SARS-CoV-2-specific T cells are rapidly expanded for therapeutic use and target conserved regions of the membrane protein.

T-cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals. In this report, we demonstrate that SARS-CoV-2-specific T cells can be expanded from convalescent donors and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleocapsid. Following in vitro expansion using a good manufacturing practice-compliant methodology (designed to allow the rapid translation of this novel SARS-CoV-2 T-cell therapy to the clinic), membrane, spike, and nucleocapsid peptides elicited interferon-γ production, in 27 (59%), 12 (26%), and 10 (22%) convalescent donors (respectively), as well as in 2 of 15 unexposed controls. We identified multiple polyfunctional CD4-restricted T-cell epitopes within a highly conserved region of membrane protein, which induced polyfunctional T-cell responses, which may be critical for the development of effective vaccine and T-cell therapies. Hence, our study shows that SARS-CoV-2 directed T-cell immunotherapy targeting structural proteins, most importantly membrane protein, should be feasible for the prevention or early treatment of SARS-CoV-2 infection in immunocompromised patients with blood disorders or after bone marrow transplantation to achieve antiviral control while mitigating uncontrolled inflammation.

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 miRNA-laden 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.

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

BACKGROUND: Chronic 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). METHODS: MEEs 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). RESULTS: Mucoid samples showed a global tendency of increased pro-inflammatory mediators. Interleukin-1ß (IL-1ß) 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. CONCLUSION: This study demonstrates divergent immune responses in children with COME by effusion quality.

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.

Physico-chemical characterization of African urban aerosols (Bamako in Mali and Dakar in Senegal) and their toxic effects in human bronchial epithelial cells: description of a worrying situation.

BACKGROUND: The involvement of particulate matter (PM) in cardiorespiratory diseases is now established in developed countries whereas in developing areas such as Africa with a high level of specific pollution, PM pollution and its effects are poorly studied. Our objective was to characterize the biological reactivity of urban African aerosols on human bronchial epithelial cells in relation to PM physico-chemical properties to identify toxic sources. METHODS: Size-speciated aerosol chemical composition was analyzed in Bamako (BK, Mali, 2 samples with one having desert dust event BK1) and Dakar (DK; Senegal) for Ultrafine UF, Fine F and Coarse C PM. PM reactivity was studied in human bronchial epithelial cells investigating six biomarkers (oxidative stress responsive genes and pro-inflammatory cytokines). RESULTS: PM mass concentrations were mainly distributed in coarse mode (60%) and were impressive in BK1 due to the desert dust event. BK2 and DK samples showed a high content of total carbon characteristic of urban areas. The DK sample had huge PAH quantities in bulk aerosol compared with BK that had more water soluble organic carbon and metals. Whatever the site, UF and F PM triggered the mRNA expression of the different biomarkers whereas coarse PM had little or no effect. The GM-CSF biomarker was the most discriminating and showed the strongest pro-inflammatory effect of BK2 PM. The analysis of gene expression signature and of their correlation with main PM compounds revealed that PM-induced responses are mainly related to organic compounds. The toxicity of African aerosols is carried by the finest PM as with Parisian aerosols, but when considering PM mass concentrations, the African population is more highly exposed to toxic particulate pollution than French population. Regarding the prevailing sources in each site, aerosol biological impacts are higher for incomplete combustion sources resulting from two-wheel vehicles and domestic fires than from diesel vehicles (Dakar). Desert dust events seem to produce fewer biological impacts than anthropogenic sources. DISCUSSION: Our study shows that combustion sources contribute to the high toxicity of F and UF PM of African urban aerosols, and underlines the importance of emission mitigation and the imperative need to evaluate and to regulate particulate pollution in Africa.

Fine PM induce airway MUC5AC expression through the autocrine effect of amphiregulin.

Particulate pollution is suspected to contribute to obstructive lung diseases characterized by chronic inflammation, mucus hypersecretion and bronchial remodeling. Our aim was to study the effect of real-world particulate matter (PM) on the expression of a mucin, MUC5AC, focusing on the role of the epidermal growth factor receptor (EGFR) pathway. MUC5AC induction was studied in vivo in mice trachea and in vitro in human bronchial epithelial cells (HBEC) exposed to urban fine PM. Fine PM were able to induce MUC5AC mRNA in mice trachea after 48 h of exposure (50 µg PM/mouse), and MUC5AC mRNA and protein in HBEC after 24 h of exposure (from 5 µg PM/cm(2)). It was associated with the increased expression of amphiregulin (AREG), an EGFR ligand. Experiments with conditioned media (media from PM-treated cells) demonstrated the involvement of AREG on MUC5AC induction as MUC5AC induction by media from PM-treated cells was prevented in the presence of either EGFR- or AREG-neutralizing antibodies. The effect of an inhibitor of a metalloprotease involved in the AREG shedding confirmed the autocrine loop made by AREG leading to MUC5AC induction by fine PM. We also demonstrated that IL-8 pro-inflammatory cytokine induction was dependent on the same autocrine mechanisms. We demonstrate for the first time that MUC5AC expression and production is increased by short-term exposure to fine PM through an autocrine effect of AREG. Our study provides mechanistic explanations to the exacerbation of obstructive lung diseases induced by particulate pollution characterized by mucus hypersecretion and chronic inflammation.

In vitro assessment of the pulmonary toxicity and gastric availability of lead-rich particles from a lead recycling plant.

Epidemiological studies in urban areas have linked increasing respiratory and cardiovascular pathologies with atmospheric particulate matter (PM) from anthropic activities. However, the biological fate of metal-rich PM industrial emissions in urban areas of developed countries remains understudied. Lead toxicity and bioaccessibility assessments were therefore performed on emissions from a lead recycling plant, using complementary chemical acellular tests and toxicological assays, as a function of PM size (PM(10-2.5), PM(2.5-1) and PM(1)) and origin (furnace, refining and channeled emissions). Process PM displayed differences in metal content, granulometry, and percentage of inhalable fraction as a function of their origin. Lead gastric bioaccessibility was relatively low (maximum 25%) versus previous studies; although, because of high total lead concentrations, significant metal quantities were solubilized in simulated gastrointestinal fluids. Regardless of origin, the finest PM(1) particles induced the most significant pro-inflammatory response in human bronchial epithelial cells. Moreover, this biological response correlated with pro-oxidant potential assay results, suggesting some biological predictive value for acellular tests. Pulmonary effects from lead-rich PM could be driven by thiol complexation with either lead ions or directly on the particulate surface. Finally, health concern of PM was discussed on the basis of pro-inflammatory effects, accellular test results, and PM size distribution.

Role of size and composition of traffic and agricultural aerosols in the molecular responses triggered in airway epithelial cells.

The increased levels of fine particles in the atmosphere are suspected of aggravating cardiopulmonary diseases, but the determinants of particle toxicity are poorly understood. This work aims at studying the role of composition and size in the toxicity of size-segregated particulate matter (PM) collected at different sites on human bronchial epithelial cells. PM were sampled at a traffic urban site (Urb S) and a rural site (Rur S) during the pesticide-spreading period. Ultrafine (UF), fine (F), and coarse (C) PM were characterized by their shape and chemical composition. Whatever the site, the finest PM (UF and F) induced the mRNA expression of CYP1A1, a biomarker of polyaromatic hydrocarbons (PAH) exposure, NQO-1 and heme HO-1, two antioxidant responsive element-driven genes; and two effect biomarkers, GM-CSF, a proinflammatory cytokine and amphiregulin (AR), a growth factor. C PM have a low or no effect. Interestingly, AR is more strongly induced by rural PM at the same mass exposure. These discrepancies suggest involvement of PM chemical composition: rural PM bearing the characteristics of aged aerosols with a high content of water-soluble components, and PM at urban kerbside sites containing mainly water-insoluble components. To conclude, we provide evidence that the finest PM fractions, whatever their origin, are more prone to induce exposure and effect biomarkers. The AR differential expression suggests a source-dependent effect requiring further investigation because of the role of this growth factor in airway remodeling, a characteristic feature of chronic lung respiratory diseases exacerbated by particulate pollution.

MUC5B induces in vitro neutrophil extracellular trap formation: Implication in otitis media.

BACKGROUND: Chronic otitis media (COM) is characterized by middle ear fluid predominantly containing cytokines, Nontypeable haemophilus influenzae (NTHi), the mucin MUC5B, and neutrophil extracellular traps (NETs). NETs consist of extracellular DNA coated with antibacterial proteins such as myeloperoxidase (MPO) and citrullinated histone 3 (CitH3). NETs can damage tissues and sustain inflammation. Our study aimed to develop an in vitro model of NETosis, testing COM inductors. METHODS: NETosis was evaluated in fresh blood human neutrophils attached to collagen-coated plates and in suspension exposed to phorbol myristate acetate (PMA) as a control, and COM relevant mediators. Confocal microscopy, DNA fluorescence assay and flow cytometry were used to quantify NETosis. RESULTS: PMA exposure induced DNA, MPO, and CitH3 by immunofluorescence (IF) most significantly at 3 hours (3.8-fold for DAPI, 7.6-fold for MPO, and 6.9-fold for CitH3, all P < .05). IL-8 and TNF-α cytokines showed milder increases of DAPI, MPO, and CitH3 positive cells. NTHi had no effect on these NETs markers. Purified salivary MUC5B (10 to 40 µg/mL) produced potent increases, comparable to PMA. A composite NET score summing the fold-increases for DAPI, MPO, and CitH3 demonstrated PMA at 13.6 to 19 relative to control set at 1; and MUC5B at 8.6 to 16.3 (all P < .05). IL-8 and TNF-α showed scores of 5.4 and 3, respectively, but these were not statistically significant. CONCLUSION: We developed a reliable in vitro assay for NETosis which demonstrated that salivary MUC5B is a potent inductor of NETs whereas IL-8, TNF-α, live and lyzed NTHi demonstrated minimal to no NETosis. LEVEL OF EVIDENCE: NA.

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.

Carbon black and titanium dioxide nanoparticles induce pro-inflammatory responses in bronchial epithelial cells: need for multiparametric evaluation due to adsorption artifacts.

The initiation of an inflammatory process is the main adverse effect observed following the exposure of the airway epithelium to nanoparticles (NPs). This study was designed to explore the pro-inflammatory potential of two different NPs of similar size but of different compositions (CB 13 nm and TiO(2) 15 nm) on a human bronchial epithelial cell line (16HBE14o-). The expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL-6), and tumor necrosis factor alpha (TNFalpha) was evaluated in terms of mRNA, intracellular proteins, and released cytokines. Exposure to NPs induced a dose-dependent expression of all these cytokines, depending upon the chemical composition of NPs. The released cytokines appeared to be an inaccurate methodology to evaluate the pro-inflammatory response. Indeed, NPs adsorbed cytokines, and the binding was dependent on the nature of both the cytokine and NPs. Furthermore, addition of fetal calf serum or bovine serum albumin improved the detection of cytokines but also reduced cellular responses. Use of different detergents (Tween, Triton, and NP40) demonstrated limited efficiency to desorb cytokines from NPs. Thus, this study demonstrated the pro-inflammatory potential for CB and TiO(2) NP but underlines the methodological artifacts faced during the in vitro evaluation of cytokine release that necessitates a multiparametric evaluation.

Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media.

OBJECTIVES: Otitis media (OM) is a ubiquitous pediatric disease leading to a significant health care burden. There is no medication beneficial to resolving COM fluid, highlighting the need for research in the field. Crucially, current human middle ear epithelial cell models are transformed cells not recapitulating physiological functions. Herein, we describe a new method to proliferate and differentiate pediatric primary middle ear epithelial cells (pMEEC) from patients as a physiological model for the study of OM. METHODS: We adapted a cell reprogramming protocol using irradiated fibroblast feeder medium in addition to Rho kinase inhibitor to proliferate pMEEC collected during cochlear implant surgery. Cells were plated on transwell membranes, proliferated with conditionally reprogrammed culture medium, and transferred to air-liquid interface (ALI). Cultures were maintained for 4 weeks at ALI, photos were taken and cell lysates and secretions were collected over time for characterization analysis using quantitative polymerase chain reaction, Western bolt, and proteomics. Keratins, MUC5B and MUC5AC mucins, and beta tubulin (TUBB) were analyzed at the mRNA and protein level. RESULTS: Cultures took a mean of 2 weeks to proliferate before transwell plating and forming a tight epithelium at ALI from 2 to 4 weeks. Although mRNA expression of MUC5B, MUC5AC, TUBB, and keratin 5 (KRT5) were variable depending on the differentiation stage and the patient, both TUBB and KRT5 proteins were detected until week 2. CONCLUSION: We demonstrate a novel method to proliferate and differentiate pMEECs that express epithelial markers and that are able to secrete mucins for the study of OM. LEVEL OF EVIDENCE: NA.

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.

Panel 8: Report on Recent Advances in Molecular and Cellular Biochemistry.

Objectives To update the medical literature on recent cellular and molecular advances in otitis media disease models with a principal focus on developments in the past 5 years. We also aim to explain recent translational advances in cellular and molecular biology that have influenced our understanding and management of otitis media. Data Sources PubMed-indexed peer-reviewed articles. Review Methods A comprehensive review of the literature was conducted with the term otitis media and the following search terms: molecular biology, cell biology, innate immunity, oxidative stress, mucins, molecular diagnostics. Included articles were published in the English language from January 1, 2010, to July 31, 2015. Implications for Practice The molecular understanding of otitis media disease progression has rapidly advanced over the last 5 years. The roles of inflammation, mucins, and cell signaling mechanisms have been elucidated and defined. Advances in the field provide a plethora of opportunities for innovative molecular targeting in the development of novel therapeutic strategies for otitis media.

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.

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

BACKGROUND: Chronic otitis media (COM) is one of the most common childhood diseases. Its pathophysiology is complex and multifactorial. The role of specific mucin glycoprotein subtypes in OM is only recently being elucidated. OBJECTIVE: To determine the relationship between middle ear fluid mucins and clinical variables of patients needing tympanostomy tubes (TT). METHODS: Middle ear effusions (MEE) from children receiving TT were collected over a 2-year period. Western blot characterization of mucins MUC5B and MUC5AC along with chart review of age, gender, effusion viscosity, hearing loss >30 dB, history of allergies, and/or respiratory disease were performed. RESULTS: MEE samples from 58 patients were available for analysis. Overall, MUC5B was significantly more often detected in middle ear fluid relative to MUC5AC (90% vs. 51%, p = 0.005). MUC5B presence was statistically associated with mucoid effusions relative to serous effusions (100% vs. 57%, p = 0.0064), MUC5AC presence was not significantly different in mucoid and serous fluid (55.1% vs. 37.5%, p = 0.447). Patients younger than 48 months were more likely to present with mucoid effusion, compared to those older than 48 months of age (p = 0.038). Finally, patients with effusions positive for MUC5B were younger than those with effusions negative for MUC5B (35.1 vs. 76 months, p = 0.045). No other variables correlated to either effusion viscosity or specific mucin content. CONCLUSION: Patients younger in age needing TT placement are more likely to present with mucoid effusions, predominantly containing MUC5B mucin. As such, we postulate a distinct pathophysiology for mucoid and serous effusions across ages in children with COM.

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.

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.