NOD2/RICK-dependent β-defensin 2 regulation is protective for nontypeable Haemophilus influenzae-induced middle ear infection.

Middle ear infection, otitis media (OM), is clinically important due to the high incidence in children and its impact on the development of language and motor coordination. Previously, we have demonstrated that the human middle ear epithelial cells up-regulate β-defensin 2, a model innate immune molecule, in response to nontypeable Haemophilus influenzae (NTHi), the most common OM pathogen, via TLR2 signaling. NTHi does internalize into the epithelial cells, but its intracellular trafficking and host responses to the internalized NTHi are poorly understood. Here we aimed to determine a role of cytoplasmic pathogen recognition receptors in NTHi-induced β-defensin 2 regulation and NTHi clearance from the middle ear. Notably, we observed that the internalized NTHi is able to exist freely in the cytoplasm of the human epithelial cells after rupturing the surrounding membrane. The human middle ear epithelial cells inhibited NTHi-induced β-defensin 2 production by NOD2 silencing but augmented it by NOD2 over-expression. NTHi-induced β-defensin 2 up-regulation was attenuated by cytochalasin D, an inhibitor of actin polymerization and was enhanced by α-hemolysin, a pore-forming toxin. NOD2 silencing was found to block α-hemolysin-mediated enhancement of NTHi-induced β-defensin 2 up-regulation. NOD2 deficiency appeared to reduce inflammatory reactions in response to intratympanic inoculation of NTHi and inhibit NTHi clearance from the middle ear. Taken together, our findings suggest that a cytoplasmic release of internalized NTHi is involved in the pathogenesis of NTHi infections, and NOD2-mediated β-defensin 2 regulation contributes to the protection against NTHi-induced otitis media

Murine Model of Buckwheat Allergy by Intragastric Sensitization with Fresh Buckwheat Flour Extract

Food allergies affect about 4% of the Korean population, and buckwheat allergy is one of the most severe food allergies in Korea. The purpose of the present study was to develop a murine model of IgE-mediated buckwheat hypersensitivity induced by intragastric sensitization. Young female C3H/HeJ mice were sensitized and challenged intragastricly with fresh buckwheat flour (1, 5, 25 mg/dose of proteins) mixed in cholera toxin, followed by intragastric challenge. Anaphylactic reactions, antigen-specific antibodies, splenocytes proliferation assays and cytokine productions were evaluated. Oral buckwheat challenges of sensitized mice provoked anaphylactic reactions such as severe scratch, perioral/periorbital swellings, or decreased activity. Reactions were associated with elevated levels of buckwheat-specific IgE antibodies. Splenocytes from buckwheat allergic mice exhibited significantly greater proliferative responses to buckwheat than non-allergic mice. Buckwheat-stimulated IL-4, IL-5, and INF-γ productions were associated with elevated levels of buckwheat-specific IgE in sensitized mice. In this model, 1 mg and 5 mg dose of sensitization produced almost the same degree of Th2-directed immune response, however, a 25 mg dose showed blunted antibody responses. In conclusion, we developed IgE-mediated buckwheat allergy by intragastric sensitization and challenge, and this model could provide a good tool for future studies

Effect of Cosensitization with Buckwheat Flour Extract on the Production of House Dust Mite-specific IgE

There are studies reporting food sensitization in infancy increases the risk of sensitization to inhalants later in life. We performed a study to evaluate whether cosensitization with buckwheat (BW) has an effect on the production of house dust mite-IgE. C3H/HeJ mice (4 weeks, female) were sensitized with house dust mite (HDM)/Al (OH)3, intraperitoneally on day 0, followed by 4 intranasal sensitizations (on days 14, 15, 16, and 21). Group 1 was cosensitized intragastrically with BW/cholera toxin (CT) (on days 0, 1, 2, 7, and 18) during sensitization with HDM, group 2 was cosensitized intragastrically with CT only (on days 0, 1, 2, 7, and 18), and group 3 was used as controls. HDM- and BW-IgE and antigen-specific T-cell proliferation and cytokine production were evaluated. In Group 1, BW-IgE levels were highest at week 4, and the HDM-IgE at week 3 (98.45±64.37 ng/mL and 169.86±55.54 ng/mL, respectively). In Group 2, HDM-IgE levels reached a peak at week 3, remarkably higher (810.52±233.29 ng/mL) compared to those of Group 1 (169.86±55.54 ng/mL). The interleukin (IL)-4 and interferon (IFN)-γ in the HDM-stimulated culture supernatants of splenocytes were not significantly different among groups. We postulate that the cosensitization with BW may down-regulate the specific IgE response to HDM

Spiral ligament fibrocyte-derived MCP-1/CCL2 contributes to inner ear inflammation secondary to nontypeable H. influenzae-induced otitis media

<p>Abstract</p> <p>Background</p> <p>Otitis media (OM), one of the most common pediatric infectious diseases, causes inner ear inflammation resulting in vertigo and sensorineural hearing loss. Previously, we showed that spiral ligament fibrocytes (SLFs) recognize OM pathogens and up-regulate chemokines. Here, we aim to determine a key molecule derived from SLFs, contributing to OM-induced inner ear inflammation.</p> <p>Methods</p> <p>Live NTHI was injected into the murine middle ear through the tympanic membrane, and histological analysis was performed after harvesting the temporal bones. Migration assays were conducted using the conditioned medium of NTHI-exposed SLFs with and without inhibition of MCP-1/CCL2 and CCR2. qRT-PCR analysis was performed to demonstrate a compensatory up-regulation of alternative genes induced by the targeting of MCP-1/CCL2 or CCR2.</p> <p>Results</p> <p>Transtympanic inoculation of live NTHI developed serous and purulent labyrinthitis after clearance of OM. THP-1 cells actively migrated and invaded the extracellular matrix in response to the conditioned medium of NTHI-exposed SLFs. This migratory activity was markedly inhibited by the viral CC chemokine inhibitor and the deficiency of MCP-1/CCL2, indicating that MCP-1/CCL2 is a main attractant of THP-1 cells among the SLF-derived molecules. We further demonstrated that CCR2 deficiency inhibits migration of monocyte-like cells in response to NTHI-induced SLF-derived molecules. Immunolabeling showed an increase in MCP-1/CCL2 expression in the cochlear lateral wall of the NTHI-inoculated group. Contrary to the <it>in vitro </it>data, deficiency of MCP-1/CCL2 or CCR2 did not inhibit OM-induced inner ear inflammation <it>in vivo</it>. We demonstrated that targeting MCP-1/CCL2 enhances NTHI-induced up-regulation of MCP-2/CCL8 in SLFs and up-regulates the basal expression of CCR2 in the splenocytes. We also found that targeting CCR2 enhances NTHI-induced up-regulation of MCP-1/CCL2 in SLFs.</p> <p>Conclusions</p> <p>Taken together, we suggest that NTHI-induced SLF-derived MCP-1/CCL2 is a key molecule contributing to inner ear inflammation through CCR2-mediated recruitment of monocytes. However, deficiency of MCP-1/CCL2 or CCR2 alone was limited to inhibit OM-induced inner ear inflammation due to compensation of alternative genes.</p

NOD2/RICK-dependent β-defensin 2 regulation is protective for nontypeable Haemophilus influenzae-induced middle ear infection.

Middle ear infection, otitis media (OM), is clinically important due to the high incidence in children and its impact on the development of language and motor coordination. Previously, we have demonstrated that the human middle ear epithelial cells up-regulate β-defensin 2, a model innate immune molecule, in response to nontypeable Haemophilus influenzae (NTHi), the most common OM pathogen, via TLR2 signaling. NTHi does internalize into the epithelial cells, but its intracellular trafficking and host responses to the internalized NTHi are poorly understood. Here we aimed to determine a role of cytoplasmic pathogen recognition receptors in NTHi-induced β-defensin 2 regulation and NTHi clearance from the middle ear. Notably, we observed that the internalized NTHi is able to exist freely in the cytoplasm of the human epithelial cells after rupturing the surrounding membrane. The human middle ear epithelial cells inhibited NTHi-induced β-defensin 2 production by NOD2 silencing but augmented it by NOD2 over-expression. NTHi-induced β-defensin 2 up-regulation was attenuated by cytochalasin D, an inhibitor of actin polymerization and was enhanced by α-hemolysin, a pore-forming toxin. NOD2 silencing was found to block α-hemolysin-mediated enhancement of NTHi-induced β-defensin 2 up-regulation. NOD2 deficiency appeared to reduce inflammatory reactions in response to intratympanic inoculation of NTHi and inhibit NTHi clearance from the middle ear. Taken together, our findings suggest that a cytoplasmic release of internalized NTHi is involved in the pathogenesis of NTHi infections, and NOD2-mediated β-defensin 2 regulation contributes to the protection against NTHi-induced otitis media

IL-10/HMOX1 Signaling Modulates Cochlear Inflammation via Negative Regulation of MCP-1/CCL2 Expression in Cochlear Fibrocytes

Cochlear inflammatory diseases, such as tympanogenic labyrinthitis, are associated with acquired sensorineural hearing loss. Although otitis media is extremely frequent in children, tympanogenic labyrinthitis is not commonly observed, which suggests the existence of a potent anti-inflammatory mechanism modulating cochlear inflammation. In this study, we aimed to determine the molecular mechanism involved in cochlear protection from inflammation-mediated tissue damage, focusing on IL-10 and hemoxygenase-1 (HMOX1) signaling. We demonstrated that IL-10Rs are expressed in the cochlear lateral wall of mice and rats, particularly in the spiral ligament fibrocytes (SLFs). The rat SLF cell line was found to inhibit nontypeable Haemophilus influenzae (NTHi)-induced upregulation of monocyte chemotactic protein-1 (MCP-1; CCL2) in response to IL-10. This inhibition was suppressed by silencing IL-10R1 and was mimicked by cobalt Protoporphyrin IX and CO-releasing molecule-2. In addition, IL-10 appeared to suppress monocyte recruitment through reduction of NTHi-induced rat SLF cell line-derived chemoattractants. Silencing of HMOX1 was found to attenuate the inhibitory effect of IL-10 on NTHi-induced MCP-1/CCL2 upregulation. Chromatin immunoprecipitation assays showed that IL-10 inhibits NTHi-induced binding of p65 NF-κB to the distal motif in the promoter region of MCP-1/CCL2, resulting in suppression of NTHi-induced NF-κB activation. Furthermore, IL-10 deficiency appeared to significantly affect cochlear inflammation induced by intratympanic injections of NTHi. Taken together, our results suggest that IL-10/HMOX1 signaling is involved in modulation of cochlear inflammation through inhibition of MCP-1/CCL2 regulation in SLFs, implying a therapeutic potential for a CO-based approach for inflammation-associated cochlear diseases

Membrane pore formation enhances NOD2-dependent human β-defensin 2 up-regulation.

<p>(A) Fluorescent microscopic images showing that α-hemolysin, a pore-forming toxin, enhances internalization of live NTHi (green) in the HMEEC cells. (B) An influx of calcein AM into the HMEEC cells was increased by α-hemolysin. (C) Quantitative RT-PCR analysis shows that α-hemolysin markedly enhances human β-defensin 2 up-regulation induced by a suboptimal dose (1 µg/ml) of NTHi lysate in the HMEEC cells. DEFB4: human β-defensin 2. (D) Note that silencing of NOD2 inhibits α-hemolysin-mediated enhancement of NTHi lysate-induced human β-defensin 2 up-regulation in the HMEEC cells. NC: a control group transfected with a nonspecific siRNA, KD: a group transfected with a gene-specific siRNA. Results were expressed as fold-induction, taking the value of the non-treated group as 1. The experiments were performed in triplicate and repeated twice. Values are given as the mean ± standard deviation (n = 3). *: <i>p</i><0.05.</p

NTHi exists as a free or entrapped form after internalization into the human epithelial cells.

<p>(A) Transmission electron microscopic image shows that NTHi is found either surrounded by an enclosing membrane (a) or free in the cytoplasm (b). (B) The membrane (arrow) surrounding the bacterial cell is partially ruptured, and NTHi (arrowhead) appears to be in the process of being released into the cytoplasm of the Chang cells. Scale bar for both images: 500 nm. (C, D) Note that NTHi (asterisks) exists with or without a peribacterial space (white arrows) in the cytoplasm of the HMEEC cells. Scale bar for both images: 200 nm.</p