Human Immunology xxx (2014) xxx–xxx

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Attenuated TLRs in middle ear mucosa contributes to susceptibility of chronic suppurative otitis media Yu Si a, Zhi Gang Zhang a,⇑, Sui Jun Chen a, Yi Qing Zheng a, Yu Bin Chen a, Yi Liu a, Huaili Jiang a, Lian Qiang Feng b,c, Xi Huang b,c,⇑ a b c

Department of Otolaryngology Head and Neck Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China Department of Immunology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou 510080, China Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou 510080, China

a r t i c l e

i n f o

Article history: Received 26 November 2013 Accepted 18 May 2014 Available online xxxx Keywords: TLR Otitis media Recovery

a b s t r a c t The variability in the recovery of otitis media (OM) is not well understood. Recent data have shown a critical role for toll-like receptors (TLRs) in inflammatory responses to bacteria. It remains unclear whether TLRs-mediated mucosal immunity plays a role in the OM recovery. The etiology, pathological profile, expression levels of TLR2, TLR4, TLR5, TLR9 and proinflammatory cytokines were measured in human middle-ear mucosae sampled from three subject groups: non-OM group, chronic otitis-media (COM) group, and chronic suppurative otitis-media (CSOM) group. Of the 72 ears, 86.11% CSOM patients were positive for bacteria. The cellular makeup of the middle ear mucosa differs among the three groups. Mucosae from the CSOM group presented chronic inflammation or suppurative inflammation in the rudimentary stroma, mainly with infiltration of monocytes and macrophages. The mRNA and protein levels of TLR2, TLR4, and TLR5 exhibited no difference between the non-OM and COM groups but were significantly lower in the CSOM group. Conversely, there was no significant difference in the TLR9 level among the three groups. Furthermore, proinflammatory cytokines TNF-a, IL-1b, IFN-c, IL-6 were up-regulated in the CSOM group. This study provides evidence that the variability in clinical otitis media recovery might be associated with the variability in the expression of mucosal TLRs. Reduced TLR levels in the middle-ear mucosa might cause weak host response to bacteria, persistent inflammation and susceptibility to CSOM. Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

1. Introduction Otitis media (OM) is a common disease, particularly in developing countries, that causes otalgia, otorrhea, deafness, and dizziness and therefore contributes to a major public health problem [1]. Acute bacterial infections of the middle ear occur in 80% of children from 1 to 6 years old, causing AOM [2]. AOM typically resolves quickly [3]; however, some AOM cases recur several times, eventually leading to dry ear, called chronic otitis media (COM) with a series of sequelae, including adhesive otitis media, tympanic membrane perforation and tympanosclerosis. Furthermore, other cases might result in chronic suppurative otitis media (CSOM) with

⇑ Corresponding authors. Address: Sun Yat-sen University, Sun Yat-sen Memorial Hospital, 107 Yangjiang Road, Guangzhou 510120, China (Z.G. Zhang). Address: Sun Yat-sen University, Zhongshan School of Medicine, 74 Zhongshan 2nd Road, Guangzhou 510080, China (X. Huang). E-mail addresses: [email protected] (Z.G. Zhang), [email protected]. edu.cn (X. Huang).

persistent or recurrent otorrhea, and even intracranial infection despite the widespread use of antibiotics [4,5,6]. Although the development of OM is clearly understood, as depicted in Fig. 1, the underlying mechanisms of OM remain unresolved. OM is a multifaceted disease. Bacterial infection is a dominant factor in most cases of OM [7]. Bacterial infection swiftly activates the host mucosal immune response, inducing leukocytic infiltration, mucosal hyperplasia and effusion of the middle ear [8]. The innate mucosal immune system, characterized by epithelial and other mucosal cells, has both anti-infectious and barrier functions [9]. The middle-ear mucosa acts as a major barrier in OM pathology, as the middle-ear mucosa is the first line of defense against bacteria [8]. The innate mucosal immune system plays an anti-infective role by recognizing pathogen-associated molecular patterns (PAMPs) via pattern-recognition receptors (PRR), such as Toll-like receptors (TLRs) [10]. The activation of TLRs mobilizes the innate immune response, including the infiltration of inflammatory cells, the production of inflammatory cytokines, and defense against bacterial

http://dx.doi.org/10.1016/j.humimm.2014.05.009 0198-8859/Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

Please cite this article in press as: Si Y et al. Attenuated TLRs in middle ear mucosa contributes to susceptibility of chronic suppurative otitis media. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.05.009

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Y. Si et al. / Human Immunology xxx (2014) xxx–xxx

Fig. 1. Flow chart of otitis-media recovery. AOM might resolve and recur, or might lead to otitis media with effusion (OME), to irreversible pathology of COM, such as adhesive otitis media and tympanosclerosis, or to chronic otorrhea through a perforated tympanic membrane, referred to as CSOM. OME reflects the presence of chronic effusion in the middle ear cleft, regardless of effusion type. OME might progress to COM, silent OM or CSOM in the disease continuum.

infection [11]. At least 10 human and 13 murine TLRs have been identified, each mediating distinct functions in pathogen recognition. TLRs (TLR2-TLR9) are expressed by multiple cell types, including dendritic cells, lymphocytes, epithelial cells, and endothelial cells, acting as the first line of host defense against infection [12]. Among the classical TLRs are TLR2, TLR4, TLR5, and TLR9, which primarily sense peptidoglycans or peptidoglycan-associated proteins [13], lipopolysaccharide (LPS) from Gram-negative bacteria [14], bacteria flagellin and CpG-DNA [15] respectively. Recent data have shown a critical role for TLRs in inflammatory responses to bacteria in the ear, and these receptors are particularly important during the recovery from OM in mice. For example, a TLR2 mutation delays the clearance of bacteria from the mouse middle ear and could lead to sepsis and death [16]. C3H/HeJ mice with TLR4 gene defects tend to develop spontaneous chronic otitis media [17]. The deletion of Toll-like receptor 9 significantly prolonged the NTHi-induced inflammatory response in the middle ear and delayed bacterial clearance [18]. However, it remains unclear whether these receptors play a role in the variable treatment outcomes in human otitis media. The aim of the present study was to reveal the role of mucosal immunity in OM pathogenesis and recovery through an investigation of the TLR profiles in normal and diseased middle-ear mucosa.

Table 1 Demographic data of included patients. Sex

Group I Group II Group III

Female

Male

39 41 35

29 30 37

Average age (years)

Median age (years)

Median medical history (years)

35.5 37.5 40.2

40 (14–56) 33 (13–57) 39 (12–70)

7 (1–25) 8 (1–35) 6 (1–40)

patients with selected infections (e.g., tuberculosis, Treponema pallidum or HIV), systemic diseases, and congenital or acquired immunodeficiencies were excluded, and patients meeting the above criteria were enrolled in our study. Detailed demographics are described in Table 1. 2.3. Preoperative middle ear swab culture For the CSOM group, a middle-ear swab culture was performed, as previously described [19]. Briefly, the external auditory canal was cleaned, and the middle-ear discharge was collected under head mirrors using cotton swabs. Subsequently, the cotton was placed in a sterile tube and subjected to bacteriological examination within 4 h. 2.4. Tissues collection

2. Materials and methods

Written informed consent from all patients and approval from the Local Ethics Committee of Sun Yat-sen University were obtained before the study (IRB No. 201010).

During each operation, the middle-ear mucosa was exposed via tympanotomy or mastoidectomy. The mucosae located in the epitympanum were collected using a microcurette and placed in a 2-ml Eppendorf tube. The samples were stored in a HetoUltra Freeze (Thermo, USA) for further use in downstream applications, such as RT-PCR and Western blot analyses.

2.2. Patient selection

2.5. Real-time PCR

The research subjects were selected from three patient groups: Group I (non-OM), Group II (chronic otitis-media, COM), and Group III (chronic suppurative otitis-media, CSOM). All enrolled patients were subjected to surgery between February 2011 and December 2013 in the Department of Otolaryngology Head and Neck Surgery at Sun Yat-sen Memorial Hospital, and the following inclusive criteria were considered: (1) the non-OM group: patients diagnosed with otosclerosis or ossicular-chain deformities, subjected to hearing reconstruction operations. The patients had no history of otitis media. Both the mastoid and tympanic cavity appeared normal on a CT scan; (2) the COM group: patients diagnosed with COM sequelae, such as tympanosclerosis or adhesive otitis media, who required further middle ear reconstruction. (3) The CSOM group: patients experiencing recurrent otorrhea with a clinical course longer than 1 year, as demonstrated through otoscopy. Middle ear swabs were collected and subjected to preoperative bacteriological examination in the laboratory. In all three groups, all the

Total RNA was isolated from the middle-ear mucosae for analysis. TRIzol (Invitrogen) reagent was used according to the manufacturer’s instructions. Spectrophotometry (260 nm) was used for quantification, and the ratio of 260/280 nm spectrophotometry should be between 1.8 and 2.0. A 1-lg sample of total RNA was reverse-transcribed to cDNA, and 2 ll of cDNA (1:10 diluted) were amplified in a 20-ll PCR reaction using SYBR Green Master Mix (Bio-Rad, Hercules, CA, USA). The primer sequences used to amplify TLR2, TLR4, TLR5, and TLR9, TNF-a, IL-1b, IFN-c, IL-6 are shown in Table 2. The quantitative real-time RT-PCR reactions were performed using the CFX96 Real-Time PCR System (Bio-Rad). Relative mRNA levels were calculated after normalization to b-actin.

2.1. Ethics statement

2.6. Western blot Pooled specimens from each group were immersed in lysis buffer (2% SDS, 10% glycerol, 5% mercaptoethanol) in 2-ml Eppen-

Please cite this article in press as: Si Y et al. Attenuated TLRs in middle ear mucosa contributes to susceptibility of chronic suppurative otitis media. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.05.009

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Y. Si et al. / Human Immunology xxx (2014) xxx–xxx Table 2 Sequences of primers used in the PCR.

3. Results

Primer

Oligonucleotide sequence (50 –30 )

Actin (F) Actin (R)

GCTCCTCCTGAGCGCAAG CATCTGCTGGAAGGTGGACA

TLR2 (F) TLR2 (R)

CCGTAGATGAAGTCAGCTCACCGATG CCTCCGACAGTTCCAAGA TGT AACGC

130

TLR4 (F) TLR4 (R)

GAGGACCGACACCAATGATG GAACGAATGG AATGTGCAAC ACC

126

TLR5 (F) TLR5 (R)

TGCTCAAACACCTGGATGCTCACTAC ACAGCCGCCTGGATGTTGGAGATATG

122

TLR9 (F) TLR9 (R)

ACCTTCCATCACCTGAGCCATCTG GC CGCTGAAGTCAAG AAACCTCAC

115

TNF-a (F) TNF-a (R)

CCAGGCAGTCAGATCATCTTC GTTATCTCTCAGCTCCACGC

IL-1b (F) IL-1b (R)

CGCAGCAGCACATCAACAAGAGC TGTCCTCATCCTGGAAGGTCCACG

122

IFN-c (F) IFN-c (R)

GTTACTGCCACGGCACAGTCATTG ACCATCCTTTTGCCAGTTCCTCCAG

132

IL-6 (F) IL-6 (R)

TTCCTCTCTGCAAGAGACTTCCATC GCCTCCGACTTGTGAAGTGGTATAG

153

Size (BP)

3.1. Types and proportions of preoperatively cultured pathogens

75

To determine the middle ear microenvironment of CSOM patients, we performed bacteriological examination. Of the 72 ears, 62 (86.11%) were positive for bacteria, and 1 (1.38%) was positive for fungi, whereas no pathogens were detected in other 9 (12.50%) upon culture of middle ear discharge. Among cultured bacteria, Staphylococcus aureus was the most common species, followed by Pseudomonas aeruginosa (Table 3). 3.2. Pathological profile of mucosae from COM and CSOM patients

95

dorf tubes and lysed using TissueLyser II (Retsch, Germany). The cellular debris was centrifuged at 10,000g for 10 min, and the protein concentration of the supernatant was measured using the Quick Start Bradford protein assay (Bio-Rad). 20-lg of the protein sample were loaded onto each lane and separated through 10% SDS–PAGE before transfer to a supported PVDF membrane (Bio-Rad). After blocking, the blots were incubated with primary rabbit anti-human TLR2, TLR4, TLR5, and TLR9 antibodies (Ab; 0.2 lg/ml, Imgenex) at 4 °C overnight. Subsequently, the samples were washed three times for 5 min each with PBST and PBS and incubated with secondary goat anti-rabbit IgG (H + L) Ab (1:5000, Li-COR Biosciences, Lincoln, NE) for 1 h at room temperature, followed by additional washing and detection using the Odyssey Infrared Imaging System (Li-COR Biosciences), according to the manufacturer’s protocol. The results of the Western blots were quantified through the band intensity using Image-Pro Plus 6.0 software.

We observed that the cellular make-up of the middle-ear mucosa differs among the three groups (Fig. 2). Mucosae from the normal group showed a layer of simple ciliated columnar epithelium and a rudimentary stroma without inflammatory cells, and the COM group presented mild mucosal epithelial hyperplasia, combined with calcification or/and fibrosis in the rudimentary stroma, but without inflammatory cells infiltration; however, mucosae from the CSOM group presented chronic inflammation or suppurative inflammation in the rudimentary stroma, mainly with monocytes and macrophages infiltration, and (commonly) granulation in the tissues. 3.3. TLR2, TLR4, and TLR5 levels were significantly lower in the CSOM patients To identify whether TLRs associated to otitis media pathogenesis and recovery, the TLR2, TLR4, TLR5, and TLR9 mRNA levels were determined in middle ear mucosae obtained from non-OM, COM, and CSOM patients. The real-time PCR data showed that the TLR2, TLR4, and TLR5 mRNA levels exhibited no difference between the non-OM and COM groups, while the CSOM group exhibited lower levels of TLR2, TLR4, and TLR5 mRNA than the other two groups. The expression levels of TLR2, TLR4, TLR5 in non-OM group were approximately 14.7-fold higher, 28.53-fold higher and 5.74-fold higher when compared with CSOM group (all p < 0.05, Fig. 3A–C). The expression of TLR9 showed no significant difference

2.7. Histological analysis The paraffin specimen were also used for the histological analysis. The sections (5 lm) were cut, mounted on glass slides, and visualized using hematoxylin/eosin staining. Two pathologists evaluated the histopathological profile of each specimen. 2.8. ELISA Proinflammatory cytokines protein levels were selectively tested by ELISA kits (CST). Mucosae from the three groups were re-collected. Mucosae were homogenized in 0.5 ml of PBS with 0.1% Tween 20. All samples were centrifuged for 5 min at 13,000g and the supernatant was tested in duplicate for TNF-a and IL-1b protein according to the manufacturer’s instruction. The reported sensitivity of these assays is C within the gene encoding TLR4 [25], as well as Arg753Gln, Arg677 Trp for human TLR2 were clearly a focus to investigate [26]. SNPs with TLRs may occurred in CSOM patients, causing deficient TLRs expression. TLRs mediate innate immune response by recognizing pathogen associated molecular patterns through cell-surface receptors. TLR activation initiates intracellular nuclear factor kappa B signaling and cytokine gene expression that ultimately leads to bacterial clearance [15]. TLRs defects may turn patients unable to mount

Please cite this article in press as: Si Y et al. Attenuated TLRs in middle ear mucosa contributes to susceptibility of chronic suppurative otitis media. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.05.009

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an effective immune response against bacteria infection, leading to chronic infection [12]. Recent studies have shown that mice deficient in TLR signaling molecules exhibited weak host defense and delays in bacterial clearance, mucosal recovery and the retreat of inflammation, resulting in the transition from self-limiting, acute otitis media to persistent suppurative otitis media [27,28]. It is important to note that TNF-a, IL-1b and IL-6, apart from being inflammatory mediators that promote bacterial killing also facilitate soft and hard tissue destruction [29]. The expression of TNFa, IL-1b in chronic otitis media had a strong positive correlation between the degree of bone destruction [30]. Bacterial clearance and host inflammatory response play an important role in determine the disease outcome of infection disease [31]. In the present study, we found that 86.11% CSOM patients were positive for bacteria upon culture of middle ear discharge and presented increased proinflammatory cytokines TNF-a, IL-1b, IL-6 and IFN-c. The down-regulation of TLR2, TLR4, and TLR5 in CSOM patients might lead to inefficient host defense in the middle ear, causing repeated infections and prolonged inflammation response, turning the middle ear at risk for tissue destruction and remodeling, and increasing susceptibility to CSOM. M. Sakagami study suggested that the hearing level deteriorated when acute inflammation occurred and ear discharge was present [32]. So surgical treatment to remove the granulations is essential for elimination of disease, improvement of healing and an infection free ear. Consistently, the normal levels of TLR2, TLR4, and TLR5 in the COM group indicate that TLR2, TLR4, and TLR5 are important in the mucosae of patients with COM, which serves as a normal barrier to maintain a sterile and dry ear. TLR9 recognizes CpG DNA motifs in bacteria and viral DNA and is mainly expressed on B cells and dendritic cells [33]. In the present study, we observed that there are few B cells and dendritic cells in the mucosae from non-OM, COM and CSOM patients, therefore the non-significant difference in the TLR9 levels among all three patient groups suggested that TLR9 might not play a significant role in the local immunity of the middle ear mucosa. Taken together, these data demonstrated that the diversity in the TLR expression levels might be associated with the variability observed in otitis media recovery. Deficiencies in TLR2, TLR4, and TLR5 expression in the middle ear mucosa might lead to an obstacle in bacterial clearance and resolution of inflammation. 5. Conclusions Individual variability in TLR expression might be associated with variability in otitis media recovery. The down-regulation of TLR2, TLR4, and TLR5 expression in CSOM patients might indicate inefficient host defense in the middle ear, causing repeated infections and persistent inflammations, eventually leading to recurrent, persistent suppurative chronic middle ear diseases. Acknowledgment The authors would like to thank Professor Qing Liu for assistance with the statistical analysis. This work was supported by Natural Science Foundation of China (81371082,30972763,V0832006), Guangdong science and technology plan projects (2010B080701083), The introduction of innovative R&D team program of Guangdong Province (NO.2009010058), Guangdong Natural Science (7001684), Foundation Guangdong Province Universities and Colleges Pearl River School Funded Scheme (NO.2009), Doctoral Fund of Ministry of Education of China (2010017111004). References [1] Lalwani Anil K. Current diagnosis and treatment in otolaryngology – head and neck surgery; 2004. p. 701–3.

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Please cite this article in press as: Si Y et al. Attenuated TLRs in middle ear mucosa contributes to susceptibility of chronic suppurative otitis media. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.05.009

Attenuated TLRs in middle ear mucosa contributes to susceptibility of chronic suppurative otitis media.

The variability in the recovery of otitis media (OM) is not well understood. Recent data have shown a critical role for toll-like receptors (TLRs) in ...
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