RESUMO
The NFκB transcription factor family facilitates the activation of dendritic cells (DCs) and CD4(+) T helper (Th) cells, which are important for protective adaptive immunity. Inappropriate activation of these immune cells may cause inflammatory disease, and NFκB inhibitors are promising anti-inflammatory drug candidates. Here, we investigated whether inhibiting the NFκB-inducing kinase IKK2 can attenuate crescentic GN, a severe DC- and Th cell-dependent kidney inflammatory disease. Prophylactic pharmacologic IKK2 inhibition reduced DC and Th cell activation and ameliorated nephrotoxic serum-induced GN in mice. However, therapeutic IKK2 inhibition during ongoing disease aggravated the nephritogenic immune response and disease symptoms. This effect resulted from the renal loss of regulatory T cells, which have been shown to protect against crescentic GN and which require IKK2. In conclusion, although IKK2 inhibition can suppress the induction of nephritogenic immune responses in vivo, it may aggravate such responses in clinically relevant situations, because it also impairs regulatory T cells and thereby, unleashes preexisting nephritogenic responses. Our findings argue against using IKK2 inhibitors in chronic GN and perhaps, other immune-mediated diseases.
Assuntos
Glomerulonefrite/induzido quimicamente , Glomerulonefrite/prevenção & controle , Oxazinas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Animais , Progressão da Doença , Masculino , Camundongos , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Índice de Gravidade de Doença , Quinase Induzida por NF-kappaBRESUMO
Kidney dendritic cells (DCs) regulate nephritogenic T cell responses. Most kidney DCs belong to the CD11b+ subset and promote crescentic GN (cGN). The function of the CD103+ subset, which represents <5% of kidney DCs, is poorly understood. We studied the role of CD103+ DCs in cGN using several lines of genetically modified mice that allowed us to reduce the number of these cells. In all lines, we detected a reduction of FoxP3+ intrarenal regulatory T cells (Tregs), which protect against cGN. Mice lacking the transcription factor Batf3 had a more profound reduction of CD103+ DCs and Tregs than did the other lines used, and showed the most profound aggravation of cGN. The conditional reduction of CD103+ DC numbers by 50% in Langerin-DTR mice halved Treg numbers, which did not suffice to significantly aggravate cGN. Mice lacking the cytokine Flt3L had fewer CD103+ DCs and Tregs than Langerin-DTR mice but exhibited milder cGN than did Batf3-/- mice presumably because proinflammatory CD11b+ DCs were somewhat depleted as well. Conversely, Flt3L supplementation increased the number of CD103+ DCs and Tregs, but also of proinflammatory CD11b+ DCs. On antibody-mediated removal of CD11b+ DCs, Flt3L supplementation ameliorated cGN. Mechanistically, CD103+ DCs caused cocultured T cells to differentiate into Tregs and produced the chemokine CCL20, which is known to attract Tregs into the kidney. Our findings show that CD103+ DCs foster intrarenal FoxP3+ Treg accumulation, thereby antagonizing proinflammatory CD11b+ DCs. Thus, increasing CD103+ DC numbers or functionality might be advantageous in cGN.
Assuntos
Antígenos CD/imunologia , Células Dendríticas/imunologia , Glomerulonefrite/imunologia , Cadeias alfa de Integrinas/imunologia , Interleucina-10/imunologia , Rim/citologia , Linfócitos T Reguladores/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Pathologic proliferation of mesangial and parietal epithelial cells (PECs) is a hallmark of various glomerulonephritides. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that mediates inflammation by engagement of a receptor complex involving the components CD74, CD44, CXCR2, and CXCR4. The proliferative effects of MIF may involve CD74 together with the coreceptor and PEC activation marker CD44. Herein, we analyzed the effects of local glomerular MIF/CD74/CD44 signaling in proliferative glomerulonephritides. MIF, CD74, and CD44 were upregulated in the glomeruli of patients and mice with proliferative glomerulonephritides. During disease, CD74 and CD44 were expressed de novo in PECs and colocalized in both PECs and mesangial cells. Stress stimuli induced MIF secretion from glomerular cells in vitro and in vivo, in particular from podocytes, and MIF stimulation induced proliferation of PECs and mesangial cells via CD74. In murine crescentic GN, Mif-deficient mice were almost completely protected from glomerular injury, the development of cellular crescents, and the activation and proliferation of PECs and mesangial cells, whereas wild-type mice were not. Bone marrow reconstitution studies showed that deficiency of both nonmyeloid and bone marrow-derived Mif reduced glomerular cell proliferation and injury. In contrast to wild-type mice, Cd74-deficient mice also were protected from glomerular injury and ensuing activation and proliferation of PECs and mesangial cells. Our data suggest a novel molecular mechanism and glomerular cell crosstalk by which local upregulation of MIF and its receptor complex CD74/CD44 mediate glomerular injury and pathologic proliferation in GN.
Assuntos
Antígenos de Diferenciação de Linfócitos B/fisiologia , Glomerulonefrite/etiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Animais , Proliferação de Células , Células Cultivadas , Feminino , Glomerulonefrite/patologia , Glomérulos Renais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Transgenic mice expressing the diphtheria toxin receptor (DTR) in specific cell types are key tools for functional studies in several biological systems. B6.FVB-Tg(Itgax-DTR/EGFP)57Lan/J (CD11c.DTR) and B6.Cg-Tg(Itgax-DTR/OVA/EGFP)1Gjh/Crl (CD11c.DOG) mice express the DTR in CD11c(+) cells, allowing conditional depletion of dendritic cells. We report that dendritic-cell depletion in these models caused polymorphonuclear neutrophil (PMN) release from the bone marrow, which caused chemokine-dependent neutrophilia after 6-24 h and increased bacterial clearance in a mouse pyelonephritis model. We present a transgenic mouse line, B6.Cg-Tg(Itgax-EGFP-CRE-DTR-LUC)2Gjh/Crl (CD11c.LuciDTR), which is unaffected by early neutrophilia. However, CD11c.LuciDTR and CD11c.DTR mice showed late neutrophilia 72 h after dendritic cell depletion, which was independent of PMN release and possibly resulted from increased granulopoiesis. Thus, the time point of dendritic cell depletion and the choice of DTR transgenic mouse line must be considered in experimental settings where neutrophils may be involved.
Assuntos
Antígeno CD11c/imunologia , Neutrófilos/imunologia , Animais , Antígeno CD11c/genética , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Células Dendríticas/patologia , Toxina Diftérica/farmacologia , Modelos Animais de Doenças , Feminino , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Imunológicos , Neutrófilos/citologia , Pielonefrite/imunologia , Pielonefrite/microbiologia , Pielonefrite/patologia , Escherichia coli Uropatogênica/imunologia , Escherichia coli Uropatogênica/fisiologiaRESUMO
In vitro studies identified Y-box-binding protein (YB)-1 as a key regulator of inflammatory mediators. In this study, we observed increased levels of secreted YB-1 in sera from sepsis patients. This led us to investigate the in vivo role of YB-1 in murine models of acute peritonitis following LPS injection, in sterile renal inflammation following unilateral ureteral obstruction, and in experimental pyelonephritis. LPS injection enhanced de novo secretion of YB-1 into the urine and the peritoneal fluid of LPS-treated mice. Furthermore, we could demonstrate a significant, transient upregulation and posttranslational modification (phosphorylation at serine 102) of YB-1 in renal and inflammatory cells. Increased renal cytoplasmic YB-1 amounts conferred enhanced expression of proinflammatory chemokines CCL2 and CCL5. Along these lines, heterozygous YB-1 knockout mice (YB-1(+/d)) that display 50% reduced YB-1 levels developed significantly lower responses to both LPS and sterile inflammation induced by unilateral ureteral obstruction. This included diminished immune cell numbers due to impaired migration propensities and reduced chemokine expression. YB-1(+/d) mice were protected from LPS-associated mortality (20% mortality on day 3 versus 80% in wild-type controls); however, immunosuppression in YB-1(+/d) animals resulted in 50% mortality. In conclusion, our findings identify YB-1 as a major, nonredundant mediator in both systemic and local inflammatory responses.
Assuntos
Inflamação/imunologia , Sepse/imunologia , Fatores de Transcrição/imunologia , Animais , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Imunoprecipitação , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nefrite/imunologia , Nefrite/metabolismo , Peritonite/imunologia , Peritonite/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The Th17 immune response appears to contribute to the pathogenesis of human and experimental crescentic GN, but the cell types that produce IL-17A in the kidney, the mechanisms involved in its induction, and the IL-17A-mediated effector functions that promote renal tissue injury are incompletely understood. Here, using a murine model of crescentic GN, we found that CD4(+) T cells, γδ T cells, and a population of CD3(+)CD4(-)CD8(-)γδT cell receptor(-)NK1.1(-) T cells all produce IL-17A in the kidney. A time course analysis identified γδ T cells as a major source of IL-17A in the early phase of disease, before the first CD4(+) Th17 cells arrived. The production of IL-17A by renal γδ T cells depended on IL-23p19 signaling and retinoic acid-related orphan receptor-γt but not on IL-1ß or IL-6. In addition, depletion of dendritic cells, which produce IL-23 in the kidney, reduced IL-17A production by renal γδ T cells. Furthermore, the lack of IL-17A production in γδ T cells, as well as the absence of all γδ T cells, reduced neutrophil recruitment into the kidney and ameliorated renal injury. Taken together, these data suggest that γδ T cells produce IL-17A in the kidney, induced by IL-23, promoting neutrophil recruitment, and contributing to the immunopathogenesis of crescentic GN.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Glomerulonefrite/metabolismo , Interleucina-17/metabolismo , Rim/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Animais , Linfócitos T CD4-Positivos/patologia , Modelos Animais de Doenças , Glomerulonefrite/patologia , Interleucina-23/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologia , Células Th17/patologia , Fatores de TempoRESUMO
Immature renal dendritic cells (DCs) are protective early in murine crescentic GN, but the mechanisms underlying this protection are unknown. Here, depletion of DCs reduced the recruitment of invariant natural killer T (iNKT) cells, which attenuate GN, into the kidney in the early stage of experimental crescentic GN. More than 90% of renal iNKT cells expressed the chemokine receptor CXCR6, and renal DCs produced high amounts of the cognate ligand CXCL16 early after induction of nephritis, suggesting that renal DC-derived CXCL16 might attract protective CXCR6(+) iNKT cells. Consistent with this finding, CXCR6-deficient mice exhibited less iNKT cell recruitment and developed nephritis that was more severe, similar to the aggravated nephritis observed in mice depleted of immature DCs. Finally, adoptive transfer of CXCR6-competent NKT cells ameliorated nephritis. Taken together, these results suggest an immunoprotective mechanism involving immature DCs, CXCL16, CXCR6, and regulatory iNKT cells, which might stimulate the development of new therapeutic strategies for GN.
Assuntos
Quimiocina CXCL6/metabolismo , Células Dendríticas/fisiologia , Glomerulonefrite/imunologia , Leucócitos Mononucleares/fisiologia , Receptores CXCR/metabolismo , Animais , Quimiocina CXCL16 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR6 , OvinosRESUMO
Dendritic cells (DCs) are the most abundant immune cells in the kidney and form an intricate network in the tubulointerstitium, suggesting that they may play an important role in interstitial infections such as pyelonephritis. Here, we optimized a murine pyelonephritis model by instilling uropathogenic Escherichia coli two times at a 3-hour interval, which produced an infection rate of 84%. By 3 hours after the second instillation, resident kidney DCs began secreting the chemokine CXCL2, which recruits neutrophilic granulocytes. During the time studied, DCs remained responsible for most of the CXCL2 production. Neutrophils began infiltrating the kidney 3 hours after the second instillation and phagocytozed bacteria. Macrophages followed 3 hours later and contributed much less to both CXCL2 production and bacterial phagocytosis. To investigate whether DCs recruit neutrophils into the kidney for antibacterial defense, we used CD11c.DTR mice allowing conditional depletion of CD11c(+) dendritic cells. The absence of CD11c(+) DCs markedly delayed neutrophil recruitment and bacterial clearance. In conclusion, these findings suggest that the tubulointerstitial dendritic cell network serves an innate immune sentinel function against bacterial pyelonephritis.
Assuntos
Células Dendríticas/citologia , Rim/patologia , Pielonefrite/imunologia , Pielonefrite/microbiologia , Animais , Antígeno CD11c/biossíntese , Células Dendríticas/imunologia , Escherichia coli/metabolismo , Humanos , Imunidade Inata , Incidência , Rim/citologia , Rim/microbiologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fagócitos/citologia , Sistema Urinário/patologia , Fatores de VirulênciaRESUMO
CCR2 is thought to recruit monocytes to sites of infection. Two subpopulations of murine blood monocytes differing in Gr1 and CCR2 expression have been described. The exact role of CCR2 in migration of CCR2(low)Gr1(low) and CCR2(high)Gr1(high) monocytes into nonlymphoid tissue is controversial. In this study, we have addressed this question in a murine model of bacterial urinary tract infection. Only Gr1(high) monocytes were recruited into the infected bladder. CCR2 deficiency reduced their frequency in this organ, indicating a requirement of this chemokine receptor. Importantly, CCR2-deficient mice also showed reduced Gr1(high) monocyte numbers in the blood, but not in the bone marrow (BM), indicating that CCR2 acted at the step of monocyte release into the circulation. The same was found also in noninfected mice, indicating a further involvement of CCR2 in steady-state BM egress. An additional requirement of CCR2 in monocyte recruitment from the blood into the bladder was excluded by tracking particle-labeled endogenous monocytes and by adoptive transfer of BM-derived monocyte subsets. These findings demonstrate that CCR2 governs homeostatic and infection-triggered release of Gr1(high) monocytes from the BM into the blood but is dispensable for recruitment into a nonlymphoid tissue.
Assuntos
Antígenos Ly/imunologia , Infecções Bacterianas/imunologia , Regulação da Expressão Gênica/imunologia , Homeostase/imunologia , Monócitos/imunologia , Receptores CCR2/imunologia , Bexiga Urinária/imunologia , Infecções Urinárias/imunologia , Animais , Antígenos Ly/genética , Infecções Bacterianas/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/genética , Homeostase/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Masculino , Camundongos , Camundongos Knockout , Receptores CCR2/genética , Bexiga Urinária/microbiologia , Infecções Urinárias/genéticaRESUMO
This unit describes a simple way to induce pyelonephritis in female mice using uropathogenic Escherichia coli (UPEC). Methods for culturing and preparing working stocks of UPEC are provided. Protocols to measure the bacterial load in the murine kidney following the establishment of pyelonephritis by determining the bacterial colony forming units (CFU) are also included. This assay can be performed with kidney homogenates if the bacterial load is the sole read-out or if cellular intactness is not required for subsequent assays, or from kidney single cell suspensions prepared by enzymatic digestion. A support protocol describes methods for isolating cells for flow cytometry, ex vivo culture, and other assays.
Assuntos
Modelos Animais de Doenças , Infecções por Escherichia coli/patologia , Rim/patologia , Pielonefrite/patologia , Animais , Contagem de Colônia Microbiana , Infecções por Escherichia coli/microbiologia , Feminino , Humanos , Rim/química , Rim/microbiologia , Camundongos , Cultura Primária de Células , Pielonefrite/microbiologia , Análise de Célula Única , Escherichia coli Uropatogênica/fisiologiaRESUMO
DCs and macrophages both express the chemokine receptor CX3CR1. Here we demonstrate that its ligand, CX3CL1, is highly expressed in the murine kidney and intestine. CX3CR1 deficiency markedly reduced DC numbers in the healthy and inflamed kidney cortex, and to a lesser degree in the kidney medulla and intestine, but not in other organs. CX3CR1 also promoted influx of DC precursors in crescentic glomerulonephritis, a DC-dependent aggressive type of nephritis. Disease severity was strongly attenuated in CX3CR1-deficient mice. Primarily CX3CR1-dependent DCs in the kidney cortex processed antigen for the intrarenal stimulation of T helper cells, a function important for glomerulonephritis progression. In contrast, medullary DCs played a specialized role in inducing innate immunity against bacterial pyelonephritis by recruiting neutrophils through rapid chemokine production. CX3CR1 deficiency had little effect on the immune defense against pyelonephritis, as medullary DCs were less CX3CR1 dependent than cortical DCs and because recruited neutrophils produced chemokines to compensate for the DC paucity. These findings demonstrate that cortical and medullary DCs play specialized roles in their respective kidney compartments. We identify CX3CR1 as a potential therapeutic target in glomerulonephritis that may involve fewer adverse side effects, such as impaired anti-infectious defense or compromised DC functions in other organs.
Assuntos
Células Dendríticas/fisiologia , Glomerulonefrite/patologia , Receptores de Quimiocinas/metabolismo , Animais , Apresentação de Antígeno , Receptor 1 de Quimiocina CX3C , Células Cultivadas , Progressão da Doença , Feminino , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunidade Inata , Rim/imunologia , Rim/metabolismo , Rim/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pielonefrite/imunologia , Pielonefrite/metabolismo , Pielonefrite/microbiologia , Receptores de Quimiocinas/genética , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
PURPOSE: To investigate potential inhibitory effects of three polyphenolic agents, epigallocatechin gallate (EGCG; from green tea), resveratrol (from red wine), and curcumin (from turmeric), on the proliferation of human retinal pigment epithelial (RPE) cells and to elucidate unwanted effects. METHODS: ARPE19 cells and primary human RPE cells were cultured in the presence of various concentrations of EGCG, resveratrol, or curcumin, and compared with controls. The number of viable cells was determined after 24, 48, and 72 hr by flow cytometrical enumeration. Furthermore, cell division was measured by dye dilution assay using carboxyfluorescein succinimidyl ester (CFSE), cell death by Hoechst 33258 staining, and apoptosis by staining for active caspase 3/7 and 8. RESULTS: The three drugs inhibited the increase of RPE cell numbers at all time points, with resveratrol being the most efficient and curcumin being the least efficient. EGCG inhibited cell proliferation with intermediate efficiency, and showed little induction of cell death. Resveratrol almost completely suppressed cell proliferation, and induced RPE cell necrosis and caspase 3/7- and caspase 8-dependent apoptosis. Curcumin inhibited RPE cell increase exclusively by inducing caspase 3/7-dependent but caspase 8-independent cell death and necrosis. CONCLUSIONS: All three polyphenols tested reduced the absolute number of cells, but had different effects on cell proliferation, apoptosis, and necrosis. Resveratrol was most potent and EGCG induced the least cell death. These polyphenols may aid treatment of proliferative vitreoretinopathy (PVR).