Clin Exp Otorhinolaryngol.  2008 Dec;1(4):189-195.

Toll-like Receptors 2 and 4 and Their Mutations in Patients with Otitis Media and Middle Ear Effusion

• Lee YC
• Kim C
• Shim JS
• Byun JY
• Park MS
• Cha CI
• Kim YI
• Lee JW
• Yeo SG

Abstract

OBJECTIVES
Toll-like receptors (TLRs) detect microbial infections and they can directly induce innate host defense responses. TLR 2 has been shown to be primarily involved in the recognition of peptidoglycans and lipoteichoic acid of gram positive bacteria. TLR 4 recognizes lipopolysaccharides and lipoteichoic acids from both gram-negative and gram-positive bacteria. Both mutations lead a reduced capacity to elicit inflammation and they increase the risk for gram-positive and negative infections. This study was performed to investigate the expressions of TLR 2 and 4 and their mutations in patients suffering with otitis media and middle ear effusion. METHODS: Middle ear fluid samples were collected from 40 otitis media effusion (OME) patients who had ventilating tubesinserted. Bacteria in the effusion fluid were detected by standard bacterial culture. The secreted IgG, IgA and IgM were measured by Enzyme-linked immunosorbent assay. TLR 2 and 4 were assessed by performing RT-PCR. The genomic DNA from each patient was isolated from the middle ear fluid samples that were collected from 60 OME patients, and the presence of mutations was determined by performing restriction digestion and DNA sequencing analysis. RESULTS: Among the 40 middle ear fluid samples, bacteria were detected in 13 middle ear fluid samples. The amounts of IgM, IgA, and IgG were 151.20+/-60.94 ng/mL, 21.59+/-7.96 ng/mL and 11.55+/-16.98 ng/mL, respectively. TLR 2 and 4 were expressed in the middle ear fluid and the expression of TLR 2 was higher than that of TLR 4. However, there was no correlation between the expressions of TLR 2 and 4, and the concentration of immunoglobulin or the presence of bacteria (P>0.05). There ware no mutations of TLR 2 (Arg753Gln, Arg677Trp) and TLR 4 (Asp299Gly, Thr399Ile). CONCLUSION: TLR 2 and 4 were expressed in all the middle ear fluid samples of OME, but the mutations of TLR 2 and 4 were not detected. TLR 2 and 4 may play a vital role in the immunological responses of patients with OME.

Keyword

Otitis media; Toll-like receptor; Mutation

MeSH Terms

Bacteria
Digestion
DNA
Ear, Middle
Enzyme-Linked Immunosorbent Assay
Gram-Positive Bacteria
Humans
Immunoglobulin A
Immunoglobulin G
Immunoglobulin M
Immunoglobulins
Inflammation
Lipopolysaccharides
Otitis
Otitis Media
Otitis Media with Effusion
Peptidoglycan
Sequence Analysis, DNA
Stress, Psychological
Teichoic Acids
Toll-Like Receptors
DNA
Immunoglobulin A
Immunoglobulin G
Immunoglobulin M
Immunoglobulins
Lipopolysaccharides
Peptidoglycan
Teichoic Acids
Toll-Like Receptors

Figure

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  Fig. 1 Concentrations of IgA, IgG, and IgM in the effusion fluid of OME according to the culture results.

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  Fig. 2 Comparison of the TLR 2, TLR 4, and TLR 9 expressions according to the culture results.

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  Fig. 3 The expression of TLR2, TLR4, and TLR9 mRNA in the middle ear fluid from patients with otitis media with effusion. The mRNA expression was measured by RT-PCR and it was resolved on 2% agarose gel by electrophoresis.Lane 1: 100 bp DNA ladder; lane 2: normal PBL; lane 3-7: patients. TLR 2, TLR 4, and TLR 9 and β-actin mRNA were expressed in all the patients.

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  Fig. 4 Polymorphism of the TLR2 (Arg677Trp and Arg753Gln) and TLR4 (Asp299Gly and Thr399Ile) genes in the middle ear fluid from patients with OME. The genomic DNA was amplified by PCR, and the products were digested with MwoI (TLR2 Arg677Trp), MsPI (TLR2 Arg753Gln), NcoI (TLR4 Asp299Gly), and HinfI (TLR4 Thr399Ile) restriction enzymes, and then they were separated on 3% agarose gel by electrophoresis.Lane 1: normal PBL; lane 2-5: patients; lane 6: 100 bp DNA ladder; lane 7: normal PBL; lane 8-11: patients. TLR2 (Arg677Trp and Arg753Gln) and TLR4 (Asp299Gly and Thr399Ile) were absent from all the patients.

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