Stéphanie Val

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

Background Chronic 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. Objective This study aimed at characterizing the proteome of MEEs from children with COM with the goal of elucidating important innate immune responses. Method MEEs 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. Results 109 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. Conclusion Proteomic 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.

Airway Secretory microRNAome Changes during Rhinovirus Infection in Early Childhood.

Background Innate immune responses are fine-tuned by small noncoding RNA molecules termed microRNAs (miRs) that modify gene expression in response to the environment. During acute infections, miRs can be secreted in extracellular vesicles (EV) to facilitate cell-to-cell genetic communication. The purpose of this study was to characterize the baseline population of miRs secreted in EVs in the airways of young children (airway secretory microRNAome) and examine the changes during rhinovirus (RV) infection, the most common cause of asthma exacerbations and the most important early risk factor for the development of asthma beyond childhood. Methods Nasal airway secretions were obtained from children (≤3 yrs. old) during PCR-confirmed RV infections (n = 10) and age-matched controls (n = 10). Nasal EVs were isolated with polymer-based precipitation and global miR profiles generated using NanoString microarrays. We validated our in vivo airway secretory miR data in an in vitro airway epithelium model using apical secretions from primary human bronchial epithelial cells (HBEC) differentiated at air-liquid interface (ALI). Bioinformatics tools were used to determine the unified (nasal and bronchial) signature airway secretory miRNAome and changes during RV infection in children. Results Multiscale analysis identified four signature miRs comprising the baseline airway secretory miRNAome: hsa-miR-630, hsa-miR-302d-3p, hsa- miR-320e, hsa-miR-612. We identified hsa-miR-155 as the main change in the baseline miRNAome during RV infection in young children. We investigated the potential biological relevance of the airway secretion of hsa-mir-155 using in silico models derived from gene datasets of experimental in vivo human RV infection. These analyses confirmed that hsa-miR-155 targetome is an overrepresented pathway in the upper airways of individuals infected with RV. Conclusions Comparative analysis of the airway secretory microRNAome in children indicates that RV infection is associated with airway secretion of EVs containing miR-155, which is predicted in silico to regulate antiviral immunity. Further characterization of the airway secretory microRNAome during health and disease may lead to completely new strategies to treat and monitor respiratory conditions in all ages.

Middle Ear Response of Muc5ac and Muc5b Mucins to Nontypeable Haemophilus influenzae.

IMPORTANCE Chronic otitis media with effusion is characterized by middle ear secretion of mucin glycoproteins, predominantly MUC5B; MUC5AC, the other secretory mucin studied frequently, has also been identified in the middle ear. Emerging evidence suggests a dichotomous role for these mucins in innate immune responses. We hypothesized that MUC5AC is an acute responder and MUC5B is expressed at later time points, reflecting a chronic situation. OBJECTIVE To determine middle ear regulation of MUC5B and MUC5AC following in vitro bacterial and cytokine exposure. DESIGN, SETTING, AND SAMPLES An in vitro cell-based model of mucin gene regulation was conducted in a basic science laboratory at a tertiary pediatric hospital. The study was conducted from July 1, 2014, to June 30, 2015; data analysis was performed in July 2015. INTERVENTIONS Nontypeable Haemophilus influenzae (NTHi) lysates were generated and used to stimulate mouse middle ear epithelial cells (mMEECs) for 2 hours during 3 weeks. MAIN OUTCOMES AND MEASURES Real-time quantitative polymerase chain reaction, luciferase assays, Western blot assay, and immunofluorescence techniques were performed to determine Muc5ac and Muc5b expression over time, Cxcl2 chemokine response, and nuclear factor-κB activation. Luciferase reporter assays were performed to evaluate specific promoter responses after NTHi exposure. RESULTS Nontypeable H influenzae lysates (200 μg/mL) drove differential mucin gene activation, with Muc5ac being induced up to 2.04 fold at 24 hours and 2.79 fold at 96 hours (P < .05) and Muc5b being induced only at more long-term points: 1.61 fold at 96 hours, 1.41 fold at 1 week, and 1.53 fold at 3 weeks (P < .05). Although NTHi lysates induced robust, early nuclear factor-κB nuclear translocation with nuclear factor-κB-dependent induction of Cxlc2 expression, the lysates had minimal to no effect on Muc5ac and Muc5b promoter activity. However, in contrast to NTHi lysates, CXCL2 induced significant transcription of both Muc5b and Muc5ac as early as 24 hours. CONCLUSIONS AND RELEVANCE Nontypeable H influenzae lysates activate differential mucin gene activation in mMEECs. Although Muc5ac is an early response mucin gene, Muc5b appears to react as a chronic response mucin.

Impact of Staphylococcus Epidermidis Lysates on Middle Ear Epithelial Proinflammatory and Mucogenic Response

Background Chronic otitis media with effusion (COME) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Staphylococcus epidermidis, typically considered a commensal organism, is very frequently recovered in chronic middle ear fluid and in middle ear biofilms. Although it has been shown to drive inflammation in sinonasal epithelium, the impact of S. epidermidis on COME is markedly understudied. The goal of this study was to examine the in vitro effects of S. epidermidis lysates on murine and human middle ear epithelial cells. Methods Staphylococcus epidermidis lysates were generated and used to stimulate submerged and differentiated human and murine epithelial cells (MEECs) for 24 to 48 hours. Quantitative real time-polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, and immunocytochemistry techniques were performed to interrogate the mucin gene MUC5AC and MUC5B expression and protein production, chemokine response, as well as NF-κB activation. Luciferase reporter assays were performed to further evaluate nuclear factor κB (NF-κB) activation and query specific promoter responses after S. epidermidis exposure. Results Staphylococcus epidermidis induced a time- and dose-dependent MUC5AC and MUC5B overexpression along with a parallel overexpression of Cxcl2 in mouse MEEC and IL-8 in human MEEC. Further investigations in mMEEC showed a 1.3 to 1.5 induction of the MUC5AC and MUC5B promoters. As potential mechanisms for these responses, induction of an oxidative stress marker, along with early nuclear translocation and activation of NF-κB, was found. Finally, chronic exposure induced marked epithelial thickening of cells differentiated at the air liquid interface. Conclusions Staphylococcus epidermidis lysates activate a proinflammatory response in MEEC, including mucin gene expression and protein production. Although typically considered a nonpathogenic commensal organism in the ear, these results suggest that they may play a role in the perpetuation of an inflammatory and mucogenic response in COME.

Relationship of the Middle Ear Effusion Microbiome to Secretory Mucin Production in Pediatric Patients With Chronic Otitis Media.

Background: Acute otitis media, an infection of the middle ear, can become chronic after multiple episodes. Microbial influence on chronic otitis media remains unclear. It has been reported that mucin glycoproteins are required for middle ear immune defense against pathogens. We aim to characterize the middle ear effusion (MEE) microbiome using high-throughput sequencing and assess potential associations in microbiome diversity with the presence of the secretory mucins MUC5B and MUC5AC. We hypothesize that MEEs containing MUC5B will exhibit a microbiome largely devoid of typical acute otitis media bacteria. Methods: Fifty-five MEEs from children undergoing myringotomy at Children’s National Health System were recovered. Mucin was semiquantitatively determined through Western blot analysis. DNA was subjected to 16S rRNA amplicon sequencing using the Illumina MiSeq platform. Raw data were processed in mothur (SILVA reference database). Alpha- and beta-diversity metrics were determined. Abundance differences between sample groups were estimated. Results: MUC5B was present in 94.5% and MUC5AC in 65.5% of MEEs. Sequencing revealed 39 genera with a relative abundance ≥0.1%. Haemophilus (22.54%), Moraxella (11.11%) and Turicella (7.84%) were the most abundant. Turicella and Pseudomonas proportions were greater in patients older than 24 months of age. In patients with hearing loss, Haemophilus was more abundant, while Turicella and Actinobacteria were less abundant. Haemophilus was also more abundant in samples containing both secretory mucins. Conclusions: The microbiome of MEEs from children with chronic otitis media differs according to specific clinical features, such as mucin content, age and presence of hearing loss. These associations provide novel pathophysiologic insights across the spectrum of otitis media progression.

SuperSILAC Quantitative Proteome Profiling of Murine Middle Ear Epithelial Cell Remodeling with NTHi

Background Chronic Otitis Media with effusion (COME) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Non-typeable Haemophilus influenzae (NTHi), the most common acute Otitis Media (OM) pathogen, is postulated to promote middle ear epithelial remodeling in the progression of OM from acute to chronic. The goals of this study were to examine histopathological and quantitative proteomic epithelial effects of NTHi challenge in a murine middle ear epithelial cell line. Methods NTHi lysates were generated and used to stimulate murine epithelial cells (mMEEC) cultured at air-liquid interface over 48 hours– 1 week. Conditional quantitative Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) of cell lysates was performed to interrogate the global protein production in the cells, using the SuperSILAC technique. Histology of the epithelium over time was done to measure bacterial dependent remodeling. Results Mass spectrometry analysis identified 2,565 proteins across samples, of which 74 exhibited differential enrichment or depletion in cell lysates (+/-2.0 fold-change; p value<0.05). The key molecular functions regulated by NTHi lysates exposure were related to cell proliferation, death, migration, adhesion and inflammation. Finally, chronic exposure induced significant epithelial thickening of cells grown at air liquid interface. Conclusions NTHi lysates drive pathways responsible of cell remodeling in murine middle ear epithelium which likely contributes to observed epithelial hyperplasia in vitro. Further elucidation of these mediators will be critical in understanding the progression of OM from acute to chronic at the molecular level.

A proteomic characterization of NTHi lysates

BACKGROUND Non-typeable Haemophilus influenzae (NTHi) is a ubiquitous bacterial pathogen which accounts for a majority of human upper respiratory tract infections. Laboratory lysate preparations from this bacterium are commonly utilized to investigate the promulgation of inflammatory responses in respiratory and middle ear epithelium both in vivo and in vitro. We undertook an unbiased proteomics based analysis of NTHi lysate preps to: (a) identify abundant bacterial proteins present in these lysates that could play a role in NTHi biological effects and (b) determine the protein content variability in different lysate prep batches from the same NTHI strain. STUDY DESIGN Proteomic analysis of laboratory NTHi lysate preparations from clinical strain 12. METHODS NTHi lysates were denatured, gel-fractionated, digested by trypsin and the generated peptides were identified using a liquid chromatography tandem mass spectrometry (LC-MS/MS). Western blot analyses for the important proinflammatory enhancer, outer membrane protein 6 (OMP6), was performed to validate the MS findings. Luciferase assays for NF-kB activation were used to measure the pro-inflammatory biologic effects from each NTHi lysate preparation. RESULTS The MS identified 793 unique NTHi proteins. Most common and abundant proteins found have been described to either contribute to biofilm formation, elude the innate immune system, or activate epithelial pro-inflammatory pathways such as Toll Like Receptor 2 (TLR-2) signaling and NF-kB transcription factor. Strong positive signal for OMP6 was found in each of the NTHi lysate preparations. Significant NF-kB promoter response activation as expected with NTHi stimulation over control was also noted for each NTHi lysate preparation. CONCLUSIONS Proteomics was a successful technique to broadly define the protein content of NTHi lysates. This is the first report of the proteome of NTHi lysates widely used in laboratories to study the biological effect of NTHi. Despite the variability of the protein composition from different preps, all the batches of NTHi lysates induced similar NFκB activation. LEVEL OF EVIDENCE NA.

Purification and characterization of microRNAs within middle ear fluid exosomes: implication in otitis media pathophysiology.

Background:Otitis media (OM) is characterized by acute infection progressing to chronic middle ear effusion (MEE). Extracellular secretion of microRNAs (miRNAs) in exosomes is a newly discovered mechanism for cells exerting distant cell genetic regulation. Whether MEE contains exosomes with specific miRNAs is unknown. This study aimed to purify and characterize the exosomal and miRNA content of MEE.Method:MEEs were subjected to Exoquick exosomal purification and EXOCET exosomal quantification. Extracted vesicles were analyzed by dynamic light scattering (DLS), transmission electron microscopy (TEM), and immunoblotting of HSP-70. NanoString hybridization was performed to profile miRNAs. Exosomal protein content was profiled by Liquid chromatography tandem mass spectrometry (LC-MS/MS).Results:EXOCET assays showed presence of exosomes (0–0.5 × 107/ml) in MEEs. DLS confirmed exosomal size between 10 and 200 nm. Western blot analysis showed presence of HSP-70. Twenty-nine miRNAs were found to be unique to MEEs. The most abundant miRNA was miR-223, a miRNA typically secreted by neutrophils. Proteomics demonstrated typical neutrophil markers as well as common innate immune molecules. Conclusion:To our knowledge, this the first report demonstrating the presence of exosomes transporting miRNAs in MEEs. These findings open a broad and novel area of research in OM pathophysiology as driven by miRNA cell communication.

Characterization of mucoid and serous middle ear effusions from patients with chronic otitis media: implication of different biological mechanisms?

BackgroundChronic otitis media with effusion (COME) is characterized by persistent middle ear effusions that are in most cases highly viscous, but some patients present with serous fluid. This study aimed at comprehensively characterizing the macromolecular composition of mucoid vs. serous middle ear effusions (MEEs).MethodsMEEs from patients with COME were analyzed for proteins by mass spectrometry (MS) and western blot techniques, total DNA quantity, bacterial DNA (16S sequencing), and cytokine content. Proteomics datasets were studied in Ingenuity Pathway Analysis (IPA).ResultsMucoid samples showed a global tendency of increased pro-inflammatory mediators. Interleukin-1β (IL-1β) and IL-10 were significantly more abundant in serous samples (p < 0.01). Mucoid samples had higher DNA quantity (p = 0.04), more likely to be positive in MUC5B protein (p = 0.008) and higher peptide counts (12,786 vs. 2225), as well as an overall larger number of identified proteins (331 vs. 177), compared to serous. IPA found the mucoid sample dataset to be related to immune cell function and epithelial remodeling, whereas the serous sample dataset showed acute responses and blood-related proteins. Interestingly, serous samples showed more bacterial DNA than mucoid ones, with less bacterial genera variability.ConclusionThis study demonstrates divergent immune responses in children with COME by effusion quality.

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

Nontypeable Haemophilus influenzae (NTHi), one of the most common acute otitis media (OM) pathogens, is postulated to promote middle‐ear epithelial remodeling in the progression of OM from acute to chronic. The goal of this study was to examine early quantitative proteomic secretome effects of NTHi lysate exposure in a human middle‐ear epithelial cell (HMEEC) line. NTHi lysates were used to stimulate HMEEC, and conditional quantitative stable isotope labeling with amino acids in cell culture of cell secretions was performed. Mass spectrometry analysis identified 766 proteins across samples. Of interest, several heterogeneous nuclear ribonucleoproteins (hnRNPs) were regulated by NTHi lysate treatment, especially hnRNP A2B1 and hnRNP Q, known to be implicated in microRNA (miRNA) packaging in exosomes. After purification, the presence of exosomes in HMEEC secretions was characterized by dynamic light scattering (<100 nm), transmission electron microscopy, and CD63/heat shock protein 70 positivity. hnRNP A2B1 and hnRNP Q were confirmed to be found in exosomes by Western blot and proteomic analysis. Finally, exosomal miRNA content comprised 110 unique miRNAs, with 5 found to be statistically induced by NTHi lysate (miR‐378a‐3p+miR‐378i, miR‐200a‐3p, miR‐378g, miR30d‐5p, and miR‐222‐3p), all known to target innate immunity genes. This study demonstrates that NTHi lysates promote release of miRNAladen exosomes from middle‐ear epithelium in vitro.— Val, S., Krueger, A., Poley, M., Cohen, A., Brown, K., Panigrahi, A., Preciado, D. Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle‐ear epithelial cells. FASEB J. 32, 1855–1867 (2018). www.fasebj.org