- Email: [email protected]
Expression of Toll-like receptors on peripheral blood white cells in acute otitis media
otolaryngologia polska 68 (2014) 77–82
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/otpol
Original research article/Artykuł oryginalny
Expression of Toll-like receptors on peripheral blood white cells in acute otitis media Krzysztof Trzpis 1,*, Edwina Kasprzycka 2, Bożena Skotnicka 1, Elżbieta Hassmann-Poznańska 1, Jolanta Wysocka 2 1 2
Department of Pediatric Otolaryngology, Medical University, Bialystok, Poland Department of Pediatric Laboratory Diagnostics Medical University, Bialystok, Poland
article info
abstract
Article history:
Objective: From 10 to 15% of children suffer from recurrent acute otitis media (AOM). An
Received: 07.02.2013
association between polymorphism in TLRs and their co-receptor CD14 with otitis media
Accepted: 24.06.2013
proneness has been described in children. Moreover, the experiments on animal models
Available online: 28.06.2013
have shown that TLRs and their signaling molecules are critical for timely resolution of
Keywords: Expression
and TLR4 on lymphocytes, monocytes and granulocytes in peripheral blood in children
Toll-like receptors Acute otitis media
25 children hospitalized for recurrent AOM, failures of previous treatments and/or acute
bacterial otitis. Aim: The aim of this study was to determine the expression of TLR1, TLR2 with recurrent or persistent AOM. Methods: The study was performed on a group of mastoiditis. The results were compared to the control group of healthy children at the same age. The expression of TLRs on peripheral blood white cells was measured by flow Słowa kluczowe: ekspresja receptory Toll-podobne ostre zapalenie ucha środkowego
cytometric analysis. The results were expressed as mean fluorescence intensity (MFI). The statistical analysis was performed using the Mann–Whitney U test. Results: The highest expression of TLR was found on monocytes, the lowest on lymphocytes in both groups of children (AOM and the control one). The expression of TLR1 was the lowest and expression of TLR4 was the highest on all examined cells. The expression of all examined TLRs on monocytes was significantly higher in the AOM group. Conclusions: Peripheral blood monocytes are characterized by increased expression of TLRs in the course of recurrent AOM. © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Acute otitis media (AOM) is the most common bacterial infection in preschool children. Largest part of the episodes occur in the first years of life. The majority of AOM cases resolve spontaneously, but approximately 10–15% of children experience four or more episodes in the following year,
which allow the diagnosis of recurrent otitis media. AOM may also occasionally result in serious complications, as mastoiditis. There are several known risk factors for recurrent or persistent AOM and the disease is considered to be multifacto-
Please, cite this article in press as: Trzpis K, et al. Expression of Toll-like receptors on peripheral blood white cells in acute otitis media. Otolaryngol Pol. (2013), doi:10.1016/j.otpol.2013.06.006 * Corresponding author at: Klinika Otolaryngologii Dziecięcej, UM, ul. Waszyngtona 17, 15-274 Białystok, Poland. Tel.: +48 85 7 450 832; fax: +48 85 7 450 832. E-mail address: [email protected] (K. Trzpis). 0030-6657/$ – see front matter © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
http://dx.doi.org/10.1016/j.otpol.2013.06.006
78
otolaryngologia polska 68 (2014) 77–82
rial. Environmental factors (day-care attendance, poor socioeconomic status), diseases associated with poor Eustachian tube function, genetic and immune factors all contribute to predisposition for recurrent AOM. Although it is apparent that AOM is commonly associated with viral infection of upper respiratory tract, bacterial infection is considered to be the most significant in the etiology of this disease. The most frequent bacterial pathogens of AOM are Streptococcus pneumoniae and Haemophilus influenzae. The human immune system is comprised of two interrelated branches: innate and adaptive immunity. Those branches use different strategies to recognize pathogens. Whereas the innate, immediate, immune system detects molecular structures unique to microbes using a limited number of encoded pattern recognition receptors (PRR), the adaptive immune system uses clonally expressed, highly specific receptors [1, 2]. Humans cells are provided with various PRRs, including Toll-like receptors (TLRs) expressed on cells of the innate immune system as macrophages and dendritic cells but they are also present on many other cells including epithelial [3]. TLR2 responds to the cell wall components of Gram-positive bacteria as peptoglycans, lipoteichoic acid and lipoproteins. TLR4 binds to the toxic pneumolysin ligand produced by Gram-positive bacteria, as well as to lipopolisaccharide (LPS), the major component of Gram negative bacteria cell walls [4, 5]. TLR4 has also been shown to respond to the fusion protein of respiratory syncytial virus [6]. Activation of TLRs, leads to activation of the complement cascade and proinflammatory cytokine production [7–9]. TLRs induced signals also control adaptive immunity at various checkpoints regulating the selection of antigens presented by dendritic cells, type of response, its magnitude and duration and the production of long term memory [1, 10]. Increased expression of those receptors have been found in the mucosa and effusion from middle ears during the course of experimental forms of otitis media [11–14]. Therefore, it can be presumed that increased expression of those molecules would also be found on circulating immune cells from children with recurrent otitis media. Accordingly, the study was undertaken to define the expression of TLR 1, 2 and 4 on peripheral blood monocytes, lymphocytes and granulocytes in patients suffering from recurrent or persistent otitis media and compare them with other parameters of inflammatory reaction. The aim of this study was to explain the role of TLR's in the immunological response and their potential influence on the intensity of inflammatory reaction. This may provide new insight into the pathology of AOM and possibly modify required intervention.
Materials and methods Twenty-five pediatric patients (mean age 3.5 years) hospitalized because of acute otitis media (group AOM) in the Department of Pediatric Otorhinolaryngology, Medical University, Bialystok, participated in the study. The reasons for hospitalizations were recurrent acute otitis media (3 and more episodes during 6 months or 4 or more during 1 year),
persistent otitis media (failures of previous treatment) or complications of acute otitis media (mastoiditis). All patients fulfill the AAP criteria for diagnosis of AOM (acute onset of signs and symptoms, presence of middle-ear inflammation as indicated by earache and distinct erythema of the tympanic membrane and presence of effusion in the middle ear indicated by bulging of tympanic membrane or otorrhea). The control group consisted of 25 healthy children (mean age 3.54 years), with no history of recurrent upper respiratory tract and ear infections, admitted to the Department of Pediatric Surgery for planned minor surgical procedures. The blood samples in this group was taken before the surgery, together with the probes for routine examinations. The study was approved by the Ethics Committee of Medical University, Bialystok. The purpose of the study was explained to the parents and written informed consent was obtained from all of them. On the day of admission, before starting antibiotic and/or surgical treatment or up to 8 h after it, from all patients peripheral blood samples were obtained (1.8 ml) to test-tubes with K3EDTA anticoagulant for the purpose of assessing the expression of TLRs on granulocytes, monocytes and lymphocytes of peripheral blood. Also blood count in peripheral blood smear, erythrocyte sedimentation rate and C-reactive protein (CRP) level were assessed in all collected samples. C-reactive protein (CRP) level was performed using Cobas Integra 800 Roche, blood count in peripheral blood smear using Sysmex XT2000i hematologic analyzer and erythrocyte sedimentation rate using Roller 20 Alifax. Criteria for normal CRP values (ranged between 0 and 0.5 mg/dl) used in our Department of Pediatric Laboratory Diagnostics were defined for Cobas Integra 800 (turbidimetric immunoassay) by Roche. Flow cytometry was performed on all the blood samples (using Cytomics FC500 by Beckman Coulter cytometer). Prior to staining the cells for cytometric analysis, cell viability was assessed by trypan blue exclusion. Only the samples with at least 98% of cells viability were selected for the study. 10 thousand cells were assessed in every collected sample. All the blood samples were incubated for 15 min in the room temperature, in the darkness, using monoclonal antibodies conjugated with proper fluorochromes (anti-human CD 281 (TLR1) PE, IgG1, clone CD2. F4; anti-human CD 282 (TLR2) PE, IgG2a, clone TL2.1; anti-human CD 284 (TLR4) PE, IgG2a, clone HT A 125 produced by eBioscience company and anti-human CD14 FITC, IgG2a clone TUK4 produced by DakoCytomation company). The negative control was applied in accordance with the class of used monoclonal antibodies. After the incubation all the samples underwent the process of lysis in automatic lysis station (Epics Immunology Work Station) using Multi-Q-Prep set. Lymphocytes and granulocytes were separated using the gate in FS/SS system, monocytes were signed with the usage of anti-CD14 antibodies. All the cytometric analyzes were performed in maximum 60 min after the incubation. The results were given as MFI (mean fluorescence intensity), that indirectly equals the level of receptors' density on the cell surface. All the analyses were performed at maximum 2 h after collecting the sample. Statistical analysis of the results was performed using of non-parametrical Mann–Whitney U test in Statistica 5.0.
79
otolaryngologia polska 68 (2014) 77–82
Table I – Characteristics of patients from AOM group Patient no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Age (yr)
Diagnosis
CRP (mg/dl)
WBC (103/mL)
ESR (mm/h)
4 5 1.7 1.2 1.2 1.3 1.1 3.5 1.7 0.5 6 9 2.2 3.5 3.5 1.1 4 2.5 4 6.9 3.1 6.8 8.1 3.5 3
pAOM rAOM Mastoiditis rAOM rAOM rAOM rAOM rAOM Mastoiditis rAOM rAOM pAOM pAOM rAOM rAOM rAOM rAOM rAOM rAOM rAOM rAOM Mastoiditis rAOM pAOM rAOM
0.1 2.6 5.2 0.9 3.7 0.1 4.8 2.9 5.7 1.5 4.6 0.2 10.2 1.2 1.5 4.1 0.1 2.1 2.8 1.5 0.7 5.6 1.6 0.8 13.7
7.00 13.20 21.19 13.20 17.70 9.55 15.30 11.20 15.40 23.60 8.80 12.90 9.50 9.28 7.09 14.80 11.20 11.10 20.38 8.94 5.80 18.70 6.70 7.44 8.81
44 45 44 35 44 26 42 28 90 47 15 37 55 17 25 28 8 30 9 26 14 42 10 83 78
pAOM: persistent acute otitis media; rAOM: recurrent acute otitis media.
Results In AOM group CRP extended from 0.1 to 13.7 mg/dl. In four cases CRP level was within the normal range (below 0.5 mg/dl),
in all of the others – average level was 3.7 mg/dl. ESR level ranged from 7 to 90 mm/h, average 37 mm/h. The clinical and laboratory characteristics for the AOM and control groups are presented in Tables I and II, respectively.
Table II – Characteristics of patients from control group Patient no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Age (yr) 8 1 9 1.2 1 3 1.2 1 2.5 3.5 0.9 2 1.5 4.2 5.3 1.1 3.2 7 3.5 1.5 4 6 5.4 7 4.5
Diagnosis GUA GUA SL IH SL IH IH GUA GUA IH IH GUA IH GUA IH GUA SL IH SL IH GUA GUA IH IH SL
SL: skin lesions; GUA: genitourinary anomalies; IH: inguinal hernia.
CRP (mg/dl)
WBC (103/mL)
ESR (mm/h)
0.3 0.0 0.1 0.0 0.0 0.1 0.0 0.1 0.2 0.0 0.1 0.0 0.2 0.0 0.1 0.1 0.3 0.1 0.0 0.2 0.0 0.0 0.0 0.1 0.2
4.70 14.30 17.60 8.30 8.36 14.47 11.92 7.26 6.36 10.25 11.18 6.59 10.24 7.28 8.56 10.25 6.32 6.11 9.36 11.32 6.33 6.36 8.35 7.25 7.18
4 6 5 3 4 4 6 3 5 4 3 7 4 3 7 7 4 3 5 4 3 3 5 4 6
80
otolaryngologia polska 68 (2014) 77–82
Table III – Average fluorescence intensity (MFI) of TLR1, TLR2 and TLR4 on monocytes in control group and OMA group Monocytes MFI TLR1 MFI TLR2 MFI TLR4
Control group (N = 25)
OMA group (N = 25)
P
3.25 1.57 5.15 2.24 10.76 3.95
4.55 1.75 7.06 2.04 14.61 7.41
0.013 0.006 0.032
Table IV – Average fluorescence intensity (MFI) of TLR1, TLR2 and TLR4 on lymphocytes in control group and OMA group Lymphocytes MFI TLR1 MFI TLR2 MFI TLR4
Control group (N = 25)
OMA group (N = 25)
P
1.03 0.2 1.27 0.17 3.72 0.58
1.14 0.3 1.24 0.35 3.93 1.02
0.181 0.719 0.399
Table V – Average fluorescence intensity (MFI) of TLR1, TLR2 and TLR4 on granulocytes in control group and OMA group Granulocytes MFI TLR1 MFI TLR2 MFI TLR4
Control group (N = 25)
OMA group (N = 25)
P
1.66 0.28 2.06 0.39 6.18 1.25
1.85 0.35 2.24 0.63 7.19 1.22
0.057 0.253 0.0098
The monocytes showed the highest level of expression of all the studied TLRs (TLR1, TLR2, TLR4) in AOM and the control group as well (Table III). The lowest expression was detected on lymphocytes also in both groups (Table IV). In the control and OMA group on all types of examined cells: monocytes, lymphocytes and granulocytes TLR1 expression was the lowest, and TLR4 the highest. The expression of all examined TLRs was significantly higher on monocytes in AOM group. TLR4 was the only one among the TLRs which showed significantly higher expression on granulocytes in AOM group. The expression of TLR1 and TLR2 was also higher on granulocytes in this group, but the difference did not reach statistical significance (Table V). The expression of all TLRs on lymphocytes was not significantly different between AOM and the control group. No correlation was found between the markers of inflammatory response – CRP and ESR and the expression of TLRs on the elements of white cell system in peripheral blood.
Discussion In healthy middle ear mucosa only very few lymphocytes are present and most probably the first response against infection is mediated by an innate immunity system [14]. The studies on animal models showed that strains deficient of TLR 4, TLR2 or their signaling molecules showed more severe inflammation, reduced clearance of middle ear bacteria and spontaneously developed chronic otitis media [14–16]. In the healthy human middle ear mucosa presence
of at least 6 PPRs, among them TLR4, was demonstrated by immunohistochemical and RT PCR studies [17]. Samples of mucosa from patients with a chronic middle ear disease exhibited lower mRNA levels of TLRs (4, 5 and 7) [17]. Several TLRs (2, 4, 5, 9) were also detected in effusion collected from children with secretory otitis media [3, 13]. Some studies showed that certain polymorphisms in the TLR4 gene were associated with otitis media susceptibility [19], however, this was not confirmed in latest studies [20, 21]. As presented above, in recent years great progress in the understanding of TLRs function in the response of immunological system to infection of middle ear was observed. However, the publications presenting attempts to utilize this knowledge in clinical assessment or monitoring of inflammatory reaction are scarce probably because of difficulties in obtaining adequate material from the middle ear in routine practice. We undertook the study to examine if the similar up-regulation of TLRs takes place on the monocytes, granulocytes and lymphocytes of peripheral blood in patients with severe form of AOM. If this hypothesis would be confirmed TLRs could be used as a marker of inflammatory response, potentially modifying future treatment decisions. The performed studies showed that the expression of all examined TLRs on monocytes and TLR4 on granulocytes was significantly higher in the AOM group. Observed changes might express the variations in innate PPR-expression profiles, or more probably, be a sign of stimulation by bacterial antigens. Although recurrent otitis media is not usually accompanied by bacteraemia, it is characterized by prolonged presence of pathogenic bacteria (Gram-positive and Gram-negative) inside the tympanic cavity, increased nasopharyngeal bacterial colonization and recurrent upper respiratory tract infections. Bacterial products from those sites may provide the stimulus for TLRs expression on peripheral blood white cells. Also proinflammatory cytokines released to the blood stream during localized inflammatory process may contribute to the regulation of TLRs expression on monocytes and neutrophils [22]. Higher expression of TLRs on both monocytes and granulocytes result in enhanced activation of those cells by ligands of bacterial origin. Activation of TLR4 leads to enhanced synthesis and release of proinflammatory cytokines IL-1b, -6, -8, -12 and TNFa. Moreover TLR4 stimulation improve phagocytic capabilities of monocytes and expression of costymulatory molecules which makes presentation of antigen to lymphocytes T more effective [23]. TLR4 regulate also important granulocyte functions, including adhesion, generation of reactive oxygen species and inhibition of apoptosis [9]. TLR2 was up-regulated only on monocytes. Its activation triggers gene transcription of cytokines, mainly interferon's [24]. Observed in our study up-regulation of TLRs expression on monocytes and granulocytes may initiate the inflammatory cascade. Although in our study CRP level in AOM group was not very high (average 3.7 mg/dl), it was within normal range in only four cases, which speak up for the presence of general inflammatory reaction in the course of AOM. The other parameters of inflammatory response as ESR and WBC counts were also
otolaryngologia polska 68 (2014) 77–82
higher in AOM group. However no correlation was found between examined parameters of inflammatory reaction and TLR's expression. This may be the result of the fact that the expression of TLR's on studied cells showed great variability in the control as well as in the AOM groups as expressed by the values of standard deviation. It should also be taken into account that the inflammatory reaction is not the direct result of TLR's expression. Activation of several mechanisms influenced by various factors are involved in this complex and not easily predicted process. In the studies performed in the middle ear effusion also no correlation between TLR 2 and 4 expressions and the characteristics of effusion, presence of bacteria in the exudates or frequency of ventilation tube insertion was found [13, 18]. In the group of patients with AOM the monocytes were the cells which showed most distinct difference in TLRs expression comparing with the group of healthy children. On granulocytes such difference was observed only in the expression of TLR4. This observation is in the agreement with the fact that both this cells are involved in innate immune responses. No difference of expression was observed on lymphocytes participating mainly in the adaptive immune system. Gomes et al. [25], showed increased expression TLR4 on human monocytes 6 h after stimulation with LPS. However the expression of TLR4 on granulocytes was not similarly modulated by LPS which shows that the reaction of granulocytes on stimulation is different [25]. It is believed that TLR2 responds mainly to gram-positive and TLR4 to Gram negative bacteria. The findings of Zang et al. [26] demonstrate that during bacterial infection in vivo, newborns selectively and coordinately amplify the TLR2 pathway in Gram-positive bacterial infection and the TLR4 pathway in Gram negative bacterial infection. These results indicate that various bacteria differ in their dose-dependent patterns of induction of TLRs [24, 26]. However studies mostly performed in vitro, in laboratory models or in newborns, may not reflect the true situation during infectious diseases because pathogens are equipped with various ligands which may activate multiple signaling pathways. In cases of recurrent AOM we are usually dealing with polymicrobial infection which makes the interpretation of the role of specific bacteria in TLRs expression difficult. Differential expression of TLRs was described in various diseases [27–30]. Xu et al. [27] observed significantly higher expression of genes encoding five TLRs (2,3,4,7,8) on peripheral blood cells of patients with acute rotavirus diarrhea. Patients with irritable bowel syndrome presented increased expression of TLR2 on monocytes [28]. Pons et al. [29] have found up-regulated expression of TLR2 on peripheral blood monocytes from chronic obstructive pulmonary disease patients. In children with cystic fibrosis TLR4 expression was significantly higher compared to healthy controls but did not vary according to the presence or absence of pulmonary infection with Gram-negative or Gram-positive bacteria, so the authors consider the observed difference to be related to having CF per se and not related to current pulmonary infection [30]. The enhanced expression of TLR 1, TLR2, TLR4 on the monocytes of peripheral blood in the course of recurrent or persistent acute otitis media shows that the innate
81
immunological reaction is not limited to the middle ear and nasopharyngeal mucosa. This supports the importance of innate immunity in developing the general immunological response to infection. However the lack of correlation between TLRs expression and CRP, ESR and WBC limits theirs role as potential marker of inflammatory response.
Authors' contributions/Wkład autorów KT – data collection and interpretation, acceptance of final manuscript version, literature search. EK – data collection, statistical analysis, acceptance of final manuscript version. BS – study design, data interpretation, acceptance of final manuscript version, literature search. EH-P – study design, data interpretation, acceptance of final manuscript version, funds collection. JW – data interpretation, acceptance of final manuscript version, literature search.
Financial support/Finansowanie The manuscript was funded from grant no. 3-67743L.
Conflict of interest/Konflikt interesu None declared.
Ethics/Etyka The work described in this article have been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans; EU Directive 2010/63/EU for animal experiments; Uniform Requirements for manuscripts submitted to Biomedical journals.
references/pi smiennictwo
[1] Palm NW, Medzhitov R. Pattern recognition receptors and control of adaptive immunity. Immunol Rev 2009;227:221–233. [2] Turvey SE, Broide DH. Innate immunity. J Allergy Clin Immunol 2010;125:24–32. [3] Kim MG, Park DC, Shim JS, Jung H, Park MS, Kim YI, et al. TLR-9, NOD-1, NOD-2, RIG-I and immunoglobulins in recurrent otitis media with effusion. Int J Pediatr Otorhinolaryngol 2010;74:1425–1429. [4] Turvey SE, Hawn TR. Towards subtlety: understanding the role of Toll-like receptor signaling in susceptibility to human infections. Clin Immunol 2006;120:1–9. [5] Leichtle A, Lai Y, Wollenberg B, Wasserman SI, Ryan AF. Innate signaling in otitis media: pathogenesis and recovery. Curr Allergy Asthma Rep 2011;11:78–84. [6] Dabbagh K, Lewis DB. Toll-like receptors and T-helper-1/Thelper-2 responses. Curr Opin Infect Dis 2003;16:199–204. [7] Uematsu S, Akira S. Toll-like receptors and innate immunity. J Mol Med 2006;84:712–725.
82
otolaryngologia polska 68 (2014) 77–82
[8] Parker LC, Prince LR, Sabroe I. Translational mini-review series on toll-like receptors: networks regulated by Toll-like receptors mediate innate and adaptive immunity. Clin Exp Immunol 2007;147:199–207. [9] Sabroe I, Dower SK, Whyte MK. The role of toll-like receptors in the regulation of neutrophil migration, activation, and apoptosis. Clin Infect Dis 2005;15(Suppl 7):S421–S426. [10] Mundford RS. Sensing Gram-negative bacterial lipopolysaccharides: a human disease determinant? Infect Immun 2008;76:454–465. [11] Song JJ, Cho JG, Woo JS, Lee HM, Hwang SJ, Chae SW. Differential expression of toll-like receptors 2 and 4 in rat middle ear. Int J Pediatr Otorhinolaryngol 2009;73:821–824. [12] Komori M, Nakamura Y, Ping J, Feng L, Toyama K, Kim Y, et al. Pneumococcal peptidoglycan-polysaccharides regulate Toll-like receptor 2 in the mouse middle ear epithelial cells. Pediatr Res 2011;69:101–105. [13] Lee SY, Ryu EW, Kim JB, Yeo SG. Clinical approaches for understanding the expression levels of pattern recognition receptors in otitis media with effusion. Clin Exp Otorhinolaryngol 2011;4:163–167. [14] Leichtle A, Hernandez M, Pak K, Yamasaki K, Cheng CF, Webster NJ, et al. TLR4-mediated induction of TLR2 signaling is critical in the pathogenesis and resolution of otitis media. Innate Immun 2009;15:205–215. [15] Hirano T, Kodama S, Fujita K, Maeda K, Suzuki M. Role of Toll-like receptor 4 in innate immune responses in a mouse model of acute otitis media. FEMS Immunol Med Microbiol 2007;49:75–83. [16] Hernandez M, Leichtle A, Pak K, Ebmeyer J, Euteneuer S, Obonyo M, et al. Myeloid differentiation primary response gene 88 is required for the resolution of otitis media. J Infect Dis 2008;15:1862–1869. [17] Granath A, Cardell LO, Uddman R, Harder H. Altered Tolland Nod-like receptor expression in human middle ear mucosa from patients with chronic middle ear disease. J Infect 2011;63:174–176. [18] Lee YC, Kim C, Shim JS, Byun JY, Park MS, Cha CI, et al. Tolllike receptors 2 and 4 and their mutations in patients with otitis media and middle ear effusion. Clin Exp Otorhinolaryngol 2008;1:189–195. [19] Emonts M, Veenhoven RH, Wiertsema SP, HouwingDuistermaat JJ, Walraven V, de Groot R, et al. Genetic polymorphisms in immunoresponse genes TNFA, IL6, IL10, and TLR4 are associated with recurrent acute otitis media. Pediatrics 2007;120:814–823.
[20] Carroll SR, Zald PB, Soler ZM, Milczuk HA, Trune DR, Macarthur CJ. Innate immunity gene single nucleotide polymorphisms and otitis media. Int J Pediatr Otorhinolaryngol 2012;76:976–979. [21] Sale MM, Chen WM, Weeks DE, Mychaleckyj JC, Hou X, Marion M, et al. Evaluation of 15 functional candidate genes for association with chronic otitis media with effusion and/ or recurrent otitis media (COME/ROM). PLoS ONE 2011;6: e22297. [22] O'Mahony DS, Pham U, Iyer R, Hawn TR, Liles WC. Differential constitutive and cytokine-modulated expression of human Toll-like receptors in primary neutrophils, monocytes, and macrophages. Int J Med Sci 2008;5:1–8. [23] Majewska M, Szczepanik M. Rola receptorów tollpodobnych (TLR) w odporności wrodzonej I nabytej oraz ich funkcja w regulacji odpowiedzi immunologicznej. Postępy Hig Med Dosw 2006;60:52–63. [24] Gomes NE, Brunialti MKC, Mendes ME, Freudenberg M, Galanos C, Salomão R. Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood. Braz J Med Biol Res 2010;43:853–859. [25] Oliveira-Nascimento L, Massari P, Wetzler LM. The role of TLR2 in infection and immunity. Front Immunol 2012;3:79. [26] Zhang JP, Yang Y, Levy O, Chen C. Human neonatal peripheral blood leukocytes demonstrate pathogen-specific coordinate expression of TLR2, TLR4/MD2, and MyD88 during bacterial infection in vivo. Pediatr Res 2010; 68:479–483. [27] Xu J, Yang Y, Sun J, Ding Y, Su L, Shao C, et al. Expression of Toll-like receptors and their association with cytokine responses in peripheral blood mononuclear cells of children with acute rotavirus diarrhoea. Clin Exp Immunol 2006;144:376–381. [28] Ohman L, Lindmark AC, Isaksson S, Posserud I, Strid H, Sjövall H, et al. Increased TLR2 expression on blood monocytes in irritable bowel syndrome patients. Eur J Gastroenterol Hepatol 2012;24:398–405. [29] Pons J, Sauleda J, Regueiro V, Santos C, López M, Ferrer J, et al. Expression of Toll-like receptor 2 is up-regulated in monocytes from patients with chronic obstructive pulmonary disease. Respir Res 2006;10:64. [30] Sturges NC, Wikström ME, Winfield KR, Gard SE, Brennan S, Sly PD, et al. Monocytes from children with clinically stable cystic fibrosis show enhanced expression of Toll-like receptor 4. Pediatr Pulmonol 2010;45:883–889.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/otpol
Original research article/Artykuł oryginalny
Expression of Toll-like receptors on peripheral blood white cells in acute otitis media Krzysztof Trzpis 1,*, Edwina Kasprzycka 2, Bożena Skotnicka 1, Elżbieta Hassmann-Poznańska 1, Jolanta Wysocka 2 1 2
Department of Pediatric Otolaryngology, Medical University, Bialystok, Poland Department of Pediatric Laboratory Diagnostics Medical University, Bialystok, Poland
article info
abstract
Article history:
Objective: From 10 to 15% of children suffer from recurrent acute otitis media (AOM). An
Received: 07.02.2013
association between polymorphism in TLRs and their co-receptor CD14 with otitis media
Accepted: 24.06.2013
proneness has been described in children. Moreover, the experiments on animal models
Available online: 28.06.2013
have shown that TLRs and their signaling molecules are critical for timely resolution of
Keywords: Expression
and TLR4 on lymphocytes, monocytes and granulocytes in peripheral blood in children
Toll-like receptors Acute otitis media
25 children hospitalized for recurrent AOM, failures of previous treatments and/or acute
bacterial otitis. Aim: The aim of this study was to determine the expression of TLR1, TLR2 with recurrent or persistent AOM. Methods: The study was performed on a group of mastoiditis. The results were compared to the control group of healthy children at the same age. The expression of TLRs on peripheral blood white cells was measured by flow Słowa kluczowe: ekspresja receptory Toll-podobne ostre zapalenie ucha środkowego
cytometric analysis. The results were expressed as mean fluorescence intensity (MFI). The statistical analysis was performed using the Mann–Whitney U test. Results: The highest expression of TLR was found on monocytes, the lowest on lymphocytes in both groups of children (AOM and the control one). The expression of TLR1 was the lowest and expression of TLR4 was the highest on all examined cells. The expression of all examined TLRs on monocytes was significantly higher in the AOM group. Conclusions: Peripheral blood monocytes are characterized by increased expression of TLRs in the course of recurrent AOM. © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Acute otitis media (AOM) is the most common bacterial infection in preschool children. Largest part of the episodes occur in the first years of life. The majority of AOM cases resolve spontaneously, but approximately 10–15% of children experience four or more episodes in the following year,
which allow the diagnosis of recurrent otitis media. AOM may also occasionally result in serious complications, as mastoiditis. There are several known risk factors for recurrent or persistent AOM and the disease is considered to be multifacto-
Please, cite this article in press as: Trzpis K, et al. Expression of Toll-like receptors on peripheral blood white cells in acute otitis media. Otolaryngol Pol. (2013), doi:10.1016/j.otpol.2013.06.006 * Corresponding author at: Klinika Otolaryngologii Dziecięcej, UM, ul. Waszyngtona 17, 15-274 Białystok, Poland. Tel.: +48 85 7 450 832; fax: +48 85 7 450 832. E-mail address: [email protected] (K. Trzpis). 0030-6657/$ – see front matter © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
http://dx.doi.org/10.1016/j.otpol.2013.06.006
78
otolaryngologia polska 68 (2014) 77–82
rial. Environmental factors (day-care attendance, poor socioeconomic status), diseases associated with poor Eustachian tube function, genetic and immune factors all contribute to predisposition for recurrent AOM. Although it is apparent that AOM is commonly associated with viral infection of upper respiratory tract, bacterial infection is considered to be the most significant in the etiology of this disease. The most frequent bacterial pathogens of AOM are Streptococcus pneumoniae and Haemophilus influenzae. The human immune system is comprised of two interrelated branches: innate and adaptive immunity. Those branches use different strategies to recognize pathogens. Whereas the innate, immediate, immune system detects molecular structures unique to microbes using a limited number of encoded pattern recognition receptors (PRR), the adaptive immune system uses clonally expressed, highly specific receptors [1, 2]. Humans cells are provided with various PRRs, including Toll-like receptors (TLRs) expressed on cells of the innate immune system as macrophages and dendritic cells but they are also present on many other cells including epithelial [3]. TLR2 responds to the cell wall components of Gram-positive bacteria as peptoglycans, lipoteichoic acid and lipoproteins. TLR4 binds to the toxic pneumolysin ligand produced by Gram-positive bacteria, as well as to lipopolisaccharide (LPS), the major component of Gram negative bacteria cell walls [4, 5]. TLR4 has also been shown to respond to the fusion protein of respiratory syncytial virus [6]. Activation of TLRs, leads to activation of the complement cascade and proinflammatory cytokine production [7–9]. TLRs induced signals also control adaptive immunity at various checkpoints regulating the selection of antigens presented by dendritic cells, type of response, its magnitude and duration and the production of long term memory [1, 10]. Increased expression of those receptors have been found in the mucosa and effusion from middle ears during the course of experimental forms of otitis media [11–14]. Therefore, it can be presumed that increased expression of those molecules would also be found on circulating immune cells from children with recurrent otitis media. Accordingly, the study was undertaken to define the expression of TLR 1, 2 and 4 on peripheral blood monocytes, lymphocytes and granulocytes in patients suffering from recurrent or persistent otitis media and compare them with other parameters of inflammatory reaction. The aim of this study was to explain the role of TLR's in the immunological response and their potential influence on the intensity of inflammatory reaction. This may provide new insight into the pathology of AOM and possibly modify required intervention.
Materials and methods Twenty-five pediatric patients (mean age 3.5 years) hospitalized because of acute otitis media (group AOM) in the Department of Pediatric Otorhinolaryngology, Medical University, Bialystok, participated in the study. The reasons for hospitalizations were recurrent acute otitis media (3 and more episodes during 6 months or 4 or more during 1 year),
persistent otitis media (failures of previous treatment) or complications of acute otitis media (mastoiditis). All patients fulfill the AAP criteria for diagnosis of AOM (acute onset of signs and symptoms, presence of middle-ear inflammation as indicated by earache and distinct erythema of the tympanic membrane and presence of effusion in the middle ear indicated by bulging of tympanic membrane or otorrhea). The control group consisted of 25 healthy children (mean age 3.54 years), with no history of recurrent upper respiratory tract and ear infections, admitted to the Department of Pediatric Surgery for planned minor surgical procedures. The blood samples in this group was taken before the surgery, together with the probes for routine examinations. The study was approved by the Ethics Committee of Medical University, Bialystok. The purpose of the study was explained to the parents and written informed consent was obtained from all of them. On the day of admission, before starting antibiotic and/or surgical treatment or up to 8 h after it, from all patients peripheral blood samples were obtained (1.8 ml) to test-tubes with K3EDTA anticoagulant for the purpose of assessing the expression of TLRs on granulocytes, monocytes and lymphocytes of peripheral blood. Also blood count in peripheral blood smear, erythrocyte sedimentation rate and C-reactive protein (CRP) level were assessed in all collected samples. C-reactive protein (CRP) level was performed using Cobas Integra 800 Roche, blood count in peripheral blood smear using Sysmex XT2000i hematologic analyzer and erythrocyte sedimentation rate using Roller 20 Alifax. Criteria for normal CRP values (ranged between 0 and 0.5 mg/dl) used in our Department of Pediatric Laboratory Diagnostics were defined for Cobas Integra 800 (turbidimetric immunoassay) by Roche. Flow cytometry was performed on all the blood samples (using Cytomics FC500 by Beckman Coulter cytometer). Prior to staining the cells for cytometric analysis, cell viability was assessed by trypan blue exclusion. Only the samples with at least 98% of cells viability were selected for the study. 10 thousand cells were assessed in every collected sample. All the blood samples were incubated for 15 min in the room temperature, in the darkness, using monoclonal antibodies conjugated with proper fluorochromes (anti-human CD 281 (TLR1) PE, IgG1, clone CD2. F4; anti-human CD 282 (TLR2) PE, IgG2a, clone TL2.1; anti-human CD 284 (TLR4) PE, IgG2a, clone HT A 125 produced by eBioscience company and anti-human CD14 FITC, IgG2a clone TUK4 produced by DakoCytomation company). The negative control was applied in accordance with the class of used monoclonal antibodies. After the incubation all the samples underwent the process of lysis in automatic lysis station (Epics Immunology Work Station) using Multi-Q-Prep set. Lymphocytes and granulocytes were separated using the gate in FS/SS system, monocytes were signed with the usage of anti-CD14 antibodies. All the cytometric analyzes were performed in maximum 60 min after the incubation. The results were given as MFI (mean fluorescence intensity), that indirectly equals the level of receptors' density on the cell surface. All the analyses were performed at maximum 2 h after collecting the sample. Statistical analysis of the results was performed using of non-parametrical Mann–Whitney U test in Statistica 5.0.
79
otolaryngologia polska 68 (2014) 77–82
Table I – Characteristics of patients from AOM group Patient no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Age (yr)
Diagnosis
CRP (mg/dl)
WBC (103/mL)
ESR (mm/h)
4 5 1.7 1.2 1.2 1.3 1.1 3.5 1.7 0.5 6 9 2.2 3.5 3.5 1.1 4 2.5 4 6.9 3.1 6.8 8.1 3.5 3
pAOM rAOM Mastoiditis rAOM rAOM rAOM rAOM rAOM Mastoiditis rAOM rAOM pAOM pAOM rAOM rAOM rAOM rAOM rAOM rAOM rAOM rAOM Mastoiditis rAOM pAOM rAOM
0.1 2.6 5.2 0.9 3.7 0.1 4.8 2.9 5.7 1.5 4.6 0.2 10.2 1.2 1.5 4.1 0.1 2.1 2.8 1.5 0.7 5.6 1.6 0.8 13.7
7.00 13.20 21.19 13.20 17.70 9.55 15.30 11.20 15.40 23.60 8.80 12.90 9.50 9.28 7.09 14.80 11.20 11.10 20.38 8.94 5.80 18.70 6.70 7.44 8.81
44 45 44 35 44 26 42 28 90 47 15 37 55 17 25 28 8 30 9 26 14 42 10 83 78
pAOM: persistent acute otitis media; rAOM: recurrent acute otitis media.
Results In AOM group CRP extended from 0.1 to 13.7 mg/dl. In four cases CRP level was within the normal range (below 0.5 mg/dl),
in all of the others – average level was 3.7 mg/dl. ESR level ranged from 7 to 90 mm/h, average 37 mm/h. The clinical and laboratory characteristics for the AOM and control groups are presented in Tables I and II, respectively.
Table II – Characteristics of patients from control group Patient no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Age (yr) 8 1 9 1.2 1 3 1.2 1 2.5 3.5 0.9 2 1.5 4.2 5.3 1.1 3.2 7 3.5 1.5 4 6 5.4 7 4.5
Diagnosis GUA GUA SL IH SL IH IH GUA GUA IH IH GUA IH GUA IH GUA SL IH SL IH GUA GUA IH IH SL
SL: skin lesions; GUA: genitourinary anomalies; IH: inguinal hernia.
CRP (mg/dl)
WBC (103/mL)
ESR (mm/h)
0.3 0.0 0.1 0.0 0.0 0.1 0.0 0.1 0.2 0.0 0.1 0.0 0.2 0.0 0.1 0.1 0.3 0.1 0.0 0.2 0.0 0.0 0.0 0.1 0.2
4.70 14.30 17.60 8.30 8.36 14.47 11.92 7.26 6.36 10.25 11.18 6.59 10.24 7.28 8.56 10.25 6.32 6.11 9.36 11.32 6.33 6.36 8.35 7.25 7.18
4 6 5 3 4 4 6 3 5 4 3 7 4 3 7 7 4 3 5 4 3 3 5 4 6
80
otolaryngologia polska 68 (2014) 77–82
Table III – Average fluorescence intensity (MFI) of TLR1, TLR2 and TLR4 on monocytes in control group and OMA group Monocytes MFI TLR1 MFI TLR2 MFI TLR4
Control group (N = 25)
OMA group (N = 25)
P
3.25 1.57 5.15 2.24 10.76 3.95
4.55 1.75 7.06 2.04 14.61 7.41
0.013 0.006 0.032
Table IV – Average fluorescence intensity (MFI) of TLR1, TLR2 and TLR4 on lymphocytes in control group and OMA group Lymphocytes MFI TLR1 MFI TLR2 MFI TLR4
Control group (N = 25)
OMA group (N = 25)
P
1.03 0.2 1.27 0.17 3.72 0.58
1.14 0.3 1.24 0.35 3.93 1.02
0.181 0.719 0.399
Table V – Average fluorescence intensity (MFI) of TLR1, TLR2 and TLR4 on granulocytes in control group and OMA group Granulocytes MFI TLR1 MFI TLR2 MFI TLR4
Control group (N = 25)
OMA group (N = 25)
P
1.66 0.28 2.06 0.39 6.18 1.25
1.85 0.35 2.24 0.63 7.19 1.22
0.057 0.253 0.0098
The monocytes showed the highest level of expression of all the studied TLRs (TLR1, TLR2, TLR4) in AOM and the control group as well (Table III). The lowest expression was detected on lymphocytes also in both groups (Table IV). In the control and OMA group on all types of examined cells: monocytes, lymphocytes and granulocytes TLR1 expression was the lowest, and TLR4 the highest. The expression of all examined TLRs was significantly higher on monocytes in AOM group. TLR4 was the only one among the TLRs which showed significantly higher expression on granulocytes in AOM group. The expression of TLR1 and TLR2 was also higher on granulocytes in this group, but the difference did not reach statistical significance (Table V). The expression of all TLRs on lymphocytes was not significantly different between AOM and the control group. No correlation was found between the markers of inflammatory response – CRP and ESR and the expression of TLRs on the elements of white cell system in peripheral blood.
Discussion In healthy middle ear mucosa only very few lymphocytes are present and most probably the first response against infection is mediated by an innate immunity system [14]. The studies on animal models showed that strains deficient of TLR 4, TLR2 or their signaling molecules showed more severe inflammation, reduced clearance of middle ear bacteria and spontaneously developed chronic otitis media [14–16]. In the healthy human middle ear mucosa presence
of at least 6 PPRs, among them TLR4, was demonstrated by immunohistochemical and RT PCR studies [17]. Samples of mucosa from patients with a chronic middle ear disease exhibited lower mRNA levels of TLRs (4, 5 and 7) [17]. Several TLRs (2, 4, 5, 9) were also detected in effusion collected from children with secretory otitis media [3, 13]. Some studies showed that certain polymorphisms in the TLR4 gene were associated with otitis media susceptibility [19], however, this was not confirmed in latest studies [20, 21]. As presented above, in recent years great progress in the understanding of TLRs function in the response of immunological system to infection of middle ear was observed. However, the publications presenting attempts to utilize this knowledge in clinical assessment or monitoring of inflammatory reaction are scarce probably because of difficulties in obtaining adequate material from the middle ear in routine practice. We undertook the study to examine if the similar up-regulation of TLRs takes place on the monocytes, granulocytes and lymphocytes of peripheral blood in patients with severe form of AOM. If this hypothesis would be confirmed TLRs could be used as a marker of inflammatory response, potentially modifying future treatment decisions. The performed studies showed that the expression of all examined TLRs on monocytes and TLR4 on granulocytes was significantly higher in the AOM group. Observed changes might express the variations in innate PPR-expression profiles, or more probably, be a sign of stimulation by bacterial antigens. Although recurrent otitis media is not usually accompanied by bacteraemia, it is characterized by prolonged presence of pathogenic bacteria (Gram-positive and Gram-negative) inside the tympanic cavity, increased nasopharyngeal bacterial colonization and recurrent upper respiratory tract infections. Bacterial products from those sites may provide the stimulus for TLRs expression on peripheral blood white cells. Also proinflammatory cytokines released to the blood stream during localized inflammatory process may contribute to the regulation of TLRs expression on monocytes and neutrophils [22]. Higher expression of TLRs on both monocytes and granulocytes result in enhanced activation of those cells by ligands of bacterial origin. Activation of TLR4 leads to enhanced synthesis and release of proinflammatory cytokines IL-1b, -6, -8, -12 and TNFa. Moreover TLR4 stimulation improve phagocytic capabilities of monocytes and expression of costymulatory molecules which makes presentation of antigen to lymphocytes T more effective [23]. TLR4 regulate also important granulocyte functions, including adhesion, generation of reactive oxygen species and inhibition of apoptosis [9]. TLR2 was up-regulated only on monocytes. Its activation triggers gene transcription of cytokines, mainly interferon's [24]. Observed in our study up-regulation of TLRs expression on monocytes and granulocytes may initiate the inflammatory cascade. Although in our study CRP level in AOM group was not very high (average 3.7 mg/dl), it was within normal range in only four cases, which speak up for the presence of general inflammatory reaction in the course of AOM. The other parameters of inflammatory response as ESR and WBC counts were also
otolaryngologia polska 68 (2014) 77–82
higher in AOM group. However no correlation was found between examined parameters of inflammatory reaction and TLR's expression. This may be the result of the fact that the expression of TLR's on studied cells showed great variability in the control as well as in the AOM groups as expressed by the values of standard deviation. It should also be taken into account that the inflammatory reaction is not the direct result of TLR's expression. Activation of several mechanisms influenced by various factors are involved in this complex and not easily predicted process. In the studies performed in the middle ear effusion also no correlation between TLR 2 and 4 expressions and the characteristics of effusion, presence of bacteria in the exudates or frequency of ventilation tube insertion was found [13, 18]. In the group of patients with AOM the monocytes were the cells which showed most distinct difference in TLRs expression comparing with the group of healthy children. On granulocytes such difference was observed only in the expression of TLR4. This observation is in the agreement with the fact that both this cells are involved in innate immune responses. No difference of expression was observed on lymphocytes participating mainly in the adaptive immune system. Gomes et al. [25], showed increased expression TLR4 on human monocytes 6 h after stimulation with LPS. However the expression of TLR4 on granulocytes was not similarly modulated by LPS which shows that the reaction of granulocytes on stimulation is different [25]. It is believed that TLR2 responds mainly to gram-positive and TLR4 to Gram negative bacteria. The findings of Zang et al. [26] demonstrate that during bacterial infection in vivo, newborns selectively and coordinately amplify the TLR2 pathway in Gram-positive bacterial infection and the TLR4 pathway in Gram negative bacterial infection. These results indicate that various bacteria differ in their dose-dependent patterns of induction of TLRs [24, 26]. However studies mostly performed in vitro, in laboratory models or in newborns, may not reflect the true situation during infectious diseases because pathogens are equipped with various ligands which may activate multiple signaling pathways. In cases of recurrent AOM we are usually dealing with polymicrobial infection which makes the interpretation of the role of specific bacteria in TLRs expression difficult. Differential expression of TLRs was described in various diseases [27–30]. Xu et al. [27] observed significantly higher expression of genes encoding five TLRs (2,3,4,7,8) on peripheral blood cells of patients with acute rotavirus diarrhea. Patients with irritable bowel syndrome presented increased expression of TLR2 on monocytes [28]. Pons et al. [29] have found up-regulated expression of TLR2 on peripheral blood monocytes from chronic obstructive pulmonary disease patients. In children with cystic fibrosis TLR4 expression was significantly higher compared to healthy controls but did not vary according to the presence or absence of pulmonary infection with Gram-negative or Gram-positive bacteria, so the authors consider the observed difference to be related to having CF per se and not related to current pulmonary infection [30]. The enhanced expression of TLR 1, TLR2, TLR4 on the monocytes of peripheral blood in the course of recurrent or persistent acute otitis media shows that the innate
81
immunological reaction is not limited to the middle ear and nasopharyngeal mucosa. This supports the importance of innate immunity in developing the general immunological response to infection. However the lack of correlation between TLRs expression and CRP, ESR and WBC limits theirs role as potential marker of inflammatory response.
Authors' contributions/Wkład autorów KT – data collection and interpretation, acceptance of final manuscript version, literature search. EK – data collection, statistical analysis, acceptance of final manuscript version. BS – study design, data interpretation, acceptance of final manuscript version, literature search. EH-P – study design, data interpretation, acceptance of final manuscript version, funds collection. JW – data interpretation, acceptance of final manuscript version, literature search.
Financial support/Finansowanie The manuscript was funded from grant no. 3-67743L.
Conflict of interest/Konflikt interesu None declared.
Ethics/Etyka The work described in this article have been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans; EU Directive 2010/63/EU for animal experiments; Uniform Requirements for manuscripts submitted to Biomedical journals.
references/pi smiennictwo
[1] Palm NW, Medzhitov R. Pattern recognition receptors and control of adaptive immunity. Immunol Rev 2009;227:221–233. [2] Turvey SE, Broide DH. Innate immunity. J Allergy Clin Immunol 2010;125:24–32. [3] Kim MG, Park DC, Shim JS, Jung H, Park MS, Kim YI, et al. TLR-9, NOD-1, NOD-2, RIG-I and immunoglobulins in recurrent otitis media with effusion. Int J Pediatr Otorhinolaryngol 2010;74:1425–1429. [4] Turvey SE, Hawn TR. Towards subtlety: understanding the role of Toll-like receptor signaling in susceptibility to human infections. Clin Immunol 2006;120:1–9. [5] Leichtle A, Lai Y, Wollenberg B, Wasserman SI, Ryan AF. Innate signaling in otitis media: pathogenesis and recovery. Curr Allergy Asthma Rep 2011;11:78–84. [6] Dabbagh K, Lewis DB. Toll-like receptors and T-helper-1/Thelper-2 responses. Curr Opin Infect Dis 2003;16:199–204. [7] Uematsu S, Akira S. Toll-like receptors and innate immunity. J Mol Med 2006;84:712–725.
82
otolaryngologia polska 68 (2014) 77–82
[8] Parker LC, Prince LR, Sabroe I. Translational mini-review series on toll-like receptors: networks regulated by Toll-like receptors mediate innate and adaptive immunity. Clin Exp Immunol 2007;147:199–207. [9] Sabroe I, Dower SK, Whyte MK. The role of toll-like receptors in the regulation of neutrophil migration, activation, and apoptosis. Clin Infect Dis 2005;15(Suppl 7):S421–S426. [10] Mundford RS. Sensing Gram-negative bacterial lipopolysaccharides: a human disease determinant? Infect Immun 2008;76:454–465. [11] Song JJ, Cho JG, Woo JS, Lee HM, Hwang SJ, Chae SW. Differential expression of toll-like receptors 2 and 4 in rat middle ear. Int J Pediatr Otorhinolaryngol 2009;73:821–824. [12] Komori M, Nakamura Y, Ping J, Feng L, Toyama K, Kim Y, et al. Pneumococcal peptidoglycan-polysaccharides regulate Toll-like receptor 2 in the mouse middle ear epithelial cells. Pediatr Res 2011;69:101–105. [13] Lee SY, Ryu EW, Kim JB, Yeo SG. Clinical approaches for understanding the expression levels of pattern recognition receptors in otitis media with effusion. Clin Exp Otorhinolaryngol 2011;4:163–167. [14] Leichtle A, Hernandez M, Pak K, Yamasaki K, Cheng CF, Webster NJ, et al. TLR4-mediated induction of TLR2 signaling is critical in the pathogenesis and resolution of otitis media. Innate Immun 2009;15:205–215. [15] Hirano T, Kodama S, Fujita K, Maeda K, Suzuki M. Role of Toll-like receptor 4 in innate immune responses in a mouse model of acute otitis media. FEMS Immunol Med Microbiol 2007;49:75–83. [16] Hernandez M, Leichtle A, Pak K, Ebmeyer J, Euteneuer S, Obonyo M, et al. Myeloid differentiation primary response gene 88 is required for the resolution of otitis media. J Infect Dis 2008;15:1862–1869. [17] Granath A, Cardell LO, Uddman R, Harder H. Altered Tolland Nod-like receptor expression in human middle ear mucosa from patients with chronic middle ear disease. J Infect 2011;63:174–176. [18] Lee YC, Kim C, Shim JS, Byun JY, Park MS, Cha CI, et al. Tolllike receptors 2 and 4 and their mutations in patients with otitis media and middle ear effusion. Clin Exp Otorhinolaryngol 2008;1:189–195. [19] Emonts M, Veenhoven RH, Wiertsema SP, HouwingDuistermaat JJ, Walraven V, de Groot R, et al. Genetic polymorphisms in immunoresponse genes TNFA, IL6, IL10, and TLR4 are associated with recurrent acute otitis media. Pediatrics 2007;120:814–823.
[20] Carroll SR, Zald PB, Soler ZM, Milczuk HA, Trune DR, Macarthur CJ. Innate immunity gene single nucleotide polymorphisms and otitis media. Int J Pediatr Otorhinolaryngol 2012;76:976–979. [21] Sale MM, Chen WM, Weeks DE, Mychaleckyj JC, Hou X, Marion M, et al. Evaluation of 15 functional candidate genes for association with chronic otitis media with effusion and/ or recurrent otitis media (COME/ROM). PLoS ONE 2011;6: e22297. [22] O'Mahony DS, Pham U, Iyer R, Hawn TR, Liles WC. Differential constitutive and cytokine-modulated expression of human Toll-like receptors in primary neutrophils, monocytes, and macrophages. Int J Med Sci 2008;5:1–8. [23] Majewska M, Szczepanik M. Rola receptorów tollpodobnych (TLR) w odporności wrodzonej I nabytej oraz ich funkcja w regulacji odpowiedzi immunologicznej. Postępy Hig Med Dosw 2006;60:52–63. [24] Gomes NE, Brunialti MKC, Mendes ME, Freudenberg M, Galanos C, Salomão R. Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood. Braz J Med Biol Res 2010;43:853–859. [25] Oliveira-Nascimento L, Massari P, Wetzler LM. The role of TLR2 in infection and immunity. Front Immunol 2012;3:79. [26] Zhang JP, Yang Y, Levy O, Chen C. Human neonatal peripheral blood leukocytes demonstrate pathogen-specific coordinate expression of TLR2, TLR4/MD2, and MyD88 during bacterial infection in vivo. Pediatr Res 2010; 68:479–483. [27] Xu J, Yang Y, Sun J, Ding Y, Su L, Shao C, et al. Expression of Toll-like receptors and their association with cytokine responses in peripheral blood mononuclear cells of children with acute rotavirus diarrhoea. Clin Exp Immunol 2006;144:376–381. [28] Ohman L, Lindmark AC, Isaksson S, Posserud I, Strid H, Sjövall H, et al. Increased TLR2 expression on blood monocytes in irritable bowel syndrome patients. Eur J Gastroenterol Hepatol 2012;24:398–405. [29] Pons J, Sauleda J, Regueiro V, Santos C, López M, Ferrer J, et al. Expression of Toll-like receptor 2 is up-regulated in monocytes from patients with chronic obstructive pulmonary disease. Respir Res 2006;10:64. [30] Sturges NC, Wikström ME, Winfield KR, Gard SE, Brennan S, Sly PD, et al. Monocytes from children with clinically stable cystic fibrosis show enhanced expression of Toll-like receptor 4. Pediatr Pulmonol 2010;45:883–889.