Mucosal Regulatory System for the Balanced Immunity in the Middle Ear and Nasopharynx

C H A P T E R

18 Mucosal Regulatory System for the Balanced Immunity in the Middle Ear and Nasopharynx Mucosal Immunity of Middle Ear and Nasopharynx Hideyuki Kawauchi Department of Otorhinolaryngology, Faculty of Medicine, Shimane University, Izumo City, Japan

I. INTRODUCTION Bacteria and their components, such as lipopolysaccharide (LPS) or teichoic acid (TA), induce middle ear or nasopharyngeal inflammation, which can become a so-called vicious circle in the auditory tube and tympanic cavity and the paranasal sinus. Innate and adaptive immune cells recognize microbes via various innate and antigen-specific receptors, respectively. The former includes toll-like receptors (TLRs) expressed on dendritic cells, macrophages, endothelial cells, and γδ T cells. Once the ostium blockade occurs, mucosal swelling and paranasal sinus inflammation can persist. The vicious circle in the middle ear cleft or paranasal sinus had been proposed and

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00018-3

classically explained by the complement pathway. A revised consideration now takes into account the interactions between microbial products and TLRs present on resident epithelial cells and/or recruited inflammatory cells. These various TLR-expressing cells can secrete different inflammatory cytokines and/or chemokines in response to the continuous stimulation by molecules with pathogen-associated molecular patterns (PAMPs) [1 4]. In this chapter, innate and adaptive immunity of the middle ear and nasopharynx are discussed. We discuss the clinical impact of mucosal regulatory system to modify inflammatory diseases such as otitis media and allergic rhinitis, using a number of our experimental studies as examples.

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II. INNATE AND ACQUIRED IMMUNITY OF MIDDLE EAR AND NASOPHARYNX A. Distribution of Toll-Like Receptors in Human Epithelial Cells in the Middle Ear and Changes That Result From the Ensuing Middle Ear Inflammatory Response to Microbial Infection At least ten different TLRs have been detected on the epithelial cell surface in the human middle ear. TLRs contain an N-terminal extracellular leucine-rich repeat domain and a cytoplasmic toll/IL-1 receptor domain. TLR molecules provide protection against infection by recognizing infectious agents through their invariant PAMPs, resulting in the mobilization of appropriate immune defenses [5,6]. The activation of most TLRs results in downstream activation of the mitogen-activated protein kinase or the nuclear factor kappa B (NF-κB)-dependent cell signaling cascades, thus leading to further activation of immune responses [7]. In otitis media, recognition of nontypable Haemophilus influenzae (NTHi) is associated with several PAMPs acting as TLR ligands. NTHi cell surface peptidoglycans and the associated proteins, such as outer membrane protein P6, serve as TLR2 ligands [8]. Another notable example is lipooligosaccharide, which serves as a ligand for TLR2 and TLR4 [9]. Not surprisingly, polymorphisms in the gene encoding for TLR4 have been associated with recurrent acute otitis media [10]. When infected with NTHi, TLR4knockout mice exhibit a depressed mucosal immune response compared to wild-type mice [11]. These TLR4-knockout mice display inferior immune responses with regard to mucosal IgA, systemic IgG, and T helper 1 (Th1) cells [11]. NTHi and its various immunogenic molecules have been shown not only to directly activate

TLR, but also to upregulate TLR2 gene expression in middle ear epithelial cell lines [12]. Patients with chronic middle ear disease, such as chronic secretory otitis media (CSOM), show lower mRNA levels for TLR4, TLR5, and TLR7 than the control group [13]. A recent report has further confirmed these findings, demonstrating lower mRNA and protein levels for TLR2, TLR4, and TLR5 in the middle ear mucosa of CSOM patients [14]. The downregulation of TLR expression during otitis media can lead to inefficient host defense in the middle ear. This can cause repeated infections and persistent inflammation, eventually leading to recurrent, persistent chronic middle ear diseases.

B. Distribution of Toll-Like Receptors in Human Epithelial Cells in Nasopharyngeal Mucosae and Its Modification of Type I Allergic Inflammation in Nasal Mucosae In our recent studies, we examined the role of TLRs of upper respiratory tract mucosal epithelial cells in chemokine (interleukin-8, IL-8) and cytokine (interleukin-15, IL-15) induction and intracellular signaling pathway and modification of inflammatory response by antiinflammatory agents. Northern blot analysis and reverse transcription polymerase chain reaction (RT-PCR) were done to determine the TLR distribution by cultured human nasal epithelial cells (HNECs). Both TLR4 and TLR 9 mRNA expression were undetectable. Respiratory epithelial cells constitutively expressed mRNA for TLR2, TLR 3, and TLR6 but not for TLR4 and TLR9 (Fig. 18.1). Northern blot analysis revealed IL-15-specific mRNA being strongly expressed after lipoprotein stimulation. In contrast, it was not found following TLR4 agonist stimulation. Lipoprotein significantly induced IL-8 production by both A549 cells and HNECs, whereas

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II. INNATE AND ACQUIRED IMMUNITY OF MIDDLE EAR AND NASOPHARYNX

FIGURE 18.1 Expression of TLR mRNA by macrophages and nasal epithelial cells. Expression of TLR mRNA by A549 cells, HNEC-1, and HNEC-2 was examined by RT-PCR. Both A549 cells and HNECs showed TLR2 and TLR3 mRNA expression; however, HNECs expressed no TLR4 mRNA. A549 cells and HNEC1 expressed TLR6 mRNA, but A549 cells and HNECs expressed no TLR9 mRNA. TLR1, TLR5, TLR7, TLR8, and TLR10 mRNAs were also not expressed by A549 cells and HNECs. U937, macrophage lineage cells used as controls, expressed markedly high levels of TLR2, TLR3, TLR4, TLR6, and TLR9 mRNA. RT-PCR analysis of beta-actin expression confirmed the quality of all RNA preparations used for RT-PCR. No band was detected in the non-RT sample by PCR.

stimulation of these cells with lipid A resulted in no induction of IL-8 production (Fig. 18.2). The IL-15 concentration in the supernatants of CCL185 cells was also upregulated after lipoprotein stimulation in a dose-dependent manner. Lipoprotein induced IL-15 and IL-8 production by respiratory epithelial cells, which are strictly dependent upon TLR2 stimulation.

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Lipoprotein-induced IL-15 production by respiratory epithelial cells was abolished upon NF-κB inhibition. Exposure to airborne endotoxin in infancy may protect against asthma by promoting enhanced Th1-type response and tolerance to allergens. On the other hand, later in life, exposure to airborne endotoxin adversely affects patients with asthma. Mast cells, the key player for eliciting allergic rhinitis, produce Th2 cytokines subsequent to in vitro LPS stimulation via TLR4, but in vivo studies remain to be performed [15 17]. LPS inhalation exacerbates allergic airway inflammation by activating mast cells via TLR4 and promoting Th2 responses. To investigate the effect of LPS on eliciting murine allergic rhinitis, an experimental model was tested. At the elicitation phase, ovalbumin (OVA) was administered nasally for 7 consecutive days without or with LPS, and on the final challenge, sneezing rates were measured along with nasal tissue analysis and Th2 cytokine. As a result (Fig. 18.3), LPS aggravated the induction of type 1 allergic reaction [18]. Furthermore, a significant difference in sneezing rates between C3H/HeN mice challenged with OVA alone and those challenged with OVA plus LPS was found, but this difference was not detected in TLR4mutant C3H/HeJ mice. Eosinophil infiltration was more prominent in C3H/HeN mice challenged with OVA and LPS in comparison to that in mice challenged with OVA alone. By Western blot analysis, IL-5, IL-10, and IL-13 expression was detected in both groups, but IL-5 expression was upregulated in mice challenged with OVA plus LPS. However, there was no significant difference in eosinophil infiltration and Th2 cytokine expression between C3H/HeJ mice challenged with OVA alone and OVA plus LPS. Collectively, these data suggest that LPS aggravates nasal symptoms, upregulating Th2 cytokine production of mast cells in a TLR4dependent fashion.

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FIGURE 18.2 IL-8 production from A549, HNEC-1, and HNEC-2 cells activated with lipoprotein or lipid A. IL-8-producing activities in A549 cells and HNECs after stimulation with lipoprotein (as a TLR2 ligand) and lipid A (as a TLR4 ligand) were examined. Lipoprotein significantly induced IL-8 production in both A549 cells and HNECs, whereas stimulation of these cells with lipid A resulted in no induction of IL-8 production.

FIGURE 18.3 Effect of intranasal LPS administration at eliciting phase of allergic rhinitis in murine model. At the elicitation phase, ovalbumin (OVA) antigens alone or with LPS were introduced intranasally for 7 consecutive days. On the final challenge, sneezing rates were counted in a 10-min period. A significant difference in sneezing rates between C3H/ HeN mice challenged with OVA alone and those challenged with OVA and LPS was found.

III. IMMUNOMODULATION OF MIDDLE EAR AND NASOPHARYNGEAL MUCOSAE AND ITS CLINICAL IMPACT The pathogenesis of acute otitis media or otitis media with effusion (OME) was extensively elucidated in the 1980s and 1990s. Bacterial or

viral infections and their products in the middle ear were found to be responsible for triggering middle ear acute otitis media or OME [19 22]. Suppression of immune-mediated OME by mucosa-derived suppressor T cells was first reported by Ueyama et al. [23], suggesting a mucosal regulatory system via antigen-specific

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IV. INNOVATIVE IMMUNOTHERAPY FOR ATTENUATING NASAL SYMPTOMS

immunomodulation. In their study, the effects of mucosa-derived suppressor T cells in mice were investigated upon induction of IgGmediated OME, since antigen antibody reactions in the tympanic cavity are pathogenic mechanisms of OME. Splenic T suppressor cells from orally OVA-dosed C3H/HeN female mice were transferred intravenously to syngeneic mice. The recipients were then immunized intraperitoneally with OVA and later challenged with OVA into the tympanic cavity. Nine of ten control mice, which received splenic T cells from saline-fed mice, developed OME, while OME was seen in only one of ten recipients receiving splenic T suppressor cells from OVA-fed mice. The results showed that IgG-mediated OME can be suppressed to a certain extent by the induction of antigen-specific, mucosa-derived T suppressor cells. Allergic rhinitis is well elucidated as a type 1 allergy of the nasal mucosa. Sublingual immunotherapy (SLIT) has been recently employed as a painless and effective therapeutic treatment modality for allergic rhinitis. So far, its mechanism of action has been elucidated by using immune serum and lymphocytes in an antigenspecific fashion [24 26]. Because of the limitations in sampling human tissues, there is still controversy among many reports between clinical efficacy and experimental results. To ascertain the mechanism of action, further studies in promising rodents and nonhuman primates need to be pursued. To this end, we successfully developed an effective murine model for SLIT for allergic rhinitis. Mice were treated with OVA sublingually followed by intraperitoneal sensitization and nasal challenge [27 28]. Sublingually treated mice showed significantly decreased OVA-specific IgE responses as well as suppressed Th2 cell immune responses. Nasal symptoms and eosinophil recruitment into the nasal mucosa were significantly diminished in sublingually treated mice. Sublingual OVA administration did not alter the frequency of CD41CD251 regulatory T cells (Tregs), but led

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to upregulation of Foxp3- and IL-10-specific mRNAs in the Tregs of the cervical lymph nodes (CLNs), which strongly suppressed Th2-type cytokine production from CD41CD252 effector T cells in vitro. Furthermore, sublingual administration of plasmids, encoding the lymphoid chemokines CCL19 and CCL21-Ser DNA together with OVA, suppressed allergic responses. These results suggest that IL-10-expressing CD41CD251Foxp31 Tregs in the CLNs are involved in the suppression of allergic responses and that CCL19/CCL21 may contribute to it in mice that received SLIT.

IV. INNOVATIVE IMMUNOTHERAPY FOR ATTENUATING NASAL SYMPTOMS OF CEDAR POLLINOSIS VIA THE MUCOSAL ROUTE WITH TRANSGENIC RICE SEEDS CONTAINING HYPOALLERGENIC CRYJ1 AND CRYJ2 T CELL EPITOPES Allergen-specific subcutaneous immunotherapy with Japanese cedar pollen extract has been employed to desensitize patients with cedar pollinosis. SLIT for allergy to Japanese cedar pollen is allowed in Japan and has been determined to be a clinically safe and effective method of treatment. However, its mechanism of action remains to be determined, and the question of adverse consequences of SLIT needs to be further studied. We examined the effect of sublingual administration of protein bodies (PB) of transgenic (Tg) rice seeds (see further description of plant-based vaccines and immunotherapeutics in Chapters 20: Plant-Based Mucosal Vaccine Delivery Systems and Chapter 21: Plant-Based Mucosal Immunotherapy: Challenges for Commercialization) expressing hypoallergenic whole T cell epitopes of Cryj1 and Cryj2 (PB Tg rice) in a murine model of cedar pollinosis [29]. BALB/c mice were

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sublingually dosed with PB Tg rice, followed by an intraperitoneal sensitization and nasal challenge of cedar pollen extract. For sublingual treatment, 20 or 100 mg of PB Tg rice powder was introduced on the sublingual mucosae of anesthetized mice every 4 days for 3 weeks. For an induction of a murine model of cedar pollinosis, mice were systemically sensitized by intraperitoneal injections of 100 μg of Cryj antigens with 5 mg of alum once a week for 3 weeks and then were intranasally challenged with 1 mg of Cryj for 14 days. Clinical symptoms were evaluated by counting the number of sneezes and scratches in 2 minutes at the final intranasal challenge of Cryj antigens. For an analysis of cytokine production from CLNs or spleen cells, mononuclear cells were cultured in vitro, and cytokine levels in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Gene expression was analyzed by a quantitative real-time RT-PCR. The number of

sneezes after the final intranasal challenge in sublingually treated mice with 20 and 100 mg of PB Tg rice powder were significantly diminished when compared to mice that had been given no sublingual treatment (Fig. 18.4). Nasal scratching also decreased among sublingually treated mice. Corresponding histopathological findings showed that the number of eosinophils infiltrating into the nasal mucosa decreased and that damage to the epithelium was less in the sublingually treated mice (Fig. 18.5). Cryjspecific cytokine productions by cultured spleen cells were evaluated by an ELISA. IL-13 levels in the splenic culture supernatants were significantly reduced subsequent to sublingual treatment, but no difference in IL-5 production was observed. CLN IL-13 and IL-5 production diminished significantly in sublingually treated mice with concomitant increase in interferon gamma (IFNγ) production (Fig. 18.6). The results from testing this murine model of cedar

FIGURE 18.4 The effect of sublingual administration of protein body fraction from Tg rice in a murine model of cedar pollinosis. BALB/c mice were sublingually administered PB Tg rice followed by an intraperitoneal sensitization and nasal challenge of cedar pollen extract. For sublingual treatment, 20 or 100 mg of PB Tg rice powder was introduced on the sublingual mucosae of anesthetized mice every 4 days for 3 weeks. For an induction of a murine model of cedar pollinosis, mice were systemically sensitized by intraperitoneal injections of 100 μg of Cryj antigens with 5 mg of alum once a week for 3 weeks and then were intranasally challenged with 1 mg of Cryj for 14 days. Clinical symptoms were evaluated by counting the number of sneezes and scratches in 2 min at the final intranasal challenge of Cryj antigens. Sublingual immunotherapy with 20 and 100 mg of Tg rice seed powder containing protein bodies significantly attenuated the number of scratches and sneezes. III. MUCOSAL MODULATIONS FOR INDUCTION OF EFFECTIVE IMMUNITY

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V. CONCLUDING REMARKS AND FUTURE PERSPECTIVES

FIGURE 18.5 Number of eosinophils infiltrating into the nasal mucosa in a murine model of cedar pollinosis. Sublingual immunotherapy with 20 and 100 mg of Tg rice seed powder containing protein bodies significantly decreased the number of eosinophils infiltrating into nasal mucosa.

IL-13

IL-5

(pg/mL)

600

IFN-γγ

(pg/mL)

(pg/mL)

90

90

** 400

60

200

60

*

* 30

30

** 0

**

0 Control sl. 20 mg

sl. 100 mg

0 Control sl. 20 mg

sl. 100 mg

Control sl. 20 mg

sl. 100 mg

* **

pollinosis showed its suitability in recapitulating nasal symptoms and examining the effect of SLIT. Sublingually treated mice with PB Tg rice powder showed significantly decreased nasal symptoms and suppressed Th2-type immune responses in the nasal mucosa and draining CLNs. These results suggest that sublingual administration with PB-Tg rice expressing whole T cell epitopes of Cryj1 and Cryj2 suppress cedar pollinosis in this murine model.

V. CONCLUDING REMARKS AND FUTURE PERSPECTIVES Notably, TLRs expressed on epithelial cells, mast cells, and macrophages residing in the

FIGURE 18.6 Cryj-specific cytokine production of T cells from CLN cells in murine model of cedar pollinosis. Cryj-specific cytokine production by cultured CLN cells was evaluated by an ELISA. IL-13 and IL-5 production were reduced in sublingually treated mice, while interferon gamma (IFNγ) production increased.

P < .05 P < .01

upper respiratory tract mucosae have an important role in the pathogenesis of persistent inflammation in the nasopharyngeal cavity and middle ear cleft. Persistent inflammation of the paranasal sinus or middle ear might be explained by such an interaction between bacterial products and TLRs on residing on epithelial cells and/or recruited inflammatory cells. Innate immune cells can nonspecifically remove nasopharyngeal or middle ear pathogens recognized by the TLRs expressed on epithelial cells and/or recruited inflammatory cells into the paranasal sinus or middle ear. To this end, our results may lead us to new therapeutic strategies that may include immunotherapies, TLR antagonists, signal transduction inhibitors, or

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antisense therapy to downregulate the stagnant inflammation in nasopharyngeal or tubotympanal mucosae. I have no conflict of interest with anyone as regard this manuscript preparation.

[13]

[14]

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