Table 1.
30 most abundant proteins detected by MS/MS*.
Table 2.
Complement and immunoglogulin (Ig) proteins detected by MS/MS.
Table 3.
Proteins implicated in NETs detected by MS/MS.
Fig 1.
A. Visualization of innate immunity and neutrophil markers by confocal microscopy after immunolabelling. Citrullinated H3 (CitH3), Neutrophil elastase (NE), lysozyme (LYZ), S100A8, S100A9, lactotransferrin (LTF), short Palate Lung And Nasal Epithelium Clone Protein (SPLUNC) were labeled using specific antibodies. The DNA was stained using DAPI. Mounted slides were then observed with a confocal microscope. Each row represnets a separate middle ear effusion sample. B. Visualization of NET extracellular DNA association with mucin MUC5B. The DNA was stained using DAPI. MUC5B was found to associate with extravasated, but not nuclear DNA. The top two rows are from one MEE, while the bottom two are from a separate MEE sample.
Table 4.
Summary of IF results.
Fig 2.
A. 10μl of middle ear effusions (MEEs) were loaded in a polyacrylamide gel before electrophoresis. Proteins were transferred on a nitrocellulose membrane and exposed to specific antibodies to reveal proteins of interest (as listed in the figure axis). A consistent presence of typical neutrophil markers was noted across tested samples, validating the proteomic results. Of note, not all mediators were identified in each effusion presumably due to protein degradation. B. 10μl of middle ear effusions (MEEs) were loaded in an agarose gel and electrophoresed before transfer to a PVDF membrane and exposed to aMUC5B antibody, revealing a uniform presence of this mucin glycoprotien across tested MEE samples (as expected). C. 10μl of MEEs, neutrophil lysates (PMNs) or PBS were incubated in Substrate Elastase I for 6 hours. The optical density (O.D.) was then read at 410nm to determine the apparition of the resultant chromophore. This demonstrates increased neutrophil elastase (NE) activity in MEE over background diluant levels.
Fig 3.
Middle ear effusions (MEEs) DNA gel electrophoresis.
DNA of twelve MEEs and one serum sample (considered as a negative control) were extracted with the QiaAmp DNA mini kit. The DNA concentration was measured with the Qubit double stranded DNA kit, and 1μg of each sample (except the serum because of its very low DNA concentration) was loaded in each well as well as 10μl of DNA ladder in a 1% agarose gel containing ethidium bromide. After 2hours of electrophoresis at 100V the gel was observed in a UV box.
Table 5.
Summary of DNA origin in MEE by next generation sequencing.
Fig 4.
Relationships between proteins detected in the middle ear effusions (MEEs) and functions associated, determined by Ingenuity Pathway Analysis (IPA).
A. 10 most abundant proteins realted to the pro-inflammatory cytokine IL-8 and associated molecular and cellular functions. The relationships between IL-8 and the proteins detected in MEE samples are represented by lines and arrows as detailed in the legend. The functions assigned by IPA along with the number of molecules implicated and the p value are listed on top. (LYZ lysozyme; AZU1 azurocidin; PRTN3 proteinase 3; CTSG Cathepsin G; CAMP cAMP receptor protein; MPO myeloperoxidase; LTF lactoferrin; MUC5AC mucin 5AC; MUC5B mucin 5B; MUC2B mucin 2B; ELANE neutrophil elastase; BPIFA1 BPI Fold Containing Family A, Member 1; ITGAM integrin alpha M; SERPINB1 leukocyte elastase inhibitor; S100A8/9 S100 calcium-binding protein A8/9; PIGR Polymeric immunoglobulin receptor; DMBT1 Deleted in Malignant Brain Tumors-1. B. Neutrophil proteins detected in the MEEs centered/focused on ELANE. Relationships between these and other non-neutrophilic proteins are represented by lines an arrows as detailed the legend.
Fig 5.
Inflammatory mediator concentrations in 49 middle ear effusions (MEEs) from COM patients.
A Luminex multiplex assay was performed with 50 different MEEs to determine the concentration of 10 inflammatory mediators: IL1B/6/8/10/13/17A interleukin 1B/6/8/10/13/17A; MDC macrophage derived cytokine; RANTES regulated on activation, normal T cell expressed and secreted; TNFα tumor necrosis factor alpha; VEGF vascular endothelial growth factor. The individual results are represented as shapes of each mediator and the mean concentration is listed below the graph. IL8 concentration was statistically higher by ANOVA (p<0.0001) than the concentration of the other mediators.
Fig 6.
Acute bacterial infection (A) leads to the secretion of factors by the middle ear epithelium (shown by arrows), setting up a primarily neutrophilic inflammatory response, predominantly mediated by the chemotaxis effect of IL8 (B). Recruited neutrophils kill bacteria both through phagocytosis and NETosis (C). The resulting infected fluid can persist and recurrent inflammation results in epithelial mucin glycoprotein overproduction and secretion, predominantly MUC5B, which in turn associates with accumulated NETs (D).