RESUMEN
The innate immune system recognizes nucleic acids during infection and tissue damage. Whereas viral RNA is detected by endosomal toll-like receptors (TLR3, TLR7, TLR8) and cytoplasmic RIG-I and MDA5, endosomal TLR9 and cytoplasmic DAI bind DNA, resulting in the activation of nuclear factor-kappaB and interferon regulatory factor transcription factors. However, viruses also trigger pro-inflammatory responses, which remain poorly defined. Here we show that internalized adenoviral DNA induces maturation of pro-interleukin-1beta in macrophages, which is dependent on NALP3 and ASC, components of the innate cytosolic molecular complex termed the inflammasome. Correspondingly, NALP3- and ASC-deficient mice display reduced innate inflammatory responses to adenovirus particles. Inflammasome activation also occurs as a result of transfected cytosolic bacterial, viral and mammalian (host) DNA, but in this case sensing is dependent on ASC but not NALP3. The DNA-sensing pro-inflammatory pathway functions independently of TLRs and interferon regulatory factors. Thus, in addition to viral and bacterial components or danger signals in general, inflammasomes sense potentially dangerous cytoplasmic DNA, strengthening their central role in innate immunity.
Asunto(s)
Proteínas Portadoras/inmunología , Proteínas del Citoesqueleto/inmunología , Citosol/metabolismo , Citosol/virología , ADN/inmunología , Inmunidad Innata/inmunología , Inflamación/inmunología , Adenoviridae/genética , Adenoviridae/inmunología , Adenoviridae/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Línea Celular , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Citosol/microbiología , ADN Viral/inmunología , Humanos , Inflamación/virología , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Procesamiento Proteico-PostraduccionalRESUMEN
Adeno-associated virus (AAV) vectors are associated with relatively mild host immune responses in vivo. Although AAV induces very weak innate immune responses, neutralizing antibodies against the vector capsid and transgene still occur. To understand further the basis of the antiviral immune response to AAV vectors, studies were performed to characterize AAV interactions with macrophages. Primary mouse macrophages and human THP-1 cells transduced in vitro using an AAV serotype 2 (AAV2) vector encoding green fluorescent protein did not result in measurable transgene expression. An assessment of internalized vector genomes showed that AAV2 vector uptake was enhanced in the presence of normal but not heat-inactivated or C3-depleted mouse/human serum. Enhanced uptake in the presence of serum coincided with increased macrophage activation as determined by the expression of NF-kappaB-dependent genes such as macrophage inflammatory protein 2 (MIP-2), interleukin-1beta (IL-1beta), IL-8, and MIP-1beta. AAV vector serotypes 1 and 8 also activated human and mouse macrophages in a serum-dependent manner. Immunoprecipitation studies demonstrated the binding of iC3b complement protein to the AAV2 capsid in human serum. AAV2 did not activate the alternative pathway of the complement cascade and lacked cofactor activity for factor I-mediated degradation of C3b to iC3b. Instead, our results suggest that the AAV capsid also binds complement regulatory protein factor H. In vivo, complement receptor 1/2- and C3-deficient mice displayed impaired humoral immunity against AAV2 vectors, with a delay in antibody development and significantly lower neutralizing antibody titers. These results show that the complement system is an essential component of the host immune response to AAV.
Asunto(s)
Proteínas del Sistema Complemento/fisiología , Dependovirus/inmunología , Vectores Genéticos/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Línea Celular , Dependovirus/genética , Expresión Génica , Humanos , Inmunoprecipitación , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/virología , Ratones , Ratones Endogámicos C57BLRESUMEN
The hallmark of progressive chronic kidney disease is the deposition of extracellular matrix proteins and tubulointerstitial fibrosis. Integrins mediate cell-extracellular matrix interaction and may play a role tubular epithelial injury. Murine primary tubular epithelial cells (TECs) express alpha(5)-integrin, a fibroblast marker and the natural receptor for fibronectin. Microscopy localized alpha(5)-integrin on E-cadherin-positive cells, confirming epithelial expression. The expression of alpha(5)-integrin increased in TECs grown on fibronectin and occurred in parallel with an upregulation of alpha-smooth muscle actin (alphaSMA), a marker of epithelial-mesenchymal transition (EMT). Exposure of TECs to transforming growth factor (TGF)-beta also increased TEC alpha(5)-integrin expression in association with alphaSMA and EMT. Knock-down of alpha5-integrin expression with short interfering RNA attenuated the TGF-beta induction of alphaSMA but did not alter morphologic EMT. Rather, alpha5-integrin was necessary for epithelial cell migration on fibronectin but not type IV collagen during cell spreading and epithelial wound healing in vitro. Immunohistochemistry revealed basolateral tubular epithelial alpha(5)-integrin expression in mouse kidneys after unilateral ureteric obstruction but not in contralateral control kidneys. In patient biopsies of nondiabetic kidney disease, alpha(5)-integrin expression was increased significantly in the renal interstitium. Focal basolateral staining was also detected in injured, but not in normal, tubular epithelium. In summary, these data show that TECs are induced to express alpha(5)-integrin during EMT and tubular epithelial injury in vitro and in vivo. These results increase our understanding of the biology of integrins during EMT and tubular injury in chronic kidney disease.
Asunto(s)
Integrina alfa5/biosíntesis , Fallo Renal Crónico/metabolismo , Túbulos Renales/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Epitelio/metabolismo , Regulación de la Expresión Génica , Humanos , Integrina alfa5/fisiología , Ratones , Ratones Endogámicos C57BL , Factor de Crecimiento Transformador beta/farmacología , Obstrucción Ureteral/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
In gene therapy, the innate immune system is a significant barrier to the effective application of adenovirus (Ad) vectors. In kidney epithelium-derived (REC) cells, serotype 5 Ad vectors induce the expression of the chemokine CXCL10 (IP-10), a response that is dependent on NFkappaB. Compared to the parental vector AdLuc, transduction with the RGD-deleted vector AdL.PB resulted in reduced CXCL10 activation despite increasing titers, implying that RGD-alpha(V) integrin interactions contribute to adenovirus induction of inflammatory genes. Akt, a downstream effector of integrin signaling, was activated within 10 min of transduction with Ad vectors in a dose-dependent manner. Akt activation was not present following transduction with AdL.PB, confirming the importance of capsid-alpha(V) integrin interactions in Ad vector Akt activation. Inhibition of the phosphoinositide-3-OH kinase/Akt pathway by Wortmannin or Ly294002 compounds decreased Ad vector induction of CXCL10 mRNA. Similarly, adenovirus-mediated overexpression of the dominant negative AktAAA decreased CXCL10 mRNA expression compared to the reporter vector AdLacZ alone. The effect of Akt on CXCL10 mRNA expression occurred via NFkappaB-dependent transcriptional activation, since AktAAA overexpression and Ly294002 both inhibited CXCL10 and NFkappaB promoter activation in luciferase reporter experiments. These results show that Akt plays a role in the Ad vector activation of NFkappaB and CXCL10 expression. Understanding the mechanism underlying the regulation of host immunomodulatory genes by adenovirus vectors will lead to strategies that will improve the efficacy and safety of these agents for clinical use.
Asunto(s)
Adenoviridae/fisiología , Quimiocinas CXC/metabolismo , Regulación de la Expresión Génica , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Adenoviridae/genética , Adenoviridae/inmunología , Quimiocina CXCL10 , Quimiocinas CXC/genética , Vectores Genéticos , FN-kappa B/inmunología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Increasing proteinuria in kidney disease is associated with an increased risk of renal failure. Urinary proteins such as albumin induce inflammatory signaling and gene expression in tubular epithelial cells (TECs). Fibronectin is an extracellular matrix protein that can exist in soluble form and is excreted in the urine of patients with glomerular disease. METHODS: To explore the impact of soluble fibronectin on tubular epithelium, murine TECs were stimulated with soluble fibronectin and chemokine mRNA was determined by RNase protection assay. RESULTS: Fibronectin induced the expression of inflammatory chemokine genes, including monocyte chemoattractant protein-1 (MCP-1) (CCL2) and macrophage inflammatory protein-2 (MIP-2) within 2 hours in a dose-dependent manner. Phosphorylation of Src family tyrosine kinases was also increased in TECs following exposure to fibronectin. Src tyrosine kinases were involved in the fibronectin activation of MCP-1 since the Src inhibitors SU6656 and PP2 effectively reduced the induction of this chemokine. Fibronectin also induced the phosphorylation of extracellular signal-regulated protein kinase (ERK1/2) within minutes in TECs. The ERK kinase (MEK1/2) inhibitor U0126 inhibited the fibronectin induction of MCP-1 mRNA suggesting that ERK1/2 was also involved in this inflammatory pathway. Furthermore, fibronectin also induced phosphorylation of IkappaBalpha within 20 minutes in TECs. The nuclear factor-kappaB (NF-kappaB) inhibitors N-acetyl-L-cysteine (NAC) and pyrrolidinecarbodithioic acid (PDTC) effectively blocked fibronectin induction of MCP-1 mRNA. CONCLUSION: Soluble fibronectin activates MCP-1 gene expression in TECs via Src tyrosine kinases, ERK1/2 and NF-kappaB. These data provide further support to the concept that proteinuria per se contributes to the tubulointerstitial injury observed in glomerular disease.
Asunto(s)
Quimiocinas/genética , Células Epiteliales/efectos de los fármacos , Fibronectinas/farmacología , Túbulos Renales/citología , Animales , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CXCL2 , Células Epiteliales/citología , Células Epiteliales/inmunología , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Solubilidad , Familia-src Quinasas/metabolismoRESUMEN
Helper-dependent adenovirus (HD-Ad) vectors with all adenoviral genes deleted mediate very long-term expression of therapeutic transgenes in a variety of animal models of disease. These vectors are associated with reduced toxicity and improved safety relative to traditional early region 1 deletion first-generation Ad (FG-Ad) vectors. Many studies have clearly demonstrated that FG-Ad vectors induce innate and adaptive immune responses in vivo; however, a comprehensive analysis of host immune responses to HD-Ad vectors has not yet been performed. In DBA/2 mice, intravenous injection of HD-Ad vectors encoding LacZ (HD-AdLacZ) or a murine secreted alkaline phosphatase (HD-AdSEAP) induced an early expression of inflammatory cytokine and chemokine genes in the liver, including interferon-inducible protein 10, macrophage inflammatory protein 2, and tumor necrosis factor alpha, and were expressed in a pattern similar to that induced by FG-Ad vectors encoding AdSEAP. Like AdSEAP, and consistent with the pattern of cellular gene expression, HD-AdLacZ and HD-AdSEAP induced the recruitment of CD11b-positive leukocytes to the transduced liver within hours of administration. AdSEAP also induced a second phase of liver inflammation, consisting of inflammatory gene expression and CD3-positive lymphocytic infiltrates 7 days posttransduction. In contrast, beyond 24 h no infiltrates or expression of inflammatory genes was detected in the livers of mice receiving HD-AdSEAP. Despite the lack of liver inflammation at 7 days, Ad-specific cytotoxic T lymphocytes could be detected in mice receiving HD-AdSEAP. This lack of liver inflammation was not due to reduced transduction since levels of transgene expression and the amounts of vector DNA in the liver were equivalent in mice receiving HD-AdSEAP and AdSEAP. These results demonstrate that HD-Ad vectors induce intact innate but attenuated adaptive immune responses in vivo.