RESUMO
A subset of CC chemokine receptor-6(+) (CCR6(+)), γδ-low (GDL) T cells that express Th17 cytokines in mouse skin participates in IL-23-induced psoriasiform dermatitis. We use CCR6-deficient (knockout, KO) and wild-type (WT) mice to analyze skin trafficking patterns of GDL T cells and function-blocking mAbs to determine the role of CCR6 in IL-23-mediated dermatitis. Herein, CCL20 was highly upregulated in IL-23-injected WT mouse ear skin as early as 24 hours after initial treatment, and large numbers of CCR6(+) cells were observed in the epidermis of IL-23-injected WT mice. Anti-CCL20 mAbs reduced psoriasiform dermatitis and blocked recruitment of GDL T cells to the epidermis. In CCR6 KO mice, GDL T cells failed to accumulate in the epidermis after IL-23 treatment, but the total numbers of GDL T cells in the dermis of WT and CCR6 KO mice were equivalent. There was an â¼70% reduction in the proportion of IL-22(+) GDL T cells in the dermis of CCR6 KO mice (vs WT mice), suggesting that effector function and epidermal recruitment of GDL T cells are impaired in CCR6-deficient mice. Thus, these data show that CCR6 regulates epidermal trafficking of γδ-T-cell subsets in the skin and suggest the potential of CCR6 as a therapeutic target for psoriasis.
Assuntos
Dermatite/imunologia , Epiderme/imunologia , Interleucina-23/efeitos adversos , Psoríase/imunologia , Receptores CCR6/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Anticorpos Bloqueadores/farmacologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Quimiocina CCL20/imunologia , Dermatite/patologia , Epiderme/efeitos dos fármacos , Epiderme/patologia , Interleucinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Psoríase/induzido quimicamente , Psoríase/patologia , Receptores de Antígenos de Linfócitos T gama-delta/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores CCR6/genética , Subpopulações de Linfócitos T/efeitos dos fármacos , Interleucina 22RESUMO
BACKGROUND: Lentivector-mediated gene delivery into the dorsal root ganglion (DRG) is a promising method for exploring pain pathophysiology and for genetic treatment of chronic neuropathic pain. In this study, a series of modified lentivector particles with different cellular promoters, envelope glycoproteins, and viral accessory proteins were generated to evaluate the requirements for efficient transduction into neuronal cells in vitro and adult rat DRG in vivo. RESULTS: In vitro, lentivectors expressing enhanced green fluorescent protein (EGFP) under control of the human elongation factor 1α (EF1α) promoter and pseudotyped with the conventional vesicular stomatitis virus G protein (VSV-G) envelope exhibited the best performance in the transfer of EGFP into an immortalized DRG sensory neuron cell line at low multiplicities of infection (MOIs), and into primary cultured DRG neurons at higher MOIs. In vivo, injection of either first or second-generation EF1α-EGFP lentivectors directly into adult rat DRGs led to transduction rates of 19 ± 9% and 20 ± 8% EGFP-positive DRG neurons, respectively, detected at 4 weeks post injection. Transduced cells included a full range of neuronal phenotypes, including myelinated neurons as well as both non-peptidergic and peptidergic nociceptive unmyelinated neurons. CONCLUSION: VSV-G pseudotyped lentivectors containing the human elongation factor 1α (EF1α)-EGFP expression cassette demonstrated relatively efficient transduction to sensory neurons following direct injection into the DRG. These results clearly show the potential of lentivectors as a viable system for delivering target genes into DRGs to explore basic mechanisms of neuropathic pain, with the potential for future clinical use in treating chronic pain.
Assuntos
Envelhecimento/metabolismo , Gânglios Espinais/metabolismo , Técnicas de Transferência de Genes , Lentivirus/genética , Animais , Comportamento Animal , Linhagem Celular Transformada , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Injeções , Glicoproteínas de Membrana/metabolismo , Músculos/metabolismo , Dor/metabolismo , Dor/patologia , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Regiões Promotoras Genéticas/genética , Vírus da Raiva/metabolismo , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismo , Transdução Genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
The activation of heterotrimeric G protein signaling is a key feature in the pathophysiology of polycystic kidney diseases (PKD). In this study, we report abnormal overexpression of activator of G protein signaling 3 (AGS3), a receptor-independent regulator of heterotrimeric G proteins, in rodents and humans with both autosomal recessive and autosomal dominant PKD. Increased AGS3 expression correlated with kidney size, which is an index of severity of cystic kidney disease. AGS3 expression localized exclusively to distal tubular segments in both normal and cystic kidneys. Short hairpin RNA-induced knockdown of endogenous AGS3 protein significantly reduced proliferation of cystic renal epithelial cells by 26 +/- 2% (P < 0.001) compared with vehicle-treated and control short hairpin RNA-expressing epithelial cells. In summary, this study suggests a relationship between aberrantly increased AGS3 expression in renal tubular epithelia affected by PKD and epithelial cell proliferation. AGS3 may play a receptor-independent role to regulate Galpha subunit function and control epithelial cell function in PKD.
Assuntos
Proteínas de Transporte/fisiologia , Células Epiteliais/patologia , Doenças Renais Policísticas/patologia , Animais , Proteínas de Transporte/genética , Proliferação de Células , Células Cultivadas , Expressão Gênica , Inibidores de Dissociação do Nucleotídeo Guanina , HumanosRESUMO
Angiotensin converting enzyme (ACE) inhibition is a common therapeutic modality in the treatment of autosomal recessive polycystic kidney disease (ARPKD). This study was designed to investigate whether chronic inhibition of ACE would have a therapeutic effect in attenuating the progression of renal cystogenesis in an orthologous rat model of ARPKD, the polycystic kidney (PCK) rat. Lisinopril (3 mg/kg per day) was administered orally for a period of 12 weeks, beginning at post-natal week 4. Lisinopril treatment resulted in an approximately 30% improvement in the collecting duct cystic indices (CT CI) of PCK animals. Activation of extracellular signal-regulated kinase 1 (ERK1) and 2 (ERK2), proliferative signaling markers, and proliferating cell nuclear antigen (PCNA), an end-point marker for proliferation, was reduced following chronic treatment with lisinopril compared to that in vehicle-treated PCK rats. To assess whether apoptotic pathways were altered due to chronic ACE inhibition, we examined p38 mitogen activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), which are markers of apoptotic signaling cascades. p38 MAPK was significantly reduced (P < 0.0001) following chronic treatment with lisinopril, but no change in the activation of SAPK/JNK could be detected by immunoblot analysis. Lisinopril treatment resulted in a significant reduction (P < 0.01) in cleaved caspase-7 levels, but not caspase-3 activity, in PCK rat kidneys compared to the vehicle-treated PCK rat kidneys. Proteinuria was completely ameliorated in the presence of chronic ACE inhibition in the lisinopril-treated rats compared with the vehicle-treated PCK rats. In all, these findings demonstrated that chronic ACE inhibition can beneficially alter proliferative and apoptotic pathways to promote therapeutic reductions in renal cyst development in ARPKD.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Lisinopril/farmacologia , Rim Policístico Autossômico Recessivo/tratamento farmacológico , Animais , Western Blotting , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
20-Hydroxyeicosatetraenoic acid (20-HETE) has been implicated as a potential mediator in epithelial cell proliferation and cyst formation in polycystic kidney disease (PKD). In the present study, we studied the effects of chronic blockade of 20-HETE synthesis in an orthologous rodent model of autosomal recessive polycystic kidney disease (ARPKD), the PCK rat. RT-PCR analysis indicated that the expression of CYP4A1, CYP4A2, CYP4A3, and CYP4A8 mRNA was increased two- to fourfold in cystic PCK compared with noncystic Sprague-Dawley rat kidneys. Daily administration of a 20-HETE synthesis inhibitor, HET-0016 (10 mg x kg(-1) x day(-1) ip) for 4-7 wk significantly reduced kidney size by 24% from 4.95 +/- 0.19 g in vehicle-treated PCK rats to 3.76 +/- 0.15 g (n = 4). Collecting tubule morphometric cystic indices were reduced in HET-0016-treated PCK rats (2.1 +/- 0.2; n = 4) compared with vehicle-treated PCK rats (4.4 +/- 0.1; n = 4). The cellular mechanism by which 20-HETE may play a role in cyst formation has not been well characterized, but there was a significantly lower (P < 0.05) level of intracellular cAMP and decreased phosphorylation (activation) of ERK1/2 protein in PCK rat kidneys (n = 3) treated with HET-0016 . These studies indicate a potential role of 20-HETE in cyst formation in the orthologous rodent PCK model of ARPKD.
Assuntos
Ácidos Hidroxieicosatetraenoicos/metabolismo , Rim/patologia , Rim Policístico Autossômico Recessivo/metabolismo , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Amidinas/farmacologia , Animais , AMP Cíclico/metabolismo , Citocromo P-450 CYP4A/antagonistas & inibidores , Modelos Animais de Doenças , Ativação Enzimática , Compostos de Epóxi/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Microssomos/metabolismo , Tamanho do Órgão , Rim Policístico Autossômico Recessivo/patologia , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Polycystic kidney diseases are characterized by abnormal proliferation of renal epithelial cells. In this study, the role of 20-hydroxyeicosatetraenoic acid (20-HETE), an endogenous cytochrome P450 metabolite of arachidonic acid with mitogenic properties, was evaluated in cystic renal disease. Daily administration of HET-0016, an inhibitor of 20-HETE synthesis, significantly reduced kidney size by half in the BPK mouse model of autosomal recessive polycystic kidney disease. In addition, compared with untreated BPK mice, this treatment significantly reduced collecting tubule cystic indices and approximately doubled survival. For evaluation of the role of 20-HETE as a mediator of epithelial cell proliferation, principal cells isolated from cystic BPK and noncystic Balb/c mice were genetically modified using lentiviral vectors. Noncystic Balb/c cells overproducing Cyp4a12 exhibited a four- to five-fold increase in cell proliferation compared with control Balb/c cells, and this increase was completely abolished when 20-HETE synthesis was inhibited; therefore, this study suggests that 20-HETE mediates proliferation of epithelial cells in the formation of renal cysts.
Assuntos
Proliferação de Células , Células Epiteliais/fisiologia , Ácidos Hidroxieicosatetraenoicos/fisiologia , Doenças Renais Policísticas/etiologia , Doenças Renais Policísticas/patologia , Amidinas/farmacologia , Animais , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/fisiologia , Família 4 do Citocromo P450 , Modelos Animais de Doenças , Ácidos Hidroxieicosatetraenoicos/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Doenças Renais Policísticas/metabolismoRESUMO
20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-PK(1) cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK(1) cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release (P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 microM) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 microM) also inhibited cytotoxicity significantly (P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase (P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells (P < 0.05). This was abolished in the presence of HET-0016 (P < 0.05) or MnTMPyP (P < 0.01). These results demonstrate that 20-HETE overexpression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals.
Assuntos
Apoptose/fisiologia , Citocromo P-450 CYP4A/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Túbulos Renais Proximais/fisiologia , Traumatismo por Reperfusão/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Caspase 3/metabolismo , Linhagem Celular , Citocromo P-450 CYP4A/genética , Células Epiteliais/metabolismo , Expressão Gênica , Vetores Genéticos , Isoenzimas/metabolismo , Túbulos Renais Proximais/metabolismo , Lentivirus , Camundongos , Superóxidos/metabolismo , Suínos , Transdução Genética , VesiculovirusRESUMO
11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which regulates the local level of glucocorticoids, has been suggested to be involved in the development of obesity. A definitive functional role for 11beta-HSD1 in adipogenesis, however, remains to be established. We developed 3T3-L1 cell lines stably transfected with a small hairpin RNA (shRNA) targeting 11beta-HSD1. A shRNA containing two nucleotide substitutions was used as a control. Silencing of 11beta-HSD1 substantially attenuated the accumulation of lipid droplets and the expression of adipogenesis marker genes, which was induced by a mixture containing either corticosterone or dexamethasone. Silencing of 11beta-HSD1 increased the concentration of 11-dehydrocorticosterone in the culture supernatant but did not significantly affect the levels of corticosterone or dexamethasone. Translocation of glucocorticoid receptors to the nucleus in response to glucocorticoids was significantly attenuated by silencing 11beta-HSD1. The number of cells entering the S phase of the cell cycle following the induction of adipogenesis was significantly reduced by silencing 11beta-HSD1. 11beta-HSD1 shRNA delivered by lentiviral vectors after the induction of differentiation, however, did not affect the progression of adipogenesis. These results indicate that 11beta-HSD1 plays a significant functional role in the initiation of 3T3-L1 adipogenesis and provide new mechanistic insights into the role of 11beta-HSD1 in the development of obesity and related diseases.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Células 3T3-L1/metabolismo , Adipogenia/genética , Interferência de RNA , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/fisiologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/biossíntese , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , Células 3T3-L1/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Sequência de Bases , Proteína alfa Estimuladora de Ligação a CCAAT/biossíntese , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Corticosterona/análogos & derivados , Corticosterona/biossíntese , Corticosterona/metabolismo , Corticosterona/farmacologia , Dexametasona/análogos & derivados , Dexametasona/metabolismo , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica , Vetores Genéticos/genética , Lentivirus/genética , Camundongos , Dados de Sequência Molecular , PPAR gama/biossíntese , PPAR gama/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Transdução GenéticaRESUMO
BACKGROUND: Lentiviral vectors have been designed with complex RNA export sequences in both the integrating and packaging plasmids in order to co-ordinate efficient vector production. Recent studies have attempted to replace the existing complex rev/RRE system with a more simplistic RNA export system from simple retroviruses to make these vectors in a rev-independent manner. RESULTS: Towards this end, lentiviral transfer plasmids were modified with various cis-acting DNA elements that co-ordinate RNA export during viral production to determine their ability to affect the efficiency of vector titer and transduction in different immortalized cell lines in vitro. It was found that multiple copies of the constitutive transport element (CTE) originating from different simian retroviruses, including simian retrovirus type 1 (SRV-1) and type-2 (SRV-2) and Mason-Pfizer (MPV) could be used to eliminate the requirement for the rev responsive element (RRE) in the transfer and packaging plasmids with titers >106 T.U./mL (n = 4-8 preparations). The addition of multiple copies of the murine intracisternal type A particle, the woodchuck post-regulatory element (WPRE), or single and dual copies of the simian CTE had minimal effect on viral titer. Immortalized cell lines from different species were found to be readily transduced by VSV-G pseudotyped lentiviral vectors containing the multiple copies of the CTE similar to the findings in HeLa cells, although the simian-derived CTE were found to have a lower infectivity into murine cell lines compared to the other species. CONCLUSION: These studies demonstrated that the rev-responsive element (RRE) could be replaced with other constitutive transport elements to produce equivalent titers using lentivectors containing the RRE sequence in vitro, but that concatemerization of the CTE or the close proximity of RNA export sequences was needed to enhance vector production.