Proteomic Characterization of Middle Ear Fluid Confirms Neutrophil Extracellular Traps as a Predominant Innate Immune Response in Chronic Otitis Media

Abstract

BackgroundChronic Otitis Media (COM) is characterized by middle ear effusion (MEE) and conductive hearing loss. MEE reflect mucus hypersecretion, but global proteomic profiling of the mucosal components are limited.ObjectiveThis study aimed at characterizing the proteome of MEEs from children with COM with the goal of elucidating important innate immune responses.MethodMEEs were collected from children (n = 49) with COM undergoing myringotomy. Mass spectrometry was employed for proteomic profiling in nine samples. Independent samples were further analyzed by cytokine multiplex assay, immunoblotting, neutrophil elastase activity, next generation DNA sequencing, and/or immunofluorescence analysis.Results109 unique and common proteins were identified by MS. A majority were innate immune molecules, along with typically intracellular proteins such as histones and actin. 19.5% percent of all mapped peptide counts were from proteins known to be released by neutrophils. Immunofluorescence and immunoblotting demonstrated the presence of neutrophil extracellular traps (NETs) in every MEE, along with MUC5B colocalization. DNA found in effusions revealed unfragmented DNA of human origin.ConclusionProteomic analysis of MEEs revealed a predominantly neutrophilic innate mucosal response in which MUC5B is associated with NET DNA. NETs are a primary macromolecular constituent of human COM middle ear effusions.

Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle‐ear epithelial cells

“…This miRNA has been postulated to induce apoptosis and cell migration (48). The most abundant miRNA in MEEs, miRNA‐223, was not found in HMEEC exosomes, which reinforces our hypothesis that this miRNA is produced by the abundant neutrophils found in COM effusions (10).…”

“…This inflammation is thought to participate in the conversion of middle‐ear mucosa to one permissive of chronic mucin secretion (9), but the mechanisms driving the remodeling of the middle‐ear epithelium still have to be elucidated. We previously showed the predominance of neutrophil extracellular traps in chronic OM (COM) effusion (10). NETs are characterized by partial decompaction of nuclear DNA, which is then emitted as long filaments into the extracellular space.…”

Younger patients with COME are more likely to have mucoid middle ear fluid containing mucin MUC5B

“…Moreover, it has been postulated that these molecules, particularly MUC5B, may become associated with neutrophil extracellular traps, which are commonly found within middle ear effusion. Presumably, these complexes further contribute to the marked viscosity of the effusions 16 . Finally, some have posited that MUC5AC is secreted earlier upon infectious/inflammatory stimuli, in a more robust fashion, relative to MUC5B which not only responds in a comparatively mutated fashion, but at much later time points after an initial infectious insult 17 .…”

Subversion of host immune responses by otopathogens during otitis media

“…Neutrophils are typically the first responders and form neutrophil extracellular traps (NETs) to kill pathogens. In AOM, animal models demonstrated that NETs and fibrin formation are initial host defenses in middle ear infections . However, a persistence of NETs and fibrin contribute to biofilm formation, and hence chronic disease and effusion .…”

“…This can be attributed to the fact that NETs lead to a thicker effusion, which creates a more fertile ground for biofilm formation . The end result is an association between NETs, COM and middle ear effusions . Respiratory pathogens, such as S. pneumoniae and S. aureus appear to prevent the release of NETs, at least in part due to nucleases impeding antibacterial proteins and breaking down the NETs .…”