The immune response is an important component of the cochlear response to stress. As an important player in the cochlear immune system, the basilar membrane immune cells reside on the surface of the scala tympani side of the basilar membrane. At present, the immune cell properties in this region and their responses to stress are not well understood. Here, we investigated the functional role of these immune cells in the immune response to acoustic overstimulation. This study reveals that tissue macrophages are present in the entire length of the basilar membrane under steady-state conditions. Notably, these cells in the apical and the basal sections of the basilar membrane display distinct morphologies and immune protein expression patterns. Following acoustic trauma, monocytes infiltrate into the region of the basilar membrane, and the infiltrated cells transform into macrophages. While monocyte infiltration and transformation occur in both the apical and the basal sections of the basilar membrane, only the basal monocytes and macrophages display a marked increase in the expression of MHC II and CIITA, a MHC II production cofactor, suggesting the site-dependent activation of antigen-presenting function. Consistent with the increased expression of the antigen-presenting proteins, CD4+ T cells, the antigen-presenting partner, infiltrate into the region of the basilar membrane where antigen-presenting proteins are upregulated. Further pathological analyses revealed that the basal section of the cochlea displays a greater level of sensory cell damage, which is spatially correlated with the region of antigen-presenting activity. Together, these results suggest that the antigen-presenting function of the mononuclear phagocyte population is activated in response to acoustic trauma, which could bridge the innate immune response to adaptive immunity.

BackgroundChronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood.Methodology/Principal FindingsWe found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47phox−/− mice and gp91phox-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-κB activation, and elevated downstream pro-inflammatory cytokines (TNF-α, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-κB activation.Conclusions/SignificanceThese studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD.

The breast cancer resistance protein (BCRP) is abundant in the placenta and protects the fetus by limiting placental drug penetration. We hypothesize that pregnancy-specific hormones regulate BCRP expression. Hence, we examined the effects of progesterone (P 4) and 17␤-estradiol (E2) on BCRP expression in the human placental BeWo cells. P 4 and E2 significantly increased and decreased BCRP protein and mRNA, respectively. Likewise, treatment with P 4 and E2 increased and decreased, respectively, fumitremorgin C-inhibitable mitoxantrone efflux activity of BeWo cells. Reduction in BCRP expression by E 2 was abrogated by the estrogen receptor (ER) antagonist ICI-182,780. However, the progesterone receptor (PR) antagonist RU-486 had no effect on P 4-mediated induction of BCRP. P4 together with E2 further increased BCRP protein and mRNA compared with P 4 treatment alone. This combined effect on BCRP expression was abolished by RU-486, ICI-182,780, or both. Further analysis revealed that E 2 significantly decreased ER␤ mRNA and strongly induced PRB mRNA in a dose-dependent manner but had no effect on PR A and ER␣. P4 alone had no significant effect on mRNA of ER␣, ER␤, PRA, and PR B. E2 in combination with P4 increased PRB mRNA, but the level of induction was significantly reduced compared with E 2 treatment alone. Taken together, these results indicate that E 2 by itself likely downregulates BCRP expression through an ER, possibly ER␤. P 4 alone upregulates BCRP expression via a mechanism other than PR. P 4 in combination with E2 further increases BCRP expression, presumably via a nonclassical PR-and/or E 2-mediated synthesis of PRB. hormonal regulation; ATP-binding cassette transporter; pregnancy THE BREAST CANCER RESISTANCE PROTEIN (BCRP) is the second member (gene symbol ABCG2) of the subfamily G of the large ATP-binding cassette (ABC) transporter superfamily (1, 9, 25). BCRP is highly expressed in many normal tissues, including the epithelium of the small intestine and the liver canalicular membrane (22). Therefore, in addition to conferring resistance in cancer cells to chemotherapeutic agents such as mitoxantrone (MX), topotecan, and methotrexate (8,9,25,36), BCRP has been shown to mediate apically-directed drug transport and play a significant role in absorption, distribution, and elimination of BCRP substrates (4,19,21,32,35). Of interest is that BCRP is also abundantly expressed in the apical membrane of placental syncytiotrophoblasts (22). Whereas the precise physiological role of BCRP in the placenta is still unclear, existing data suggest that BCRP may protect the fetus against toxic substances/drugs and metabolites by extruding them across the placental barrier. For example, Bcrp1, the murine homolog of BCRP, has been shown to significantly alter fetal distribution of topotecan, a BCRP substrate. The fetus/plasma ratio of topotecan was increased twofold in pregnant mice treated with the BCRP inhibitor GF-120918 compared with the vehicle-treatment control (19).Distribution of drugs that are BCRP substrate...

Dipyridamole and several dihydropyridines are effective BCRP inhibitors, but bepridil, diltiazem, and verapamil are not. We also identified a new BCRP substrate, dipyridamole.

BackgroundThe cochlea is the sensory organ of hearing. In the cochlea, the organ of Corti houses sensory cells that are susceptible to pathological insults. While the organ of Corti lacks immune cells, it does have the capacity for immune activity. We hypothesized that resident cells in the organ of Corti were responsible for the stress-induced immune response of the organ of Corti. This study profiled the molecular composition of the immune system in the organ of Corti and examined the immune response of non-immune epithelial cells to acoustic overstimulation.MethodsUsing high-throughput RNA-sequencing and qRT-PCR arrays, we identified immune- and inflammation-related genes in both the cochlear sensory epithelium and the organ of Corti. Using bioinformatics analyses, we cataloged the immune genes expressed. We then examined the response of these genes to acoustic overstimulation and determined how changes in immune gene expression were related to sensory cell damage.ResultsThe RNA-sequencing analysis reveals robust expression of immune-related genes in the cochlear sensory epithelium. The qRT-PCR array analysis confirms that many of these genes are constitutively expressed in the resident cells of the organ of Corti. Bioinformatics analyses reveal that the genes expressed are linked to the Toll-like receptor signaling pathway. We demonstrate that expression of Toll-like receptor signaling genes is predominantly from the supporting cells in the organ of Corti cells. Importantly, our data demonstrate that these Toll-like receptor pathway genes are able to respond to acoustic trauma and that their expression changes are associated with sensory cell damage.ConclusionThe cochlear resident cells in the organ of Corti have immune capacity and participate in the cochlear immune response to acoustic overstimulation.

Acoustic overstimulation traumatizes the cochlea, resulting in auditory dysfunction. As a consequence of acoustic injury, the immune system in the cochlea is activated, leading to the production of inflammatory mediators and the infiltration of immune cells. However, the molecular mechanisms responsible for initiating these immune responses remain unclear. Here, we investigate the functional role of Toll-like receptor 4 (Tlr4), a cellular receptor that activates the innate immune system, in the regulation of cochlear responses to acoustic overstimulation. Using a Tlr4 knockout mouse model, we examined how Tlr4 deficiency affects sensory cell pathogenesis, auditory dysfunction and cochlear immune activity. We demonstrate that Tlr4 knockout does not affect sensory cell viability under physiological conditions, but reduces the level of sensory cell damage and cochlear dysfunction after acoustic injury. Together, these findings suggest that Tlr4 promotes sensory cell degeneration and cochlear dysfunction after acoustic injury. Acoustic injury provokes a site-dependent inflammatory response in both the organ of Corti and the tissues of the lateral wall and basilar membrane. Tlr4 deficiency affects these inflammatory responses in a site-dependent manner. In the organ of Corti, loss of Tlr4 function suppresses the production of interleukin 6 (Il6), a pro-inflammatory molecule, after acoustic injury. By contrast, the production of inflammatory mediators, including Il6, persists in the lateral wall and basilar membrane. In addition to immune molecules, Tlr4 knockout inhibits the expression of major histocompatibility complex class II, an antigen-presenting molecule, in macrophages, suggesting that Tlr4 participates in the antigen-presenting function of macrophages after acoustic trauma. Together, these results suggest that Tlr4 regulates multiple aspects of the immune response in the cochlea and contributes to cochlear pathogenesis after acoustic injury.

These results indicate that cyclosporin A, tacrolimus and sirolimus are effective inhibitors but not substrates of BCRP. These findings could explain the altered pharmacokinetics of BCRP substrate drugs when co-administered with the immunosuppressants and suggest that pharmacokinetic modulation by the immunosuppressants may improve the therapeutic outcome of these drugs.

The response of the prostate tissue microenvironment to androgen deprivation (AD) represents a critical component in the treatment of benign prostatic hyperplasia and prostate cancer (CaP). Primary xenografts of human benign and CaP tissue transplanted to immunocompromized SCID mice were used to characterize the response of the prostate vasculature during the initial 14 days of AD. Microvessel density and vascular lumen diameter in the prostate xenografts decreased rapidly after AD, reached a nadir on days 2-4, and recovered between days 4 and 14. The number of apoptotic endothelial cells peaked on day 2 after AD and decreased to precastration levels over days 4-7. Leakage of vascular contents in the interstitial space was apparent between days 1 and 3 after AD; however, the vascular permeability barrier reestablished between days 7 and 14. Expression of vascular endothelial growth factor (VEGF)-A, VEGF receptor-2, and basic fibroblast growth factor protein increased in endothelial cells between days 2 and 4 after AD, which preceded vascular recovery and appeared to be a direct and specific response of the endothelial cells to AD. Lack of comparable upregulation of these genes in primary cultures of human prostate endothelial cells in response to AD suggests a role for paracrine signaling mediated through stromal or epithelial cells. VEGF-A expression by prostate endothelial cells appears to represent a key facilitator of the vascular rebound in human prostate tissue induced by removal of circulating testicular androgens.