This application is a response to The National Institute on Deafness and Other Communication Disorders (NIDCD) issued PA-09-228 encouraging R01 applications that focus on Proteomics in Auditory Developmental and Disease Processes. Otitis media (OM), the most prevalent chronic childhood disorder, is associated with staggering public health care costs. It is a disease of the middle ear space characterized by acute infectious injury and inflammation, acute OM (AOM), progressing to chronic epithelial mucoid fluid secretion, i.e. chronic OM (COM). The OM continuum from acute to chronic is triggered by infectious or noxious stimuli that result in thickening of middle ear epithelia (MEE) and leads to self-sustaining chronic inflammation and mucous hypersecretion. This in turn often leads to medical treatment failure, and the placement of surgical tympanostomy tubes - the most common surgical procedure of children. In published and preliminary data, we have begun to characterize the molecular progression of OM events. We have recently shown that the hyperviscous mucus in COM is characterized by an overabundance of MUC5B mucin, a major airway secreted mucin. Through a gene expression profiling approach in a mouse model of acute OM based on middle ear inoculation of Non-typeable Hemophilus Influenza (NTHi) (currently the most common human AOM pathogen), we identified the pro-inflammatory cytokine Cxcl2 as markedly and acutely upregulated. Additionally, we observed that the middle ear epithelium chronically trans-differentiated into a thicker, pseudostratified state after repeatd NTHi lysate exposure. Here, we propose an interdisciplinary approach to understanding, and then intervening in, the molecular pathobiology of COM. We hypothesize that AOM, triggered by an inflammatory event (NTHi), activates expression of Cxcl2 and downstream expression of Muc5b/MUC5B, resulting in progression to chronic otitis media.
We first aim to use a proteomic and cytokine secretome profiling approach to determine the in vitro effects of NTHi on a middle ear epithelial pro- inflammatory response, gaining important information that would complement our preliminary in vivo data. Secondly, we will analyze the effects of key inflammatory mediators identified in Aim 1 (predictably Cxcl2 and/or NF?B) on the expression of Muc5b. Finally, we will then aim to intervene at key molecular steps in the process (predictably Cxcl2, and/or NF?B) to halt progression to COM and middle ear Muc5b over-expression. The combination of our findings should result in a better understanding of pathways in OM pathophysiology and are likely to reveal novel molecular therapeutic targets that when modulated may help abate progression of AOM to COM.

Public Health Relevance

Otitis Media (OM) is one of the most frequent disorders in children requiring physician visits. It is a disease of the middle ear space characterized by acute infectious injury and inflammation, acute OM (AOM), progressing to chronic epithelial mucoid fluid secretion, i.e. chronic OM (COM). In this application, using a proteomic profiling approach, we aim to interrogate how pathologically relevant acute infectious stimuli result in a cascade of inflammatory mediator up- regulation which in turn ultimately lead to middle ear epithelial metaplasia and inappropriate over-expression of mucins. Currently no medications exist to treat COM effectively. An understanding of the molecular mechanisms behind the progression of acute middle ear infection to chronic OM may radically change the way the disease is treated, especially if novel molecular targets are identified. Continuation Page

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National Institute on Deafness and Other Communication Disorders (NIDCD)
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Clinical Research and Field Studies of Infectious Diseases Study Section (CRFS)
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Watson, Bracie
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Children's Research Institute
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Val, Stéphanie; Poley, Marian; Anna, Krueger et al. (2018) Characterization of mucoid and serous middle ear effusions from patients with chronic otitis media: implication of different biological mechanisms? Pediatr Res 84:296-305
Val, Stéphanie; Krueger, Anna; Poley, Marian et al. (2018) Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle-ear epithelial cells. FASEB J 32:1855-1867
Val, Stéphanie; Jeong, Stephanie; Poley, Marian et al. (2017) Purification and characterization of microRNAs within middle ear fluid exosomes: implication in otitis media pathophysiology. Pediatr Res 81:911-918
Krueger, Anna; Val, Stéphanie; Pérez-Losada, Marcos et al. (2017) Relationship of the Middle Ear Effusion Microbiome to Secretory Mucin Production in Pediatric Patients With Chronic Otitis Media. Pediatr Infect Dis J 36:635-640
Duah, Vanessa; Huang, Zhen; Val, Stephanie et al. (2016) Younger patients with COME are more likely to have mucoid middle ear fluid containing mucin MUC5B. Int J Pediatr Otorhinolaryngol 90:133-137
Val, Stephanie; Poley, Marian; Brown, Kristy et al. (2016) Proteomic Characterization of Middle Ear Fluid Confirms Neutrophil Extracellular Traps as a Predominant Innate Immune Response in Chronic Otitis Media. PLoS One 11:e0152865
Preciado, Diego; Poley, Marian; Tsai, Stephanie et al. (2016) A proteomic characterization of NTHi lysates. Int J Pediatr Otorhinolaryngol 80:8-16
Val, Stéphanie; Burgett, Katelyn; Brown, Kristy J et al. (2016) SuperSILAC Quantitative Proteome Profiling of Murine Middle Ear Epithelial Cell Remodeling with NTHi. PLoS One 11:e0148612
Val, Stéphanie; Kwon, Hyung-Joo; Rose, Mary C et al. (2015) Middle Ear Response of Muc5ac and Muc5b Mucins to Nontypeable Haemophilus influenzae. JAMA Otolaryngol Head Neck Surg 141:997-1005
Val, Stéphanie; Mubeen, Humaira; Tomney, Amarel et al. (2015) Impact of Staphylococcus epidermidis lysates on middle ear epithelial proinflammatory and mucogenic response. J Investig Med 63:258-66

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