RESUMO
Meniere's disease (MD) is a chronic disease that causes episodic vertigo, fluctuating hearing loss, and aural fullness, initially managed by dietary salt reduction, and use of diuretics. Our prior research in autoimmune inner ear disease (AIED) demonstrated that in peripheral blood mononuclear cell (PBMC) from corticosteroid-resistant AIED patients, increased production, processing and release of interleukin-1ß (IL-1ß) is observed and hearing could be improved with use of anakinra, an interleukin-1 receptor antagonist. We have further identified that in these AIED patients, IL-1ß is uniquely processed to a 28 kDa pro-inflammatory product by caspase-7. In the present study, we characterize the production, processing and release of the pro-inflammatory cytokines IL-1ß and IL-6 from PBMC of MD (n = 14) patients in response to sodium chloride (NaCl), and determined the effect of the diuretic triamterene-hydrocholothiazide (T-HCTZ), or anakinra in these patients. We observed that PBMC cultured with NaCl from MD patients show processing of IL-1ß to the 28 kDa product, and that this product is abrogated with T-HCTZ. Our observations are consistent with other autoimmune diseases where high concentrations of NaCl caused release of pro-inflammatory cytokines and may provide further insight as to the mechanism of disease progression in MD patients.
Assuntos
Doenças Autoimunes , Doença de Meniere , Citocinas/metabolismo , Humanos , Hidroclorotiazida/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-1beta/farmacologia , Leucócitos Mononucleares/metabolismo , Doença de Meniere/tratamento farmacológico , Cloreto de Sódio/farmacologiaRESUMO
HYPOTHESIS: Autoimmune inner ear disease (AIED) patients will differentially express interleukin (IL)-6 based on corticosteroid responsiveness. BACKGROUND: AIED is characterized by periods of acute sensorineural hearing loss (SNHL). In a majority of patients corticosteroid responsiveness is lost over time. The mechanisms that control corticosteroid responsiveness have not been fully elucidated. METHODS: Thirty-five AIED patients and 13 age-matched control subjects were enrolled in this study. Steroid responsive (nâ=â15) and steroid nonresponsive AIED patients (nâ=â20) were characterized based on audiometry before and after treatment for acute SNHL. Plasma and peripheral blood mononuclear cells (PBMC) were obtained at the time of acute SNHL to quantify plasma IL-6, soluble IL-6 receptor (sIL-6R), and C-C Motif Chemokine Ligand 3 (CCL3). PBMCs were stimulated with dexamethasone and release of soluble IL-6, sIL-6R, and CCL3 protein into conditioned supernatants was measured. Plasma IL-6 was also correlated to serum c-reactive protein (CRP), cardiac CRP, erythrocyte sedimentation rate. RESULTS: Statistically significant differences were observed in the plasma IL-6 between AIED patients and controls (2.37 versus 2.03âpg/ml, pâ<â0.01), plasma IL-6, and CCL3 between responders and nonresponders (0.136 versus 3.84âpg/ml, pâ<â0.005; 30.5 versus 32.4, pâ<â0.05) and released IL-6 from dexamethasone stimulated PBMC in AIED patients compared with controls (0.54 versus 1.12âpg/ml, pâ<â0.001). There was a correlation between plasma IL-6 levels of AIED patients to both serum CRP and cardiac CRP (R2â=â0.83, R2â=â0.88). CONCLUSIONS: We observed AIED patients, specifically nonresponders expressed greater levels of IL-6. Elevated IL-6 levels in AIED patients correlated with CRP levels, providing a commonly available laboratory test that may aid in rapid clinical decision-making in these patients.
Assuntos
Doenças Autoimunes , Interleucina-6/sangue , Labirintite , Corticosteroides/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Estudos de Casos e Controles , Humanos , Leucócitos MononuclearesRESUMO
Interleukin-1ß (IL-1ß) is a key proinflammatory cytokine involved in the progression of many autoinflammatory and autoimmune diseases, including autoimmune inner ear disease (AIED). IL-1ß inhibition has been shown to result in clinical hearing improvement in a small cohort of corticosteroid-resistant patients with AIED. Canonical processing of pro-IL-1ß by caspase-1 generates an active 17-kDa fragment, capable of instigating a proinflammatory microenvironment. However, in response to LPS, PBMCs from patients with AIED uniquely express a 28-kDa IL-1ß fragment, as compared with PBMCs from control subjects. We synthesized and compared the biologic activity of the 28-kDa fragment to the 17-kDa IL-1ß product and the pro-IL-1 31-kDa protein. The 28-kDa IL-1ß fragment induces IL-6, TNF-α, and CCL3 in PBMCs. Uniquely, only caspase-7 treatment showed a dose- and time-dependent increase in 28-kDa band generation. Mass spectrometry confirmed the putative caspase-7 cleavage site of pro-IL-1ß, which was used to generate the 28-kDa fragment used for PBMC stimulation studies. Collectively, these results provide insight into the function of a poorly understood, processed 28-kDa form of IL-1ß in patients with AIED that is uniquely generated by caspase-7 and is capable of activating further downstream proinflammatory cytokines. Further investigation may provide novel pharmacologic targets for the treatment of this rare disease.
Assuntos
Doenças Autoimunes/metabolismo , Caspase 7/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Doenças do Labirinto/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , ProteóliseRESUMO
Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a protein implicated in the control of inflammation in a number of autoimmune diseases. We hypothesized that the balance of TIMP-1 and matrix metalloproteinase-9 (MMP-9) may influence the control or perpetuation of inflammation in corticosteroid-responsive (RES) and corticosteroid-resistant (NR) autoimmune inner ear disease (AIED) patients. In the present study, we observed that plasma from AIED patients exhibited greater levels of TIMP-1 values compared with normal healthy controls. TIMP-1 abrogates lipopolysaccharide-mediated interleukin (IL)-1ß release from peripheral blood mononuclear cells in a dose-dependent manner. RES AIED patients have higher basal TIMP-1 levels and produce more TIMP-1 in response to IL-1ß. Conversely, consistent with our previous studies, we found that NR patients have higher basal MMP-9 levels and produce more MMP-9 levels in response to IL-1ß.
Assuntos
Doenças Autoimunes/metabolismo , Doenças do Labirinto/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Corticosteroides/uso terapêutico , Adulto , Idoso , Doenças Autoimunes/sangue , Doenças Autoimunes/tratamento farmacológico , Microambiente Celular , Feminino , Humanos , Inflamação/patologia , Interleucina-1beta/sangue , Doenças do Labirinto/sangue , Doenças do Labirinto/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Inibidor Tecidual de Metaloproteinase-1/sangueRESUMO
OBJECTIVES: Autoinflammatory diseases are a family of immune-mediated, rare diseases, some of which, exhibit sensorineural hearing loss (SNHL), suggesting potentially similar mechanisms of molecular pathogenesis between autoinflammatory-mediated hearing loss and autoimmune inner ear disease (AIED) may exist. The purpose of this review is to compare the clinical features of autoimmune and autoinflammatory diseases that affect hearing, discuss the limitations of our knowledge, and highlight potential new disease mechanisms and therapeutics. DATA SOURCES: Pubmed Literature Review; Google Scholar Literature review. REVIEW METHODS: A focused comparison of AIED with a number of autoinflammatory diseases that manifest with sensorineural hearing loss was performed. The pathogenesis of these diseases is reviewed in the context of the innate and adaptive immune system, cytokine expression and genetic polymorphisms. RESULTS: AIED, since first described by Cogan and Lehnhardt and first clinically characterized by McCabe, has remained an enigmatic disease, with limited advances in both new diagnostics and new therapeutics. Since the discovery of autoinflammatory diseases, a number of systemic autoimmune diseases have either been re-classed as autoinflammatory diseases or identified to have features of autoinflammatory disease. CONCLUSION: AIED has clinical features of both autoimmune and autoinflammatory disease. It is critical that autoinflammatory diseases be correctly identified, as failure to do so may result in systemic amyloidosis and kidney damage.
RESUMO
Autoimmune inner ear disease (AIED) is a poorly understood disease marked by bilateral, rapidly progressive hearing loss triggered by unknown stimuli, which is corticosteroid responsive in 60 % of patients. Although the mechanism of the disease is not precisely understood, a complex interaction of cytokines is believed to contribute toward the inflammatory disease process and hearing loss. Previously, we showed the role of TNF-α in steroid-sensitive and IL-1ß in steroid-resistant immune-mediated hearing loss. N-Acetylcysteine (NAC), a broad spectrum antioxidant, has been effective in other autoimmune disorders. Other studies have shown NAC to have a protective adjunct role in human idiopathic sudden hearing loss, where the addition of NAC resulted in better hearing recovery than with steroids alone, although the mechanism of this protection was not elucidated. In the present study, we observed PBMCs from AIED patients exhibited higher baseline TNF-α and MPO levels compared with normal healthy controls. NAC effectively abrogates LPS-mediated TNF-α release from PBMC of both AIED patients and controls. We demonstrated that in AIED patients, the TNF-α downstream signaling pathway appears aberrantly regulated, influencing both MPO and IL-8 expression. Given that NAC effectively abrogated LPS-mediated TNF-α release and exerted minimal effects on the downstream targets of this pathway, we feel NAC may be a rational adjunct therapy for this enigmatic disease, worthy of clinical exploration.
Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Doenças Autoimunes/tratamento farmacológico , Doenças do Labirinto/tratamento farmacológico , Leucócitos Mononucleares/efeitos dos fármacos , Doenças Autoimunes/imunologia , Humanos , Interleucina-8/metabolismo , Doenças do Labirinto/imunologia , Leucócitos Mononucleares/imunologia , Terapia de Alvo Molecular , Peroxidase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Autoimmune inner ear disease (AIED) is a rare disease that results in progressive sensorineural hearing loss. Patients with AIED initially respond to corticosteroids; however, many patients become unresponsive to this treatment over time, and there is no effective alternative therapy for these individuals. METHODS: We performed a phase I/II open-label, single-arm clinical trial of the IL-1 receptor antagonist anakinra in corticosteroid-resistant AIED patients. Given that the etiology of corticosteroid resistance is likely heterogeneous, we used a Simon 2-stage design to distinguish between an unacceptable (≤10%) and an acceptable (≥30%) response rate to anakinra therapy. Subjects received 100 mg anakinra by subcutaneous injection for 84 days, followed by a 180-day observational period. RESULTS: Based on patient responses, the Simon 2-stage rule permitted premature termination of the trial after 10 subjects completed the 84-day drug period, as the target efficacy for the entire trial had been achieved. Of these 10 patients, 7 demonstrated audiometric improvement, as assessed by pure tone average (PTA) and word recognition score (WRS). In these 7 responders, reduced IL-1ß plasma levels correlated with clinical response. Upon discontinuation of treatment, 3 subjects relapsed, which correlated with increased IL-1ß plasma levels. CONCLUSION: We demonstrated that IL-1ß inhibition in corticosteroid-resistant AIED patients was effective in a small cohort of patients and that IL-1ß plasma levels associated with both clinical hearing response and disease relapse. These results suggest that a larger phase II randomized clinical trial of IL-1ß inhibition is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT01267994. FUNDING: NIH, Merrill & Phoebe Goodman Otology Research Center, and Long Island Hearing & Speech Society.
Assuntos
Corticosteroides/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Perda Auditiva Neurossensorial/tratamento farmacológico , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Doenças do Labirinto/tratamento farmacológico , Adolescente , Adulto , Idoso , Feminino , Humanos , Análise de Intenção de Tratamento , Proteína Antagonista do Receptor de Interleucina 1/efeitos adversos , Interleucina-1beta/sangue , Masculino , Pessoa de Meia-IdadeRESUMO
Autoimmune Inner Ear Disease (AIED) is characterized by bilateral, fluctuating sensorineural hearing loss with periods of hearing decline triggered by unknown stimuli. Here we examined whether an environmental exposure to mold in these AIED patients is sufficient to generate a pro-inflammatory response that may, in part, explain periods of acute exacerbation of disease. We hypothesized that molds may stimulate an aberrant immune response in these patients as both several Aspergillus species and penecillium share homology with the LCCL domain of the inner ear protein, cochlin. We showed the presence of higher levels of anti-mold IgG in plasma of AIED patients at dilution of 1:256 (p = 0.032) and anti-cochlin IgG 1:256 (p = 0.0094 and at 1:512 p = 0.024) as compared with controls. Exposure of peripheral blood mononuclear cells (PBMC) of AIED patients to mold resulted in an up-regulation of IL-1ß mRNA expression, enhanced IL-1ß and IL-6 secretion, and generation of IL-17 expressing cells in mold-sensitive AIED patients, suggesting mold acts as a PAMP in a subset of these patients. Naïve B cells secreted IgM when stimulated with conditioned supernatant from AIED patients' monocytes treated with mold extract. In conclusion, the present studies indicate that fungal exposure can trigger autoimmunity in a subset of susceptible AIED patients.
Assuntos
Aspergilose/imunologia , Aspergillus/imunologia , Doenças Autoimunes/imunologia , Doenças do Labirinto/imunologia , Leucócitos Mononucleares/imunologia , Adulto , Idoso , Anticorpos Antifúngicos/sangue , Antígenos de Fungos/imunologia , Células Cultivadas , Reações Cruzadas , Citocinas/genética , Citocinas/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Imunidade Inata , Imunoglobulina G/sangue , Leucócitos Mononucleares/microbiologia , Masculino , Pessoa de Meia-Idade , Homologia de Sequência de AminoácidosRESUMO
HIV-associated nephropathy (HIVAN) is the manifestation of HIV gene expression by kidney cells in the presence of specific host factors. Recently, rapamycin (sirolimus) has been demonstrated to modulate the progression of HIVAN. We hypothesized that rapamycin would modulate the progression of HIVAN by attenuating HIV gene expression. To test our hypothesis, three weeks old Tg26 mice (n=6) were administered either vehicle or rapamycin (5 mg/kg, every other day, intraperitoneal) for eight weeks. At the end of the experimental period, the kidneys were harvested. In in vitro studies, human podocytes were transduced with either HIV-1 (NL4-3) or empty vector (EV), followed by treatment with either vehicle or rapamycin. Total RNA and proteins were extracted from renal tissues/cellular lysates and HIV gene transcription/translation was measured by real time PCR and Western blotting studies. Renal histological slides were graded for glomerular sclerosis and tubular dilatation with microcyst formation. Rapamycin attenuated both glomerular and tubular lesions in Tg26 mice. Rapamycin decreased transcription of HIV genes both in renal tissues as well as in HIV-1 transduced podocytes. Our data strongly indicate that HIV-1 long terminal repeat-mediated transcriptional activity was targeted by rapamycin. Rapamycin enhanced podocyte NF-κB and CREB activities but then it decreased AP-1 binding activity. Since expression of HIV genes by kidney cells has been demonstrated to be the key factor in the development HIVAN, it appears that rapamycin-induced altered transcription of HIV genes might have partly contributed to its disease modulating effects.
Assuntos
Nefropatia Associada a AIDS/tratamento farmacológico , Nefropatia Associada a AIDS/virologia , HIV-1/genética , Rim/efeitos dos fármacos , Sirolimo/farmacologia , Transcrição Gênica/efeitos dos fármacos , Nefropatia Associada a AIDS/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Progressão da Doença , HIV-1/efeitos dos fármacos , Humanos , Rim/patologia , Rim/virologia , Glomérulos Renais/patologia , Glomérulos Renais/virologia , Túbulos Renais/patologia , Túbulos Renais/virologia , Camundongos , NF-kappa B/metabolismo , Podócitos/efeitos dos fármacos , Podócitos/virologia , Esclerose , Fator de Transcrição AP-1/metabolismoRESUMO
OBJECTIVES: To characterize levels of tumor necrosis factor (TNF; formerly known as tumor necrosis factor α), a well-established proinflammatory cytokine, in patients with immune-mediated sensorineural hearing loss (IM-SNHL) and to determine the role of this cytokine in identifying steroid-responsive hearing loss. DESIGN: Prospective case-control study. SETTING: Tertiary care academic medical center. PATIENTS: A total of 11 control subjects and 85 patients with clinical and audiometric characteristics of IM-SNHL (autoimmune inner ear disease and sudden SNHL combined) treated with corticosteroids were enrolled in the study. Patients were categorized as steroid responders (n = 47) and steroid nonresponders (n = 38). Peripheral venous blood was used to determine the total amount of plasma TNF by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cells (PBMCs) were isolated and treated with in vitro dexamethasone. Treated and untreated PBMCs were then analyzed for release of soluble TNF protein into conditioned supernatants as well as expression of TNF messenger RNA (mRNA). MAIN OUTCOME MEASURES: Mean plasma levels of TNF, unstimulated and dexamethasone-stimulated PBMC-secreted levels of TNF, and TNF mRNA levels in unstimulated and dexamethasone-stimulated PBMCs. RESULTS: Steroid nonresponders had the highest mean baseline plasma levels of TNF compared with steroid responders and control subjects (27.6, 24.1, and 14.4 pg/mL, respectively) (P = .03). For patients with IM-SNHL with a high baseline plasma levels of TNF (>14.4 pg/mL), the mean TNF secreted by PBMCs was 59.1 pg/mL, which decreased to 7.2 pg/mL with in vitro dexamethasone stimulation in the responder group, while the mean TNF secreted by PBMCs was 11.2 pg/mL, which slightly increased to 11.7 pg/mL with in vitro dexamethasone stimulation in the nonresponder group (P = .04). CONCLUSIONS: The level of TNF can be used as both a diagnostic and prognostic cytokine for IM-SNHL. For patients presenting with a sudden change in hearing threshold, a high baseline plasma TNF from the peripheral circulation is supportive of the diagnosis if it is greater than 18.8 pg/mL, with a positive predictive value higher than 97%. In addition, this study demonstrates that for patients with IM-SNHL and high plasma levels of TNF, their clinical response to oral glucocorticoids can be predicted by their in vitro PBMC response to dexamethasone. This algorithm may further guide optimal medical treatment and possibly avoid the deleterious adverse effects of administering glucocorticoids to those patients who would not benefit from their effect.
Assuntos
Perda Auditiva Neurossensorial/sangue , Perda Auditiva Neurossensorial/tratamento farmacológico , Leucócitos Mononucleares/imunologia , Fator de Necrose Tumoral alfa/sangue , Centros Médicos Acadêmicos , Adolescente , Adulto , Idoso , Análise de Variância , Biomarcadores/sangue , Estudos de Casos e Controles , Células Cultivadas/efeitos dos fármacos , Dexametasona/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Feminino , Glucocorticoides/uso terapêutico , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Índice de Gravidade de Doença , Estatísticas não Paramétricas , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
Human immunodeficiency virus (HIV)-1 has been reported to cause tubular cell injury both in in vivo and in vitro studies. In the present study, we evaluated the role of oxidative stress in the induction of apoptosis in HIV gene expressing mouse tubular cells in in vivo (Tg26, a transgenic mouse model of HIV-associated nephropathy) and in vitro (tubular cells were transduced with pNL4-3: ΔG/P-GFP, VSV.G psueudo typed virus) studies. Although Tg26 mice showed enhanced tubular cell reactive oxygen species (ROS) generation and apoptosis, renal tissue did not display a robust antioxidant response in the form of enhanced free radical scavenger (MnSOD/catalase) expression. Tg26 mice not only showed enhanced tubular cell expression of phospho-p66ShcA but also displayed nuclear Foxo3a translocation to the cytoplasm. These findings indicated deactivation of tubular cell Foxo3A-dependent redox-sensitive stress response program (RSSRP) in Tg26 mice. In in vitro studies, NL4-3 (pNL4-3: ΔG/P-GFP, VSV.G pseudotyped virus)-transduced mouse proximal tubular cells (NL4-3/MPTEC) displayed enhanced phosphorylation of p66ShcA. NL4-3/MPTECs also displayed greater (P < 0.01) ROS generation when compared with empty vector-transduced tubular cells; however, both diminution of p66ShcA and N-acetyl cysteine attenuated NL4-3-induced tubular cell ROS generation as well as apoptosis. In addition, both antioxidants and free radical scavengers partially inhibited HIV-induced tubular cell apoptosis. NL4-3/MPTEC displayed deactivation of RSSRP in the form of enhanced phosphorylation of Foxo3A and attenuated expression of superoxide dismutase (SOD) and catalase. Since both SOD and catalase were able to provide protection against HIV-1-induced tubular cell apoptosis, it suggests that HIV-1-induced proapoptotic effect may be a consequence of the deactivated RSSRP.
Assuntos
Nefropatia Associada a AIDS/etiologia , Apoptose/efeitos dos fármacos , Túbulos Renais/fisiopatologia , Estresse Oxidativo/fisiologia , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Animais , Antioxidantes/farmacologia , Catalase/biossíntese , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Infecções por HIV/genética , HIV-1 , Camundongos , Camundongos Transgênicos , Oxirredução , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Superóxido Dismutase/biossínteseRESUMO
Autoimmune inner ear disease is an enigmatic disorder characterized by recurring episodes of sudden or progressive sensorineural hearing loss. Hearing loss can be improved by timely corticosteroid administration, but only half of those treated respond, and for many responders, that response is lost over time. The mechanisms that control corticosteroid responsiveness in this disorder are largely uncharacterized. We have previously identified that the induction by dexamethasone of IL-1R type II (IL-1R2) expression in PBMC predicts corticosteroid responsiveness in this disorder. In this study, we asked whether IL-1ß was overexpressed, and whether clinical corticosteroid responders differentially regulated IL-1ß expression or release in response to dexamethasone, as compared with nonresponders. IL-1ß has been reported to induce matrix metalloproteinase-9 (MMP-9) expression. Given that metalloproteinases can cleave IL-1R2, we also asked whether MMP-9 expression was altered in this disorder. In this study, we demonstrate that corticosteroid nonresponders have elevated plasma levels of IL-1ß and MMP-9 as compared with clinically responsive patients (p = 0.0008 and p = 0.037, respectively). Increasing MMP-9 expression correlated with increasing IL-1ß concentration, suggesting that IL-1ß expression regulates MMP-9 expression. As expected, monocytes were the predominant producers of IL-1ß. In vitro exposure of PBMC to dexamethasone from clinical corticosteroid responders suppressed IL-1ß release. PBMC of corticosteroid nonresponders have substantially higher release of IL-1ß into the conditioned media, and when exposed to dexamethasone, failed to repress IL-1ß release (p = 0.05). Treatment of PBMC from clinical corticosteroid nonresponders with anakinra resulted in repression of IL-1ß release, suggesting that IL-1ß blockade may be a viable therapy for these patients.
Assuntos
Doenças Autoimunes/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Glucocorticoides/uso terapêutico , Interleucina-1beta/biossíntese , Administração Oral , Adulto , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/genética , Células Cultivadas , Implante Coclear , Dexametasona/farmacologia , Feminino , Perda Auditiva Neurossensorial/tratamento farmacológico , Perda Auditiva Neurossensorial/imunologia , Perda Auditiva Neurossensorial/metabolismo , Humanos , Imunossupressores/uso terapêutico , Interleucina-1beta/sangue , Interleucina-1beta/genética , Masculino , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/sangue , Inibidores de Metaloproteinases de Matriz , Prednisona/uso terapêutico , Estudos ProspectivosRESUMO
Angiotensin II (Ang II) has been reported to play an important role in both the development and progression of renal injury. Many of the downstream effects of Ang II are mediated through the activation of nuclear factor-kappaB (NF-kappaB). In the present study, we evaluated the effect of Ang II on the activation of Ets-1 (a transcription factor) in tubular cells. In addition, we studied the expression of pro-inflammatory molecules transcribed by Ets-1 in response to Ang II. Mice receiving Ang II infusion showed enhanced renal cortical mRNA expression of Ets-1. Immunolabeling studies localized the expression of Ets-1 in distal tubular cells of mice receiving Ang II. However, this effect of Ang II was mitigated by telmisartan, an AT1-receptor blocker. Mice receiving Ang II infusion also showed increased tubular cell expression of macrophage chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1), and p21 when compared with control mice; nevertheless, this effect of Ang II was attenuated by telmisartan. In in vitro studies, Ang II enhanced mRNA expression of Ets-1 by MDCK cells. However, this effect of Ang II was inhibited by losartan, an AT1-receptor blocker. Losartan also inhibited Ang II-induced PAI-1 and p21 expression by MDCK cells. These findings indicate that Ang II-induced tubular cell expression Ets-1 and associated downstream signaling is mediated through AT1 receptors.
Assuntos
Angiotensina II/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Proteína Proto-Oncogênica c-ets-1/metabolismo , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Vasoconstritores/farmacologia , Antagonistas de Receptores de Angiotensina/farmacologia , Animais , Técnicas de Cultura de Células , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Proteína Proto-Oncogênica c-ets-1/genética , RNA Mensageiro/metabolismoRESUMO
Apoptosis is central to the interaction between pathogenic mycobacteria and host macrophages. Caspase-8-dependent apoptosis of infected macrophages, which requires activation of the mitogen-activated protein (MAP) kinase p38, lowers the spread of mycobacteria. Here we establish a link between the release of tumor necrosis factor (TNF) and mycobacteria-mediated macrophage apoptosis. TNF activated a pathway involving the kinases ASK1, p38 and c-Abl. This pathway led to phosphorylation of FLIP(S), which facilitated its interaction with the E3 ubiquitin ligase c-Cbl. This interaction triggered proteasomal degradation of FLIP(S), which promoted activation of caspase-8 and apoptosis. Our findings identify a previously unappreciated signaling pathway needed for Mycobacterium tuberculosis-triggered macrophage cell death.
Assuntos
Apoptose/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Macrófagos/fisiologia , Mycobacterium tuberculosis/fisiologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Caspase 8/metabolismo , Linhagem Celular , Proliferação de Células , Humanos , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 5/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-cbl/genética , Transdução de Sinais , Ubiquitinação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Apolipoprotein E (apoE) has been demonstrated to play an important role in providing protection against mesangial cell injury. In the present study, we evaluated the role of apoE and its associated downstream effects in human immunodeficiency virus (HIV)-associated nephropathy (HIVAN). Control (n = 6) and age- and sex-matched HIV-1 transgenic mice (Tg26, n = 6) were evaluated for their renal cortical expression of apoE. Renal tissue from Tg26 mice not only showed decreased apoE expression but also displayed downregulation of perlecan mRNA expression. In in vitro studies, conditionally immortalized human podocytes (CIHPs) were transduced with either NL4-3HIV (an HIV-1 construct lacking gag and pol, used for the development of Tg26 mouse model; NL4-3/CIHP) or empty vector (EV/CIHP); NL4-3/CIHPs and EV/CIHPs were studied for apoE mRNA expression. NL4-3/CIHPs showed reduction in apoE expression compared with EV/CIHPs. To evaluate the role of HIV-1 genes in the modulation of apoE expression, conditionally immortalized mouse podocytes (CIMPs) were transduced with individual HIV-1 gene constructs. Only nef-transduced CIMPs showed a decrease in apoE expression. To confirm this effect of nef in CIHPs, microarray analysis was performed in stable colonies of nef/CIHPs and EV/CIHPs. nef/CIHPs showed a 60% decrease in apoE and a 90% reduction in heparan sulfate mRNA expression. Moreover, nef transgenic mice showed a decrease in renal tissue expression of both apoE and perlecan. Both Tg26 and nef transgenic mice also showed areas of mesangial cell proliferation. These findings suggest that HIV-1-induced reduction in podocyte apoE expression and associated downregulation of podocyte perlecan might be contributing to mesangial cell (MC) phenotype in HIVAN.
Assuntos
Nefropatia Associada a AIDS/metabolismo , Apolipoproteínas E/metabolismo , HIV-1/genética , Células Mesangiais/metabolismo , Podócitos/metabolismo , Nefropatia Associada a AIDS/patologia , Nefropatia Associada a AIDS/virologia , Animais , Linhagem Celular Transformada , Proliferação de Células , Regulação para Baixo , Genótipo , HIV-1/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Células Mesangiais/patologia , Células Mesangiais/virologia , Camundongos , Camundongos Transgênicos , Fenótipo , Podócitos/patologia , Podócitos/virologia , RNA Mensageiro/metabolismo , Transdução Genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The pathophysiology of Helicobacter pylori-associated gastroduodenal diseases, ulcerogenesis, and carcinogenesis is intimately linked to activation of epidermal growth factor receptor (EGFR) and production of vascular endothelial growth factor (VEGF). Extracellular virulence factors, such as CagA and VacA, have been proposed to regulate EGFR activation and VEGF production in gastric epithelial cells. We demonstrate that the H. pylori secretory protein, HP0175, by virtue of its ability to bind TLR4, transactivates EGFR and stimulates EGFR-dependent VEGF production in the gastric cancer cell line AGS. Knock-out of the hp0175 gene attenuates the ability of the resultant H. pylori strain to activate EGFR or to induce VEGF production. HP0175-induced activation of EGFR is preceded by translocation of TLR4 into lipid rafts. In lipid rafts, the Src kinase family member Lyn interacts with TLR4, leading to tyrosine phosphorylation of TLR4. Knockdown of Lyn prevents HP0175-induced activation of EGFR and VEGF production. Tyrosine-phosphorylated TLR4 interacts with EGFR. This interaction is necessary for the activation of EGFR. Disruption of lipid rafts with methyl beta-cyclodextrin prevents HP0175-induced tyrosine phosphorylation of TLR4 and activation of EGFR. This mechanism of transactivation of EGFR is novel and distinct from that of metalloprotease-dependent shedding of EGF-like ligands, leading to autocrine activation of EGFR. It provides new insight into our understanding of the receptor cross-talk network.
Assuntos
Proteínas de Bactérias/metabolismo , Células Epiteliais/metabolismo , Helicobacter pylori/metabolismo , Neoplasias Gástricas/microbiologia , Receptor 4 Toll-Like/metabolismo , Transativadores/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Microdomínios da Membrana/química , Modelos Biológicos , Fosforilação , Neoplasias Gástricas/metabolismo , Transativadores/fisiologia , Ativação Transcricional , Tirosina/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases da Família src/genéticaRESUMO
Matrix metalloproteinases (MMPs) contribute to the matrix-degrading phenotype of mycobacterial diseases. Considering that MMPs could contribute to the mutual exacerbation of both Mycobacterium avium and HIV in coinfections, it is of importance to understand the mechanisms of M. avium-induced MMP induction. Focusing on MMP-9, our work demonstrates that a cyclooxygenase-2 (COX-2)-dependent signalling loop is critical for activation of MMP-9 transcription in RAW264.7 cells and murine bone marrow-derived macrophages. M. avium-stimulated MMP-9 induction involves the p65 and p50 subunits of NF-kappaB and the c-Fos and c-jun subunits of AP-1. The c-Fos gene is upregulated in a MEK1-dependent manner in M. avium-challenged macrophages. M. avium-induced MMP-9 gene induction requires the histone acetyltransferase p300 and chromatin modifications involving phosphorylation of p65 at serine 276 and its acetylation at lysines 221 and 310. At the same time, histone H3 modified by mitogen and stress-activated protein kinase 1 (MSK1)-dependent phosphorylation on serine 10 and by acetylation on lysine 14, typical signatures linked to transcriptional activation, also associates with the MMP-9 promoter following M. avium challenge. Taken together, our results show that co-ordinated post-translational modifications of p65 and histone H3 involving phosphorylation and acetylation drive COX-2-dependent transcriptional activation of the MMP-9 gene in response to challenge of macrophages with M. avium.
Assuntos
Cromatina/metabolismo , Ciclo-Oxigenase 2/fisiologia , Metaloproteinase 9 da Matriz/biossíntese , Mycobacterium avium/imunologia , Mycobacterium avium/fisiologia , Regulação para Cima/fisiologia , Acetilação , Animais , Linhagem Celular , Células Cultivadas , Histonas/metabolismo , Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Fosforilação , Fator de Transcrição AP-1/biossíntese , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Expression of early secreted antigenic target protein 6 (ESAT-6) by Mycobacterium tuberculosis is associated with lower innate immune responses to infection. Here we show that ESAT-6 inhibited activation of transcription factor NF-kappaB and interferon-regulatory factors (IRFs) after Toll-like receptor (TLR) signaling; inhibition of TLR signaling by ESAT-6 required the kinase Akt. Direct binding of ESAT-6 to TLR2 activated Akt and prevented interaction between the adaptor MyD88 and 'downstream' kinase IRAK4, thus abrogating NF-kappaB activation. The six carboxy-terminal amino acid residues of ESAT-6 were required and sufficient for the TLR2-mediated inhibitory effect. A critical function for the carboxy-terminal peptide of ESAT-6 in restricting MyD88-dependent TLR signaling emphasizes the possibility that mimetic inhibitory peptides could be used to restrict innate immune responses in situations in which prolonged TLR signaling has deleterious effects.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Macrófagos/metabolismo , Mycobacterium tuberculosis/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Humanos , Fatores Reguladores de Interferon/metabolismo , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Subunidade p40 da Interleucina-12/biossíntese , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Tempo , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genéticaRESUMO
Combating tuberculosis requires a detailed understanding of how mycobacterial effectors modulate the host immune response. The role of the multigene PE family of proteins unique to mycobacteria in the pathogenesis of tuberculosis is still poorly understood, although certain PE_PGRS genes have been linked to virulence. Tumor necrosis factor-alpha (TNF-alpha) is essential for successfully combating tuberculosis. In this study we provide evidence that PE_PGRS33, a surface exposed protein, elicits TNF-alpha release from macrophages in a TLR2 (Toll-like receptor 2)-dependent manner. ASK1 (apoptosis signal-regulating kinase 1) is activated downstream of TLR2. ASK1 activates the MAPKs p38 and JNK. PE_PGRS33-induced signaling leads to enhanced expression of TNF-alpha and TNF receptor I (TNFRI) genes. Mycobacterium smegmatis expressing PE_ PGRS33 elicits the same effects as purified PE_PGRS33. TNF-alpha release occurs even when internalization of the bacteria is blocked by cytochalasin D, suggesting that interaction of PE_ PGRS33 with TLR2 is sufficient to trigger the effects described. Release of TNF-alpha plays the determining role in triggering apoptosis in macrophages challenged with PE_PGRS33. The death receptor-dependent signals are amplified through classical caspase 8-dependent mitochondrial release of cytochrome c, leading to the activation of caspases 9 and 3. An important aspect of our findings is that deletions within the PGRS domain (simulating those occurring in clinical strains) attenuate the TNF-alpha-inducing ability of PE_PGRS33. These results provide the first evidence that variations in the polymorphic repeats of the PGRS domain modulate the innate immune response.