Single-Cell Transcriptome Profiling Identifies Phagocytosis-Related Dual-Feature Cells in A Model of Acute Otitis Media in Rats.

Background: The molecular mechanisms of acute otitis media (AOM) development, and the intercellular crosstalk within the multicellular ecosystem of AOM, are not clear. Methods: We established a model of AOM in rats (with normal rats as controls) and undertook single-cell RNA sequencing (scRNA-seq) for the middle-ear mucosa (MEM). Cell clustering and trajectory analyses were undertaken using Seurat and Monocle 2 packages in R software. Pathway analyses were done by gene set enrichment analysis (GSEA). Cell-cell interactions were inferred by CellChat. Cell scores were calculated to identify cells with dual-feature. Results: A total of 7023 cells from three samples of inflamed MEM and 5258 cells from three samples of healthy MEM underwent scRNA-seq, which identified 20 cell clusters belonging to eight major cell types. After exposure to lipopolysaccharide, the MEM underwent significant conversion of cell types characterized by rapid infiltration of macrophages and neutrophils. M2 macrophages seemed to play a key part in inflammatory intercellular crosstalk, which facilitated the maintenance and proliferation of macrophages, cell chemotaxis, and regulation of the proinflammatory activities of cytokines. Three rare cell clusters with phagocytosis-related dual-feature were also identified. They coexisted with professional phagocytes in the MEM, and displayed distinct immunoregulatory functions by maintaining a normal immune microenvironment or influencing inflammation progression. Conclusions: Macrophages might be the "master" initiators and regulators of the inflammatory response of the MEM to external stimuli. And their functions are fulfilled by a specific polarization status (M2) and sophisticated intercellular crosstalk via certain signaling pathways. Besides, the coexistence of professional phagocytes and non-professional phagocytes as well as their interplay in the MEM provides new clues for deciphering the underlying pathogenic mechanisms of AOM.

Glycolytic Metabolism Is Critical for the Innate Antibacterial Defense in Acute Streptococcus pneumoniae Otitis Media.

Objective: Streptococcus pneumoniae (S.pn) is a common respiratory pathogen and a frequent cause of acute otitis media (AOM) in children. However, little is known about the immunometabolism during AOM. This study was to assess the presence of glucose metabolic reprogramming during AOM and its underlying mechanism affecting inflammatory response and middle ear injury. Methods: The levels of glycolytic metabolism were evaluated by measuring the expression of glycolysis-related genes and the production of metabolites. HE stain, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and Western blot were performed to measure the effect of glucose metabolic reprogramming on inflammatory response, pneumococcal clearance, hypoxia-inducible factor 1 alpha (HIF-1α) expression and cytokine secretion during AOM, respectively. Results: The analysis of microarray revealed an increase of the expression of glycolysis-related genes during S.pn-induced AOM, which was verified by real-time PCR. Increased glycolysis promoted the production of IL-1ß and TNF-α and facilitated the clearance of S.pn by enhancing phagocytosis and killing capability of neutrophils, but also aggravated the middle ear injury. Furthermore, these pathogenic effects could be reversed after glycolytic inhibitor 2DG treatment. Additionally, HIF-1α was observed to involve in glycolytic metabolism during AOM. Conclusion: S.pn infection induced increased glycolysis conversion during AOM, which promoted inflammatory responses and bacterial clearance, but also aggravated tissue damage.

Suppression of lncRNA MALAT1 Reduces LPS- or IL-17A-Induced Inflammatory Response in Human Middle Ear Epithelial Cells via the NF-κB Signaling Pathway.

Otitis media (OM) is a common inflammatory disease of the middle ear cavity and mainly occurs in children. As a critical regulator of inflammation response, the nuclear factor kappa B (NF-κB) pathway has been found to play an essential role in the pathogenesis of various human diseases. The aim of this study was to explore the potential mechanism under the inflammatory response of human middle ear epithelial cells (HMEECs). We established in vitro models of OM by treating HMEECs with lipopolysaccharide (LPS) or interleukin 17A (IL-17A). Enzyme-linked immunosorbent assay and western blot analysis were used to measure the inflammatory response of HMEECs under LPS or IL-17A stimulation. The results revealed that the concentrations of proinflammatory cytokines (p < 0.001) and protein levels of mucin (MUC) (for MUC5AC, p = 0.002, p = 0.004; for MUC8, p = 0.004, p < 0.001) were significantly elevated by LPS or IL-17A stimulation in HMEECs. Moreover, we found that LPS or IL-17A treatment promoted the phosphorylation of IκBα (for p-IκBα, p = 0.018, p = 0.002; for IκBα, p = 0.238, p = 0.057) and the translocation of p65 from cytoplasm to nucleus in HMEECs (for nucleus p65, p = 0.01; for cytoplasm p65, p < 0.001). In addition, RT-qPCR analysis revealed that long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was verified to be upregulated in LPS- or IL-17A-stimulated HMEECs (p < 0.001). Western blot analysis and immunofluorescence staining assay revealed that that MALAT1 knockdown significantly suppressed the activation of the NF-κB pathway by reducing phosphorylated IκBα levels and inhibiting the nuclear translocation of p65 (p < 0.001) in LPS- or IL-17A-stimulated HMEECs (for p-IκBα, p < 0.001; for IκBα, p = 0.242, p = 0.647). Silence of MALAT1 decreased the proinflammatory cytokine production and MUC protein levels (p < 0.001). Furthermore, rescue assays revealed that the increase of proinflammatory cytokine production (for TNF-α, p = 0.002, p = 0.015; for IL-1ß, p < 0.001, p = 0.006; for IL-6, p = 0.002, p < 0.001) and MUC protein levels (for MUC5AC, p = 0.001, p < 0.001; for MUC8, p < 0.001, p = 0.001) induced by MALAT1 overexpression was neutralized by 4-N-[2-(4-phenoxyphenyl) ethyl] quinazoline-4, 6-diamine (QNZ) treatment in LPS- or IL-17A-stimulated HMEECs. In conclusion, MALAT1 promotes inflammatory response in LPS- or IL-17A- stimulated HMEECs via the NF-κB signaling pathway, which may provide a potential novel insight for the treatment of OM.

Phase variation with altering phosphorylcholine expression of nontypeable Haemophilus influenzae affects bacteria clearance and mucosal immune response in the middle ear and nasopharynx.

OBJECTIVES: Nontypeable Haemophilus influenzae (NTHi) is a chief pathogen in both acute otitis media and otitis media with effusion. Phosphorylcholine (ChoP) is expressed on lipooligosaccharides, and ChoP has phase variation, which is related to its adhesion to and invasion of epithelial cells in the upper airway. However, little is known about the role of ChoP expression. We examined the kinetics of the mucosal clearance of NTHi from the nose and middle ear and the mucosal immune response to NTHi infection by comparing ChoP(+) and ChoP(-) strains in a mouse model of middle ear and nasal challenge. METHODS: Six-week-old male BALB/c mice were subjected to bacterial challenge in the middle ear and nasopharynx. Mice were inoculated with a suspension of a ChoP(+) strain or ChoP(-) strain of NTHi. On days 1, 3, and 7 after inoculation, the middle ear wash (MEW) and nasal wash (NW) were harvested from each group. The samples were used for bacterial counts and the supernatant was used to measure the level of cytokines and C-reactive protein (CRP). RESULTS: MEWs in the ChoP(+) strain group had significantly higher bacterial counts than those in the ChoP(-) strain group on day 1. However, bacteria were eradicated in the ChoP(+) strain group on day 7. NWs in the ChoP(+) strain group had higher bacterial counts than those in the ChoP(-) strain group during the experiment, however, there was no significant difference between the two strains. The levels of cytokines were significantly higher in the ChoP(-) strain group than in the ChoP(+) strain group in MEWs, but these cytokine levels were low in NWs. The CRP concentration in the ChoP(-) group was high on day 7 in the MEWs. In NWs, the CRP concentration was low in all groups during the experiment. CONCLUSION: ChoP expression of NTHi changes the organism susceptible to killing by CRP, and the ChoP(+) strain might be gradually eradicated from the middle ear via the CRP-complement cascade, but not from nasopharynx. Based on our findings, phase variation by altering Phosphorylcholine expression of nontypeable Haemophilus influenzae affects bacteria clearance and mucosal immune response in the middle ear and nasopharynx.

Analysis of Inflammatory and Homeostatic Roles of Tissue-resident Macrophages in the Progression of Cholesteatoma by RNA-Seq.

BACKGROUND: Tissue-resident macrophages (TRMØs) can act as innate-immune sentinels to protect body against microbe invaders and stimulating materials such as cholesterol crystals in cholesteatoma, as well as to preserve tissue integrity by cleaning unwanted cellular debris. METHODS: TRMØs in the incised middle ear tissues were obtained from the patients with cholesteatoma as an experimental group and the patients without cholesteatoma as a control group. Differential gene expression profiling of TRMØs was conducted between two groups by analyzing GO processes, KEGG and GSEA pathways of inflammation, tissue repair and homeostasis. RESULTS: The current study showed that 145 of 7060 genes were significantly up-regulated (logFC>2 and FDR <0.05) when compared with the patients without cholesteatoma. GO process, GSEA and Cytoscape analysis of the over-expressed genes illustrated the boosted inflammatory and anti-infection functions of TRMØs existed neutrophil function, leukocyte migration, and adaptive immune response involved receptors and signaling pathways. Whereas the homeostasis and repair functions of TRMØs were affected from up-regulated genes, such as over-expressed keratin-13 that helped form the outer keratinising squamous epithelial layer, and over-expressed MMPs that activated the extracellular matrix molecules to promote inflammation and disturb tissue remodeling. Additionally, 74 down-regulated genes (logFC<-2 and FDR <0.05) also affected the homeostasis and repair functions by affecting extracelluar matrix structure and contractile fibres in TRMØs. CONCLUSIONS: The cellular and molecular levels in cholesteatoma is attributable to chronic infection and several disturbed cellular biological processes involving cell integrity and tissue remodeling.

Inflammatory Mediator Profiles in Secretory Otitis Media in Relationship to Viable Bacterial Pathogens and Bacterial and Viral Nucleic Acids.

Secretory otitis media (SOM) is characterized by persistence of fluid in the middle ear, often following an episode of acute otitis media. Our hypothesis is that failure to eliminate bacterial or viral pathogens may result in persistent low-grade inflammation. In this study, we analyzed inflammatory mediators in middle ear fluids from 67 children with SOM. This was combined with determinations of viable bacteria by culture along with detection of bacterial and viral genetic material by real-time polymerase chain reaction (PCR). The inflammatory mediators found at the highest concentrations (>30 ng/mL) were stem cell growth factor-ß (median 110 ng/mL), CXCL1, IL-16, IL-8, migration inhibitory factor, CXCL10, and CXCL9. Among bacterial pathogens, Moraxella catarrhalis and Haemophilus influenzae dominated, regardless of detection methods, while rhinovirus dominated among viral pathogens. Middle ear fluid levels of interleukin (IL)-1α, IL-17, IL-1ß, fibroblast growth factor basic, and tumor necrosis factor correlated strongly with presence of bacteria detected either by culture or PCR, while IL-1RA, IL-3, IL-6, IL-8, CCL3, CCL4, and granulocyte-colony stimulating factor correlated significantly with real-time PCR values. CXCL10, CXCL9, CCL2, and TRAIL correlated significantly with viral nucleic acid levels. To conclude, persistence of viral and bacterial pathogens may fuel persistent inflammation in SOM. Bacteria caused a broad inflammatory response, while viruses chiefly elicited the interferon-induced chemokines CXCL9 and CXCL10.

Extensive qPCR analysis reveals altered gene expression in middle ear mucosa from cholesteatoma patients.

The middle ear is a small and hard to reach compartment, limiting the amount of tissue that can be extracted and the possibilities for studying the molecular mechanisms behind diseases like cholesteatoma. In this paper 14 reference gene candidates were evaluated in the middle ear mucosa of cholesteatoma patients and two different control tissues. ACTB and GAPDH were shown to be the optimal genes for the normalisation of target gene expression when investigating middle ear mucosa in multiplex qPCR analysis. Validation of reference genes using c-MYC expression confirmed the suitability of ACTB and GAPDH as reference genes and showed an upregulation of c-MYC in middle ear mucosa during cholesteatoma. The occurrence of participants of the innate immunity, TLR2 and TLR4, were analysed in order to compare healthy middle ear mucosa to cholesteatoma. Analysis of TLR2 and TLR4 showed variable results depending on control tissue used, highlighting the importance of selecting relevant control tissue when investigating causes for disease. It is our belief that a consensus regarding reference genes and control tissue will contribute to the comparability and reproducibility of studies within the field.

Niche- and Gender-Dependent Immune Reactions in Relation to the Microbiota Profile in Pediatric Patients with Otitis Media with Effusion.

Otitis media with effusion (OME) is a common inflammatory disease that primarily affects children. OME is defined as a chronic low-grade inflammation of the middle ear (ME), without any signs of infection and with effusion persisting in the ME for more than 3 months. The precise pathogenesis is, however, not fully understood. Here, we comprehensively characterized and compared the host immune responses (inflammatory cells and mediators) and the overall microbial community composition (microbiota) present in matched middle ear effusion (MEE) samples, external ear canal (EEC) lavages, and nasopharynx (NPH) samples from children with OME. Female patients had significantly increased percentages of T lymphocytes and higher levels of a wide array of inflammatory mediators in their MEE compared to that of male patients, which were unrelated to microbiota composition. The relative abundances of identified microorganisms were strongly associated with their niche of origin. Furthermore, specific inflammatory mediators were highly correlated with certain bacterial species. Interestingly, some organisms displayed a niche-driven inflammation pattern in which presence of Haemophilus spp. and Corynebacterium propinquum in MEE was accompanied by proinflammatory mediators, whereas their presence in NPH was accompanied by anti-inflammatory mediators. For Turicella and Alloiococcus, we found exactly the opposite results, i.e., an anti-inflammatory profile when present in MEE, whereas their presence in the the NPH was accompanied by a proinflammatory profile. Together, our results indicate that immune responses in children with OME are highly niche- and microbiota-driven, but gender-based differences were also observed, providing novel insight into potential pathogenic mechanisms behind OME.

Effect of probiotics in experimental otitis media with effusion.

OBJECTIVES: The article's aim was to investigate the effects of probiotics in the experimental otitis media with effusion. MATERIALS AND METHODS: Twenty-four male Wistar albino rats were used. They were divided into four groups. Experimental otitis media with effusion was created by intratympanic histamine injection. The effusion was confirmed by otomicroscopic examination 24 h after injection. Group 1; did not receive any treatment, group 2; received probiotics for 7 days after the detection of effusion, group 3; received probiotics for 7 days prior to injection of histamine, group 4; received probiotics for 7 days before injection of histamine and 7 days after detection of effusion. After detection of effusion, animals were sacrificed. Otomicroscopic evaluation was done to determine the effusion. In histopathological examination neutrophil leukocyte counts were determined in 25 areas of the sub-mucosa of the temporal bulla. RESULTS: The otomicroscopic ear effusions' healing rate in group 1 was 10%, in group 2 was 25%, in group 3 was 50%, and in group 4 was 100% (p < 0,013). The mean counts of submucosal neutrophil leukocyte from 25 areas of the temporal bulla of group 1 was 86,8 ± 24, group 2 was 66,5 ± 21, group 3 was 66,2 ± 16, and group 4 was 26,3 ± 6,5 (p < 0,001). CONCLUSION: Probiotics have a curative effect on the prevention and treatment of otitis media with effusion. This result may be related to their anti-inflammatory effects. Therefore, probiotics can be widely used in the age group at risk for otitis media with effusion as a complementary therapy by dietary supplements. LEVEL OF EVIDENCE: NA.

Vinpocetine Suppresses Streptococcus pneumoniae-Induced Inflammation via Inhibition of ERK1 by CYLD.

Otitis media (OM) is the most common bacterial infection in children. It remains a major health problem and a substantial socioeconomic burden. Streptococcus pneumoniae (S. pneumoniae) is one of the most common bacterial pathogens causing OM. Innate inflammatory response plays a critical role in host defense against bacterial pathogens. However, if excessive, it has a detrimental impact on the middle ear, leading to middle ear inflammation, a hallmark of OM. Currently, there has been limited success in developing effective therapeutic agents to suppress inflammation without serious side effects. In this study, we show that vinpocetine, an antistroke drug, suppressed S. pneumoniae-induced inflammatory response in cultured middle ear epithelial cells as well as in the middle ear of mice. Interestingly, vinpocetine inhibited S. pneumoniae-induced inflammation via upregulating a key negative regulator cylindromatosis (CYLD). Moreover, CYLD suppressed S. pneumoniae-induced inflammation via inhibiting the activation of ERK. Importantly, the postinfection administration of vinpocetine markedly inhibited middle ear inflammation induced by S. pneumoniae in a well-established mouse OM model. These studies provide insights into the molecular mechanisms underlying the tight regulation of inflammation via inhibition of ERK by CYLD and identified vinpocetine as a potential therapeutic agent for suppressing the inflammatory response in the pathogenesis of OM via upregulating negative regulator CYLD expression.

Panel 2- recent advance in otitis media bioinformatics.

OBJECTIVES: To update the medical literature on recent large-scale studies employing bioinformatics data analysis tools in otitis media (OM) disease models with a principal focus on developments in the past 5 years. DATA SOURCES: Pubmed indexed peer-reviewed articles. REVIEW METHODS: Comprehensive review of the literature using the following search terms: 'genomics, inflammasome, microRNA, proteomics, transcriptome, bioinformatics' with the term 'otitis media', and 'middle ear'. Included articles published in the English language from January 1, 2015-April 1, 2019. IMPLICATIONS FOR PRACTICE: Large scale bioinformatics tools over the past five years lend credence to the paradigm of innate immune response playing a critical role in host defense against bacteria contributing to Otitis Media (OM) progression from acute to chronic. In total, genomic, miRNAomic, and proteomic analyses all point to the need for a tightly regulated innate immune and inflammatory response in the middle ear. Currently, there is an urgent need for developing novel therapeutic strategies to control immunopathology and tissue damage, improve hearing and enhance host defense for both acute and chronic OM based on full understanding of the basic molecular pathogenesis of OM.

High concentrations of middle ear antimicrobial peptides and proteins and proinflammatory cytokines are associated with detection of middle ear pathogens in children with recurrent acute otitis media.

Recurrent and chronic otitis media (OM) are often refractory to antibiotics due to bacterial persistence in biofilm within the middle ear. In vitro and in vivo studies have demonstrated that antimicrobial proteins and peptides (AMPs) are bactericidal against otopathogens, indicating potential therapeutic value for recalcitrant OM. We measured concentrations of 6 AMPs and 14 cytokines in middle ear effusion (MEE) from 67 children undergoing ventilation tube insertion for recurrent acute OM. Sixty one percent of children had bacterial otopathogens detected in their MEE, 39% by PCR and 22% by PCR and culture. Groups were defined as: PCR-negative/culture-negative (absence of bacterial otopathogen), n = 26; PCR-positive/culture-negative (presence of nonculturable bacterial otopathogen), n = 26; PCR-positive/culture-positive (presence of culturable bacterial otopathogen), n = 15. Age, antibiotic usage, day-care attendance, presence of respiratory viruses in MEE and number of AOM episodes were similar between groups. AMP and cytokine concentrations were higher in children with bacterial otopathogens in their MEE compared to those with no bacterial otopathogens. Median concentrations of AMPs (except HBD2) were 3 to 56-fold higher in MEE from children with bacterial otopathogens detected in their MEE (P ≤ 0.01). Similarly, median cytokine concentrations (except TGFß) were >16-fold higher in MEE with bacterial otopathogens detected (P ≤ 0.001). This is the first study to measure AMPs in MEE and together with the cytokine data, results suggest that elevated AMPs and cytokines in MEE are a marker of inflammation and bacterial persistence. AMPs may play an important role in OM pathogenesis.

Lack of the hyaluronan receptor CD44 affects the course of bacterial otitis media and reduces leukocyte recruitment to the middle ear.

BACKGROUND: CD44 is a multifunctional molecule that plays major roles in both leukocyte recruitment and tissue proliferation. Since mucosal hyperplasia and leukocyte infiltration of the middle ear cavity are major features of otitis media, we evaluated the role of CD44 in the pathophysiology and course of this disease in a mouse model of middle ear infection. Expression of genes related to CD44 function were evaluated using gene arrays in wild-type mice. The middle ears of mice deficient in CD44 were inoculated with non-typeable Haemophilus influenzae. Histopathology and bacterial clearance were compared to that seen in wild-type controls. RESULTS: We observed strong up-regulation of CD44 and of genes related to its role in leukocyte extravasation into the middle ear, during the course of acute otitis media. Mice deficient in CD44 exhibited reduced early mucosal hyperplasia and leukocyte recruitment, followed by delayed resolution of infection and persistent inflammation. CONCLUSIONS: CD44 plays an important role in OM pathogenesis by altering the mucosal growth and neutrophil enlistment. Targeted therapies based on CD44 could be useful adjuncts to the treatment of middle ear infections.

Effect of a pneumococcal whole cell vaccine on influenza A-induced pneumococcal otitis media in infant mice.

The pneumococcus remains a common cause of otitis media (OM) despite the widespread introduction of pneumococcal conjugate vaccines. In mice, a pneumococcal whole cell vaccine (WCV) induces serotype-independent protection against pneumococcal colonisation and invasive disease via TH17- and antibody-mediated immunity, respectively. We investigated the effect of WCV on influenza A-induced pneumococcal OM in an infant mouse model. C57BL/6 mice were immunised subcutaneously with a single dose of WCV or adjuvant at 6 days of age, infected with pneumococci (EF3030 [serotype 19F] or PMP1106 [16F]) at 12 days of age, and given influenza A virus (A/Udorn/72/307 [H3N2], IAV) at 18 days of age to induce pneumococcal OM. Pneumococcal density in middle ear and nasopharyngeal tissues was determined 6 and 12 days post-virus. Experiments were repeated in antibody (B6.µMT-/-)- and CD4+ T-cell-deficient mice to investigate the immune responses involved. A single dose of WCV did not prevent the development of pneumococcal OM, nor accelerate pneumococcal clearance compared with mice receiving adjuvant alone. However, WCV reduced the density of EF3030 in the middle ear at 6 days post-viral infection (p = 0.022), and the density of both isolates in the nasopharynx at 12 days post-viral infection (EF3030, p = 0.035; PMP1106, p = 0.011), compared with adjuvant alone. The reduction in density in the middle ear required antibodies and CD4+ T cells: WCV did not reduce EF3030 middle ear density in B6.µMT-/- mice (p = 0.35) nor in wild-type mice given anti-CD4 monoclonal antibody before and after IAV inoculation (p = 0.91); and WCV-immunised CD4+ T cell-deficient GK1.5 mice had higher levels of EF3030 in the middle ear than their adjuvant-immunised counterparts (p = 0.044). A single subcutaneous dose of WCV reduced pneumococcal density in the middle ears of co-infected mice in one of two strains tested, but did not prevent OM from occurring in this animal model.

Subversion of host immune responses by otopathogens during otitis media.

Otitis media (OM) is one of the most common ear diseases affecting humans. Children are at greater risk and suffer most frequently from OM, which can cause serious deterioration in the quality of life. OM is generally classified into two main types: acute and chronic OM (AOM and COM). AOM is characterized by tympanic membrane swelling or otorrhea and is accompanied by signs or symptoms of ear infection. In COM, there is a tympanic membrane perforation and purulent discharge. The most common pathogens that cause AOM are Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis whereas Pseudomonas aeruginosa and Staphylococcus aureus are commonly associated with COM. Innate and adaptive immune responses provide protection against OM. However, pathogens employ a wide arsenal of weapons to evade potent immune responses and these mechanisms likely contribute to AOM and COM. Immunologic evasion is multifactorial, and involves damage to host mucociliary tract, genetic polymorphisms within otopathogens, the number and variety of different otopathogens in the nasopharynx as well as the interaction between the host's innate and adaptive immune responses. Otopathogens utilize host mucin production, phase variation, biofilm production, glycans, as well as neutrophil and eosinophilic extracellular traps to induce OM. The objective of this review article is to discuss our current understanding about the mechanisms through which otopathogens escape host immunity to induce OM. A better knowledge about the molecular mechanisms leading to subversion of host immune responses will provide novel clues to develop effective treatment modalities for OM.

Changes in the serotype distribution of Streptococcus pneumoniae causing otitis media after PCV13 introduction in Spain.

One of the beneficial effects of pneumococcal conjugate vaccines (PCVs) has been a decrease in the incidence of non-invasive infections, such as otitis media (OM) caused by vaccine serotypes. In this study, we analyzed the epidemiology of pneumococcal OM before and after PCV13 introduction in 2010. Between 2008 and 2016, the middle ear exudates from 2653 children under 14 years of age with OM were studied in two Spanish provinces (Gipuzkoa and Barcelona), and S. pneumoniae was isolated in 235 (8.9%) of cases. The 204 available isolates were serotyped and distributed in three 3-year periods: one before and two after PCV13 introduction (early and late post-PCV13). A significant decrease in the rate of OM caused by S. pneumoniae was observed mainly due to a decrease in infections caused by all PCV13 serotypes, although exceptions were observed including the persistence of serotype 3 in Gipuzkoa and a weak re-emergence of serotype 19F in both regions. The rate and diversity of non-PCV13 serotypes increased in both regions and an emerging clone causing OM was detected in each region: serotype 23B ST2372 in Gipuzkoa and serotype 11A ST838/ST6521 in Barcelona. The introduction of PCV13 has been followed by a change in the epidemiology of pneumococcal OM, with a decrease in the rate of vaccine serotypes accompanied by an increase in the diversity of non-vaccine serotype and the clonal spreading of different single clones in each region.

Eosinophilic Otitis Media Treated with Anti-IgE Monoclonal Antibodies and A Bone Conduction Implant.

Eosinophilic otitis media (EOM) are intractable otitis media characterized by highly viscous secretions containing eosinophils in the middle ear. They are resistant to conventional medication and surgery. This condition occurs primarily in patients with bronchial asthma or allergic rhinitis and is often complicated by rhinosinusitis. Systemic and topical steroid therapies are effective treatments. However, long-term steroid therapy is often limited by a high risk of serious adverse effects. The use of topical steroids and otorrhea are bothersome when wearing hearing aids. Here, we report a case of intractable otitis media due to EOM. Otorrhea was controlled with topical steroids. Bone conduction hearing was stable for an extended period with anti-IgE monoclonal antibodies (omalizumab). An implantable bone conduction hearing aid was used for rehabilitation of conductive hearing loss.

Otitis Media and Nasopharyngeal Colonization in ccl3-/- Mice.

We previously found CC chemokine ligand 3 (CCL3) to be a potent effector of inflammation during otitis media (OM): exogenous CCL3 rescues the OM phenotype of tumor necrosis factor-deficient mice and the function of macrophages deficient in several innate immune molecules. To further delineate the role of CCL3 in OM, we evaluated middle ear (ME) responses of ccl3-/-mice to nontypeable Haemophilus influenzae (NTHi). CCL chemokine gene expression was evaluated in wild-type (WT) mice during the complete course of acute OM. OM was induced in ccl3-/- and WT mice, and infection and inflammation were monitored for 21 days. Phagocytosis and killing of NTHi by macrophages were evaluated by an in vitro assay. The nasopharyngeal bacterial load was assessed in naive animals of both strains. Many CCL genes showed increased expression levels during acute OM, with CCL3 being the most upregulated, at levels 600-fold higher than the baseline. ccl3-/- deletion compromised ME bacterial clearance and prolonged mucosal hyperplasia. ME recruitment of leukocytes was delayed but persisted far longer than in WT mice. These events were linked to a decrease in the macrophage capacity for NTHi phagocytosis and increased nasopharyngeal bacterial loads in ccl3-/- mice. The generalized impairment in inflammatory cell recruitment was associated with compensatory changes in the expression profiles of CCL2, CCL7, and CCL12. CCL3 plays a significant role in the clearance of infection and resolution of inflammation and contributes to mucosal host defense of the nasopharyngeal niche, a reservoir for ME and upper respiratory infections. Therapies based on CCL3 could prove useful in treating or preventing persistent disease.