RESUMEN
The metalloproteinase ADAM17 (a disintegrin and metalloprotease 17) controls EGF receptor (EGFR) signaling by liberating EGFR ligands from their membrane anchor. Consequently, a patient lacking ADAM17 has skin and intestinal barrier defects that are likely caused by lack of EGFR signaling, and Adam17(-/-) mice die perinatally with open eyes, like Egfr(-/-) mice. A hallmark feature of ADAM17-dependent EGFR ligand shedding is that it can be rapidly and posttranslationally activated in a manner that requires its transmembrane domain but not its cytoplasmic domain. This suggests that ADAM17 is regulated by other integral membrane proteins, although much remains to be learned about the underlying mechanism. Recently, inactive Rhomboid 2 (iRhom2), which has seven transmembrane domains, emerged as a molecule that controls the maturation and function of ADAM17 in myeloid cells. However, iRhom2(-/-) mice appear normal, raising questions about how ADAM17 is regulated in other tissues. Here we report that iRhom1/2(-/-) double knockout mice resemble Adam17(-/-) and Egfr(-/-) mice in that they die perinatally with open eyes, misshapen heart valves, and growth plate defects. Mechanistically, we show lack of mature ADAM17 and strongly reduced EGFR phosphorylation in iRhom1/2(-/-) tissues. Finally, we demonstrate that iRhom1 is not essential for mouse development but regulates ADAM17 maturation in the brain, except in microglia, where ADAM17 is controlled by iRhom2. These results provide genetic, cell biological, and biochemical evidence that a principal function of iRhoms1/2 during mouse development is to regulate ADAM17-dependent EGFR signaling, suggesting that iRhoms1/2 could emerge as novel targets for treatment of ADAM17/EGFR-dependent pathologies.
Asunto(s)
Proteínas ADAM/metabolismo , Proteínas Portadoras/metabolismo , Receptores ErbB/metabolismo , Proteína ADAM17 , Animales , Separación Celular , Células Madre Embrionarias/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Fibroblastos/metabolismo , Citometría de Flujo , Heterocigoto , Selectina L/metabolismo , Leucocitos/metabolismo , Ligandos , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microglía/metabolismo , Neoplasias/metabolismo , Fenotipo , Fosforilación , Regiones Promotoras Genéticas , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
CSF1R (colony stimulating factor 1 receptor) is the main receptor for CSF1 and has crucial roles in regulating myelopoeisis. CSF1R can be proteolytically released from the cell surface by ADAM17 (A disintegrin and metalloprotease 17). Here, we identified CSF1R as a major substrate of ADAM17 in an unbiased degradomics screen. We explored the impact of CSF1R shedding by ADAM17 and its upstream regulator, inactive rhomboid protein 2 (iRhom2, gene name Rhbdf2), on homeostatic development of mouse myeloid cells. In iRhom2-/- mice, we found constitutive accumulation of membrane-bound CSF1R on myeloid cells at steady state, although cell numbers of these populations were not altered. However, in the context of mixed bone marrow (BM) chimera, under competitive pressure, iRhom2-/- BM progenitor-derived monocytes, tissue macrophages and lung DCs showed a repopulation advantage over those derived from wild-type (WT) BM progenitors, suggesting enhanced CSF1R signaling in the absence of iRhom2. In vitro experiments indicate that iRhom2-/- Lin- SCA-1+ c-Kit+ (LSKs) cells, but not granulocyte-macrophage progenitors (GMPs), had faster growth rates than WT cells in response to CSF1. Our results shed light on an important role of iRhom2/ADAM17 pathway in regulation of CSF1R shedding and repopulation of monocytes, macrophages and DCs.
Asunto(s)
Proteína ADAM17/metabolismo , Células de la Médula Ósea/fisiología , Proteínas Portadoras/metabolismo , Células Progenitoras Mieloides/fisiología , Mielopoyesis , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Proteína ADAM17/genética , Animales , Proteínas Portadoras/genética , Células Cultivadas , Células Dendríticas/fisiología , Femenino , Regulación de la Expresión Génica , Pulmón/patología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Transducción de Señal , Quimera por TrasplanteRESUMEN
Cholinergic neural output has been shown to modulate innate immune responses to infection, injury and ischemia through stimulation of α7 nicotinic acetylcholine receptors (α7nAChR) on mononuclear phagocytes. We tested the hypothesis that cholinergic neurotransmitters, similar to those released through activation of a neural reflex, regulate responses to products of the adaptive immune system, specifically immune complex (IC)-mediated activation of effector cells. In this study, we show that stimulation of α7nAChR on human polymorphonuclear neutrophils (PMNs) and blood mononuclear phagocytes in vitro attenuates C5aR- and FcγR-triggered generation of reactive oxygen species, expression of leukocyte markers involved in cell recruitment and adhesion, and release of TNF-α and other proinflammatory cytokines. We show that this pathway is operative in vivo. Ligation of cholinergic receptors blunts IC-triggered responses in the reverse peritoneal Arthus reaction in mice. The selective 7nAChR agonist GTS21 decreased PMN accumulation and release of cytokines and chemokines at sites of IC deposition. In addition, mice lacking α7nAChR had exaggerated responses to reverse peritoneal Arthus reaction characterized by increased infiltration of PMNs and elevated of levels of TNF-α and CXCL1 in peritoneal fluid compared with wild-type mice. Taken together, these findings suggest that cholinergic output has the potential to exert tonic inhibitory activity that dampens responses to ICs and C5a and thus may be a target to minimize tissue damage in autoimmune diseases.
Asunto(s)
Enfermedades del Complejo Inmune/inmunología , Neutrófilos/inmunología , Fagocitos/inmunología , Receptor Nicotínico de Acetilcolina alfa 7/inmunología , Acetilcolina/farmacología , Animales , Líquido Ascítico/química , Líquido Ascítico/inmunología , Compuestos de Bencilideno/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL1/biosíntesis , Quimiocina CXCL1/genética , Quimiotaxis de Leucocito/efectos de los fármacos , Complemento C5a/genética , Complemento C5a/farmacología , Citocinas/metabolismo , Femenino , Humanos , Quinasa I-kappa B/metabolismo , Inflamación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/efectos de los fármacos , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Fagocitos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Piridinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Receptores de IgG/antagonistas & inhibidores , Proteínas Recombinantes/farmacología , Estallido Respiratorio/efectos de los fármacos , Estallido Respiratorio/fisiología , Organismos Libres de Patógenos Específicos , Receptor Nicotínico de Acetilcolina alfa 7/agonistasRESUMEN
Complement activation modulates DC-mediated T-cell activation, but whether complement affects DC-mediated priming of NK cells is unknown. Here, we demonstrated that conventional DCs (cDCs) from C3(-/-) and C5aR(-/-) mice are hyperresponsive to polyI:C, a TLR3 ligand, leading to enhanced NK-cell activation. We found that cDCs lack C5a receptor (C5aR) and do not respond to C5a directly. Depletion of Gr-1(+) myeloid cells augments polyI:C-induced cDC activation in WT but not in C3(-/-) or C5aR(-/-) mice, indicating that the effect of complement activation on cDCs is indirectly mediated through C5aR-expressing Gr-1(+) myeloid cells. We further demonstrated that the mechanism by which Gr-1(+) myeloid cells regulate the activity of cDCs involves C5a-dependent TGF-ß1 production in Gr-1(+) myeloid cells. C5a enhances and blocking C5aR decreases TGF-ß1 production in cultured bone marrow Gr-1(+) CD11b(+) cells. C5aR deficiency is associated with reduced circulating TGF-ß1 levels, while depleting Gr-1(+) myeloid cells abrogates this difference between WT and C5aR(-/-) mice. Lastly, we showed that enhanced cDC-NK-cell activity in C3(-/-) mice led to delayed melanoma tumor growth. Thus, complement activation indirectly regulates cDC-NK-cell activation in response to inflammatory stimuli such as TLR3 by promoting TGF-ß1 production in Gr-1(+) myeloid cells at steady state.
Asunto(s)
Complemento C5a/inmunología , Células Dendríticas/inmunología , Células Asesinas Naturales/inmunología , Células Mieloides/inmunología , Receptor de Anafilatoxina C5a/inmunología , Factor de Crecimiento Transformador beta1/inmunología , Animales , Complemento C3/deficiencia , Complemento C3/inmunología , Células Dendríticas/citología , Femenino , Citometría de Flujo , Células Asesinas Naturales/citología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Poli I-C/inmunología , Poli I-C/farmacología , Receptores de Quimiocina/inmunologíaRESUMEN
OBJECTIVE: The mechanism by which anti-DNA antibodies mediate lupus nephritis has yet to be conclusively determined. Previously, we found that treatment of mesangial cells with anti-DNA antibodies induced high expression of neutrophil gelatinase-associated lipocalin (NGAL), an iron-binding protein up-regulated in response to kidney injury. We undertook this study to determine whether NGAL is instrumental in the pathogenesis of nephritis, is induced as part of repair, or is irrelevant to damage/repair pathways. METHODS: To investigate the role of NGAL in antibody-mediated nephritis, we induced nephrotoxic nephritis by passive antibody transfer to 129/SyJ and C57BL/6 mice. To determine if NGAL up-regulation is instrumental, we compared the severity of renal damage in NGAL wild-type mice and NGAL-knockout mice following induction of nephrotoxic nephritis. RESULTS: We found that kidney NGAL expression, as well as urine NGAL levels, were significantly increased in mice with nephrotoxic nephritis as compared to control-injected mice. Tight correlations were observed between NGAL expression, renal histopathology, and urine NGAL excretion. NGAL-knockout mice had attenuated proteinuria and improved renal histopathology compared to wild-type mice. Similarly, following nephritis induction, NGAL injection significantly exacerbated nephritis and decreased survival. NGAL induced apoptosis via caspase 3 activation and up-regulated inflammatory gene expression in kidney cells in vitro and when injected in vivo. CONCLUSION: We conclude that kidney binding of pathogenic antibodies stimulates local expression of NGAL, which plays a crucial role in the pathogenesis of nephritis via promotion of inflammation and apoptosis. NGAL blockade may be a novel therapeutic approach for the treatment of nephritis mediated by pathogenic antibodies, including anti-glomerular basement membrane disease and lupus nephritis.
Asunto(s)
Proteínas de Fase Aguda/metabolismo , Lipocalinas/metabolismo , Nefritis/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/farmacología , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Expresión Génica , Silenciador del Gen , Mesangio Glomerular/efectos de los fármacos , Mesangio Glomerular/inmunología , Mesangio Glomerular/metabolismo , Riñón/inmunología , Riñón/metabolismo , Riñón/patología , Lipocalina 2 , Lipocalinas/genética , Lipocalinas/farmacología , Longevidad , Nefritis Lúpica/genética , Nefritis Lúpica/metabolismo , Nefritis Lúpica/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nefritis/genética , Nefritis/patología , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/farmacología , ARN Interferente Pequeño/genética , Regulación hacia ArribaRESUMEN
OBJECTIVE: To construct a humanized mouse model of systemic lupus erythematosus (SLE) that resembles the human disease in order to define the pathophysiology and targets for treatments. METHODS: We infused peripheral blood mononuclear cells (PBMCs) from SLE patients into BALB- RAG-2-/- IL-2Rγ-/- double-knockout (DKO) mice, which lack T cells, B cells, and natural killer cells. PBMCs from 5 SLE patients and 4 normal donors were infused intravenously/intraperitoneally at a density of 3-5×10(6) cells per animal into nonirradiated 4-5-week-old mice. We evaluated the engraftment of human CD45+ cells and monitored the plasma levels of human IgG, anti-double-stranded DNA (anti-dsDNA) antibody, and anticardiolipin antibody (aCL), as well as proteinuria and kidney histology. RESULTS: There was 100% successful engraftment in 40 DKO mice infused with human PBMCs. In the PBMC fraction from SLE PBMC-infused DKO (SLE-DKO) mice and normal donor PBMC-infused DKO (ND-DKO) mice, an average of 41% and 53% human CD45+ cells, respectively, were observed at 4 weeks postengraftment, with 70-90% CD3+ cells. There were fewer CD3+CD4+ cells (mean±SEM 5.5±2.1%) and more CD3+CD8+ cells (79.4±3.6%) in the SLE-DKO mice as in the SLE patients from which the PBMCs were derived. CD19+ B cells and CD11c+ monocytic cells were found in the spleen, lung, liver, and bone marrow. There was no significant difference in plasma levels of human IgG and anti-dsDNA antibodies between SLE-DKO and ND-DKO mice. Levels of aCL were significantly higher in all SLE-DKO mice infused with PBMCs from an SLE patient who had high titers of aCL. SLE-DKO mice had proteinuria, human IgG deposits in the kidneys, and a shorter life span. In SLE-DKO mice engrafted with PBMCs from the aCL-positive patient, we found microthrombi and infiltration of CD3+, CD8+, and CD19+ cells in the glomeruli, recapitulating the human antiphospholipid syndrome in these mice. CONCLUSION: We established a novel humanized SLE-DKO mouse exhibiting many of the immunologic and clinical features of human SLE.
Asunto(s)
Síndrome Antifosfolípido/inmunología , Leucocitos Mononucleares/trasplante , Lupus Eritematoso Sistémico/inmunología , Adulto , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Leucocitos Mononucleares/inmunología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana EdadRESUMEN
Preeclampsia is a major cause of maternal and neonatal morbidity and mortality. In mouse models, complement activation in the placenta is associated with abnormal placental development and miscarriage, and inhibiting complement prevents fetal injury. We mated two mouse strains, DBA/2 and CBA/J, expecting that the pregnancies might show features of preeclampsia and of immunologically mediated pregnancy loss. Along with placental dysfunction, these matings resulted in proteinuria, elevated BUN, fibrin deposition, and glomerular endotheliosis. We blocked placental complement activation throughout pregnancy by administering a single dose of the C3 inhibitor CR2-Crry given on day 5 of the pregnancy. This procedure specifically targets the sites of complement activation without inducing any systemic effects. Placental complement inhibition prevented oxidative stress and placental dysfunction, as well as proteinuria and renal pathologic features of preeclampsia. Thus, local blockade of complement activation at the maternal-fetal interface rescues preeclampsia in mice, and identifies new treatments. Hence, complement triggers a feed-forward cycle of placental damage, antiangiogenic factor production, and maternal vascular damage in patients.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Riñón/efectos de los fármacos , Placenta/efectos de los fármacos , Preeclampsia/tratamiento farmacológico , Proteínas Recombinantes de Fusión/administración & dosificación , Animales , Nitrógeno de la Urea Sanguínea , Modelos Animales de Enfermedad , Femenino , Fibrina/metabolismo , Inyecciones Intravenosas , Riñón/inmunología , Riñón/metabolismo , Riñón/patología , Riñón/fisiopatología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Neovascularización Fisiológica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Placenta/inmunología , Placenta/metabolismo , Placenta/fisiopatología , Preeclampsia/inmunología , Preeclampsia/metabolismo , Preeclampsia/fisiopatología , Embarazo , Proteinuria/inmunología , Proteinuria/prevención & control , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Engineered viral vectors represent a promising strategy to trigger antigen-specific antitumor T cell responses. Arenaviruses have been widely studied because of their ability to elicit potent and protective T cell responses. Here, we provide an overview of a novel intravenously administered, replication-competent, non-lytic arenavirus-based vector technology that delivers tumor antigens to induce antigen-specific anti-cancer T cell responses. Preclinical studies in mice and cell culture experiments with human peripheral blood mononuclear cells demonstrate that arenavirus vectors preferentially infect antigen-presenting cells. This, in conjunction with a non-lytic functional activation of the infected antigen-presenting cells, leads to a robust antigen-specific CD8+ T cell response. T cell migration to, and infiltration of, the tumor microenvironment has been demonstrated in various preclinical tumor models with vectors encoding self- and non-self-antigens. The available data also suggest that arenavirus-based vector therapy can induce immunological memory protecting from tumor rechallenge. Based on promising preclinical data, a phase 1/2 clinical trial was initiated and is currently ongoing to test the activity and safety of arenavirus vectors, HB-201 and HB-202, created using lymphocytic choriomeningitis virus and Pichinde virus, respectively. Both vectors have been engineered to deliver non-oncogenic versions of the human papilloma virus 16 (HPV16) antigens E7 and E6 and will be injected intravenously with or without an initial intratumoral dose. This dose escalation/expansion study is being conducted in patients with recurrent or metastatic HPV16+ cancers. Promising preliminary data from this ongoing clinical study have been reported. Immunogenicity data from several patients demonstrate that a single injection of HB-201 or HB-202 monotherapy is highly immunogenic, as evidenced by an increase in inflammatory cytokines/chemokines and the expansion of antigen-specific CD8+ T cell responses. This response can be further enhanced by alternating injections of HB-202 and HB-201, which has resulted in frequencies of circulating HPV16 E7/E6-specific CD8+ T cells of up to 40% of the total CD8+ T cell compartment in peripheral blood in analyses to date. Treatment with intravenous administration also resulted in a disease control rate of 73% among 11 evaluable patients with head and neck cancer dosed every three weeks, including 2 patients with a partial response.
RESUMEN
Infection with human papillomavirus (HPV) is associated with a variety of cancer types and limited therapy options. Therapeutic cancer vaccines targeting the HPV16 oncoproteins E6 and E7 have recently been extensively explored as a promising immunotherapy approach to drive durable antitumor T cell immunity and induce effective tumor control. With the goal to achieve potent and lasting antitumor T cell responses, we generated a novel lymphocytic choriomeningitis virus (LCMV)-based vaccine, TT1-E7E6, targeting HPV16 E6 and E7. This replication-competent vector was stably attenuated using a three-segmented viral genome packaging strategy. Compared to wild-type LCMV, TT1-E7E6 demonstrated significantly reduced viremia and CNS immunopathology. Intravenous vaccination of mice with TT1-E7E6 induced robust expansion of HPV16-specific CD8+ T cells producing IFN-γ, TNF-α and IL-2. In the HPV16 E6 and E7-expressing TC-1 tumor model, mice immunized with TT1-E7E6 showed significantly delayed tumor growth or complete tumor clearance accompanied with prolonged survival. Tumor control by TT1-E7E6 was also achieved in established large-sized tumors in this model. Furthermore, a combination of TT1-E7E6 with anti-PD-1 therapy led to enhanced antitumor efficacy with complete tumor regression in the majority of tumor-bearing mice that were resistant to anti-PD-1 treatment alone. TT1-E7E6 vector itself did not exhibit oncolytic properties in TC-1 cells, while the antitumor effect was associated with the accumulation of HPV16-specific CD8+ T cells with reduced PD-1 expression in the tumor tissues. Together, our results suggest that TT1-E7E6 is a promising therapeutic vaccine for HPV-positive cancers.
Asunto(s)
Vacunas contra Papillomavirus , Neoplasias del Cuello Uterino , Animales , Linfocitos T CD8-positivos , Femenino , Humanos , Inmunoterapia Activa , Virus de la Coriomeningitis Linfocítica , Ratones , Ratones Endogámicos C57BL , Proteínas E7 de Papillomavirus/genética , Vacunas AtenuadasRESUMEN
The immunoproteasome (i-20S) has emerged as a therapeutic target for autoimmune and inflammatory disorders and hematological malignancies. Inhibition of the chymotryptic ß5i subunit of i-20S inhibits T cell activation, B cell proliferation, and dendritic cell differentiation in vitro and suppresses immune responses in animal models of autoimmune disorders and allograft rejection. However, cytotoxicity to immune cells has accompanied the use of covalently reactive ß5i inhibitors, whose activity against the constitutive proteasome (c-20S) is cumulative with the time of exposure. Herein, we report a structure-activity relationship study of a class of noncovalent proteasome inhibitors with picomolar potencies and 1000-fold selectivity for i-20S over c-20S. Furthermore, these inhibitors are specific for ß5i over the other five active subunits of i-20S and c-20S, providing useful tools to study the functions of ß5i in immune responses. The potency of these compounds in inhibiting human T cell activation suggests that they may have therapeutic potential.
Asunto(s)
Dipéptidos/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Sitios de Unión , Proliferación Celular/efectos de los fármacos , Dipéptidos/metabolismo , Dipéptidos/farmacología , Células HeLa , Humanos , Concentración 50 Inhibidora , Cinética , Activación de Linfocitos/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/metabolismo , Inhibidores de Proteasoma/farmacología , Unión Proteica , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Although anti-DNA antibodies have been decisively linked to the pathogenesis of lupus nephritis, the mechanisms have not been conclusively determined. Recently, we reported that anti-DNA antibodies may contribute to kidney damage by upregulation of proinflammatory genes in mesangial cells (MC), a process involving both Fc receptor-dependent and independent pathways. In investigating the mechanism by which pathogenic anti-DNA antibodies modulate gene expression in MC, we found that the pathogenic anti-DNA antibody 1A3F bound to high mobility group binding protein 1 (HMGB1), an endogenous ligand for TLR2/4 and RAGE (receptor for advanced glycation end products). Interestingly, HMGB1 treatment of MC induced a similar pattern of genes as stimulation with 1A3F. Furthermore, HMGB1 and 1A3F exhibited a synergistic proinflammatory effect in the kidney, where increased expression of HMGB1 was found in lupus patients but not in patients with other types of renal disease. TLR2/Fc and RAGE/Fc inhibited the proinflammatory effects of 1A3F on MC. Finally, we found enhanced susceptibility of lupus prone MRL-lpr/lpr (MRL/lpr) as compared to normal BALB/c derived MC to pathogenic anti-DNA antibody and LPS stimulation (in particular enhanced chemokine synthesis), in addition to significantly increased expression of TLR4. Our results suggest that gene upregulation in MC induced by nephritogenic anti-DNA antibodies is TLR2/4 and RAGE-dependent. Finally, HMGB1 may act as a proinflammatory mediator in antibody-induced kidney damage in systemic lupus erythematosus (SLE).
Asunto(s)
Anticuerpos Antinucleares/inmunología , Proteína HMGB1/inmunología , Lupus Eritematoso Sistémico/inmunología , Nefritis Lúpica/inmunología , Células Mesangiales/inmunología , Animales , Regulación hacia Abajo/inmunología , Femenino , Expresión Génica , Proteína HMGB1/metabolismo , Humanos , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , Nefritis Lúpica/genética , Nefritis Lúpica/patología , Células Mesangiales/metabolismo , Células Mesangiales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos MRL lpr , Proteínas Quinasas Activadas por Mitógenos/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
Lupus nephritis (LN) often results in progressive renal dysfunction. The inactive rhomboid 2 (iRhom2) is a newly identified key regulator of A disintegrin and metalloprotease 17 (ADAM17), whose substrates, such as TNF-α and heparin-binding EGF (HB-EGF), have been implicated in the pathogenesis of chronic kidney diseases. Here, we demonstrate that deficiency of iRhom2 protects the lupus-prone Fcgr2b-/- mice from developing severe kidney damage without altering anti-double-stranded DNA (anti-dsDNA) Ab production by simultaneously blocking HB-EGF/EGFR and TNF-α signaling in the kidney tissues. Unbiased transcriptome profiling of kidneys and kidney macrophages revealed that TNF-α and HB-EGF/EGFR signaling pathways are highly upregulated in Fcgr2b-/- mice, alterations that were diminished in the absence of iRhom2. Pharmacological blockade of either TNF-α or EGFR signaling protected Fcgr2b-/- mice from severe renal damage. Finally, kidneys from LN patients showed increased iRhom2 and HB-EGF expression, with interstitial HB-EGF expression significantly associated with chronicity indices. Our data suggest that activation of iRhom2/ADAM17-dependent TNF-α and EGFR signaling plays a crucial role in mediating irreversible kidney damage in LN, thereby uncovering a target for selective and simultaneous dual inhibition of 2 major pathological pathways in the effector arm of the disease.
Asunto(s)
Proteínas Portadoras/biosíntesis , Receptores ErbB/metabolismo , Riñón/metabolismo , Nefritis Lúpica/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Receptores ErbB/genética , Regulación de la Expresión Génica , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Riñón/patología , Nefritis Lúpica/genética , Nefritis Lúpica/patología , Ratones , Ratones Noqueados , Receptores de IgG/genética , Receptores de IgG/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Photosensitivity, or skin sensitivity to ultraviolet radiation (UVR), is a feature of lupus erythematosus and other autoimmune and dermatologic conditions, but the mechanistic underpinnings are poorly understood. We identify a Langerhans cell (LC)-keratinocyte axis that limits UVR-induced keratinocyte apoptosis and skin injury via keratinocyte epidermal growth factor receptor (EGFR) stimulation. We show that the absence of LCs in Langerin-diphtheria toxin subunit A (DTA) mice leads to photosensitivity and that, in vitro, mouse and human LCs can directly protect keratinocytes from UVR-induced apoptosis. LCs express EGFR ligands and a disintegrin and metalloprotease 17 (ADAM17), the metalloprotease that activates EGFR ligands. Deletion of ADAM17 from LCs leads to photosensitivity, and UVR induces LC ADAM17 activation and generation of soluble active EGFR ligands, suggesting that LCs protect by providing activated EGFR ligands to keratinocytes. Photosensitive systemic lupus erythematosus (SLE) models and human SLE skin show reduced epidermal EGFR phosphorylation and LC defects, and a topical EGFR ligand reduces photosensitivity. Together, our data establish a direct tissue-protective function for LCs, reveal a mechanistic basis for photosensitivity, and suggest EGFR stimulation as a treatment for photosensitivity in lupus erythematosus and potentially other autoimmune and dermatologic conditions.
Asunto(s)
Citoprotección/efectos de la radiación , Queratinocitos/citología , Queratinocitos/efectos de la radiación , Células de Langerhans/citología , Células de Langerhans/efectos de la radiación , Rayos Ultravioleta , Proteína ADAM17/metabolismo , Animales , Apoptosis/efectos de la radiación , Modelos Animales de Enfermedad , Epidermis/metabolismo , Epidermis/efectos de la radiación , Receptores ErbB/metabolismo , Humanos , Ligandos , Lupus Eritematoso Sistémico/patología , Ratones Endogámicos C57BL , Fosforilación/efectos de la radiaciónRESUMEN
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a complex pathogenesis involving multiple genetic and environmental contributions. DNA microarray technology has recently been applied to unravel some of this complexity through genomewide profiling. Early studies using microarray analysis of peripheral blood mononuclear cells (PBMCs) from SLE patients revealed dysregulation of inflammatory cytokines, chemokines, and immune response-related genes, as well as genes involved in apoptosis, signal transduction, and the cell cycle. More recently, using arrays containing many more genes, 4 independent research groups have found that interferon (IFN)-regulated genes are highly overexpressed in the peripheral blood and kidney glomeruli of SLE patients, supporting a crucial role for interferon in SLE. Future studies focusing on target tissues or organs in lupus, including the kidney, may further contribute to our understanding of lupus pathogenesis while providing new targets for therapy.
Asunto(s)
Perfilación de la Expresión Génica , Expresión Génica/fisiología , Lupus Eritematoso Sistémico/metabolismo , Animales , Humanos , Interferones/metabolismo , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/etiología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
iRHOM2, encoded by the gene Rhbdf2, regulates the maturation of the TNF-α convertase (TACE), which controls shedding of TNF-α and its biological activity in vivo. TACE is a potential target to treat TNF-α-dependent diseases, such as rheumatoid arthritis, but there are concerns about potential side effects, because TACE also protects the skin and intestinal barrier by activating EGFR signaling. Here we report that inactivation of Rhbdf2 allows tissue-specific regulation of TACE by selectively preventing its maturation in immune cells, without affecting its homeostatic functions in other tissues. The related iRHOM1, which is widely expressed, except in hematopoietic cells, supported TACE maturation and shedding of the EGFR ligand TGF-α in Rhbdf2-deficient cells. Remarkably, mice lacking Rhbdf2 were protected from K/BxN inflammatory arthritis to the same extent as mice lacking TACE in myeloid cells or Tnfa-deficient mice. In probing the underlying mechanism, we found that two main drivers of K/BxN arthritis, complement C5a and immune complexes, stimulated iRHOM2/TACE-dependent shedding of TNF-α in mouse and human cells. These data demonstrate that iRHOM2 and myeloid-expressed TACE play a critical role in inflammatory arthritis and indicate that iRHOM2 is a potential therapeutic target for selective inactivation of TACE in myeloid cells.
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Artritis Experimental/etiología , Proteínas Portadoras/fisiología , Proteínas ADAM/deficiencia , Proteínas ADAM/genética , Proteínas ADAM/fisiología , Proteína ADAM17 , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Artritis Experimental/inmunología , Artritis Experimental/fisiopatología , Artritis Reumatoide/etiología , Artritis Reumatoide/inmunología , Artritis Reumatoide/fisiopatología , Proteínas Portadoras/genética , Receptores ErbB/fisiología , Humanos , Proteínas con Dominio LIM/fisiología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
OBJECTIVE: Pathogenic monoclonal anti-double-stranded DNA (anti-dsDNA) antibodies up-regulate the expression of lipocalin-2 in glomerular mesangial cells. This study was undertaken to investigate whether polyclonal anti-dsDNA antibodies promote the local secretion of lipocalin-2 in the kidneys of patients with systemic lupus erythematosus (SLE), and whether urinary lipocalin-2 represents a marker of kidney involvement in SLE. METHODS: Hispanic, African American, and white patients with SLE and normal healthy control subjects from affiliated hospitals of the Albert Einstein College of Medicine were recruited for this cross-sectional study. Patients were classified based on the presence of active renal disease according to the SLE Disease Activity Index (SLEDAI). Correlations of clinical and laboratory data with urinary and serum levels of lipocalin-2 were assessed. RESULTS: Among SLE patients, urinary lipocalin-2 levels were significantly higher in those with lupus nephritis (LN) (median 17.1 ng/mg creatinine, interquartile range [IQR] 10.3-45.4; n = 32) than in those without LN (median 11.2 ng/mg creatinine, IQR 3.1-20.3; n = 38) (P = 0.023). Compared with the values in normal controls (median 4 ng/ml, IQR 0-11.1; n = 14), urinary levels of lipocalin-2 in SLE patients were significantly higher (non-normalized median 19.3 ng/ml, IQR 8-34.2) (P = 0.004). The presence of lipocalin-2 in the urine of patients with LN correlated significantly with the renal SLEDAI score (r = 0.452, P = 0.009), but not with extrarenal disease activity. CONCLUSION: The high prevalence of LN in SLE patients and the prognostic significance of kidney disease support the need for identifying early biomarkers to assess the risk of nephritis development and for following up patients with established disease. These findings indicate that urinary lipocalin-2 is a potential marker of the presence and severity of renal involvement in adult patients with SLE.
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Proteínas de Fase Aguda/orina , Lupus Eritematoso Sistémico/orina , Nefritis Lúpica/orina , Proteínas Proto-Oncogénicas/orina , Adulto , Negro o Afroamericano/etnología , Factores de Edad , Biomarcadores/sangre , Biomarcadores/orina , Estudios de Casos y Controles , Estudios Transversales , Femenino , Hispánicos o Latinos/etnología , Humanos , Riñón/fisiopatología , Lipocalina 2 , Lipocalinas , Lupus Eritematoso Sistémico/etnología , Lupus Eritematoso Sistémico/fisiopatología , Nefritis Lúpica/etnología , Nefritis Lúpica/fisiopatología , Masculino , Pronóstico , Proteínas Proto-Oncogénicas/sangre , Factores de Riesgo , Índice de Severidad de la Enfermedad , Factores Sexuales , Población Blanca/etnologíaRESUMEN
OBJECTIVE: Lupus-associated IgG anti-double-stranded DNA antibodies are thought to be pathogenic in the kidney due to cross-reaction with glomerular antigens, leading subsequently to immune complex formation in situ and complement activation. We undertook this study to determine if pathogenic anti-DNA antibodies may also contribute to renal damage by directly influencing mesangial gene expression. METHODS: Complementary DNA microarray gene profiling was performed in primary mesangial cells (derived from lupus-prone MRL/lpr mice) treated with pathogenic, noncomplexed anti-DNA antibodies. Significant gene up-regulation induced by anti-DNA antibodies as determined by microarray analysis was further investigated by real-time polymerase chain reaction and methods to detect the relevant proteins. Induction of proinflammatory genes by pathogenic antibodies was confirmed by comparing gene expression in glomeruli of old versus young MRL/lpr mice, and by antibody injection in vivo. RESULTS: Pathogenic, but not nonpathogenic, antibodies significantly induced a number of transcripts, including CXCL1/KC, LCN2, iNOS, CX3CL1/fractalkine, SERPINA3G, and IkappaBalpha ("marker genes"). Blocking of Fcgamma receptors or using Fcgamma chain-knockout mesangial cells had no effect on the gene regulation effect of the pathogenic antibody R4A, indicating a non-Fc-dependent mechanism. The glomerular expression of these marker genes increased over time with the development of glomerular antibody deposition and active nephritis in MRL/lpr mice. Moreover, injection of R4A into SCID mice in vivo significantly up-regulated glomerular marker gene expression. CONCLUSION: These findings indicate that the renal pathogenicity of anti-DNA antibodies may be attributed in part to their ability to directly modulate gene expression in kidney mesangial cells through both Fc-dependent and non-Fc-dependent mechanisms.
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Anticuerpos Antinucleares/efectos adversos , Anticuerpos Antinucleares/farmacología , Vasculitis por Lupus del Sistema Nervioso Central/genética , Células Mesangiales/metabolismo , Ratones Endogámicos MRL lpr/genética , Regulación hacia Arriba/efectos de los fármacos , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Animales , Células Cultivadas , Quimiocina CX3CL1 , Quimiocina CXCL1 , Quimiocinas CX3C/genética , Quimiocinas CX3C/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Femenino , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Enfermedades Renales/inducido químicamente , Enfermedades Renales/fisiopatología , Lipocalina 2 , Lipocalinas , Vasculitis por Lupus del Sistema Nervioso Central/metabolismo , Vasculitis por Lupus del Sistema Nervioso Central/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Células Mesangiales/efectos de los fármacos , Células Mesangiales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , Inhibidor NF-kappaB alfa , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serpinas/genética , Serpinas/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
Anti-DNA Abs commonly found in patients with systemic lupus erythematosus are thought to play an important pathogenic role in lupus nephritis. Anti-DNA Abs may contribute to renal disease by cross-reactivity with renal Ags, the identity of which remain elusive. To identify a target Ag for pathogenic anti-DNA Abs, we performed Western blotting and immunoprecipitations of mesangial cell lysates from the lupus-prone MRL-lpr/lpr mouse and a nonautoimmune BALB/c mouse with the pathogenic anti-DNA Ab R4A. We found that R4A (but not a nonpathogenic Ab mutant of R4A) binds to and immunoprecipitates a 100-kDa protein expressed on the cell surface and in lysates of MRL-lpr/lpr mesangial cells. DNase treatment of the lysate and of the R4A Ab did not effect binding, indicating that the binding of R4A to the 100-kDa protein was direct and not mediated by an antigenic bridge containing DNA. Binding was greatly diminished in BALB/c lysates, suggesting that Ag expression or availability at the level of the target organ may be a factor in determining susceptibility to lupus nephritis. Following identification of this 100-kDa protein as nonmuscle alpha-actinin, binding of R4A to alpha-actinin was confirmed by Western blot, ELISA, inhibition studies, and immunofluorescence. High titers of anti-alpha-actinin Abs were present in sera and kidney eluates of lupus mice with active nephritis. These results indicate that the nephritogenicity of some anti-DNA Abs may be mediated via cross-reactivity with alpha-actinin. Furthermore, variations in target Ag display between individuals may underlie differential susceptibility to anti-DNA Ab-induced renal disease.