Complement activation in pediatric patients with recurrent acute otitis media

Abstract

Objective: Otitis media (OM) is one of the most common childhood diseases. The relative contribution of complement activation in protection and pathogenesis during OM remains largely unknown. The purpose of this study was to investigate the beneficial and pathogenic contributions of complement activation in the middle ear of pediatric patients with recurrent acute otitis media (rAOM), and therefore to provide a rational approach to prevent sequelae of OM such as hearing loss.

Methods: Twenty children undergoing pressure equalization tube placement with or without adenoidectomy for rAOM were enrolled in the study. Bacterial cultures, enzyme-linked immunosorbent assay (ELISA) for complement components and cytokines and western blot for complement activation were performed on middle ear effusion (MEE) and serum samples. The levels of complement C3a, C5a and sC5-b9 in MEEs and serum samples were compared. The levels of these factors were also examined in regards to length of episode. Pearson's correlation coefficients were calculated on variables between C5a and IL-6 or IL-8. Complement gene expression in human middle ear epithelial (HMEE) cells induced by otopathogens was evaluated. Data were analyzed with Student's t test or the Mann-Whitney rank sum test. In all cases, a P value of <0.05 was set as the measure of significance.

Results: Our data demonstrated that the complement classical/lectin, alternative and terminal pathways were activated in the middle ear of children with rAOM. Increased complement components of C3a, C5a and sC5-b9 in MEEs were detected in patients with the episode lasting more than six weeks. There was a strong correlation between C5a and IL-6 or IL-8 in the MEEs. Additionally, otopathogens induced enhanced gene expression of factor B and C3 in HMEE cells, which is beneficial for host defense against invading pathogens.

Conclusion: Our studies provided important new insights on how complement activation contributes to inflammatory process during rAOM. Knowledge of the activity of the complement pathway in patients with rAOM may stimulate the development of new strategies to prevent middle ear inflammatory tissue destruction by directing treatment to specific pathways within the complement cascade.

Fig. 1

Factor B and C3 activation in MEEs collected from seven patients. (A) Factor B of the alternative pathway is activated in MEEs. Factor B fragments Bb and Ba were detected in MEEs by western blot. (B) C3 activation in MEEs. C3 fragments in MEEs detected by western blot. (C) C4a in MEEs (79.1± 16.2 ng/ml) was 1.2 log lower than that in serum samples (972.3± 47.6 ng/ml). (D) C3a in MEEs (33,000 ± 10,220 ng/ml) was 1.1 log higher than that in sera samples (2,800 ± 162 ng/ml), *, p<0.001.

Fig. 2

The terminal pathway is activated in MEEs. (A) C5a concentration in MEEs (84 ± 6.6 ng/ml) was significantly higher than in sera samples (19 ± 1.8 ng/ml), *, P < 0.01. (B) sC5b-9 concentration in MEEs (9600 ± 1,330 ng/ml) was significantly higher than in sera samples (756 ± 148 ng/ml), *, P < 0.01. (C and D) Pearson correlation analysis between C5a and IL8 and IL-6.

Fig. 3

Gene expression of complement factor B and C3. In HMEE cells. Induction of gene expression as measured by real-time PCR on total RNA samples of HMEE cells stimulated with otopathogens and TNFα. Results are the mean fold changes the transcript levels (± SEM) from two separate experiments. *, P < 0.05 compared with the values determined for the medium control cohorts.

Fig. 4

Protein production of complement factor B and C3 in HMEE cells. (A) Concentrations of C3 in the HMEE cell culture supernatant. Results are the mean concentration of C3 (± SEM) from two separate experiments. *P < 0.05 for the comparison with each medium control cohorts. (B) Levels of factor B protein and factor B fragments in HMEE cells infected with killed Spn, IAV or the combined infection. Approximately 30 μg of protein samples was subjected to SDS-PAGE. Bb and Ba were detected in HMEE cell lysates upon infection. Factor B was secreted into the supernatants upon infection. A representative of two experiments is shown. (C and D) Increased C3 and factor B immunofluorescence staining in HMEE cells at 24 h post infection with killed Spn compared with the sham control cohorts. Images are representative of three independent studies.