RESUMO
Susceptibility and protection against human autoimmune diseases, including type I diabetes, multiple sclerosis, and Goodpasture disease, is associated with particular human leukocyte antigen (HLA) alleles. However, the mechanisms underpinning such HLA-mediated effects on self-tolerance remain unclear. Here we investigate the molecular mechanism of Goodpasture disease, an HLA-linked autoimmune renal disorder characterized by an immunodominant CD4+ T-cell self-epitope derived from the α3 chain of type IV collagen (α3135-145). While HLA-DR15 confers a markedly increased disease risk, the protective HLA-DR1 allele is dominantly protective in trans with HLA-DR15 (ref. 2). We show that autoreactive α3135-145-specific T cells expand in patients with Goodpasture disease and, in α3135-145-immunized HLA-DR15 transgenic mice, α3135-145-specific T cells infiltrate the kidney and mice develop Goodpasture disease. HLA-DR15 and HLA-DR1 exhibit distinct peptide repertoires and binding preferences and present the α3135-145 epitope in different binding registers. HLA-DR15-α3135-145 tetramer+ T cells in HLA-DR15 transgenic mice exhibit a conventional T-cell phenotype (Tconv) that secretes pro-inflammatory cytokines. In contrast, HLA-DR1-α3135-145 tetramer+ T cells in HLA-DR1 and HLA-DR15/DR1 transgenic mice are predominantly CD4+Foxp3+ regulatory T cells (Treg cells) expressing tolerogenic cytokines. HLA-DR1-induced Treg cells confer resistance to disease in HLA-DR15/DR1 transgenic mice. HLA-DR15+ and HLA-DR1+ healthy human donors display altered α3135-145-specific T-cell antigen receptor usage, HLA-DR15-α3135-145 tetramer+ Foxp3- Tconv and HLA-DR1-α3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes. Moreover, patients with Goodpasture disease display a clonally expanded α3135-145-specific CD4+ T-cell repertoire. Accordingly, we provide a mechanistic basis for the dominantly protective effect of HLA in autoimmune disease, whereby HLA polymorphism shapes the relative abundance of self-epitope specific Treg cells that leads to protection or causation of autoimmunity.
Assuntos
Doença Antimembrana Basal Glomerular/imunologia , Autoimunidade/imunologia , Linfócitos T Reguladores/imunologia , Animais , Doença Antimembrana Basal Glomerular/patologia , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Colágeno Tipo IV/química , Colágeno Tipo IV/imunologia , Citocinas/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Subtipos Sorológicos de HLA-DR/imunologia , Antígeno HLA-DR1/imunologia , Humanos , Epitopos Imunodominantes , Masculino , Camundongos , Camundongos Transgênicos , Modelos MolecularesRESUMO
ANCA-associated vasculitis (AAV) is intricately linked with infections. Toll-like receptors (TLR) provide a potential link between infection and anti-myeloperoxidase (MPO) autoimmunity. TLR9 ligation has been shown to promote anti-MPO autoimmunity and glomerular vasculitis in murine MPO-AAV. This study investigates dendritic cell TLR9 ligation in murine experimental anti-MPO glomerulonephritis. We analyzed autoimmune responses to MPO following transfer of TLR9 stimulated, MPO pulsed dendritic cells and kidney injury following a sub-nephritogenic dose of sheep anti-mouse glomerular basement membrane globulin. TLR9 ligation enhanced dendritic cell activation upregulating CD40 and CD80 expression, promoting systemic anti-MPO autoimmunity and T cell recall responses and exacerbating kidney injury. CD40 upregulation by TLR9 was critical for the induction of nephritogenic autoimmunity. The presence of DEC205, which transports the TLR9 ligand to TLR9 located in the endosome, also promoted kidney injury. This confirms TLR9 mediated dendritic cell activation as a mechanism of anti-MPO autoimmunity in AAV and further defines the link between infection and the generation of MPO specific autoimmune inflammation.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Glomerulonefrite , Receptor Toll-Like 9 , Animais , Camundongos , Autoimunidade , Células Dendríticas , Glomerulonefrite/metabolismo , Peroxidase/metabolismo , Ovinos , Receptor Toll-Like 9/metabolismoRESUMO
OBJECTIVES: ANCA-associated vasculitis (AAV) is an autoimmune disease characterized by small blood vessel inflammation, commonly affecting the kidneys and respiratory tract. It is unclear why the incidence of this condition increases with age. Previous studies in a passive antibody transfer system in aged mice have implicated innate effectors. To test the hypothesis that autoimmunity to myeloperoxidase (MPO), an autoantigen responsible for AAV, increases with age, anti-MPO autoimmunity was studied in murine models of active autoimmunity and disease induced by cellular immunity. METHODS: Young (8 weeks) and aged (either 15 or 22 months) mice were immunized with whole proteins or peptides from ovalbumin, as a model foreign antigen, or MPO protein or peptides. Mice were subjected to a model of active anti-MPO glomerulonephritis. Cellular and humoral immune responses, and tissue inflammation were assessed. RESULTS: While cellular immunity to ovalbumin was diminished in aged mice, cellular autoimmunity to MPO and its immunodominant CD4+ and CD8+ T cell epitopes was increased after immunization with either MPO peptides or whole MPO protein, assessed by peptide and antigen-specific production of the pro-inflammatory cytokines IFN-γ and IL-17A. MPO-ANCA titres were not increased in aged mice compared with young mice. In experimental anti-MPO glomerulonephritis, cell-mediated injury was increased, likely due to CD4+ and CD8+ T cells, innate immunity and the increased vulnerability of aged kidneys. CONCLUSION: Heightened cellular immunity to MPO develops with ageing in mice and may contribute to the increased incidence and severity of AAV in older people.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Glomerulonefrite , Idoso , Envelhecimento , Animais , Anticorpos Anticitoplasma de Neutrófilos , Linfócitos T CD4-Positivos , Feminino , Humanos , Imunidade Celular , Inflamação/metabolismo , Masculino , Camundongos , Ovalbumina/metabolismo , PeroxidaseRESUMO
BACKGROUND: Myeloperoxidase ANCA-associated vasculitis is a major cause of ESKD. Efficacy of anti-CD20 mAb treatment was tested in a mouse model of the disease. METHODS: MPO immunization induced anti-MPO autoimmunity, and a subnephritogenic dose of sheep anti-mouse GBM globulin triggered GN. RESULTS: Anti-CD20 mAb treatment increased the numbers and immunomodulatory capacity of MPO-specific T regulatory cells (Tregs) and attenuated T cell-mediated and humoral anti-MPO autoimmunity and GN. Disabling of Tregs negated the therapeutic benefit of anti-CD20 treatment. The mechanism of enhancement of Treg activity could be attributed to anti-CD20 mAb effects on inducing B cell apoptosis. Administering anti-CD20 mAb-induced apoptotic splenocytes to mice developing anti-MPO GN was as effective as anti-CD20 mAb treatment in inducing Tregs and attenuating both anti-MPO autoimmunity and GN. A nonredundant role for splenic macrophages in mediating the anti-CD20 mAb-induced immunomodulation was demonstrated by showing that administration of anti-CD20 mAb ex vivo-induced apoptotic splenocytes to unmanipulated mice attenuated autoimmunity and GN, whereas deletion of splenic marginal zone macrophages prevented anti-CD20 mAb-induced immunomodulation and treatment efficacy. Six days after administering anti-CD20 mAb to mice with murine anti-MPO GN, cell-mediated anti-MPO responses and GN were attenuated, and Tregs were enhanced, but ANCA levels were unchanged, suggesting humoral autoimmunity was redundant at this time point. CONCLUSIONS: Collectively, these data suggest that, as well as reducing humoral autoimmunity, anti-CD20 mAb more rapidly induces protective anti-MPO Treg-mediated immunomodulation by splenic processing of anti-CD20-induced apoptotic B cells.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , Rituximab/uso terapêutico , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/etiologia , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Apoptose , Linfócitos B/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Camundongos , Peroxidase , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
Background Because of their capacity to induce antigen-specific immunosuppression, tolerogenic dendritic cells are a promising tool for treatment of autoimmune conditions, such as GN caused by autoimmunity against myeloperoxidase (MPO). METHODS: We sought to generate tolerogenic dendritic cells to suppress anti-MPO GN by culturing bone marrow cells with an NFκB inhibitor (BAY 11-7082) and exposing them to a pulse of MPO. After administering these MPO/BAY dendritic cells or saline to mice with established anti-MPO or anti-methylated BSA (mBSA) immunity, we assessed immune responses and GN. We also examined mechanisms of action of MPO/BAY dendritic cells. RESULTS: MPO/BAY dendritic cells decreased anti-MPO immunity and GN without inhibiting immune responses against mBSA; they also induced IL-10-producing regulatory T cells in MPO-immunized mice without affecting IL-10+ CD4+Foxp3- type 1 regulatory T cells or regulatory B cells. MPO/BAY dendritic cells did not inhibit anti-MPO immunity when CD4+Foxp3+ cells were depleted in vivo, showing that regulatory T cells are required for their effects. Coculture experiments with dendritic cells and CD4+Foxp3- or CD4+Foxp3+ cells showed that MPO/BAY dendritic cells generate Foxp3+ regulatory T cells from CD4+Foxp3- cells through several pathways, and induce IL-10+ regulatory T cells via inducible costimulator (ICOS), which was confirmed in vivo. Transfer of MPO/BAY dendritic cell-induced regulatory T cells in vivo, with or without anti-IL-10 receptor antibody, demonstrated that they suppress anti-MPO immunity and GN via IL-10. CONCLUSIONS: MPO/BAY dendritic cells attenuate established anti-MPO autoimmunity and GN in an antigen-specific manner through ICOS-dependent induction of IL-10-expressing regulatory T cells. This suggests that autoantigen-loaded tolerogenic dendritic cells may represent a novel antigen-specific therapeutic option for anti-MPO GN.
Assuntos
Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/terapia , Glomerulonefrite/terapia , Peroxidase/imunologia , Animais , Tolerância Imunológica , Proteína Coestimuladora de Linfócitos T Induzíveis/fisiologia , Interleucina-10/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/antagonistas & inibidores , Nitrilas/uso terapêutico , Sulfonas/uso terapêutico , Linfócitos T Reguladores/imunologia , Vasculite/terapiaRESUMO
BACKGROUND: Myeloperoxidase (MPO)-ANCA-associated GN is a significant cause of renal failure. Manipulating autoimmunity by inducing regulatory T cells is potentially a more specific and safer therapeutic option than conventional immunosuppression. METHODS: To generate MPO-specific regulatory T cells, we used a modified protein-conjugating compound, 1-ethyl-3-(3'dimethylaminopropyl)-carbodiimide (ECDI), to couple the immunodominant MPO peptide (MPO409-428) or a control ovalbumin peptide (OVA323-339) to splenocytes and induced apoptosis in the conjugated cells. We then administered MPO- and OVA-conjugated apoptotic splenocytes (MPO-Sps and OVA-Sps, respectively) to mice and compared their effects on development and severity of anti-MPO GN. We induced autoimmunity to MPO by immunizing mice with MPO in adjuvant; to trigger GN, we used low-dose antiglomerular basement membrane globulin, which transiently recruits neutrophils that deposit MPO in glomeruli. We also compared the effects of transferring CD4+ T cells from mice treated with MPO-Sp or OVA-Sp to recipient mice with established anti-MPO autoimmunity. RESULTS: MPO-Sp but not OVA-Sp administration increased MPO-specific, peripherally derived CD4+Foxp3- type 1 regulatory T cells and reduced anti-MPO autoimmunity and GN. However, in mice depleted of regulatory T cells, MPO-Sp administration did not protect from anti-MPO autoimmunity or GN. Mice with established anti-MPO autoimmunity that received CD4+ T cells transferred from mice treated with MPO-Sp (but not CD4+ T cells transferred from mice treated with OVA-Sp) were protected from anti-MPO autoimmunity and GN, confirming the induction of therapeutic antigen-specific regulatory T cells. CONCLUSIONS: These findings in a mouse model indicate that administering apoptotic splenocytes conjugated with the immunodominant MPO peptide suppresses anti-MPO GN by inducing antigen-specific tolerance.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/química , Apoptose , Glomerulonefrite/terapia , Peroxidase/química , Vasculite/imunologia , Vasculite/terapia , Animais , Autoimunidade , Linfócitos T CD4-Positivos/citologia , Modelos Animais de Doenças , Feminino , Glomerulonefrite/imunologia , Proteínas de Fluorescência Verde/metabolismo , Tolerância Imunológica , Rim/patologia , Glomérulos Renais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Peroxidase/imunologia , Baço/citologia , Linfócitos T/citologia , Linfócitos T Reguladores/imunologiaRESUMO
Renal lymphatics are implicated in renal disease, but their function in inflammatory kidney diseases is relatively poorly defined. In the current issue, Kasinath et al. examine the role of the kidney draining lymph node in experimental glomerulonephritis, as well the role of fibroblastic reticular cells within lymph nodes. Removing the kidney-draining lymph nodes prior to the induction of glomerulonephritis attenuated disease, as did interventions that affected the function of lymph nodes in general.
Assuntos
Glomerulonefrite , Nefropatias , Vasos Linfáticos , Humanos , Rim , LinfonodosRESUMO
Anti-myeloperoxidase nephritogenic autoimmunity induces severe glomerulonephritis. To assess the therapeutic potential of monoclonal antibodies targeting T helper (Th) subset differentiation determining cytokines, we studied a murine model of anti-myeloperoxidase glomerulonephritis. The temporal participation of T helper subsets was determined by quantitating gene expression of CD4+ T-cells isolated from nephritic kidneys and cytokine production by lymphocytes from nodes draining myeloperoxidase immunization sites. Th17 cytokines (IL-17A and IL-6) rose rapidly but declined as autoimmunity matured when Th1 cytokines (IL-12 and TNF) predominated. Therefore, T helper subset participation in anti-myeloperoxidase autoimmunity is biphasic, with Th17 early and Th1 late. To confirm the functional relevance of this biphasic pattern, we compared systemic anti-myeloperoxidase autoimmunity in wild type, Th17 deficient and Th1 deficient mice. Early, Th1 deficient mice developed similar autoimmunity and glomerulonephritis to wild type mice. However, Th17 deficient mice had significantly reduced anti-myeloperoxidase autoimmunity. In late autoimmunity, Th1 deficient mice developed reduced autoimmunity and were protected from anti-myeloperoxidase glomerulonephritis. The therapeutic potential of these findings were demonstrated by neutralizing monoclonal antibodies. Targeting IL-23p19 attenuated early Th17 dominated anti-myeloperoxidase autoimmunity and glomerulonephritis but not late phase disease. Targeting IL-12p35 attenuated late phase Th1 dominated anti-myeloperoxidase autoimmunity and glomerulonephritis but not early autoimmunity or glomerulonephritis. Targeting both T helper subsets with an anti-IL-12p40 monoclonal antibody was effective during both early and late phases of anti-myeloperoxidase glomerulonephritis. Thus, definition of dominant T helper differentiating subsets in anti-myeloperoxidase glomerulonephritis by renal CD4+ T-cell cytokine gene expression allows effective proper phase monoclonal antibody treatment of anti-myeloperoxidase glomerulonephritis.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Glomerulonefrite/tratamento farmacológico , Interleucina-12/imunologia , Interleucina-23/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Anticorpos Monoclonais/imunologia , Avaliação Pré-Clínica de Medicamentos , Glomerulonefrite/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxidase/imunologiaRESUMO
Goodpasture's disease manifests as rapidly progressive glomerulonephritis. Current immunosuppressive treatments do not specifically target the pathological immune response and have significant side effects. Like most autoimmune diseases, the strongest genetic association is with the HLA alleles. Inheritance of HLA-DR15 confers susceptibility, and structure-function studies have shown that HLA-DR15 plays a causative role in activating autoreactive pro-inflammatory T cells. Thus, specific inhibition of HLA-DR15 would provide a targeted therapeutic approach. We hypothesised that PV-267, an HLA-DR15-specific inhibitor, would effectively block HLA-DR15 presentation of the dominant epitope, attenuate the activation of autoreactive T cells, and limit disease. Using humanised HLA-DR15 transgenic mice, α3135-145-specific, pro-inflammatory T cell recall responses were measured using IFN-γ and IL-17A ELISPOTs and by proliferation assay. To determine if PV-267 could limit disease, experimental autoimmune anti-GBM glomerulonephritis was induced in HLA-DR15 transgenic mice (on an Fcgr2b-/- background), and functional and histological disease endpoints were measured. PV-267 effectively inhibited α3135-145-specific immune responses and disease development. Mice treated prior to immunization with α3135-145 had reduced α3135-145-specific recall responses, and limited disease by albuminuria, histological glomerular injury, IgG deposition, and inflammatory cell infiltrates. PV-267 treatment commencing after the onset of active anti-α3(IV)NC1 autoimmunity attenuated functional and histological renal injury. When treatment was administered after disease was established, PV-267 limited the severity of histological injury. In conclusion, HLA-DR15 inhibition attenuates α3(IV)NC1-specific pro-inflammatory responses and could be used as an adjunct therapy for anti-GBM disease.
Assuntos
Doença Antimembrana Basal Glomerular/terapia , Autoantígenos/imunologia , Colágeno Tipo IV/imunologia , Glomerulonefrite/terapia , Subtipos Sorológicos de HLA-DR/metabolismo , Rim/efeitos dos fármacos , Peptídeos/uso terapêutico , Linfócitos T/imunologia , Animais , Doença Antimembrana Basal Glomerular/genética , Células Cultivadas , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Glomerulonefrite/genética , Subtipos Sorológicos de HLA-DR/genética , Humanos , Rim/patologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Peptídeos/imunologia , Ligação Proteica , Receptores de IgG/genéticaRESUMO
BACKGROUND: The functional relevance of OX40 ligand (OX40L) in the effector phase of crescentic glomerulonephritis (GN) is unknown. These studies defined the role of endogenous OX40L during the effector stage of murine crescentic GN. METHODS: GN was induced by immunization with sheep globulin/adjuvant on Day 0 and injection of sheep anti-mouse glomerular basement membrane immunoglobulin (Ig) on Day 10. Rat IgG or neutralizing anti-OX40L antibody was administered on Days 10-18 and immune responses and renal injury assessed on Day 20. RESULTS: Compared with naïve animals, OX40L was upregulated in the lymph nodes (LNs) and on leucocytes and resident non-immune cells in the kidneys of mice with GN. Inhibition of OX40L in GN augmented renal injury, as indicated by increased crescent formation, proteinuria and glomerular leucocyte accumulation. In line with increased injury, anti-OX40L treatment increased proliferation and decreased apoptosis of CD4 T cells in the LNs, without affecting LN CD4 cytokine production and CD8 T-cell responses. Blockade of OX40L decreased LN regulatory T-cell (Treg) proliferation, transforming growth factor ß production and foxp3 expression. OX40L inhibition did not affect B cell expansion or circulating antibody levels. In the kidney, neutralization of OX40L augmented interferon γ (IFNγ) expression by CD4 and CD8 T cells and shifted macrophage polarization towards the pro-inflammatory M1 phenotype. CONCLUSIONS: OX40L is protective during the effector phase of murine crescentic GN by reducing the expansion of CD4 T cells and enhancing Treg responses in the LNs, and by locally inhibiting T-cell IFNγ production and pro-inflammatory macrophage phenotype in the kidney.
Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos T CD4-Positivos/imunologia , Glomerulonefrite/imunologia , Macrófagos/imunologia , Ligante OX40/imunologia , Linfócitos T Reguladores/imunologia , Animais , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Interferon gama/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores OX40/metabolismoRESUMO
BACKGROUND: The aim of this study was to determine whether a composite score of simple immune biomarkers and clinical characteristics could predict severe infections in kidney transplant recipients. METHODS: We conducted a prospective study of 168 stable kidney transplant recipients who underwent measurement of lymphocyte subsets, immunoglobulins, and renal function at baseline and were followed up for 2 years for the development of any severe infections, defined as infection requiring hospitalization. A point score was developed to predict severe infection based on logistic regression analysis of factors in baseline testing. RESULTS: Fifty-nine (35%) patients developed severe infection, 36 (21%) had two or more severe infections, and 3 (2%) died of infection. A group of 19 (11%) patients had the highest predicted infectious risk (>60%), as predicted by the score. Predictive variables were mycophenolate use, graft function, CD4+, and natural killer cell number. The level of immunosuppression score had an area under the receiver operating curve of 0.75 (95% CI: 0.67-0.83). CONCLUSION: Our level of immunosuppression score for predicting the development of severe infection over 2 years has sufficient prognostic accuracy for identification of high-risk patients. This data can inform research that examines strategies to reduce the risks of infection.
Assuntos
Infecções/diagnóstico , Transplante de Rim/efeitos adversos , Transplantados , Biomarcadores/análise , Linfócitos T CD4-Positivos/imunologia , Feminino , Humanos , Imunoglobulinas/análise , Terapia de Imunossupressão , Transplante de Rim/estatística & dados numéricos , Células Matadoras Naturais/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Análise de Regressão , Fatores de RiscoRESUMO
In antineutrophil cytoplasmic antibody-associated vasculitis (AAV), Toll-like receptors (TLRs) may be engaged by infection-associated patterns and by endogenous danger signals, linking infection and innate inflammation with this autoimmune disease. This study examined intrarenal TLR2, TLR4, and TLR9 expression and renal injury in AAV, testing the hypothesis that increased TLR expression correlates with renal injury. Patients with AAV exhibited both glomerular and tubulointerstitial expression of TLR2, TLR4, and TLR9, with TLR4 being the most prominent in both compartments. Glomerular TLR4 expression correlated with glomerular segmental necrosis and cellular crescents, with TLR2 expression correlating with glomerular segmental necrosis. The extent and intensity of glomerular and tubulointerstitial TLR4 expression and the intensity of glomerular TLR2 expression inversely correlated with the presenting estimated glomerular filtration rate. Although myeloid cells within the kidney expressed TLR2, TLR4, and TLR9, TLR2 and TLR4 colocalized with endothelial cells and podocytes, whereas TLR9 was expressed predominantly by podocytes. The functional relevance of intrarenal TLR expression was further supported by the colocalization of TLRs with their endogenous ligands high-mobility group box 1 and fibrinogen. Therefore, in AAV, the extent of intrarenal TLR4 and TLR2 expression and their correlation with renal injury indicates that TLR4, and to a lesser degree TLR2, may be potential therapeutic targets in this disease.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Glomerulonefrite/imunologia , Glomérulos Renais/imunologia , Receptor 2 Toll-Like/análise , Receptor 4 Toll-Like/análise , Idoso , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/fisiopatologia , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Feminino , Fibrinogênio/análise , Taxa de Filtração Glomerular , Glomerulonefrite/patologia , Glomerulonefrite/fisiopatologia , Proteína HMGB1/análise , Humanos , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Masculino , Pessoa de Meia-Idade , Peroxidase/imunologia , Receptor PAR-1/imunologia , Índice de Gravidade de Doença , Receptor Toll-Like 9/análiseRESUMO
The prospects for complement-targeted therapy in ANCA-associated vasculitis have been enhanced by a recent clinical trial in which C5a receptor 1 (C5aR1) inhibition safely replaced glucocorticoids in induction treatment. C5aR1 primes neutrophils for activation by anti-neutrophil cytoplasmic antibody (ANCA) and is therefore required in models of glomerulonephritis induced by anti-myeloperoxidase antibody. Although humoral and cellular autoimmunity play essential roles in ANCA-associated vasculitis, a role for C5aR1 in these responses has not been described. Here, we use murine models to dissect the role of C5aR1 in the generation of anti-myeloperoxidase autoimmunity and the effector responses resulting in renal injury. The genetic absence or pharmacological inhibition of C5aR1 results in reduced autoimmunity to myeloperoxidase with an attenuated Th1 response, increased Foxp3+ regulatory T cells and reduction in generation of myeloperoxidase-ANCA. These changes are mediated by C5aR1 on dendritic cells, which promotes activation, and thus myeloperoxidase autoimmunity and glomerulonephritis. We also use renal intravital microscopy to determine the effect of C5aR1 inhibition on ANCA induced neutrophil dysfunction. We found that myeloperoxidase-ANCA induce neutrophil retention and reactive oxygen species burst within glomerular capillaries. These pathological behaviors are abrogated by C5aR1 inhibition. Thus, C5aR1 inhibition ameliorates both autoimmunity and intra-renal neutrophil activation in ANCA-associated vasculitis.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Autoimunidade , Glomerulonefrite/imunologia , Glomérulos Renais/imunologia , Ativação de Neutrófilo , Neutrófilos/imunologia , Peroxidase/imunologia , Receptor da Anafilatoxina C5a/imunologia , Animais , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Imunidade Celular , Imunidade Humoral , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Explosão Respiratória , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th1/imunologia , Células Th1/metabolismoRESUMO
Observations in patients with ANCA-associated vasculitis suggest that CD8+ T cells participate in disease, but there is no experimental functional evidence of pathologic involvement for these cells. Myeloperoxidase (MPO) is a well defined autoantigen in ANCA-associated vasculitis. Studies in experimental models of anti-MPO GN suggest that, after ANCA-induced neutrophil localization, deposited MPO within glomeruli is recognized by autoreactive T cells that contribute to injury. We tested the hypothesis that CD8+ T cells mediate disease in experimental ANCA-associated vasculitis. CD8+ T cell depletion in the effector phase of disease attenuated injury in murine anti-MPO GN. This protection associated with decreased levels of intrarenal IFN-γ, TNF, and inflammatory chemokines and fewer glomerular macrophages. Moreover, we identified a pathogenic CD8+ T cell MPO epitope (MPO431-439) and found that cotransfer of MPO431-439-specific CD8+ T cell clones exacerbated disease mediated by MPO-specific CD4+ cells in Rag1-/- mice. Transfer of MPO431-439-specific CD8+ cells without CD4+ cells mediated glomerular injury when MPO was planted in glomeruli. These results show a pathogenic role for MPO-specific CD8+ T cells, provide evidence that CD8+ cells are a therapeutic target in ANCA-associated vasculitis, and suggest that a molecular hotspot within the MPO molecule contains important CD8+, CD4+, and B cell epitopes.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/enzimologia , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Autoantígenos/imunologia , Linfócitos T CD8-Positivos/fisiologia , Glomerulonefrite/enzimologia , Glomerulonefrite/imunologia , Glomérulos Renais/patologia , Peroxidase/imunologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Regulatory T (Treg) cells are a suppressive CD4+ T-cell subset. We generated induced Treg (iTreg) cells and explored their therapeutic potential in a murine model of rapidly progressive glomerulonephritis. Polyclonal naive CD4+ T cells were cultured in vitro with interleukin-2 (IL-2), transforming growth factor-ß1, all-trans-retinoic acid and monoclonal antibodies against interferon-γ and IL-4, generating Foxp3+ iTreg cells. To enhance their suppressive phenotype, iTreg cultures were modified with the addition of a monoclonal antibody against IL-12p40 or by using RORγt-/- CD4+ T cells. Induced Treg cells were transferred into models of delayed-type hypersensitivity and experimental glomerulonephritis. The iTreg cells exhibited comparable surface receptor expression and in vitro suppressive ability to natural Treg cells, but did not regulate antigen-specific delayed-type hypersensitivity or systemic inflammatory immune responses, losing Foxp3 expression in vivo. In glomerulonephritis, transferred iTreg cells did not prevent renal injury or modulate systemic T helper type 1 immune responses. Induced Treg cells cultured with anti-IL-12p40 had an enhanced suppressive phenotype in vitro and regulated dermal delayed-type hypersensitivity in vivo, but were not protective against renal injury, losing Foxp3 expression, especially in the transferred cells recruited to the kidney. Use of RORγt-/- CD4+ T cells or iTreg cells generated from sensitized CD4+ Foxp3- cells did not regulate renal or systemic inflammatory responses in vivo. In conclusion, iTreg cells suppress T-cell proliferation in vitro, but do not regulate experimental glomerulonephritis, being unstable in this inflammatory milieu in vivo.
Assuntos
Glomerulonefrite/imunologia , Hipersensibilidade Tardia/imunologia , Pele/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD4/metabolismo , Proliferação de Células , Células Cultivadas , Microambiente Celular , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Terapia de Imunossupressão , Masculino , Camundongos , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Pele/patologiaRESUMO
Next generation sequencing of T and B cell receptors is emerging as a valuable and effective method to diagnose and monitor hematopoietic malignancies. So far, this approach has not been fully explored in regard to autoimmune diseases. T cells develop in the thymus where they undergo positive and negative selection, and the autoimmune regulator (Aire) is central in the establishment of immunological tolerance. Loss of Aire leads to severe multiorgan autoimmune disease with infiltration of autoreactive T cells in affected organs. Here, we have utilized next generation sequencing technology to investigate the T cell receptor repertoire in autoimmunity induced by immunization of mice with a self-antigen, myeloperoxidase. By investigating the T cell receptor repertoire in peripheral blood, spleen and lumbar lymph nodes from naïve and immunized Aire -/- mice and wild type littermates, changes in the usage of V and J genes were evident. Our results identify TCR clonotypes which could be potential targets for immune therapy. Also, Aire -/- autoimmunity is driven by a variety of autoantigens where the autoimmune response is highly polyclonal, and access to the most adjacent immunologically active tissue is required to identify T cell receptor sequences that are potentially unique to the antigen in Aire-/- immunized mice.
Assuntos
Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Autoimunidade/genética , Comunicação Celular/imunologia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Doenças Autoimunes/metabolismo , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Modelos Animais de Doenças , Feminino , Genótipo , Masculino , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Análise de Sequência de DNARESUMO
Less toxic treatment options for patients with myeloperoxidase (MPO)-ANCA-associated GN are needed. Using an established murine model of focal necrotizing GN mediated by autoimmunity to MPO (autoimmune anti-MPO GN), we assessed the capacity for nasal tolerance induced by nasal insufflation of the immunodominant nephritogenic MPO peptide (MPO409-428) to attenuate this disease. Compared with mice that received an irrelevant immunodominant ovalbumin (OVA) peptide, OVA323-339, mice that received MPO409-428 were protected from the development of humoral and cell-mediated autoimmunity to full-length MPO and the development of GN. In mice with established anti-MPO autoimmunity, nasal insufflation of MPO409-428 as a therapeutic attenuated anti-MPO GN. To investigate the nature of this induced tolerance, we isolated CD4(+) T cells from the upper airway draining lymph nodes of both OVA323-339- and MPO409-428-tolerized mice. Adoptive transfer of CD4(+) T cells from MPO409-428- but not OVA323-339-tolerized mice to animals with established anti-MPO autoimmunity attenuated the subsequent development of GN, confirming that the immunosuppression induced by these T cells is antigen specific. Ex vivo studies showed that nasal tolerance to MPO is mediated by both conventional and induced T regulatory cells. The strong homology between the pathogenic human MPO B cell epitope recognized by ANCA in patients with acute vasculitis and the nephritogenic murine T cell MPO epitope emphasizes the clinical relevance of this study.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos , Glomerulonefrite/enzimologia , Glomerulonefrite/imunologia , Tolerância Imunológica , Nariz/imunologia , Peroxidase , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PeptídeosRESUMO
Observations in experimental murine myeloperoxidase (MPO)-ANCA-associated vasculitis (AAV) show mast cells degranulate, thus enhancing injury as well as producing immunomodulatory IL-10. Here we report that, compared with biopsy specimens from control patients, renal biopsy specimens from 44 patients with acute AAV had more mast cells in the interstitium, which correlated with the severity of tubulointerstitial injury. Furthermore, most of the mast cells were degranulated and spindle-shaped in patients with acute AAV, indicating an activated phenotype. We hypothesized that the mast cell stabilizer disodium cromoglycate would attenuate mast cell degranulation without affecting IL-10 production. We induced anti-MPO GN by immunizing mice with MPO and a low dose of anti-glomerular basement membrane antibody. When administered before or after induction of MPO autoimmunity in these mice, disodium cromoglycate attenuated mast cell degranulation, development of autoimmunity, and development of GN, without diminishing IL-10 production. In contrast, administration of disodium cromoglycate to mast cell-deficient mice had no effect on the development of MPO autoimmunity or GN. MPO-specific CD4(+) effector T cell proliferation was enhanced by co-culture with mast cells, but in the presence of disodium cromoglycate, proliferation was inhibited and IL-10 production was enhanced. These results indicate that disodium cromoglycate blocks injurious mast cell degranulation specifically without affecting the immunomodulatory role of these cells. Thus as a therapeutic, disodium cromoglycate may substantially enhance the regulatory role of mast cells in MPO-AAV.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Cromolina Sódica/farmacologia , Cromolina Sódica/uso terapêutico , Glomerulonefrite/tratamento farmacológico , Glomerulonefrite/imunologia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Peroxidase/imunologia , Idoso , Animais , Feminino , Humanos , Masculino , CamundongosRESUMO
Toll-like receptor 9 (TLR9) enhances proinflammatory responses, but whether it can act in a regulatory capacity remains to be established. In experimental murine AKI induced by cisplatin, Tlr9(-/-) mice developed enhanced renal injury and exhibited fewer intrarenal regulatory T cells (Tregs) compared with genetically intact mice. A series of reconstitution and depletion studies defined a role for TLR9 in maintaining Treg-mediated homeostasis in cisplatin-induced AKI. When Rag1(-/-) mice were reconstituted with nonregulatory CD25(-) splenocytes from wild-type (WT) or Tlr9(-/-) mice, AKI was similarly enhanced. However, when Rag1(-/-) mice were reconstituted with CD4(+)CD25(+) regulatory cells, WT CD4(+)CD25(+) cells were more renoprotective and localized to the kidney more efficiently than Tlr9(-/-) CD4(+)CD25(+) cells. In Treg-depleted Foxp3(DTR) mice, reconstitution with naive WT CD4(+)CD25(+) cells resulted in less severe AKI than did reconstitution with Tlr9(-/-) Tregs. Tlr9(-/-) mice were not deficient in CD4(+)CD25(+) cells, and WT and TLR9-deficient Tregs had similar suppressive function ex vivo. However, expression of adhesion molecules important in Treg trafficking was reduced on peripheral CD4(+)CD25(+) cells from Tlr9(-/-) mice. In conclusion, we identified a pathway by which TLR9 promotes renal Treg accumulation in AKI.
Assuntos
Injúria Renal Aguda/imunologia , Injúria Renal Aguda/metabolismo , Ativação Linfocitária , Linfócitos T Reguladores/imunologia , Receptor Toll-Like 9/fisiologia , Injúria Renal Aguda/induzido quimicamente , Animais , Antígeno CD11a/metabolismo , Cisplatino , Proteínas de Homeodomínio/genética , Receptores de Hialuronatos/metabolismo , Integrina alfa4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismoRESUMO
The NLRP3 inflammasome is an intracellular platform that converts the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 to their active forms in response to 'danger' signals, which can be either host or pathogen derived, and mediates a form of inflammatory cell death called pyroptosis. This component of the innate immune system was initially discovered because of its role in rare autoinflammatory syndromes called cryopyrinopathies, but it has since been shown to mediate injurious inflammation in a broad range of diseases. Inflammasome activation occurs in both immune cells, primarily macrophages and dendritic cells, and in some intrinsic kidney cells such as the renal tubular epithelium. The NLRP3 inflammasome has been implicated in the pathogenesis of a number of renal conditions, including acute kidney injury, chronic kidney disease, diabetic nephropathy and crystal-related nephropathy. The inflammasome also plays a role in autoimmune kidney disease, as IL-1ß and IL-18 influence adaptive immunity through modulation of T helper cell subsets, skewing development in favour of Th17 and Th1 cells that are important in the development of autoimmunity. Both IL-1 blockade and two recently identified specific NLRP3 inflammasome blockers, MCC950 and ß-hydroxybutyrate, have shown promise in the treatment of inflammasome-mediated conditions. These targeted therapies have the potential to be of benefit in the growing number of kidney diseases in which the NLRP3 inflammasome has been implicated.