cells or their envelope components on middle ear epithelium.

Serum antibody response to pneumococcal otitis media.

Total and immunoglobulin class-specific antibody responses to the infecting pneumococcal serotype/group in sera of 114 children, 3 months to 7 years old, who had acute pneumococcal otitis media, were measured by radioimmunoassay and enzyme-linked immunosorbent assay. Of these children 36% showed a twofold or greater increase of antibodies as a response to their infection, but the responsiveness varied from 17 to 83% among different pneumococcal types. The number and intensity of responses increased with the age of the children. In acute sera only IgG and IgM class antibodies were usually detectable. IgG and IgM class antibodies were regularly formed as a response to the infection. IgA class antibodies were frequently seen in the convalescent sera when the total antibody response was strong, and they were the first to start declining.

The Pathogenesis of Pneumococcal Otitis Media in Chinchillas and the Efficacy of Vaccination in Prophylaxis

Acute pneumococcal otitis media was produced experimentally in 100% of chinchillas whose middle ear cavities were inoculated with Streptococcus pneumoniae. Although intranasal inoculation with S. pneumoniae led to otitis media in 21% of animals, 66% of the colonized chinchillas that had negative pressure in the middle ear developed otitis media. Intranasal inoculation with S. pneumoniae followed by intranasal inoculation with influenza A virus led to otitis media in 73% of chinchillas, while inoculation with infleunza A virus alone produced otitis media in 4% of animals. Measurement of middle ear pressure by tympanometry showed the influenza virus-infected chinchillas developed negative middle ear pressure before the appearance of clinical signs of otitis media. While direct inoculation of the middle ear with pneumococci produced a purulent effusion within days after inoculation, intranasal inoculation with both pneumococci and influenza A virus resulted in the rapid appearance of a serous effusion, which became culture-positive for S. pneumoniae during the second week after inoculation. Spontaneous resolution of middle ear infection occurred within three to eight weeks after inoculation and was associated with an increase in the level of type-specific pneumococcal antibody in serum and middle ear effusion. The histopathology of pneumococcal otitis media included purulent middle ear effusion, epithelial metaplasia, and subepithelial edema and hypercellularity. The initial infiltration of the subepithelial space with polymorphonuclear leukocytes was superceded by infiltration with mononuclear leukocytes in animals that were observed for longer than eight weeks. Among untreated animals observed for long periods, production of granulation tissue, development of submucosal fibrosis, and osteoneogenesis were observed. Systemically administered pneumococcal capsular polysaccharide vaccine was effective for prevention of type-specific otitis media in chinchillas when vaccinated animals were challenged by intranasal inoculation with pneumococci followed by deflation of the middle ear. Animals that seroconverted with at least a twofold increase in the level of antibody in serum after vaccination had an 87% lower incidence of otitis media than did unvaccinated animals. Protection was associated with high levels of antibody in serum before intranasal inoculation, and higher antibody levels were found in sterile middle ear effusions than in effusions that contained pneumococci.

Lactate dehydrogenase as a measure of inflammation in experimental otitis media.

A reliable animal model for acute otitis media due to Streptococcus pneumoniae was used to study the accumulation of lactate dehydrogenase (LDH) in middle ear effusion during acute pneumococcal otitis media and after penicillin treatment. The findings indicate that LDH levels rise in the middle ear effusion during the early phase of acute infection and decrease as the infection resolves over time. Penicillin treatment affects the natural course of infection by resolving most visible signs of inflammation and by sterilizing the middle ear. However, once middle ear infection was established, penicillin treatment did not reduce the elevated levels of effusion LDH, suggesting that inflammation persists after sterilization of infected middle ears.

C1 subcomponents in acute pneumococcal otitis media in children.

Twenty children with acute pneumococcal otitis media were studied. In 6 children the infection ran a normal course and healed after the first episode and in 14 it relapsed. The serum levels of the immunoglobulins IgG, IgA and IgM were normal in all 20 children. Specific antibodies to pneumococcal polysaccharide were found in all cases, with no differences in the titers between the relapsed cases and those that healed. The complement components were quantitated with electroimmuno assay. G1q proved depressed in 60 per cent of the relapsed cases and in 16 per cent of the healed cases. C1r and C1s were disproportionally high compared with the C1q levels. Furthermore, crossed immunoelectrophoresis revealed abnormal complexes composed of C1r and C1s, and complexes composed of C1r, C1s and C1 IA. These complexes were more pronounced in sera from the children with relapsing otitis media.