Otitis media: Interactions between host and environment, immune and inflammatory responses.

OBJECTIVE: To review and highlight progress in otitis media (OM) research in the areas of immunology, inflammation, environmental influences and host-pathogen responses from 2019 to 2023. Opportunities for innovative future research were also identified. DATA SOURCES: PubMed database of the National Library of Medicine. REVIEW METHODS: Key topics were assigned to each panel member for detailed review. Search of the literature was from June 2019 until February 2023. Draft reviews were collated, circulated, and discussed among panel members at the 22nd International Symposium on Recent Advances in Otitis Media in June 2023. The final manuscript was prepared and approved by all the panel members. CONCLUSIONS: Important advances were identified in: environmental influences that enhance OM susceptibility; polymicrobial middle ear (ME) infections; the role of adaptive immunity defects in otitis-proneness; additional genes linked to OM; leukocyte contributions to OM pathogenesis and recovery; and novel interventions in OM based on host responses to infection. Innovative areas of research included: identification of novel bacterial genes and pathways important for OM persistence, bacterial adaptations and evolution that enhance chronicity; animal and human ME gene expression, including at the single-cell level; and Sars-CoV-2 infection of the ME and Eustachian tube.

Negative Cross-Talk between TLR2/4-Independent AMPKα1 and TLR2/4-Dependent JNK Regulates S. pneumoniae-Induced Mucosal Innate Immune Response.

Streptococcus pneumoniae is major cause of otitis media (OM) and life-threatening pneumonia. Overproduction of mucin, the major component of mucus, plays a critical role in the pathogenesis of both OM and pneumonia. However, the molecular mechanisms underlying the tight regulation of mucin upregulation in the mucosal epithelium by S. pneumoniae infection remain largely unknown. In this study, we show that S. pneumoniae pneumolysin (PLY) activates AMP-activated protein kinase α1 (AMPKα1), the master regulator of energy homeostasis, which is required for S. pneumoniae-induced mucin MUC5AC upregulation in vitro and in vivo. Moreover, we found that PLY activates AMPKα1 via cholesterol-dependent membrane binding of PLY and subsequent activation of the Ca2+- Ca2+/calmodulin-dependent kinase kinase ß (CaMKKß) and Cdc42-mixed-lineage protein kinase 3 (MLK3) signaling axis in a TLR2/4-independent manner. AMPKα1 positively regulates PLY-induced MUC5AC expression via negative cross-talk with TLR2/4-dependent activation of MAPK JNK, the negative regulator of MUC5AC expression. Moreover, pharmacological inhibition of AMPKα1 suppressed MUC5AC induction in the S. pneumoniae-induced OM mouse model, thereby demonstrating its therapeutic potential in suppressing mucus overproduction in OM. Taken together, our data unveil a novel mechanism by which negative cross-talk between TLR2/4-independent activation of AMPKα1 and TLR2/4-dependent activation of JNK tightly regulates the S. pneumoniae PLY-induced host mucosal innate immune response.

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.

Cleaved Cochlin: One Guard with Two Roles in Surveillance.

Since the inner ear is an important but sensitive organ of hearing, bacteria-driven immune responses must be tightly controlled. In this issue of Cell Host & Microbe, Jung et al. (2019) demonstrate that cleaved cochlin plays a dual role to trap bacteria and recruit immune cells to improve ear function.

Curcumin Inhibits NTHi-Induced MUC5AC Mucin Overproduction in Otitis Media via Upregulation of MAPK Phosphatase MKP-1.

Otitis media (OM), characterized by the presence of mucus overproduction and excess inflammation in the middle ear, is the most common childhood infection. Nontypeable Haemophilus influenzae (NTHi) pathogen is responsible for approximately one-third of episodes of bacteria-caused OM. Current treatments for bacterial OM rely on the systemic use of antibiotics, which often leads to the emergence of multidrug resistant bacterial strains. Therefore there is an urgent need for developing alternative therapies strategies for controlling mucus overproduction in OM. MUC5AC mucin has been shown to play a critical role in the pathogenesis of OM. Here we show that curcumin derived from Curcuma longa plant is a potent inhibitor of NTHi-induced MUC5AC mucin expression in middle ear epithelial cells. Curcumin inhibited MUC5AC expression by suppressing activation of p38 MAPK by upregulating MAPK phosphatase MKP-1. Thus, our study identified curcumin as a potential therapeutic for inhibiting mucin overproduction in OM by upregulating MKP-1, a known negative regulator of inflammation.

Bifunctional conjugates with potent inhibitory activity towards cyclooxygenase and histone deacetylase.

We herein disclose a series of compounds with potent inhibitory activities towards histone deacetylases (HDAC) and cyclooxygenases (COX). These compounds potently inhibited the growth of cancer cell lines consistent with their anti-COX and anti-HDAC activities. While compound 2b showed comparable level of COX-2 selectivity as celecoxib, compound 11b outperformed indomethacin in terms of selectivity towards COX-2 relative to COX-1. An important observation with our lead compounds (2b, 8, 11b, and 17b) is their enhanced cytotoxicity towards androgen dependent prostate cancer cell line (LNCaP) relative to androgen independent prostate cancer cell line (DU-145). Interestingly, compounds 2b and 17b arrested the cell cycle progression of LNCaP in the S-phase, while compound 8 showed a G0/G1 arrest, similar to SAHA. Relative to SAHA, these compounds displayed tumor-selective cytotoxicity as they have low anti-proliferative activity towards healthy cells (VERO); an attribute that makes them attractive candidates for drug development.

Nontypeable Haemophilus influenzae-Induced MyD88 Short Expression Is Regulated by Positive IKKß and CREB Pathways and Negative ERK1/2 Pathway.

Airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) are characterized by excessive inflammation and are exacerbated by nontypeable Haemophilus influenzae (NTHi). Airway epithelial cells mount the initial innate immune responses to invading pathogens and thus modulate inflammation. While inflammation is necessary to eliminate a pathogen, excessive inflammation can cause damage to the host tissue. Therefore, the inflammatory response must be tightly regulated and deciphering the signaling pathways involved in this response will enhance our understanding of the regulation of the host inflammatory response. NTHi binds to TLR2 and signal propagation requires the adaptor molecule myeloid differentiation factor 88 (MyD88). An alternative spliced form of MyD88 is called MyD88 short (MyD88s) and has been identified in macrophages and embryonic cell lines as a negative regulator of inflammation. However, the role of MyD88s in NTHi-induced inflammation in airway epithelial cells remains unknown. Here we show that NTHi induces MyD88s expression and MyD88s is a negative regulator of inflammation in airway epithelial cells. We further demonstrate that MyD88s is positively regulated by IKKß and CREB and negatively regulated by ERK1/2 signaling pathways. Taken together these data indicate that airway inflammation is controlled in a negative feedback manner involving MyD88s and suggest that airway epithelial cells are essential to maintain immune homeostasis.

Vinpocetine inhibits Streptococcus pneumoniae-induced upregulation of mucin MUC5AC expression via induction of MKP-1 phosphatase in the pathogenesis of otitis media.

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.

CYLD negatively regulates nontypeable Haemophilus influenzae-induced IL-8 expression via phosphatase MKP-1-dependent inhibition of ERK.

Nontypeable Haemophilus influenzae (NTHi), a Gram-negative bacterium, is the primary cause of otitis media in children and the exacerbation of chronic obstructive pulmonary disease in adults. A hallmark of both diseases is an overactive inflammatory response, including the upregulation of chemokines, such as interleukin-8 (IL-8). An appropriate inflammatory response is essential for eradicating pathogens. However, excessive inflammation can cause host tissue damage. Therefore, expression of IL-8 must be tightly regulated. We previously reported that NTHi induces IL-8 expression in an ERK-dependent manner. We also have shown that the deubiquitinase cylindromatosis (CYLD) suppresses NTHi-induced inflammation. However, the underlying molecular mechanism of how CYLD negatively regulates ERK-mediated IL-8 production is largely unknown. Here, we examine both human lung epithelial A549 cells and lung of Cyld-/- mice to show that CYLD specifically targets the activation of ERK. Interestingly, CYLD enhances NTHi-induced upregulation of another negative regulator, MAP Kinase Phosphatase-1 (MKP-1), which, in turn, leads to reduced ERK activation and subsequent suppression of IL-8. Taken together, the CYLD suppression of ERK-dependent IL-8 via MKP-1 may bring novel insights into the tight regulation of inflammatory responses and also lead to innovative therapeutic strategies for controlling these responses by targeting key negative regulators of inflammation.

Inhibition of PDE4B suppresses inflammation by increasing expression of the deubiquitinase CYLD.

The deubiquitinase CYLD acts as a key negative regulator to tightly control overactive inflammation. Most anti-inflammatory strategies have focused on directly targeting the positive regulator, which often results in significant side effects such as suppression of the host defence response. Here, we show that inhibition of phosphodiesterase 4B (PDE4B) markedly enhances upregulation of CYLD expression in response to bacteria, thereby suggesting that PDE4B acts as a negative regulator for CYLD. Interestingly, in Cyld-deficient mice, inhibition of PDE4B no longer suppresses inflammation. Moreover, PDE4B negatively regulates CYLD via specific activation of JNK2 but not JNK1. Importantly, ototopical post-inoculation administration of a PDE4 inhibitor suppresses inflammation in this animal model, thus demonstrating the therapeutic potential of targeting PDE4. These studies provide insights into how inflammation is tightly regulated via the inhibition of its negative regulator and may also lead to the development of new anti-inflammatory therapeutics that upregulate CYLD expression.

Phosphodiesterase 4B mediates extracellular signal-regulated kinase-dependent up-regulation of mucin MUC5AC protein by Streptococcus pneumoniae by inhibiting cAMP-protein kinase A-dependent MKP-1 phosphatase pathway.

Otitis media (OM) is the most common childhood bacterial infection and the major cause of conductive hearing loss in children. Mucus overproduction is a hallmark of OM. Streptococcus pneumoniae is the most common gram-positive bacterial pathogen causing OM. Among many mucin genes, MUC5AC has been found to be greatly up-regulated in the middle ear mucosa of human patients with OM. We previously reported that S. pneumoniae up-regulates MUC5AC expression in a MAPK ERK-dependent manner. We also found that MAPK phosphatase-1 (MKP-1) negatively regulates S. pneumoniae-induced ERK-dependent MUC5AC up-regulation. Therapeutic strategies for up-regulating the expression of negative regulators such as MKP-1 may have significant therapeutic potential for treating mucus overproduction in OM. However, the underlying molecular mechanism by which MKP-1 expression is negatively regulated during S. pneumoniae infection is unknown. In this study we show that phosphodiesterase 4B (PDE4B) mediates S. pneumoniae-induced MUC5AC up-regulation by inhibiting the expression of a negative regulator MKP-1, which in turn leads to enhanced MAPK ERK activation and subsequent up-regulation of MUC5AC. PDE4B inhibits MKP-1 expression in a cAMP-PKA-dependent manner. PDE4-specific inhibitor rolipram inhibits S. pneumoniae-induced MUC5AC up-regulation both in vitro and in vivo. Moreover, we show that PDE4B plays a critical role in MUC5AC induction. Finally, topical and post-infection administration of rolipram into the middle ear potently inhibited S. pneumoniae-induced MUC5AC up-regulation. Collectively, these data demonstrate that PDE4B mediates ERK-dependent up-regulation of mucin MUC5AC by S. pneumoniae by inhibiting cAMP-PKA-dependent MKP-1 pathway. This study may lead to novel therapeutic strategy for inhibiting mucus overproduction.

CYLD negatively regulates transforming growth factor-ß-signalling via deubiquitinating Akt.

Lung injury, whether induced by infection or caustic chemicals, initiates a series of complex wound-healing responses. If uncontrolled, these responses may lead to fibrotic lung diseases and loss of function. Thus, resolution of lung injury must be tightly regulated. The key regulatory proteins required for tightly controlling the resolution of lung injury have yet to be identified. Here we show that loss of deubiquitinase CYLD led to the development of lung fibrosis in mice after infection with Streptococcus pneumoniae. CYLD inhibited transforming growth factor-ß-signalling and prevented lung fibrosis by decreasing the stability of Smad3 in an E3 ligase carboxy terminus of Hsc70-interacting protein-dependent manner. Moreover, CYLD decreases Smad3 stability by deubiquitinating K63-polyubiquitinated Akt. Together, our results unveil a role for CYLD in tightly regulating the resolution of lung injury and preventing fibrosis by deubiquitinating Akt. These studies may help develop new therapeutic strategies for preventing lung fibrosis.

Rb/E2F1 regulates the innate immune receptor Toll-like receptor 3 in epithelial cells.

Tumor suppressor genes regulate the antiviral host defense through molecular mechanisms that are not yet well explored. Here, we show that the tumor suppressor retinoblastoma (Rb) protein positively regulates Toll-like receptor 3 (TLR3) expression, the sensing receptor for viral double-stranded RNA and poly(I · C). TLR3 expression was lower in Rb knockout (Rb(-/-)) mouse embryonic fibroblasts (MEF) and in mammalian epithelial cells transfected with Rb small-interfering RNA (siRNA) than in control cells. Consequently, induction of cytokines interleukin-8 and beta interferon after poly(I · C) stimulation was impaired in Rb(-/-) MEF and Rb siRNA-transfected cells compared to controls. TLR3 promoter analysis showed that Rb modulates the transcription factor E2F1, which directly binds to the proximal promoter of TLR3. Exogenous addition of E2F1 decreased TLR3 promoter activity, while Rb dose dependently curbed the effect of E2F1. Interestingly, poly(I · C) increased the Rb expression, and the poly(I · C)-induced TLR3 expression was impaired in Rb-depleted cells, suggesting the importance of Rb in TLR3 induction by poly(I · C). Together, these data indicated that E2F1 suppresses TLR3 transcription, but during immune stimulation, Rb is upregulated to block the inhibitory effect of E2F1 on TLR3, highlighting a role of Rb-E2F1 axis in the innate immune response in epithelial cells.

Differential regulation of Streptococcus pneumoniae-induced human MUC5AC mucin expression through distinct MAPK pathways.

Human epithelial mucin, the major glycoprotein component of mucus, plays a critical role in host innate defense response against invading microbes by facilitating the mucociliary clearance. Excess mucin production, however, overwhelms the mucociliary clearance, resulting in not only defective mucosal defense but also conductive hearing loss in the middle ear and mucus obstruction in the airway. Indeed, mucus overproduction is a hall-mark of otitis media (OM) and chronic obstructive pulmonary diseases (COPD). Thus, tight regulation of mucin production plays an important role in maintaining an appropriate balance between beneficial and detrimental outcomes. We previously reported that Streptococcus pneumoniae (S. pneumoniae) up-regulates MUC5AC mucin expression via a positive MAPK ERK1/2 and a negative JNK1/2 signaling pathway. However, the signaling components including the up-stream activators and the down-stream transcription factors involved in these two path-ways remain largely unknown. In the present study, we showed that positive regulation of MUC5AC mucin expression by ERK1/2 is dependent on Ras-Raf-1 signaling pathway, whereas the negative regulation of MUC5AC expression by JNK1/2 is dependent on MEKK3. Moreover, transcriptional factor AP-1 acts as a key regulator for both of the positive and negative regulation of MUC5AC mucin expression as evidenced by mutagenesis analysis of two AP-1 sites in the promoter region of human MUC5AC mucin gene. Ras-Raf1-ERK1/2-dependent AP-1 activation positively regulates MUC5AC mucin induction by S. pneumoniae, whereas MEKK3-JNK1/2-dependent AP-1 activation negatively regulates it. Therefore, our data unveiled a novel signaling mechanism underlying the tight regulation of MUC5AC mucin induction by S. pneumoniae and may lead to the development of new therapeutic strategy for reducing mucus overproduction in both OM and COPD.

Synergistic induction of nuclear factor-kappaB by transforming growth factor-beta and tumour necrosis factor-alpha is mediated by protein kinase A-dependent RelA acetylation.

The TGF-beta (transforming growth factor-beta) pathway represents an important signalling pathway involved in regulating diverse biological processes, including cell proliferation, differentiation and inflammation. Despite the critical role for TGF-beta in inflammatory responses, its role in regulating NF-kappaB (nuclear factor-kappaB)-dependent inflammatory responses still remains unknown. In the present study we show that TGF-beta1 synergizes with proinflammatory cytokine TNF-alpha (tumour necrosis factor-alpha) to induce NF-kappaB activation and the resultant inflammatory response in vitro and in vivo. TGF-beta1 synergistically enhances TNF-alpha-induced NF-kappaB DNA binding activity via induction of RelA acetylation. Moreover, synergistic enhancement of TNF-alpha-induced RelA acetylation and DNA-binding activity by TGF-beta1 is mediated by PKA (protein kinase A). Thus the present study reveals a novel role for TGF-beta in inflammatory responses and provides new insight into the regulation of NF-kappaB by TGF-beta signalling.

Glucocorticoids inhibit nontypeable Haemophilus influenzae-induced MUC5AC mucin expression via MAPK phosphatase-1-dependent inhibition of p38 MAPK.

Glucocorticoids are highly effective in the control of many inflammatory and immune diseases. Despite the importance of glucocorticoids in suppressing immune and inflammatory responses, the molecular basis for the inhibitory effect of glucocorticoids on mucin overproduction, a hallmark of chronic respiratory diseases, still remains unclear. Here we show that glucocorticoids markedly inhibit up-regulation of MUC5AC induced by NTHi, a major human bacterial pathogen causing chronic obstructive pulmonary disease and otitis media. Inhibition of NTHi-induced MUC5AC expression by dexamethasone occurs at the level of p38 MAPK via glucocorticoid receptor. Moreover, glucocorticoids up-regulate MKP-1 expression, which in turn leads to p38 dephosphorylation and the subsequent inhibition of NTHi-induced MUC5AC expression. These studies provide new insight into the molecular mechanism underlying glucocorticoid therapy and may lead to novel therapeutic intervention for inhibiting mucin overproduction in patients with NTHi infections.

p53 regulates Toll-like receptor 3 expression and function in human epithelial cell lines.

Toll-like receptors (TLRs) are important sensors of microbial pathogens and mediators of innate immune responses. Although the signal transduction of TLRs is well elucidated, their basal regulation is largely unexplored. Here we show that the tumor suppressor p53 positively regulates the transcription of TLR3, a receptor for viral double-stranded RNA and poly(I-C), by binding to the p53 site in the TLR3 promoter. TLR3 expression was lower in HCT116 p53(-/-) cells than in HCT116 p53(+/+) cells. Activation of p53 by 5-fluorouracil increased the TLR3 mRNA in epithelial cell lines with wild-type p53 but not in cell lines harboring mutant p53. Knockdown of p53 by small interfering RNA decreased the TLR3 expression. TLR3 mRNA was also lower in liver and intestine of p53(-/-) mice than in p53(+/+) mice. Furthermore, the poly(I-C)-induced phosphorylation of IkappaB-alpha, nuclear translocation of NF-kappaB, and phosphorylation of interferon regulatory transcription factor 3, were drastically reduced in HCT116 p53(-/-) cells, indicating a dysregulation of the two signaling pathways governed by TLR3. Consequently, induction of interleukin-8 and beta interferon after poly(I-C) stimulation was impaired in HCT116 p53(-/-) cells. These results suggest that p53 influences TLR3 expression and function and highlight a role of p53 in innate immune response in epithelial cells.