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Role of group 3 innate lymphoid cells during experimental otitis media in a rat model
International Journal of Pediatric Otorhinolaryngology 88 (2016) 146e152
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Role of group 3 innate lymphoid cells during experimental otitis media in a rat model* Chang Gun Cho a, *, Sung Ho Gong a, Hee-Bok Kim a, Jae-Jun Song b, Joo Hyun Park a, Yun-Sung Lim a, Seok-Won Park a a b
Department of Otorhinolaryngology-Head and Neck Surgery, Dongguk University Hospital, Goyang, Republic of Korea Department of Otorhinolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 April 2016 Received in revised form 1 July 2016 Accepted 2 July 2016 Available online 6 July 2016
The objective of this study was to evaluate the role of group 3 innate lymphoid cells (ILC3) in the middle ear (ME) mucosal response to bacterial infection in a rat model. To confirm the role of ILC3 in bacterially induced otitis media (OM), the serum concentrations of IL-17 and IL-22 were determined by ELISA, and the tissue expression of IL-17 and IL-22 in infected ME mucosa was assessed by immunohistochemical staining. Immunohistochemical staining of specific cell surface markers was also assessed to confirm the origin of the cells expressing IL-17 and IL-22. Twenty Sprague-Dawley rats were used in the surgicallyinduced animal model of OM. OM was induced by inoculation of non-typeable Haemophilus influenzae into the ME cavity of the rats. The rats were divided into four experimental groups: three infected groups and one control group. Infected groups were subdivided into sets of 5 rats, one for each of the three time points (1, 4 and 7 days post-inoculation). For determination of rat IL-17 and IL-22 levels in infected rats and control rats, infected or control ME mucosa sections were analyzed by immunohistochemistry with specific antibodies directed against IL-17 and IL-22. Immunohistochemical staining for CD3, RORgt, and NKp46 were also conducted on the samples to confirm the origin of cells expressing IL-17 and IL-22. IL-17 and IL-22 serum concentrations were significantly increased in the infected rats compared to control rats. Immunohistochemical staining revealed increased IL-17 and IL-22 expressions in all infected ME mucosae from the first day after inoculation. In addition, the results of tissue staining for the specific surface markers were negative for CD3 and NKp46, but were highly positive for RORgt. IL-17 and IL-22 revealed their association with the bacterially induced proliferative and hyperplastic responses of ME mucosa, which are characteristic features in pathogenesis of OM. Surface marker examination showed that the source cells for IL-17 and IL-22 seemed to be lymphoid tissue inducer (LTi) cells. The results suggest that LTi cells release IL-17 and IL-22, and play a significant role in both the early phase of OM induction and recovery from it. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Otitis media Mucosal immunity Innate immunity Haemophilus influenzae Interleukin-17 Interleukin-22
1. Introduction Responses of the immune system against various microorganisms are broadly classified as innate immunity and the acquired immunity. Unlike the acquired immune system, which features antigen specific receptors, the innate immune system has no antigen specificity or functional immunological memory. Innate
* This article was presented as a poster at the ARO 39th MidWinter Meeting in San deigo, California, USA, February 20e24, 2016, Association for Research in Otolaryngology. * Corresponding author. E-mail address: [email protected] (C.G. Cho).
http://dx.doi.org/10.1016/j.ijporl.2016.07.001 0165-5876/© 2016 Elsevier Ireland Ltd. All rights reserved.
lymphoid cells (ILCs) are morphologically similar to lymphoid cells and secrete various cytokines, but do not express antigen receptors like the T-cell receptor [1,2]. ILCs are part of the innate immune system and play a fundamental role in the immune responses to infection, repair of damaged tissues and maintenance of homeostasis [3,4]. Many cells classified as ILCs are known. Of this group, the natural killer (NK) cell is the only cell type that is directly cytotoxic to its target cells. Therefore, ILCs can be classified as cytotoxic ILCs, NK cell, and non-cytotoxic ILCs. NK cells and non-cytotoxic ILCs are further categorized into three subgroups according to their specific transcription factors, a reflection of the cytokines they produce and their specific functions within the cells [5e8].
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NK cells, a member of group 1 ILCs (ILC1), are cytotoxic like the CD8þ cytolytic T cell and secrete IFNg. IFNg-producing, noncytolytic ILC1 functions in a similar way as the T helper 1 cell (Th1) and exists in the mucosal epithelium. T-bet transcription factor is required for development and function of these cells [8e10]. Group 2 ILCs (ILC2) are involved in initial anti-parasitic immunity and allergic inflammatory response by secreting IL-4, IL-5, IL-6, and IL-13, thereby functioning in a way similar to T helper 2 (Th2) cells. The transcription factor GATA3 is crucial in development and maintenance of ILC2 [11e14]. Lymphoid tissue inducer (LTi) cells, which enhance the formation of lymphoid organs, are included in group 3 ILCs (ILC3). ILC3 under the direction of the transcription factor RORgt produce IL-17 and IL-22 in response to IL-23 and IL-1beta. In this way, ILC3 are involved in the mucosal immune response and maintenance of homeostasis [15e17]. In particular, IL-17 and IL-22 expressed by ILC3 promote inflammatory reactions, increase the expression of anti-bacterial peptides including b-defensin, and promote epithelial hyperplasia. Also, functional abnormalities of IL-17 and IL-22 are associated with many inflammatory autoimmune diseases, such as rheumatic arthritis [18,19], psoriasis [20,21], multiple sclerosis [22,23], and inflammatory bowel disease [24,25]. Recent studies of IL-17 and IL-22 have also led to a better understanding of the mechanism underlying these diseases [26e28]. Otitis media (OM) is common in children and infants. About 50%e70% of children experience an OM before the third year of life. Without proper treatment, complications due to infections, such as mastoiditis, brain abscess, and meningitis, may occur. Also, with a delay in language learning, issues with behavior and education may also occur due to prolonged hearing loss. Many factors, such as a bacterial infection and dysfunction of the Eustachian tube, may combine to cause OM. The most diagnostic pathologic change of OM is transformation and hyperplasia of the middle ear (ME) mucosa. The ME mucosa is 15e20 mm thick, but irritation including bacterial infection or trauma causes the mucosa to thicken to over 1000 mm, and mucosal epithelium transits from a simple columnar epithelium to a pseudostratified columnar epithelium having cilia and secretory function [29]. These changes are generated through the inflow of various inflammatory cells into the mucosa and are the cause of various symptoms of OM. The molecular cause-and-effect for the ME hyperplasia in OM is yet to be determined and is an active area of research. Exploration of the pathogenesis of ME mucosal change in animal models of OM has shown that mucosal change starts at the early stages of infection and the inflammatory changes disappear within 7 days and change back to the normal mucosa [30]. A variety of signaling molecules, transcription factors, and inflammatory cells are associated with the active inflammatory process of OM. But the precise mechanism of the genesis and development of OM, the exact mechanism of the healing process, and the interrelated role of the mediators have not been identified. The IL-17 and IL-22 secreted by ILC3 regulate the inflammatory process by increasing the expression of antibacterial peptides, and may play a role in the occurrence of the inflammatory process of the ME. With this hypothesis, we devised the present study to confirm the role of ILC3 in the inflammatory response of the ME mucosa in animal models and investigating the role of ILC3 in the change of the ME mucosa. We compared the serum levels of IL-17 and IL-22 before and after ME infection and confirmed the expression of these cytokines at the ME mucosa in the rat model of non-typeable Haemophilus influenzae (NTHi) strain 3655 induced acute OM. We also performed tests to identify the origin of cells secreting IL-17 and IL-22 by using specific surface antigens of CD3, RORgt and NKp46.
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2. Materials & methods 2.1. Animals Twenty Sprague-Dawley rats (7 weeks old and at 300 g in weight) were used. The breeding room was kept at a temperature of 21e23 C with 40e60% humidity and a 12-h cycle of light and darkness. On arrival, the rats were kept for one week to adjust to the laboratory environment. Sterile water and food were supplied to the rats to their fill. All care and experiments were performed in concordance with the regulations of the Animal Research Institute of Medical Science, Dongguk University, and the study protocol was approved by the Institutional Review Board (ID no. 2015-04123). 2.2. Subdivision of the groups The 20 rats were randomly divided into four groups, a control group and three experimental groups. The control group had phosphate buffered saline (PBS) injected into their bilateral bullae, and the experimental group were injected with a suspension of NTHi strain 3655 (details below). The infected group was subdivided into subgroups of five rats each. One rat was sacrificed at day 1, 4, and 7 post-inoculation. 2.3. Bacterial strain NTHi strain 3655 stored at 80 C was used to induce OM. The bacteria were streaked onto chocolate II agar (BD cat#221169). The batch was incubated overnight at 37 C with a supply of CO2. Two colonies were collected, blended with 25 mL Brain Heart Infusion Broth (Teknova, B9500) and 1 mL of Fildes enrichment (Remel, R45037) and were incubated overnight at 37 C. The mixture was centrifuged to obtain a pellet. A 1:100 diluted solution was made into a bacterial suspension of 105 NTHi cells/mL and was used in the experiment. 2.4. Induction of OM All rats were first checked for abnormalities such as middle ear effusion by otoscopy. The anesthetic used for the animals was made of 100 mg/mL of ketamine, 100 mg/mL of Xyalzine and 10 mg/mL of Acepromazine. The solution used at 0.1 mL/100 g of rat weight was injected into the rat thigh muscle. Under anesthesia, the rat was held in the supine position. The operation was performed under surgical microscopy guidance. Anterior neck was disinfected with betadine and a 3-cm vertical incision was made. The soft tissue was dissected and both bulla was exposed by pushing aside the strap muscle and the submandibular gland. A 25-gauge needle tip was used to pierce a hole at bulla tip, and 0.05 mL of bacterial suspension was injected into the hole. 2.5. Otomicroscopic findings A model AM4113TL handheld digital microscope-USB (DinoLite, New Taipei City, Taiwan) was used to check the inflammation of the ME mucosa and middle ear effusion. The same anesthetic solution was used at day 1, 3, 5, and 7 post-inoculation to take a picture of the tympanic membrane and to check the progress of OM. 2.6. Serum concentration of IL-17 and IL-22 The rats were anesthetized and 0.5e1 cc of blood was collected from the lateral tail vein at each date. Blood samples were collected in BD Microtainer® SST™ (REF 365967) and centrifuged. The
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collected sera were kept in a 70 C freezer. For determination of rat IL-17 and IL-22 levels in NTHi strain 3655 infected and control rats, mouse IL-17 and IL-22 ELISA kits (R&D Systems Inc., Quantikine ELISA #M1700 and #M2200) were used. Each serum concentration was checked using spectrophotometry. 2.7. Immunohistochemical staining After blood collection, each rat was sacrificed and the bilateral temporal bone was extracted to acquire the bullae. A total of 40 bullas were collected, with 10 for each group. The bullae were fixed in 4% neutral buffered formalin for 24 h, decalcified for over 14 days, using decalcifying solution (Sigma-Aldrich, Decalcifying SolutionLite, #D0818-1L). The tissue was then embedded in paraffin and sliced to a thickness of 5 mm. Hematoxylin (Sigma-Aldrich, #HHS32) and Eosin Y (Sigma-Aldrich, #E6003) (H&E) stains were used on the prepared slides. Photographs at 200 magnification were taken of each slide and each contained views of the ME cavity and the external ear canal. To check for the expression of IL-17 and IL-22 in the ME, immunohistochemical staining antibodies for each specific antigen (Santa Cruz Biotechnology, mouse monoclonal, #sc-374218 and #sc-14436) was used and coupled with 3,30 -diaminobenzidine (DAB) color development (Dako: Liquid DAB þ Substrate Chromogen System).
3.2. Change in serum IL-17 and IL-22 concentrations The serum concentration for IL-17 for the control group was 0.628 ± 0.233 pg/mL, and the inoculation group was 0.745 ± 0.572 pg/mL. There was no significant statistical difference in serum concentration of IL-17 in the two groups at Day 1 postinoculation (p ¼ 0.789). But at day 4, the concentration level was 2.721 ± 1.029 pg/mL and at Day 7, it was 5.415 ± 1.645 pg/mL for the inoculation group, showing a statistically significant increase in serum level with the passage of time (p < 0.0001, Fig. 2). Serum concentrations of IL-22 showed similar results between the two groups. The concentration level of IL-22 was 6.552 ± 0.960 pg/mL within the control group, 7.130 ± 2.978 pg/mL, 22.374 ± 6.175 pg/ mL, and 55.996 ± 15.402 pg/mL at day 1, 4, and 7 post-inoculation, respectively, showing statistically significant increases in serum concentration (p < 0.0001, Fig. 2).
2.8. Immunohistochemical staining of specific surface markers In order to examine the origin of cells expressing IL-17 and IL22, immunohistochemical staining of specific surface markers was performed. As CD3, RORgt, and NKp46 are specific surface antigens of T cells and ILCs, and the immunohistochemical staining was performed to confirm for presence of these cells. By quantitating the areas of positive expression in the immunohistochemical tissue stainings, the prevalence of the expression of the target cells can be made and analyzed for changes over time. 2.9. Statistical analysis Each subgroup was analyzed with the GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA) program using the ttest. The results were considered statistically significant at P < 0.05. 3. Results 3.1. Induced OM All the control rats and rats inoculated with NTHi strain 3655 were alive during the research period. The PBS injected control group showed no signs of bacterial OM, but the inoculated group started to show middle ear effusion and increased vascularity from day 1e3 post-inoculation. At day 5, the purulence decreased and partial effusion remained. After 7 days, the tympanic membrane had almost returned to normal (Fig. 1).
Fig. 2. The serum concentration of the control group and one day after the inoculation showed no differences in the serum concentration of IL-17, IL-22 for the two groups. But at day 4 and day 7 the serum concentration levels were significantly increased (p < 0.0001).
Fig. 1. Otomicroscopic findings of middle ear in bacterially induced otitis media (90 magnification). The start of middle ear effusion and increased vascularity from day 1e3 is seen. At day 5, purulency decreased and partial effusion remained. After 7 days, the tympanic membrane has almost returned to normal.
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3.3. Immunohistochemical staining The H&E stains of ME mucosa sections indicated severe thickening and cellular infiltration of the ME even one day after inoculation. The membrane thickening gradually subsided with time by day 4 and more so at day 7 post-inoculation (Fig. 3). The immunohistochemical stainings showed that IL-17 and IL-22 were expressed at the mucosal epithelium from day 1 and gradually increased with time. For the control group, IL-17 and IL-22 were not detectable for the ME sections (Fig. 4). 3.4. Immunohistochemical staining of cell specific surface antigens Cell specific surface antigen stainings of CD3, RORgt, and NKp46 were performed to investigate the origin of cells expressing IL-17 and IL-22. The CD3 and NKp46 antigen staining showed no significant differences for the control and the experimental group. But RORgt expression was strongly positive for the experimental group compared to the control group. Also the expression of the RORgt increased over time in the experimental group compared to the control group (Fig. 5). We also measured the section areas that stained positively for the above antigens and calculated the average size of the areas for each positive staining. There was no increase in CD3 and NKp46 positive areas over time, but the RORgt positive surface areas showed a significant increase (Fig. 6). 4. Discussion Many factors including bacterial infection along with ventilation disorders, such as a Eustachian tube obstruction, may combine to cause OM. The most diagnostic pathologic change of OM is transformation and hyperplasia of the ME mucosa. However, the hyperplasia of ME mucosa and the influx of various inflammatory cells into the mucosa is a reversible process; once the irritation of the mucosa ceases, the ME mucosa returns to its normal state by undergoing dedifferentiation [31]. If the transformation and hyperplasia of the ME mucosa occurs repeatedly, as in chronic OM, the structure of the mucosa changes irreversibly, and it can lead to permanent hearing loss. The molecular details in the pathogenesis of OM, particularly for the factors involved in the transformation and hyperplasia, and the recovery process are yet to be determined. Many recent reports have tried to elucidate the mechanism of the ME mucosal changes and inflammation in OM using bacteria induced OM animal models. In these studies, the role of genetics in susceptibility to OM, the role of the innate immune system, the mechanisms of the middle ear immune reaction, along with the relevant intracellular signaling mediators are being identified in various animal models included those in mice. The innate immune system is the primary line of defense against various microorganisms. Unlike the acquired immune system, it is to a certain extent more of a non-specific nature. The innate immune system is related with the occurrence of bacterial OM and any insufficiency in this defense mechanism is a major risk factor
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for OM. Kim et al. elaborated on the role of many innate immune receptors involved in the ME effusion, such as TLR9, NOD1, NOD2 [32]. Lee et al. focused on possible mutations of TLR2 and TLR4 in patients suffering from OM [33]. Animal models have shown that activation of various TLRs increases expression of a number of notable inflammatory cytokines including TNF-a and a deficit in TLRs delays the recovery from OM [34,35]. Also, many genetic deficiencies concerned with the innate immunity hinder recovery from OM [36e38]. As such, innate immunity is thought to affect the initial reaction of OM as well as the recovery and the reactivation process. Although ILCs are also a member of the innate immune system, their precise characteristics and function are not completely known. Various ILCs known so far are generally categorized into three groups depending on their cytokine secretion and specific transcription factors. Among these cells, ILC3 secretes IL-17 and IL22, which have various roles in the inflammatory process and have been proven to have a role in defense against bacterial infections. IL-17 induces the production of cytokines and chemokines, such as IL-6, TNF-a, IL-1b, GM-CSF, G-CSF, PG E2, nitric oxide, CXCL1, CXCL8, CCL2 and CCL7. As such, IL-17 acts as a typical proinflammatory mediator and functions in target cells such as epithelial cells, fibroblasts and endothelial cells [39]. IL-22 leads to expression of antimicrobial molecules like b-defensin within keratinocytes and promotes hyperplasia of the epithelium, thus playing an essential role in the immune function of the epithelial barrier [40,41]. One experiment with the colon epithelium in mice showed that IL-22 leads to secretion of mucin, forming a mucous layer that protects the epithelium from damage [42]. Many cells such as CD4þ T cells, CD8þ T cells, NK cells, and ILCs like LTi cells secrete IL-17 and IL-22, and the secretion patterns in humans and rodents differ [43e45]. The cell types secreting IL-17 and IL-22 depend on the location of the inflammation, and the extent and type of the activating pathogen. The severity of psoriasis and inflammatory bowel disease is directly proportional to the level of IL-17 and IL-22 produced in the affected tissues [41,46,47]. This study showed that the level of IL-17 and IL-22 increased within progression of OM. Immunohistochemistry showed that the expression of IL-17 and IL-22 in the ME mucosa started on the first day of infection. This suggests that IL-17 and IL-22 play a key role in initial mucosal pathology. Considering the functions of IL-17 and IL22 known to date, it may be associated with expression of antimicrobial entities such as b-defensin or other cytokines and chemokines. However, the exact roles of IL-7 and IL-22 in the pathology of ME mucosa are not known, and further research is required to dissect the potentially complex signaling of various inflammatory cells and their relevant transcription factors concerning the pathophysiology of OM. The serum concentration levels of IL-17 and IL-22 increased until day 7 and the strength of DAB immunohistochemical staining also increased with time. This indicates that IL-17 and IL-22 are associated with the recovery and the healing process of damaged tissue as well as the initial reaction of inflammation. This result is
Fig. 3. Middle ear mucosa with H&E staining (200 magnification). The ME mucosa showed severe thickening and cellular infiltration one day post bacterial inoculation. The membrane thickening gradually recovered with time, as seen with samples from day 4 and day 7.
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Fig. 4. Immunohistochemical staining shows that IL-17 and IL-22 are expressed at the mucosal epithelium from day 1 and their levels gradual increase with time as seen for the day 4 and day 7 samples.
Fig. 5. Surface markers, CD3, RORgt, and NKp46 were stained in the middle ear mucosa samples. RORgt is expressed at the mucosal epithelium from day 1 and its expression is gradually increased with time at day 4 and day 7 post-induction.
Fig. 6. The average staining area (%) of specific surface makers CD3, RORgt, and NKp46 for bacterially induced middle ear mucosa. Average area for RORgt positive cells gradually and significantly increased post-induction, whereas those for CD3 and NKp46 did not.
not surprising considering the fact that IL-17 and IL-22 play a significant role in target cells as part of the bacterial defense. TLRs, MyD88, TNF-a and TRIF are part of the components of the innate immunity, and reports show that recovery from OM is delayed in animal models which lack the genes for the above [34,35,48,49].
Further genetic research is required to define the function and mechanism of IL-17 and IL-22 concerned with recovery and relapse of OM. IL-17 and IL-22 are secreted from NK cells, LTi cells, and T helper type 17 (Th17) cells [39]. CD4þ T cells are divided into subtypes that include Th1, Th2, Th17 cells. Th17 as a subtype of CD4þ T cells, produce IL-17 and IL-22, and thus play a direct role in the pathogenesis of various inflammatory and autoimmune diseases [50,51]. Just like ILC3, Th17 cells are dependent on RORgt for development, and Th17 cells and ILC3 express similar cytokines and almost the same transcription factors are associated with these two cell types. To identify the origin of cells secreting IL-17 and IL-22 in NTHi strain 3655 induced OM, we checked the surface antigen by immunohistochemical staining. The results were negative for CD3 and NKp46, but were highly positive for RORgt, with increased expression as the severity of OM increased over time. This indicated that the origin of the increased expression of IL-17 and IL-22 was LTi cells [5], and that the LTi cells played an important role in the pathophysiology of NTHi strain 3655 induced OM. Immunohistochemical staining showed that the transcription factor RORgt was highly expressed in infected ME mucosa and its expression increased over time after inoculation. But the
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expression RORgt was high in the non-infected ME mucosa as well. RORgt is an important transcription factor for Th17 cells and ILC3 including LTi cells, and the reason RORgt was highly expressed in normal ME mucosa is not yet clear. The immunohistochemical analysis of the three cell surface antigens indicated that LTi cells were distributed in normal mucosa, and we can speculate that LTi cells may be related to the maintenance of mucosal homeostasis. As the immunohistochemical staining for CD3 was negative for our ME tissue samples, it is unlikely that Th17 cells play a role in the homeostasis and the infection response for the ME mucosa. With no expression of IL-17 and IL-22 seen in the non-infected state for the ME mucosa, the question of how LTi cells are involved in the homeostasis process has yet to be addressed. In addition, further studies are required to clearly identify the cell types secreting IL-17 and IL-22 as part of defining a role for these cytokines and the ILC3 in pathogenesis of bacterially induced OM. In summary, according to the serum concentration levels and the immunohistochemistry results, IL-17 and IL-22 expressions were related to the ME mucosal response to NTHi strain 3655 induced OM. As IL-17 and IL-22 were expressed in the ME mucosa from the first day post-induction, these cytokines play an early role in the infection process. As the serum concentration levels of these two cytokines increased gradually post-induction and the expression levels remained high at Day 7, IL-17 and IL-22 may be related to recovery and restoration of the bacterially assaulted tissue. LTi cell was the origin of the cell expressing IL-17 and IL-22 according to the immunohistochemical analysis. Further research is needed in defining how non-antigen specific ILCs control the immune response in the inflammatory disease settings, and how these cells control tissue homeostasis, and also what effects they may have on the acquired immune system. Moreover, in the pathogenesis of OM, the origin and function of IL-17 and IL-22, and role of ILC3 require further study, along with verification using a variety of animal models and pathogens.
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Acknowledgement This work was supported by the Dongguk University Research Fund of 2014.
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Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: http://www.ijporlonline.com/
Role of group 3 innate lymphoid cells during experimental otitis media in a rat model* Chang Gun Cho a, *, Sung Ho Gong a, Hee-Bok Kim a, Jae-Jun Song b, Joo Hyun Park a, Yun-Sung Lim a, Seok-Won Park a a b
Department of Otorhinolaryngology-Head and Neck Surgery, Dongguk University Hospital, Goyang, Republic of Korea Department of Otorhinolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 April 2016 Received in revised form 1 July 2016 Accepted 2 July 2016 Available online 6 July 2016
The objective of this study was to evaluate the role of group 3 innate lymphoid cells (ILC3) in the middle ear (ME) mucosal response to bacterial infection in a rat model. To confirm the role of ILC3 in bacterially induced otitis media (OM), the serum concentrations of IL-17 and IL-22 were determined by ELISA, and the tissue expression of IL-17 and IL-22 in infected ME mucosa was assessed by immunohistochemical staining. Immunohistochemical staining of specific cell surface markers was also assessed to confirm the origin of the cells expressing IL-17 and IL-22. Twenty Sprague-Dawley rats were used in the surgicallyinduced animal model of OM. OM was induced by inoculation of non-typeable Haemophilus influenzae into the ME cavity of the rats. The rats were divided into four experimental groups: three infected groups and one control group. Infected groups were subdivided into sets of 5 rats, one for each of the three time points (1, 4 and 7 days post-inoculation). For determination of rat IL-17 and IL-22 levels in infected rats and control rats, infected or control ME mucosa sections were analyzed by immunohistochemistry with specific antibodies directed against IL-17 and IL-22. Immunohistochemical staining for CD3, RORgt, and NKp46 were also conducted on the samples to confirm the origin of cells expressing IL-17 and IL-22. IL-17 and IL-22 serum concentrations were significantly increased in the infected rats compared to control rats. Immunohistochemical staining revealed increased IL-17 and IL-22 expressions in all infected ME mucosae from the first day after inoculation. In addition, the results of tissue staining for the specific surface markers were negative for CD3 and NKp46, but were highly positive for RORgt. IL-17 and IL-22 revealed their association with the bacterially induced proliferative and hyperplastic responses of ME mucosa, which are characteristic features in pathogenesis of OM. Surface marker examination showed that the source cells for IL-17 and IL-22 seemed to be lymphoid tissue inducer (LTi) cells. The results suggest that LTi cells release IL-17 and IL-22, and play a significant role in both the early phase of OM induction and recovery from it. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Otitis media Mucosal immunity Innate immunity Haemophilus influenzae Interleukin-17 Interleukin-22
1. Introduction Responses of the immune system against various microorganisms are broadly classified as innate immunity and the acquired immunity. Unlike the acquired immune system, which features antigen specific receptors, the innate immune system has no antigen specificity or functional immunological memory. Innate
* This article was presented as a poster at the ARO 39th MidWinter Meeting in San deigo, California, USA, February 20e24, 2016, Association for Research in Otolaryngology. * Corresponding author. E-mail address: [email protected] (C.G. Cho).
http://dx.doi.org/10.1016/j.ijporl.2016.07.001 0165-5876/© 2016 Elsevier Ireland Ltd. All rights reserved.
lymphoid cells (ILCs) are morphologically similar to lymphoid cells and secrete various cytokines, but do not express antigen receptors like the T-cell receptor [1,2]. ILCs are part of the innate immune system and play a fundamental role in the immune responses to infection, repair of damaged tissues and maintenance of homeostasis [3,4]. Many cells classified as ILCs are known. Of this group, the natural killer (NK) cell is the only cell type that is directly cytotoxic to its target cells. Therefore, ILCs can be classified as cytotoxic ILCs, NK cell, and non-cytotoxic ILCs. NK cells and non-cytotoxic ILCs are further categorized into three subgroups according to their specific transcription factors, a reflection of the cytokines they produce and their specific functions within the cells [5e8].
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NK cells, a member of group 1 ILCs (ILC1), are cytotoxic like the CD8þ cytolytic T cell and secrete IFNg. IFNg-producing, noncytolytic ILC1 functions in a similar way as the T helper 1 cell (Th1) and exists in the mucosal epithelium. T-bet transcription factor is required for development and function of these cells [8e10]. Group 2 ILCs (ILC2) are involved in initial anti-parasitic immunity and allergic inflammatory response by secreting IL-4, IL-5, IL-6, and IL-13, thereby functioning in a way similar to T helper 2 (Th2) cells. The transcription factor GATA3 is crucial in development and maintenance of ILC2 [11e14]. Lymphoid tissue inducer (LTi) cells, which enhance the formation of lymphoid organs, are included in group 3 ILCs (ILC3). ILC3 under the direction of the transcription factor RORgt produce IL-17 and IL-22 in response to IL-23 and IL-1beta. In this way, ILC3 are involved in the mucosal immune response and maintenance of homeostasis [15e17]. In particular, IL-17 and IL-22 expressed by ILC3 promote inflammatory reactions, increase the expression of anti-bacterial peptides including b-defensin, and promote epithelial hyperplasia. Also, functional abnormalities of IL-17 and IL-22 are associated with many inflammatory autoimmune diseases, such as rheumatic arthritis [18,19], psoriasis [20,21], multiple sclerosis [22,23], and inflammatory bowel disease [24,25]. Recent studies of IL-17 and IL-22 have also led to a better understanding of the mechanism underlying these diseases [26e28]. Otitis media (OM) is common in children and infants. About 50%e70% of children experience an OM before the third year of life. Without proper treatment, complications due to infections, such as mastoiditis, brain abscess, and meningitis, may occur. Also, with a delay in language learning, issues with behavior and education may also occur due to prolonged hearing loss. Many factors, such as a bacterial infection and dysfunction of the Eustachian tube, may combine to cause OM. The most diagnostic pathologic change of OM is transformation and hyperplasia of the middle ear (ME) mucosa. The ME mucosa is 15e20 mm thick, but irritation including bacterial infection or trauma causes the mucosa to thicken to over 1000 mm, and mucosal epithelium transits from a simple columnar epithelium to a pseudostratified columnar epithelium having cilia and secretory function [29]. These changes are generated through the inflow of various inflammatory cells into the mucosa and are the cause of various symptoms of OM. The molecular cause-and-effect for the ME hyperplasia in OM is yet to be determined and is an active area of research. Exploration of the pathogenesis of ME mucosal change in animal models of OM has shown that mucosal change starts at the early stages of infection and the inflammatory changes disappear within 7 days and change back to the normal mucosa [30]. A variety of signaling molecules, transcription factors, and inflammatory cells are associated with the active inflammatory process of OM. But the precise mechanism of the genesis and development of OM, the exact mechanism of the healing process, and the interrelated role of the mediators have not been identified. The IL-17 and IL-22 secreted by ILC3 regulate the inflammatory process by increasing the expression of antibacterial peptides, and may play a role in the occurrence of the inflammatory process of the ME. With this hypothesis, we devised the present study to confirm the role of ILC3 in the inflammatory response of the ME mucosa in animal models and investigating the role of ILC3 in the change of the ME mucosa. We compared the serum levels of IL-17 and IL-22 before and after ME infection and confirmed the expression of these cytokines at the ME mucosa in the rat model of non-typeable Haemophilus influenzae (NTHi) strain 3655 induced acute OM. We also performed tests to identify the origin of cells secreting IL-17 and IL-22 by using specific surface antigens of CD3, RORgt and NKp46.
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2. Materials & methods 2.1. Animals Twenty Sprague-Dawley rats (7 weeks old and at 300 g in weight) were used. The breeding room was kept at a temperature of 21e23 C with 40e60% humidity and a 12-h cycle of light and darkness. On arrival, the rats were kept for one week to adjust to the laboratory environment. Sterile water and food were supplied to the rats to their fill. All care and experiments were performed in concordance with the regulations of the Animal Research Institute of Medical Science, Dongguk University, and the study protocol was approved by the Institutional Review Board (ID no. 2015-04123). 2.2. Subdivision of the groups The 20 rats were randomly divided into four groups, a control group and three experimental groups. The control group had phosphate buffered saline (PBS) injected into their bilateral bullae, and the experimental group were injected with a suspension of NTHi strain 3655 (details below). The infected group was subdivided into subgroups of five rats each. One rat was sacrificed at day 1, 4, and 7 post-inoculation. 2.3. Bacterial strain NTHi strain 3655 stored at 80 C was used to induce OM. The bacteria were streaked onto chocolate II agar (BD cat#221169). The batch was incubated overnight at 37 C with a supply of CO2. Two colonies were collected, blended with 25 mL Brain Heart Infusion Broth (Teknova, B9500) and 1 mL of Fildes enrichment (Remel, R45037) and were incubated overnight at 37 C. The mixture was centrifuged to obtain a pellet. A 1:100 diluted solution was made into a bacterial suspension of 105 NTHi cells/mL and was used in the experiment. 2.4. Induction of OM All rats were first checked for abnormalities such as middle ear effusion by otoscopy. The anesthetic used for the animals was made of 100 mg/mL of ketamine, 100 mg/mL of Xyalzine and 10 mg/mL of Acepromazine. The solution used at 0.1 mL/100 g of rat weight was injected into the rat thigh muscle. Under anesthesia, the rat was held in the supine position. The operation was performed under surgical microscopy guidance. Anterior neck was disinfected with betadine and a 3-cm vertical incision was made. The soft tissue was dissected and both bulla was exposed by pushing aside the strap muscle and the submandibular gland. A 25-gauge needle tip was used to pierce a hole at bulla tip, and 0.05 mL of bacterial suspension was injected into the hole. 2.5. Otomicroscopic findings A model AM4113TL handheld digital microscope-USB (DinoLite, New Taipei City, Taiwan) was used to check the inflammation of the ME mucosa and middle ear effusion. The same anesthetic solution was used at day 1, 3, 5, and 7 post-inoculation to take a picture of the tympanic membrane and to check the progress of OM. 2.6. Serum concentration of IL-17 and IL-22 The rats were anesthetized and 0.5e1 cc of blood was collected from the lateral tail vein at each date. Blood samples were collected in BD Microtainer® SST™ (REF 365967) and centrifuged. The
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collected sera were kept in a 70 C freezer. For determination of rat IL-17 and IL-22 levels in NTHi strain 3655 infected and control rats, mouse IL-17 and IL-22 ELISA kits (R&D Systems Inc., Quantikine ELISA #M1700 and #M2200) were used. Each serum concentration was checked using spectrophotometry. 2.7. Immunohistochemical staining After blood collection, each rat was sacrificed and the bilateral temporal bone was extracted to acquire the bullae. A total of 40 bullas were collected, with 10 for each group. The bullae were fixed in 4% neutral buffered formalin for 24 h, decalcified for over 14 days, using decalcifying solution (Sigma-Aldrich, Decalcifying SolutionLite, #D0818-1L). The tissue was then embedded in paraffin and sliced to a thickness of 5 mm. Hematoxylin (Sigma-Aldrich, #HHS32) and Eosin Y (Sigma-Aldrich, #E6003) (H&E) stains were used on the prepared slides. Photographs at 200 magnification were taken of each slide and each contained views of the ME cavity and the external ear canal. To check for the expression of IL-17 and IL-22 in the ME, immunohistochemical staining antibodies for each specific antigen (Santa Cruz Biotechnology, mouse monoclonal, #sc-374218 and #sc-14436) was used and coupled with 3,30 -diaminobenzidine (DAB) color development (Dako: Liquid DAB þ Substrate Chromogen System).
3.2. Change in serum IL-17 and IL-22 concentrations The serum concentration for IL-17 for the control group was 0.628 ± 0.233 pg/mL, and the inoculation group was 0.745 ± 0.572 pg/mL. There was no significant statistical difference in serum concentration of IL-17 in the two groups at Day 1 postinoculation (p ¼ 0.789). But at day 4, the concentration level was 2.721 ± 1.029 pg/mL and at Day 7, it was 5.415 ± 1.645 pg/mL for the inoculation group, showing a statistically significant increase in serum level with the passage of time (p < 0.0001, Fig. 2). Serum concentrations of IL-22 showed similar results between the two groups. The concentration level of IL-22 was 6.552 ± 0.960 pg/mL within the control group, 7.130 ± 2.978 pg/mL, 22.374 ± 6.175 pg/ mL, and 55.996 ± 15.402 pg/mL at day 1, 4, and 7 post-inoculation, respectively, showing statistically significant increases in serum concentration (p < 0.0001, Fig. 2).
2.8. Immunohistochemical staining of specific surface markers In order to examine the origin of cells expressing IL-17 and IL22, immunohistochemical staining of specific surface markers was performed. As CD3, RORgt, and NKp46 are specific surface antigens of T cells and ILCs, and the immunohistochemical staining was performed to confirm for presence of these cells. By quantitating the areas of positive expression in the immunohistochemical tissue stainings, the prevalence of the expression of the target cells can be made and analyzed for changes over time. 2.9. Statistical analysis Each subgroup was analyzed with the GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA) program using the ttest. The results were considered statistically significant at P < 0.05. 3. Results 3.1. Induced OM All the control rats and rats inoculated with NTHi strain 3655 were alive during the research period. The PBS injected control group showed no signs of bacterial OM, but the inoculated group started to show middle ear effusion and increased vascularity from day 1e3 post-inoculation. At day 5, the purulence decreased and partial effusion remained. After 7 days, the tympanic membrane had almost returned to normal (Fig. 1).
Fig. 2. The serum concentration of the control group and one day after the inoculation showed no differences in the serum concentration of IL-17, IL-22 for the two groups. But at day 4 and day 7 the serum concentration levels were significantly increased (p < 0.0001).
Fig. 1. Otomicroscopic findings of middle ear in bacterially induced otitis media (90 magnification). The start of middle ear effusion and increased vascularity from day 1e3 is seen. At day 5, purulency decreased and partial effusion remained. After 7 days, the tympanic membrane has almost returned to normal.
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3.3. Immunohistochemical staining The H&E stains of ME mucosa sections indicated severe thickening and cellular infiltration of the ME even one day after inoculation. The membrane thickening gradually subsided with time by day 4 and more so at day 7 post-inoculation (Fig. 3). The immunohistochemical stainings showed that IL-17 and IL-22 were expressed at the mucosal epithelium from day 1 and gradually increased with time. For the control group, IL-17 and IL-22 were not detectable for the ME sections (Fig. 4). 3.4. Immunohistochemical staining of cell specific surface antigens Cell specific surface antigen stainings of CD3, RORgt, and NKp46 were performed to investigate the origin of cells expressing IL-17 and IL-22. The CD3 and NKp46 antigen staining showed no significant differences for the control and the experimental group. But RORgt expression was strongly positive for the experimental group compared to the control group. Also the expression of the RORgt increased over time in the experimental group compared to the control group (Fig. 5). We also measured the section areas that stained positively for the above antigens and calculated the average size of the areas for each positive staining. There was no increase in CD3 and NKp46 positive areas over time, but the RORgt positive surface areas showed a significant increase (Fig. 6). 4. Discussion Many factors including bacterial infection along with ventilation disorders, such as a Eustachian tube obstruction, may combine to cause OM. The most diagnostic pathologic change of OM is transformation and hyperplasia of the ME mucosa. However, the hyperplasia of ME mucosa and the influx of various inflammatory cells into the mucosa is a reversible process; once the irritation of the mucosa ceases, the ME mucosa returns to its normal state by undergoing dedifferentiation [31]. If the transformation and hyperplasia of the ME mucosa occurs repeatedly, as in chronic OM, the structure of the mucosa changes irreversibly, and it can lead to permanent hearing loss. The molecular details in the pathogenesis of OM, particularly for the factors involved in the transformation and hyperplasia, and the recovery process are yet to be determined. Many recent reports have tried to elucidate the mechanism of the ME mucosal changes and inflammation in OM using bacteria induced OM animal models. In these studies, the role of genetics in susceptibility to OM, the role of the innate immune system, the mechanisms of the middle ear immune reaction, along with the relevant intracellular signaling mediators are being identified in various animal models included those in mice. The innate immune system is the primary line of defense against various microorganisms. Unlike the acquired immune system, it is to a certain extent more of a non-specific nature. The innate immune system is related with the occurrence of bacterial OM and any insufficiency in this defense mechanism is a major risk factor
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for OM. Kim et al. elaborated on the role of many innate immune receptors involved in the ME effusion, such as TLR9, NOD1, NOD2 [32]. Lee et al. focused on possible mutations of TLR2 and TLR4 in patients suffering from OM [33]. Animal models have shown that activation of various TLRs increases expression of a number of notable inflammatory cytokines including TNF-a and a deficit in TLRs delays the recovery from OM [34,35]. Also, many genetic deficiencies concerned with the innate immunity hinder recovery from OM [36e38]. As such, innate immunity is thought to affect the initial reaction of OM as well as the recovery and the reactivation process. Although ILCs are also a member of the innate immune system, their precise characteristics and function are not completely known. Various ILCs known so far are generally categorized into three groups depending on their cytokine secretion and specific transcription factors. Among these cells, ILC3 secretes IL-17 and IL22, which have various roles in the inflammatory process and have been proven to have a role in defense against bacterial infections. IL-17 induces the production of cytokines and chemokines, such as IL-6, TNF-a, IL-1b, GM-CSF, G-CSF, PG E2, nitric oxide, CXCL1, CXCL8, CCL2 and CCL7. As such, IL-17 acts as a typical proinflammatory mediator and functions in target cells such as epithelial cells, fibroblasts and endothelial cells [39]. IL-22 leads to expression of antimicrobial molecules like b-defensin within keratinocytes and promotes hyperplasia of the epithelium, thus playing an essential role in the immune function of the epithelial barrier [40,41]. One experiment with the colon epithelium in mice showed that IL-22 leads to secretion of mucin, forming a mucous layer that protects the epithelium from damage [42]. Many cells such as CD4þ T cells, CD8þ T cells, NK cells, and ILCs like LTi cells secrete IL-17 and IL-22, and the secretion patterns in humans and rodents differ [43e45]. The cell types secreting IL-17 and IL-22 depend on the location of the inflammation, and the extent and type of the activating pathogen. The severity of psoriasis and inflammatory bowel disease is directly proportional to the level of IL-17 and IL-22 produced in the affected tissues [41,46,47]. This study showed that the level of IL-17 and IL-22 increased within progression of OM. Immunohistochemistry showed that the expression of IL-17 and IL-22 in the ME mucosa started on the first day of infection. This suggests that IL-17 and IL-22 play a key role in initial mucosal pathology. Considering the functions of IL-17 and IL22 known to date, it may be associated with expression of antimicrobial entities such as b-defensin or other cytokines and chemokines. However, the exact roles of IL-7 and IL-22 in the pathology of ME mucosa are not known, and further research is required to dissect the potentially complex signaling of various inflammatory cells and their relevant transcription factors concerning the pathophysiology of OM. The serum concentration levels of IL-17 and IL-22 increased until day 7 and the strength of DAB immunohistochemical staining also increased with time. This indicates that IL-17 and IL-22 are associated with the recovery and the healing process of damaged tissue as well as the initial reaction of inflammation. This result is
Fig. 3. Middle ear mucosa with H&E staining (200 magnification). The ME mucosa showed severe thickening and cellular infiltration one day post bacterial inoculation. The membrane thickening gradually recovered with time, as seen with samples from day 4 and day 7.
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Fig. 4. Immunohistochemical staining shows that IL-17 and IL-22 are expressed at the mucosal epithelium from day 1 and their levels gradual increase with time as seen for the day 4 and day 7 samples.
Fig. 5. Surface markers, CD3, RORgt, and NKp46 were stained in the middle ear mucosa samples. RORgt is expressed at the mucosal epithelium from day 1 and its expression is gradually increased with time at day 4 and day 7 post-induction.
Fig. 6. The average staining area (%) of specific surface makers CD3, RORgt, and NKp46 for bacterially induced middle ear mucosa. Average area for RORgt positive cells gradually and significantly increased post-induction, whereas those for CD3 and NKp46 did not.
not surprising considering the fact that IL-17 and IL-22 play a significant role in target cells as part of the bacterial defense. TLRs, MyD88, TNF-a and TRIF are part of the components of the innate immunity, and reports show that recovery from OM is delayed in animal models which lack the genes for the above [34,35,48,49].
Further genetic research is required to define the function and mechanism of IL-17 and IL-22 concerned with recovery and relapse of OM. IL-17 and IL-22 are secreted from NK cells, LTi cells, and T helper type 17 (Th17) cells [39]. CD4þ T cells are divided into subtypes that include Th1, Th2, Th17 cells. Th17 as a subtype of CD4þ T cells, produce IL-17 and IL-22, and thus play a direct role in the pathogenesis of various inflammatory and autoimmune diseases [50,51]. Just like ILC3, Th17 cells are dependent on RORgt for development, and Th17 cells and ILC3 express similar cytokines and almost the same transcription factors are associated with these two cell types. To identify the origin of cells secreting IL-17 and IL-22 in NTHi strain 3655 induced OM, we checked the surface antigen by immunohistochemical staining. The results were negative for CD3 and NKp46, but were highly positive for RORgt, with increased expression as the severity of OM increased over time. This indicated that the origin of the increased expression of IL-17 and IL-22 was LTi cells [5], and that the LTi cells played an important role in the pathophysiology of NTHi strain 3655 induced OM. Immunohistochemical staining showed that the transcription factor RORgt was highly expressed in infected ME mucosa and its expression increased over time after inoculation. But the
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expression RORgt was high in the non-infected ME mucosa as well. RORgt is an important transcription factor for Th17 cells and ILC3 including LTi cells, and the reason RORgt was highly expressed in normal ME mucosa is not yet clear. The immunohistochemical analysis of the three cell surface antigens indicated that LTi cells were distributed in normal mucosa, and we can speculate that LTi cells may be related to the maintenance of mucosal homeostasis. As the immunohistochemical staining for CD3 was negative for our ME tissue samples, it is unlikely that Th17 cells play a role in the homeostasis and the infection response for the ME mucosa. With no expression of IL-17 and IL-22 seen in the non-infected state for the ME mucosa, the question of how LTi cells are involved in the homeostasis process has yet to be addressed. In addition, further studies are required to clearly identify the cell types secreting IL-17 and IL-22 as part of defining a role for these cytokines and the ILC3 in pathogenesis of bacterially induced OM. In summary, according to the serum concentration levels and the immunohistochemistry results, IL-17 and IL-22 expressions were related to the ME mucosal response to NTHi strain 3655 induced OM. As IL-17 and IL-22 were expressed in the ME mucosa from the first day post-induction, these cytokines play an early role in the infection process. As the serum concentration levels of these two cytokines increased gradually post-induction and the expression levels remained high at Day 7, IL-17 and IL-22 may be related to recovery and restoration of the bacterially assaulted tissue. LTi cell was the origin of the cell expressing IL-17 and IL-22 according to the immunohistochemical analysis. Further research is needed in defining how non-antigen specific ILCs control the immune response in the inflammatory disease settings, and how these cells control tissue homeostasis, and also what effects they may have on the acquired immune system. Moreover, in the pathogenesis of OM, the origin and function of IL-17 and IL-22, and role of ILC3 require further study, along with verification using a variety of animal models and pathogens.
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Acknowledgement This work was supported by the Dongguk University Research Fund of 2014.
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