Proteomic Study Identifies Glycolytic and Inflammation Pathways Involved in Recurrent Otitis Media

Abstract

Recurrent acute otitis media (RAOM) in children is clinically defined as the occurrence of at least three episodes of acute otitis media over a course of 6 months. A further common pathological condition of interest in the context of pediatric otolaryngology is adenotonsillar hypertrophy (ATH), a common cause of obstructive sleep apnea syndrome. Aimed at unraveling the differential modulation of proteins in the two pathologies and at understanding the possible pathways involved in their onset, we analyzed the proteomic profile of the adenoids from 14 RAOM and ATH patients by using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The 2-DE coupled with MS allowed us to identify 23 spots with significant (p-value < 0.05) changes in protein amount, recognizing proteins involved in neutrophil degranulation and glycolysis pathways.

Keywords: 2-DE; adenotonsillar hypertrophy; proteomics; recurrent otitis.

Figure 1

Two dimensional electrophoresis map of the adenotonsillar hypertrophy (ATH) and recurrent acute otitis media (RAOM) proteome. Immobilized pH gradient pH 3–10 non-linear strips were used for the first dimension and 12% polyacrylamide gels were used for the second dimension. Number correspond to different proteins identified in Table 1.

Figure 2

PANTHER classification of differently regulated proteins in RAOM in according to their biological processes.

Figure 3

PANTHER classification of differently regulated proteins in RAOM in according to their molecular function.

Figure 4

PANTHER classification of differently regulated proteins in RAOM in accordance to their protein class.

Figure 5

PANTHER classification of differently regulated proteins in RAOM in according to their pathway classification.

Figure 6

Representative Western blotting analysis of VCP and ENO1 in ATH and RAOM. The intensities of the immunostained bands were normalized with the protein intensities measured by Red Ponceau from the same blot. The bar graph shows the relative quantitation (band density) of VCP and ENO1 in ATH and RAOM. Results are shown as a histogram (* indicates p < 0.05, while ** indicates p < 0.01 statistical difference) and each bar represents mean ± standard error.

Figure 7

Schematic diagram displaying putative pathogenic mechanisms In ROAM (left side): bacterial infections (1) triggers neutrophils degranulation (2), but this is not effective due to lower PKM and HBB levels (black arrow downwards). Defective bacterial clearing (3) causes chronic infection (through formation of biofilm) in the median ear, with a consequent increase of NETs formation (4), due to higher proteasome activity provoked by increased VCP and CAP1 levels (black arrow upwards). Chronic bacterial infection of ear tissue (5) (red) establishes a milieu in which recurrent infections (6) are facilitated. In ATH (right side), TH-17 cells are activated (1) and switch to glycolytic metabolism (gray square), with increased levels of ALDOC and ENO1 (black arrow upwards). Activation of these cells causes increasing Interleukin-17 (IL-17) (2) production. This cytokine (3) could drive epithelial expression of granulopoietic and chemotactic factors such as Interleukin-8 (IL-8), Granulocyte Colony-Stimulating Factor (G-CSF) and Macrophage Inflammatory Proteins (MIP) (big arrow downwards) that could induce swelling of adenoidal tissue (4) (green), followed by upper airway obstruction. The imperfect oxygen intake could then trigger a self-sustained cycle (5) (dotted arrow), inducing furthermore a switch toward glycolytic metabolism (created with BioRender.com).