Aqueous humor TGFß and fibrillin-1 in Tsk mice reveal clues to POAG pathogenesis.

Aqueous humor (AH) and blood levels of transforming growth factor ß (TGFß) are elevated in idiopathic primary open angle glaucoma (POAG) representing a disease biomarker of unclear status and function. Tsk mice display a POAG phenotype and harbor a mutation of fibrillin-1, an important regulator of TGFß bioavailability. AH TGFß2 was higher in Tsk than wild-type (WT) mice (by 34%; p = 0.002; ELISA); similarly, AH TGFß2 was higher in human POAG than controls (2.7-fold; p = 0.00005). As in POAG, TGFß1 was elevated in Tsk serum (p = 0.01). Fibrillin-1 was detected in AH from POAG subjects and Tsk mice where both had similar levels relative to controls (p = 0.45). 350 kDa immunoblot bands representing WT full-length fibrillin-1 were present in human and mouse AH. A 418 kDa band representing mutant full-length fibrillin-1 was present only in Tsk mice. Lower molecular weight fibrillin-1 antibody-reactive bands were present in similar patterns in humans and mice. Certain bands (130 and 32 kDa) were elevated only in human POAG and Tsk mice (p ≤ 0.04 relative to controls) indicating discrete isoforms relevant to disease. In addition to sharing a phenotype, Tsk mice and human POAG subjects had common TGFß and fibrillin-1 features in AH and also blood that are pertinent to understanding glaucoma pathogenesis.

Fibrillin-1 mutant mouse captures defining features of human primary open glaucoma including anomalous aqueous humor TGF beta-2.

Primary open angle glaucoma (POAG) features an optic neuropathy, elevated aqueous humor (AH) TGFß2, and major risk factors of central corneal thickness (CCT), increasing age and intraocular pressure (IOP). We examined Tight skin (Tsk) mice to see if mutation of fibrillin-1, a repository for latent TGFß, is associated with characteristics of human POAG. We measured: CCT by ocular coherence tomography (OCT); IOP; retinal ganglion cell (RGC) and optic nerve axon counts by microscopic techniques; visual electrophysiologic scotopic threshold responses (STR) and pattern electroretinogram (PERG); and AH TGFß2 levels and activity by ELISA and MINK epithelial cell-based assays respectively. Tsk mice had open anterior chamber angles and compared with age-matched wild type (WT) mice: 23% thinner CCT (p < 0.003); IOP that was higher (p < 0.0001), more asymmetric (p = 0.047), rose with age (p = 0.04) and had a POAG-like frequency distribution. Tsk mice also had RGCs that were fewer (p < 0.04), declined with age (p = 0.0003) and showed increased apoptosis and glial activity; fewer optic nerve axons (p = 0.02); abnormal axons and glia; reduced STR (p < 0.002) and PERG (p < 0.007) visual responses; and higher AH TGFß2 levels (p = 0.0002) and activity (p = 1E-11) especially with age. Tsk mice showed defining features of POAG, implicating aberrant fibrillin-1 homeostasis as a pathogenic contributor to emergence of a POAG phenotype.

Involvement of TNF-α and IFN-γ in Inflammation-Mediated Cochlear Injury.

OBJECTIVES: Inflammation is crucial for the pathogenesis of acquired sensorineural hearing loss, but the precise mechanism involved remains elusive. Among a number of inflammatory mediators, tumor necrosis factor-alpha (TNF-α) plays a pivotal role in cisplatin ototoxicity. However, TNF-α alone is cytotoxic to cochlear sensory cells only at the extremely high concentrations, suggesting the involvement of other factors that may sensitize cells to TNF-α cytotoxicity. Since interferon gamma (IFN-γ) importantly contributes to the cochlear inflammatory processes, we aim to determine whether and how IFN-γ affects TNF-α cytotoxicity to cochlear sensory cells. METHODS: TNF-α expression was determined with western blotting in RSL cells and immunolabeling of mouse temporal bone sections. HEI-OC1 cell viability was determined with MTT assays, cytotoxicity assays, and cytometric analysis with methylene blue staining. Cochlear sensory cell injury was determined in the organotypic culture of the mouse organ of Corti. RESULTS: Spiral ligament fibrocytes were shown to upregulate TNF-α in response to pro-inflammatory stimulants. We demonstrated IFN-γ increases the susceptibility of HEI-OC1 cells to TNF-α cytotoxicity via JAK1/2-STAT1 signaling. TNFR1-mediated Caspase-1 activation was found to mediate the sensitization effect of IFN-γ on TNF-α cytotoxicity. The combination of IFN-γ and TNF-α appeared to augment cisplatin cytotoxicity to cochlear sensory cells ex vivo. CONCLUSIONS: Taken together, these findings suggest the involvement of IFN-γ in the sensitization of cochlear cells to TNF-α cytotoxicity, which would enable us to better understand the complex mechanisms underlying inflammation-mediated cochlear injury.

Glutamate mediated metabolic neutralization mitigates propionate toxicity in intracellular Mycobacterium tuberculosis.

Metabolic networks in biological systems are interconnected, such that malfunctioning parts can be corrected by other parts within the network, a process termed adaptive metabolism. Unlike Bacillus Calmette-Guérin (BCG), Mycobacterium tuberculosis (Mtb) better manages its intracellular lifestyle by executing adaptive metabolism. Here, we used metabolomics and identified glutamate synthase (GltB/D) that converts glutamine to glutamate (Q → E) as a metabolic effort used to neutralize cytoplasmic pH that is acidified while consuming host propionate carbon through the methylcitrate cycle (MCC). Methylisocitrate lyase, the last step of the MCC, is intrinsically downregulated in BCG, leading to obstruction of carbon flux toward central carbon metabolism, accumulation of MCC intermediates, and interference with GltB/D mediated neutralizing activity against propionate toxicity. Indeed, vitamin B12 mediated bypass MCC and additional supplement of glutamate led to selectively correct the phenotypic attenuation in BCG and restore the adaptive capacity of BCG to the similar level of Mtb phenotype. Collectively, a defective crosstalk between MCC and Q → E contributes to attenuation of intracellular BCG. Furthermore, GltB/D inhibition enhances the level of propionate toxicity in Mtb. Thus, these findings revealed a new adaptive metabolism and propose GltB/D as a synergistic target to improve the antimicrobial outcomes of MCC inhibition in Mtb.

Ability of γδ T cells to modulate the Foxp3 T cell response is dependent on adenosine.

Whether γδ T cells inhibit or enhance the Foxp3 T cell response depends upon their activation status. The critical enhancing effector in the supernatant is adenosine. Activated γδ T cells express adenosine receptors at high levels, which enables them to deprive Foxp3+ T cells of adenosine, and to inhibit their expansion. Meanwhile, cell-free supernatants of γδ T cell cultures enhance Foxp3 T cell expansion. Thus, inhibition and enhancement by γδ T cells of Foxp3 T cell response are a reflection of the balance between adenosine production and absorption by γδ T cells. Non-activated γδ T cells produce adenosine but bind little, and thus enhance the Foxp3 T cell response. Activated γδ T cells express high density of adenosine receptors and have a greatly increased ability to bind adenosine. Extracellular adenosine metabolism and expression of adenosine receptor A2ARs by γδ T cells played a major role in the outcome of γδ and Foxp3 T cell interactions. A better understanding of the functional conversion of γδ T cells could lead to γδ T cell-targeted immunotherapies for related diseases.

Performance of the BacT Alert 3D System Versus Solid Media for Recovery and Drug Susceptibility Testing of Mycobacterium tuberculosis in a Tertiary Hospital in Korea.

BACKGROUND: Tuberculosis (TB) is a major health problem, and accurate and rapid diagnosis of multidrug-resistant (MDR) and extended drug-resistant (XDR) TB is important for appropriate treatment. In this study, performances of solid and liquid culture methods were compared with respect to MDR- and XDR-TB isolate recovery and drug susceptibility testing. METHODS: Sputum specimens from 304 patients were stained with Ziehl-Neelsen method. Mycobacterium tuberculosis (Mtb) isolates were tested for recovery on Löwenstein-Jensen (LJ) medium and the BacT Alert 3D system. For drug susceptibility testing of Mtb, isolates were evaluated on M-KIT plates and the BacT Alert 3D system. RESULTS: The recovery rates were 94.9% (206/217) and 98.2% (213/217) for LJ medium and the BacT Alert 3D system, respectively (kappa coefficient, 0.884). The rate of drug resistance was 13.4% for at least one or more drugs, 6.0% for MDR-TB and 2.3% for XDR-TB. M-KIT plate and BacT 3D Alert 3D system were comparable in drug susceptibility testing for isoniazid (97.7%; kappa coefficient, 0.905) and rifampin (98.6%; kappa coefficient, 0.907). Antibiotic resistance was observed using M-KIT plates for 24 of the total 29 Mtb isolates (82.8%). CONCLUSION: The liquid culture system showed greater reduction in the culture period, as compared with LJ medium; however, drug susceptibility testing using M-KIT plates was advantageous for simultaneous testing against multiple drug targets.

Seroprevalence of tuberculosis in domesticated elk (Cervus canadensis) in Korea.

Tuberculosis is a contagious disease in animals, primarily cattle, although it also affects wild animals and humans. There are few data on the state of tuberculosis in domesticated elk (Cervus canadensis) in Korea. In order to investigate tuberculosis in elk, the effectiveness of an enzyme linked immunosorbent assay (ELISA) using MPB70 and MPB83 antigens was compared with the tuberculin skin test (TST), and seroprevalence was measured with this assay using serum samples collected from domesticated elk herds in Korea. The respective sensitivities of the MPB70 and MPB83 ELISAs were 51.9% (95% CI 42.0-61.6) and 49.1% (95% CI 39.3-58.9), and their specificities were 100.0% (95% CI 92.6-100.0) and 97.9% (95% CI 88.9-100.0), respectively, in comparison with the TST. The herd prevalence ranged from 50 to 80% and the mean herd seropositive rate was 67.7% (21 of 31). Of 819 serum samples, 163 (19.9%) were seropositive, and the within-region prevalence ranged from 18.5-58.0%. In conclusion, the ELISA using the MPB70 and MPB83 antigens showed moderate sensitivity and high specificity compared to TST in elk, and tuberculosis was assumed to be fairly prevalent in domesticated elk in Korea.

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.

Therapeutic potential of adenovirus-mediated delivery of ß-defensin 2 for experimental otitis media.

Otitis media (OM), one of the most prevalent diseases in young children, is clinically important owing to its high incidence in children and its potential impact on language development and motor coordination. OM is the most common reason for the prescription of antibiotics (accounting for 25% of prescriptions) due to its extremely high incidence. A recent increase in antibiotic resistance among OM pathogens is emerging as a major public health concern globally, which led us to consider non-antibiotic approaches for the management of OM. In this study, we evaluated gene transfer of an antimicrobial peptide, human ß-defensin 2 (DEFB4), using an adenoviral vector (Ad5 with deletions of E1/E3/E4) as a potential therapeutic approach. We demonstrated that the transduction of human ß-defensin 2 induces the production of human ß-defensin 2 and suppresses non-typeable Haemophilus influenzae (NTHi) adhesion to human middle ear epithelial cells. Moreover, intratympanic inoculation of Ad-DEFB4 was found to attenuate NTHi-induced middle ear effusions without eliciting a significant immune response. Most importantly, intratympanic inoculation of Ad-DEFB4 appeared to significantly augment clearance of NTHi from middle ear cavity. Collectively, our results suggest that intratympanic gene delivery of antimicrobial molecules may serve as an alternative/adjuvant approach for the management of OM.

NOD2/RICK-dependent ß-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 ß-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 ß-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 ß-defensin 2 production by NOD2 silencing but augmented it by NOD2 over-expression. NTHi-induced ß-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 ß-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 ß-defensin 2 regulation contributes to the protection against NTHi-induced otitis media.

Distal NF-kB binding motif functions as an enhancer for nontypeable H. influenzae-induced DEFB4 regulation in epithelial cells.

Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to proinflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5'-flanking region of DEFB4 contains several NF-κB binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-κB binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-κB activation mediated by IκKα/ß-IκBα signaling. Deletion of the distal NF-κB binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-κB binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-κB binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-κB binds to the distal NF-κB binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-κB to the distal NF-κB binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells.

ERK2-dependent activation of c-Jun is required for nontypeable Haemophilus influenzae-induced CXCL2 upregulation in inner ear fibrocytes.

The inner ear, composed of the cochlea and the vestibule, is a specialized sensory organ for hearing and balance. Although the inner ear has been known as an immune-privileged organ, there is emerging evidence indicating an active immune reaction of the inner ear. Inner ear inflammation can be induced by the entry of proinflammatory molecules derived from middle ear infection. Because middle ear infection is highly prevalent in children, middle ear infection-induced inner ear inflammation can impact the normal development of language and motor coordination. Previously, we have demonstrated that the inner ear fibrocytes (spiral ligament fibrocytes) are able to recognize nontypeable Haemophilus influenzae, a major pathogen of middle ear infection, and upregulate a monocyte-attracting chemokine through TLR2-dependent NF-κB activation. In this study, we aimed to determine the molecular mechanism involved in nontypeable H. influenzae-induced cochlear infiltration of polymorphonuclear cells. The rat spiral ligament fibrocytes were found to release CXCL2 in response to nontypeable H. influenzae via activation of c-Jun, leading to the recruitment of polymorphonuclear cells to the cochlea. We also demonstrate that MEK1/ERK2 signaling pathway is required for nontypeable H. influenzae-induced CXCL2 upregulation in the rat spiral ligament fibrocytes. Two AP-1 motifs in the 5'-flanking region of CXCL2 appeared to function as a nontypeable H. influenzae-responsive element, and the proximal AP-1 motif was found to have a higher binding affinity to nontypeable H. influenzae-activated c-Jun than that of the distal one. Our results will enable us better to understand the molecular pathogenesis of middle ear infection-induced inner ear inflammation.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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 in vitro data, deficiency of MCP-1/CCL2 or CCR2 did not inhibit OM-induced inner ear inflammation in vivo. 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. CONCLUSIONS: 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.

Lysozyme M deficiency leads to an increased susceptibility to Streptococcus pneumoniae-induced otitis media.

BACKGROUND: Lysozyme is an antimicrobial innate immune molecule degrading peptidoglycan of the bacterial cell wall. Lysozyme shows the ubiquitous expression in wide varieties of species and tissues including the tubotympanum of mammals. We aim to investigate the effects of lysozyme depletion on pneumococcal clearance from the middle ear cavity. METHODS: Immunohistochemistry was performed to localize lysozyme in the Eustachian tube. Lysozyme expression was compared between the wild type and the lysozyme M-/- mice using real time quantitative RT-PCR and western blotting. Muramidase activity and bactericidal activity of lysozyme was measured using a lysoplate radial diffusion assay and a liquid broth assay, respectively. To determine if depletion of lysozyme M increases a susceptibility to pneumococal otitis media, 50 CFU of S. pneumoniae 6B were transtympanically inoculated to the middle ear and viable bacteria were counted at day 3 and 7 with clinical grading of middle ear inflammation. RESULTS: Immunolabeling revealed that localization of lysozyme M and lysozyme P is specific to some/particular cell types of the Eustachian tube. Lysozyme P of lysozyme M-/- mice was mainly expressed in the submucosal gland but not in the tubal epithelium. Although lysozyme M-/- mice showed compensatory up-regulation of lysozyme P, lysozyme M depletion resulted in a decrease in both muramidase and antimicrobial activities. Deficiency in lysozyme M led to an increased susceptibility to middle ear infection with S. pneumoniae 6B and resulted in severe middle ear inflammation, compared to wild type mice. CONCLUSION: The results suggest that lysozyme M plays an important role in protecting the middle ear from invading pathogens, particularly in the early phase. We suggest a possibility of the exogenous lysozyme as an adjuvant therapeutic agent for otitis media, but further studies are necessary.

Induction of beta defensin 2 by NTHi requires TLR2 mediated MyD88 and IRAK-TRAF6-p38MAPK signaling pathway in human middle ear epithelial cells.

BACKGROUND: All mucosal epithelia, including those of the tubotympanium, are secreting a variety of antimicrobial innate immune molecules (AIIMs). In our previous study, we showed the bactericidal/bacteriostatic functions of AIIMs against various otitis media pathogens. Among the AIIMs, human beta-defensin 2 is the most potent molecule and is inducible by exposure to inflammatory stimuli such as bacterial components or proinflammatory cytokines. Even though the beta-defensin 2 is an important AIIM, the induction mechanism of this molecule has not been clearly established. We believe that this report is the first attempt to elucidate NTHi induced beta-defensin expression in airway mucosa, which includes the middle ear. METHODS: Monoclonal antibody blocking method was employed in monitoring the TLR-dependent NTHi response. Two gene knock down methods - dominant negative (DN) plasmid and small interfering RNA (siRNA) - were employed to detect and confirm the involvement of several key genes in the signaling cascade resulting from the NTHi stimulated beta-defensin 2 expression in human middle ear epithelial cell (HMEEC-1). The student's t-test was used for the statistical analysis of the data. RESULTS: The experimental results showed that the major NTHi-specific receptor in HMEEC-1 is the Toll-like receptor 2 (TLR2). Furthermore, recognition of NTHi component(s)/ligand(s) by TLR2, activated the Toll/IL-1 receptor (TIR)-MyD88-IRAK1-TRAF6-MKK3/6-p38 MAPK signal transduction pathway, ultimately leading to the induction of beta-defensin 2. CONCLUSION: This study found that the induction of beta-defensin 2 is highest in whole cell lysate (WCL) preparations of NTHi, suggesting that the ligand(s) responsible for this up-regulation may be soluble macromolecule(s). We also found that this induction takes place through the TLR2 dependent MyD88-IRAK1-TRAF6-p38 MAPK pathway, with the primary response occurring within the first hour of stimulation. In combination with our previous studies showing that IL-1alpha-induced beta-defensin 2 expression takes place through a MyD88-independent Raf-MEK1/2-ERK MAPK pathway, we found that both signaling cascades act synergistically to up-regulate beta-defensin 2 levels. We propose that this confers an essential evolutionary advantage to the cells in coping with infections and may serve to amplify the innate immune response through paracrine signaling.

Toll-like receptor 2-dependent NF-kappaB activation is involved in nontypeable Haemophilus influenzae-induced monocyte chemotactic protein 1 up-regulation in the spiral ligament fibrocytes of the inner ear.

Inner ear dysfunction secondary to chronic otitis media (OM), including high-frequency sensorineural hearing loss or vertigo, is not uncommon. Although chronic middle ear inflammation is believed to cause inner ear dysfunction by entry of OM pathogen components or cytokines from the middle ear into the inner ear, the underlying mechanisms are not well understood. Previously, we demonstrated that the spiral ligament fibrocyte (SLF) cell line up-regulates monocyte chemotactic protein 1 (MCP-1) expression after treatment with nontypeable Haemophilus influenzae (NTHI), one of the most common OM pathogens. We hypothesized that the SLF-derived MCP-1 plays a role in inner ear inflammation secondary to OM that is responsible for hearing loss and dizziness. The purpose of this study was to investigate the signaling pathway involved in NTHI-induced MCP-1 up-regulation in SLFs. Here we show for the first time that NTHI induces MCP-1 up-regulation in the SLFs via Toll-like receptor 2 (TLR2)-dependent activation of NF-kappaB. TLR2(-/-)- and MyD88(-/-)-derived SLFs revealed involvement of TLR2 and MyD88 in NTHI-induced MCP-1 up-regulation. Studies using chemical inhibitors and dominant-negative constructs demonstrated that it is mediated by the IkappaKbeta-dependent IkappaBalpha phosphorylation and NTHI-induced NF-kappaB nuclear translocation. Furthermore, we demonstrated that the binding of NF-kappaB to the enhancer region of MCP-1 is involved in this up-regulation. In addition, we have identified a potential NF-kappaB motif that is responsive and specific to certain NTHI molecules or ligands. Further studies are necessary to reveal specific ligands of NTHI that activate host receptors. These results may provide us with new therapeutic strategies for prevention of inner ear dysfunction secondary to chronic middle ear inflammation.

Low-intensity ultrasound stimulates the viability and matrix gene expression of human articular chondrocytes in alginate bead culture.

We investigated the effects of low-intensity ultrasound (LIUS) on the activity of human articular chondrocytes isolated from osteoarthritis patients and cultured in the three-dimensional alginate beads. LIUS was treated at 0, 100, 200, and 300 mW/cm(2) for 10 min everyday for 2, 7, or 15 days. LIUS induced the viability of cells only at day 15 but not until day 7 after treatment, when examined by trypan blue exclusion and LIVE/DEAD(R) assay kit. When examined at day 7, the proliferation of cells was not changed by LIUS in the (3)H-thymine incorporation. The expression of matrix producing proteins (type II collagen and proteoglycan) was clearly induced by 200-300 mW/cm(2) LIUS in the incorporation of radioactivity and Northern blot analysis. Although the expression of MMP-1, a matrix degrading protein, was decreased, that of TIMP-1, an inhibitor of MMPs, was not affected by LIUS. Histological analysis revealed an increase in the number and size of glycosaminoglycan-positive lacunae and cellular organelles, appearing as rough endoplasmic reticulum and mitochondria by LIUS. These results showed that the viability and metabolism of human articular chondrocytes in alginate culture was induced by LIUS treatment, suggesting that they could be a promising autologous source for cartilage tissue engineering.

Involvement of Niemann-Pick type C2 protein in hematopoiesis regulation.

Niemann-Pick type C2 (NPC2) protein has been characterized as a cholesterol-binding protein. Its loss leads to NPC2 disease, an inherited neurodegenerative disorder. When analyzing gene expression profile, we noticed high expression of both NPC2 and its receptor, mannose 6-phosphate receptor (MPR), in murine hematopoietic stem cells. NPC2 protein, in the presence of thrombopoietin (TPO), causes an increase in CFU-GEMM (colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte) and a decrease in CFU-GM (colony-forming unit-granulocyte-macrophage) colony number in colony-forming cell (CFC) assays. This effect is independent of cholesterol binding but does require the presence of MPR. With M07e cells, a TPO-dependent hematopoietic leukemia cell line, NPC2 can inhibit TPO-induced differentiation and enhance TPO-mediated anti-apoptosis effects. Strikingly, these results are not observed under the standard 20% O(2) level of the standard incubator, but rather at 7% O(2), the physiological oxygen level of bone marrow. Furthermore, NPC2 protein upregulates hypoxia inducible factor 1-alpha protein level at 7% O(2), but not at 20% O(2). Our results demonstrate that NPC2 protein plays a role in hematopoiesis at the physiologic bone marrow level of O(2).