RESUMO
Therapies that promote tolerance in solid organ transplantation will improve patient outcomes by eliminating the need for long-term immunosuppression. To investigate mechanisms of rapamycin-induced tolerance, C3H/HeJ mice were heterotopically transplanted with MHC-mismatched hearts from BALB/cJ mice and were monitored for rejection after a short course of rapamycin treatment. Mice that had received rapamycin developed tolerance with indefinite graft survival, whereas untreated mice all rejected their grafts within 9 days. In vitro, splenic mononuclear cells from tolerant mice maintained primary CD4(+) and CD8(+) immune responses to donor antigens consistent with a mechanism that involves active suppression of immune responses. Furthermore, infection with lymphocytic choriomeningitis virus strain WE led to loss of tolerance suggesting that tolerance could be overcome by infection. Rapamycin-induced, donor-specific tolerance was associated with an expansion of regulatory T (Treg) cells in both the spleen and allograft and elevated plasma levels of fibrinogen-like protein 2 (FGL2). Depletion of Treg cells with anti-CD25 (PC61) and treatment with anti-FGL2 antibody both prevented tolerance induction. Tolerant allografts were populated with Treg cells that co-expressed FGL2 and FoxP3, whereas rejecting allografts and syngeneic grafts were nearly devoid of dual-staining cells. We examined the utility of an immunoregulatory gene panel to discriminate between tolerance and rejection. We observed that Treg-associated genes (foxp3, lag3, tgf-ß and fgl2) had increased expression and pro-inflammatory genes (ifn-γ and gzmb) had decreased expression in tolerant compared with rejecting allografts. Taken together, these data strongly suggest that Treg cells expressing FGL2 mediate rapamycin-induced tolerance. Furthermore, a gene biomarker panel that includes fgl2 can distinguish between rejecting and tolerant grafts.
Assuntos
Fibrinogênio/imunologia , Rejeição de Enxerto/prevenção & controle , Transplante de Coração , Imunossupressores/farmacologia , Sirolimo/farmacologia , Linfócitos T Reguladores/imunologia , Tolerância ao Transplante/efeitos dos fármacos , Aloenxertos , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Fibrinogênio/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Granzimas/genética , Granzimas/imunologia , Interferon gama/genética , Interferon gama/imunologia , Depleção Linfocítica/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Tolerância ao Transplante/genética , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
UNLABELLED: Coronaviruses express a deubiquitinating protein, the papain-like protease-2 (PLP2), that removes both ubiquitin and the ubiquitin-like interferon (IFN)-stimulated gene 15 (ISG15) protein from target proteins. ISG15 has antiviral activity against a number of viruses; therefore, we examined the effect of ISG15 conjugation (ISGylation) in a model of acute viral hepatitis induced by the murine hepatitis virus strain 3 (MHV-3) coronavirus. Mice deficient in the ISG15 deconjugating enzyme, ubiquitin-specific peptidase-18 (USP18), accumulate high levels of ISG15-conjugated proteins and are hypersensitive to type I IFN. Infecting USP18(-/-) mice with MHV-3 resulted in extended survival (8 ± 1.2 versus 4 days) and in improved liver histology, a decreased inflammatory response, and viral titers 1 to 2 logs lower than in USP18(+/+) mice. The suppression of viral replication was not due to increased IFN since infected USP18(-/-) mice had neither increased hepatic IFN-α, -ß, or -γ mRNA nor circulating protein. Instead, delayed MHV-3 replication coincided with high levels of cellular ISGylation. Decreasing ISGylation by knockdown of the ISG15 E1 enzyme, Ube1L, in primary USP18(+/+) and USP18(-/-) hepatocytes led to increased MHV-3 replication. Both in vitro and in vivo, increasing MHV-3 titers were coincident with increased PLP2 mRNA and decreased ISGylation over the course of infection. The pharmacologic inhibition of the PLP2 enzyme in vitro led to decreased MHV-3 replication. Overall, these results demonstrate the antiviral effect of ISGylation in an in vivo model of coronavirus-induced mouse hepatitis and illustrate that PLP2 manipulates the host innate immune response through the ISG15/USP18 pathway. IMPORTANCE: There have been a number of serious worldwide pandemics due to widespread infections by coronavirus. This virus (in its many forms) is difficult to treat, in part because it is very good at finding "holes" in the way that the host (the infected individual) tries to control and eliminate the virus. In this study, we demonstrate that an important host viral defense-the ISG15 pathway-is only partially effective in controlling severe coronavirus infection. Activation of the pathway is very good at suppressing viral production, but over time the virus overwhelms the host response and the effects of the ISG15 pathway. These data provide insight into host-virus interactions during coronavirus infection and suggest that the ISG15 pathway is a reasonable target for controlling severe coronavirus infection although the best treatment will likely involve multiple pathways and targets.
Assuntos
Infecções por Coronavirus/metabolismo , Citocinas/metabolismo , Hepatite Viral Animal/metabolismo , Vírus da Hepatite Murina , Papaína/metabolismo , Ubiquitina Tiolesterase/deficiência , Alanina Transaminase/sangue , Análise de Variância , Animais , Aspartato Aminotransferases/sangue , Western Blotting , Proteases Semelhantes à Papaína de Coronavírus , Primers do DNA/genética , Hepatite Viral Animal/virologia , Hepatócitos , Interferons/sangue , Macrófagos Peritoneais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Papaína/antagonistas & inibidores , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sobrevida , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMO
Several types of regulatory T cells maintain self-tolerance and control excessive immune responses to foreign antigens. The major regulatory T subsets described over the past decade and novel function in transplantation will be covered in this review with a focus on CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells. Multiple mechanisms have been proposed to explain how Treg cells inhibit effector cells but none can completely explain the observed effects in toto. Proposed mechanisms to explain suppressive activity of Treg cells include the generation of inhibitory cytokines, induced death of effector cells by cytokine deprivation or cytolysis, local metabolic perturbation of target cells mediated by changes in extracellular nucleotide/nucleoside fluxes with alterations in intracellular signaling molecules such as cyclic AMP, and finally inhibition of dendritic cell functions. A better understanding of how Treg cells operate at the molecular level could result in novel and safer therapeutic approaches in transplantation and immune-mediated diseases.
Assuntos
Células Dendríticas/fisiologia , Tolerância Imunológica , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Imunologia de Transplantes , Animais , Apoptose/imunologia , Antígenos CD4 , AMP Cíclico/imunologia , AMP Cíclico/metabolismo , Fatores de Transcrição Forkhead , Humanos , Terapia de Imunossupressão , Subunidade alfa de Receptor de Interleucina-2 , Potenciais da Membrana/imunologia , Comunicação Parácrina/imunologiaRESUMO
BACKGROUND & AIMS: Sinusoidal endothelial cell (SEC) and hepatocyte death are early, TNF-α mediated events in ischemia and reperfusion of the liver (I/Rp). We previously reported that TNF-α induced liver injury is dependent on Fibrinogen like protein 2 (FGL2/Fibroleukin) and showed that FGL2 binding to its receptor, FcγRIIB, results in lymphocyte apoptosis. In this study we examine whether I/Rp is induced by specific binding of FGL2 to FcγRIIB expressed on SEC. METHODS: Hepatic ischemia and reperfusion was induced in wild type (WT) mice and in mice with deletion or inhibition of FGL2 and FcRIIB. Liver injury was determined by AST release, necrosis and animal death. Apoptosis was evaluated with caspase 3 and TUNEL staining. RESULTS: FGL2 deletion or inhibition resulted in decreased liver injury as determined by a marked reduction in both levels of AST and ALT and hepatocyte necrosis. Caspase 3 staining of SEC (12% vs. 75%) and hepatocytes (12% vs. 45%) as well as TUNEL staining of SEC (13% vs. 60%, p=0.02) and hepatocytes (18% vs. 70%, p=0.03), markers of apoptosis, were lower in Fgl2(-/-) compared to WT mice. In vitro incubation of SEC with FGL2 induced apoptosis of SEC from WT mice, but not FcγRIIB(-/-) mice. Deletion of FcγRIIB fully protected mice against SEC and hepatocyte death in vivo. Survival of mice deficient in either Fgl2(-/-) (80%) or FcγRIIB(-/-) (100%) was markedly increased compared to WT mice (10%) which were subjected to 75min of total hepatic ischemia (p=0.001). CONCLUSIONS: FGL2 binding to the FcγRIIB receptor expressed on SEC is a critical event in the initiation of the hepatic reperfusion injury cascade through induction of SEC and hepatocyte death.
Assuntos
Fibrinogênio/fisiologia , Fígado/lesões , Fígado/patologia , Traumatismo por Reperfusão/patologia , Animais , Apoptose/fisiologia , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Fibrinogênio/genética , Hepatócitos/patologia , Fígado/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgG/antagonistas & inibidores , Receptores de IgG/deficiência , Receptores de IgG/genética , Receptores de IgG/metabolismo , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/fisiopatologiaRESUMO
Liver transplantation is now recognized as the most effective therapy for patients with end-stage acute and chronic liver failure. Despite outstanding short-term graft and patient survival, liver transplantation continues to face several major challenges, including poor long-term graft survival due to chronic rejection and major side effects of long-term immunosuppressive therapy (which is required for the prevention of rejection). The ability to produce a state of tolerance after transplantation would potentially obviate long-term immunosuppression. Self-tolerance and immune homeostasis involve both central and peripheral immunoregulatory mechanisms. To date, studies have shown that many subsets of regulatory T cells (Tregs) control immune responses to foreign and alloantigens. The identification of Tregs that are positive for CD4, CD25, and the transcription factor forkhead box (Foxp3) has resulted in major advances in our understanding of the immunology of rejection and the development of transplant tolerance. In this article, we focus on the importance of Tregs in tolerance induction in experimental models of liver transplantation. Furthermore, we discuss the therapeutic potential of Tregs for the promotion of tolerance in transplant patients and highlight recent clinical trials of Treg-based therapies.
Assuntos
Transplante de Fígado/métodos , Linfócitos T Reguladores/citologia , Animais , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/citologia , Epigênese Genética , Fatores de Transcrição Forkhead/metabolismo , Rejeição de Enxerto , Humanos , Imunossupressores/uso terapêutico , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Camundongos , Modelos Biológicos , Timo/metabolismo , Tolerância ao TransplanteRESUMO
Here we examined whether the expression of a novel immunoregulatory gene set could be used to predict outcomes in murine models of rapamycin-induced cardiac tolerance, spontaneous hepatic tolerance, and cardiac rejection. The expression of the immunoregulatory gene set was assessed with the GeXP multiplex reverse-transcription polymerase chain reaction (RT-PCR) analysis system, and it was correlated to the pathological and biochemical parameters of the allografts. In rejecting cardiac grafts, the increased expression of an inflammatory set of genes, which included CD45, CD4, CD25, suppressor of cytokine signaling 2, cytotoxic T lymphocyte-associated protein 4 (CTLA4), selectin lymphocyte, interferon-γ (IFN-γ), programmed cell death 1 (Pdcd1), and granzyme B (Gzmb), was seen 8 days after transplantation along with histological evidence of severe allograft rejection. In tolerant cardiac allografts, the expression of fibrinogen-like protein 2 (Fgl2), Pdcd1, killer cell lectin-like receptor G1 (Klrg1), CTLA4, and lymphocyte-activation gene 3 was associated with tolerance. In a model of liver allograft tolerance, the increased expression of lectin galactose-binding soluble 1, Fgl2, CD39, phosphodiesterase 3B, Klrg1, forkhead box P3 (Foxp3), and transforming growth factor ß as well as the inflammatory set of genes was observed 8 to 14 days after transplantation (ie, when there was severe inflammatory injury). At a later time when the liver allografts had been fully accepted and were histologically normal, the expression of the inflammatory set of genes returned to the baseline, but the expression of the tolerogenic set of genes was still increased. Genes that were expressed in tolerant cardiac and liver allografts included Fgl2, Klrg1, and Foxp3, whereas genes associated with rejection included CD25, Gzmb, and IFN-γ. Our data indicate that monitoring the graft expression of a novel biomarker gene set with the GeXP multiplex RT-PCR analysis system may allow differentiation between rejection and tolerance.
Assuntos
Perfilação da Expressão Gênica , Marcadores Genéticos , Rejeição de Enxerto/genética , Sobrevivência de Enxerto/genética , Transplante de Coração/imunologia , Transplante de Fígado/imunologia , Tolerância ao Transplante/genética , Animais , Perfilação da Expressão Gênica/métodos , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Rejeição de Enxerto/prevenção & controle , Imunossupressores/farmacologia , Inflamação/genética , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Sirolimo/farmacologia , Fatores de Tempo , Resultado do TratamentoRESUMO
We report here investigation into the genetic basis of mouse hepatitis virus strain 1 (MHV-1) pneumovirulence. Sequencing of the 3' one-third of the MHV-1 genome demonstrated that the genetic organization of MHV-1 was similar to that of other strains of MHV. The hemagglutinin esterase (HE) protein was truncated, and reverse transcription-PCR (RT-PCR) studies confirmed previous work that suggested that the MHV-1 HE is a pseudogene. Targeted recombination was used to select chimeric viruses containing either the MHV-1 S gene or genes encoding all of the MHV-1 structural proteins, on an MHV-A59 background. Challenge studies in mice demonstrated that expression of the MHV-1 S gene within the MHV-A59 background (rA59/S(MHV-1)) increased the pneumovirulence of MHV-A59, and mice infected with this recombinant virus developed pulmonary lesions that were similar to those observed with MHV-1, although rA59/S(MHV-1) was significantly less virulent. Chimeras containing all of the MHV-1 structural genes on an MHV-A59 background were able to reproduce the severe acute respiratory syndrome (SARS)-like pathology observed with MHV-1 and reproducibly increased pneumovirulence relative to rA59/S(MHV-1), but were still much less virulent than MHV-1. These data suggest that important determinants of pneumopathogenicity are contained within the 3' one-third of the MHV-1 genome, but additional important virulence factors must be encoded in the genome upstream of the S gene. The severity of the pulmonary lesions observed correlates better with elevated levels of inflammatory cytokines than with viral replication in the lungs, suggesting that pulmonary disease has an important immunological component.
Assuntos
Pulmão/patologia , Pulmão/virologia , Glicoproteínas de Membrana/fisiologia , Vírus da Hepatite Murina/patogenicidade , Proteínas do Envelope Viral/fisiologia , Fatores de Virulência/fisiologia , Animais , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Feminino , Ordem dos Genes , Genes Virais , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Vírus da Hepatite Murina/genética , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Viral/química , RNA Viral/genética , Recombinação Genética , Análise de Sequência de DNA , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/genética , Fatores de Virulência/genéticaRESUMO
BACKGROUND & AIMS: This report describes the use of a novel sensitive and specific ELISA for the measurement of human fibrinogen-like protein 2 (FGL2/fibroleukin), a novel effector of natural regulatory T (Treg) cells, to predict the course of chronic hepatitis C viral infection (HCV). METHODS: Plasma levels of FGL2 were measured in HCV patients and compared to healthy controls and to patients with alcoholic liver disease. RESULTS: FGL2 levels were significantly higher in HCV patients (84.3+/-89.1 ng/ml, n=80) compared to healthy controls (36.4+/-21.9 ng/ml, n=30, p<0.001), to a subset of patients who cleared HCV following anti-viral treatment (16.6+/-19.7 ng/ml, n=32, p<0.001), and to patients with inactive alcoholic liver disease (18.8+/-17.4 ng/ml, n=24, p<0.001). Among HCV patients, plasma levels of FGL2 correlated significantly with the stage of fibrosis (p=0.001) and were significantly higher in patients with cirrhosis (164.1+121.8 ng/ml, n=60) compared to non-cirrhotics (57.7+/-52.8 ng/ml, n=20, p=0.001). Genotype 1 patients had significantly higher levels of FGL2 (98.1+/-100.3 ng/ml, n=60) compared to patients with genotype 2/3 (41.5+/-38.6 ng/ml, n=20, p=0.0008). Patients with genotype 2/3 had FGL2 levels similar to healthy controls (41.5+/-38.6 vs. 36.41+/-21.9 ng/ml, p=ns). Infiltrating lymphocytes in liver biopsies of HCV patients were positive for either FGL2 or FoxP3 (a marker of Treg cells) or expressed both markers. CONCLUSIONS: This report documents the development of a sensitive ELISA for measurement of plasma levels of FGL2 an effector Treg cells, which correlates with the severity of HCV infection.
Assuntos
Fibrinogênio/análise , Hepatite C Crônica/sangue , Hepatopatias Alcoólicas/sangue , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Fibrinogênio/imunologia , Hepatite C Crônica/imunologia , Humanos , Hepatopatias Alcoólicas/imunologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Índice de Gravidade de DoençaRESUMO
Liver transplantation is now recognized as the treatment of choice for end-stage liver failure. Its success can be attributed largely to the generation of selective immunosuppressive agents, which have resulted in a dramatic reduction in the incidence of acute rejection and improvements in the short- and long-term outcomes of patients. However, the unresolved limitation of current immunosuppressive agents is long-term toxicity, which results in increases in the incidence and severity of cardiovascular, neurological, and renal diseases. Our recent understanding of the pathways of cell activation has resulted in the development of a new generation of immunosuppressive agents that may address the challenges facing transplantation today and allow the minimization or substitution of existing agents. Furthermore, advances in our understanding of the mechanisms of tolerance and the identification of biomarker signatures hold the promise that in some patients transplantation may be able to be performed without the need for long-term immunosuppression (tolerance).
Assuntos
Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Transplante de Fígado , Rejeição de Enxerto/imunologia , Hepatite C/prevenção & controle , Humanos , Incidência , Transplante de Fígado/imunologia , Fatores de Risco , Prevenção SecundáriaRESUMO
UNLABELLED: Fulminant viral hepatitis (FH) remains an important clinical problem in which the underlying pathogenesis is not well understood. Here, we present insight into the immunological mechanisms involved in FH caused by murine hepatitis virus strain 3 (MHV-3), indicating a critical role for CD4(+)CD25(+) regulatory T cells (Tregs) and production of the novel Treg effector molecule FGL2. Before infection with MHV-3, susceptible BALB/cJ mice had increased numbers of Tregs and expression of fgl2 messenger RNA (mRNA) and FGL2 protein compared with resistant A/J mice. After MHV-3 infection, plasma levels of FGL2 in BALB/cJ mice were significantly increased, correlating with increased percentage of Tregs. Treatment with anti-FGL2 antibody completely inhibited Treg activity and protected susceptible BALB/cJ mice against MHV-3-liver injury and mortality. Adoptive transfer of wild-type Tregs into resistant fgl2(-/-) mice increased their mortality caused by MHV-3 infection, whereas transfer of peritoneal exudate macrophages had no adverse effect. CONCLUSION: This study demonstrates that FGL2 is an important effector cytokine of Tregs that contributes to susceptibility to MHV-3-induced FH. The results further suggest that targeting FGL2 may lead to the development of novel treatment approaches for acute viral hepatitis infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Fibrinogênio/imunologia , Hepatite Viral Animal/imunologia , Linfócitos T Reguladores/imunologia , Animais , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos A/imunologia , Camundongos Endogâmicos BALB C , Vírus da Hepatite Murina , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Xenografts ultimately fail as a result of acute vascular rejection (AVR), a process characterized by intravascular thrombosis, fibrin deposition, and endothelial cell activation. METHODS AND RESULTS: We studied whether targeted deletion of Fgl-2, an inducible endothelial cell procoagulant, (Fgl-2-/-) in the donor prevents AVR in a mouse-to-rat cardiac xenotransplantation model. By 3 days after transplant, Fgl-2+/+ grafts developed typical features of AVR associated with increased levels of donor Fgl-2 mRNA. Grafts from Fgl-2-/- mice had reduced fibrin deposition but developed cellular rejection. Treatment with a short course of cobra venom factor and maintenance cyclosporine resulted in long-term acceptance of both Fgl-2+/+ and Fgl-2-/- grafts. On withdrawal of cyclosporine, Fgl-2+/+ grafts developed features of AVR; in contrast, Fgl-2-/- grafts again developed acute cellular rejection. Rejecting Fgl-2+/+ hearts stained positively for IgG, IgM, C3, and C5b-9, whereas rejecting Fgl-2-/- hearts had minimal Ig and complement deposition despite xenoantibodies in the serum. Furthermore, serum containing xenoantibodies failed to stain Fgl-2-/- long-term treated hearts but did stain wild-type heart tissues. Treatment of Fgl-2-/- xenografts with mycophenolate mofetil and tacrolimus, a clinically relevant immune suppression protocol, led to long-term graft acceptance. CONCLUSIONS: Deletion of Fgl-2 ameliorates AVR by downregulation of xenoantigens and may facilitate successful clinical heart xenotransplantation.
Assuntos
Fibrinogênio/genética , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/prevenção & controle , Transplante de Coração , Imunologia de Transplantes , Animais , Anticorpos Heterófilos/sangue , Antígenos Heterófilos/genética , Proteínas do Sistema Complemento/análise , Rejeição de Enxerto/imunologia , Isotipos de Imunoglobulinas/sangue , Imunossupressores/uso terapêutico , Camundongos , Camundongos Knockout , Ratos , Transplante HeterólogoRESUMO
BACKGROUND: The growth potential of the tumor-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly linked to the nature/function of the periparasitic host immune-mediated processes. We previously showed that Fibrinogen-like-protein 2 (FGL2), a novel CD4+CD25+ Treg effector molecule, was over-expressed in the liver of mice experimentally infected with E. multilocularis. However, little is known about its contribution to the control of this chronic helminth infection. METHODS/FINDINGS: Key parameters for infection outcome in E. multilocularis-infected fgl2-/- (AE-fgl2-/-) and wild type (AE-WT) mice at 1 and 4 month(s) post-infection were (i) parasite load (i. e. wet weight of parasitic metacestode tissue), and (ii) parasite cell proliferation as assessed by determining E. multilocularis 14-3-3 gene expression levels. Serum FGL2 levels were measured by ELISA. Spleen cells cultured with ConA for 48h or with E. multilocularis Vesicle Fluid (VF) for 96h were analyzed ex-vivo and in-vitro. In addition, spleen cells from non-infected WT mice were cultured with rFGL2/anti-FGL2 or rIL-17A/anti-IL-17A for further functional studies. For Treg-immune-suppression-assays, purified CD4+CD25+ Treg suspensions were incubated with CD4+ effector T cells in the presence of ConA and irradiated spleen cells as APCs. Flow cytometry and qRT-PCR were used to assess Treg, Th17-, Th1-, Th2-type immune responses and maturation of dendritic cells. We showed that AE-fgl2-/- mice exhibited (as compared to AE-WT-animals) (a) a significantly lower parasite load with reduced proliferation activity, (b) an increased T cell proliferative response to ConA, (c) reduced Treg numbers and function, and (d) a persistent capacity of Th1 polarization and DC maturation. CONCLUSIONS: FGL2 appears as one of the key players in immune regulatory processes favoring metacestode survival by promoting Treg cell activity and IL-17A production that contributes to FGL2-regulation. Prospectively, targeting FGL2 could be an option to develop an immunotherapy against AE and other chronic parasitic diseases.
Assuntos
Equinococose Hepática/imunologia , Echinococcus multilocularis/imunologia , Fibrinogênio/imunologia , Interleucina-17/biossíntese , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD4 , Concanavalina A/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Equinococose , Equinococose Hepática/parasitologia , Fibrinogênio/genética , Citometria de Fluxo , Camundongos , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologiaRESUMO
Mounting effective innate and adaptive immune responses are critical for viral clearance and the generation of long lasting immunity. It is known that production of inhibitory factors may result in the inability of the host to clear viruses, resulting in chronic viral persistence. Fibrinogen-like protein 2 (FGL2) has been identified as a novel effector molecule of CD4(+)CD25(+) Foxp3(+) regulatory T (Treg) cells that inhibits immune activity by binding to FCγRIIB expressed primarily on antigen presenting cells (APC). In this study, we show that infection of mice with Lymphocytic Choriomeningitis Virus WE (LCMV WE) leads to increased plasma levels of FGL2, which were detected as early as 2 days post-infection (pi) and persisted until day 50 pi. Mice deficient in FGL2 (fgl2(-/-)) had increased viral titers of LCMV WE in the liver early p.i but cleared the virus by day 12 similar to wild type mice. Dendritic cells (DC) isolated from the spleens of LCMV WE infected fgl2(-/-) had increased expression of the DC maturation markers CD80 and MHC Class II compared to wild type (fgl2(+/+)). Frequencies of CD8(+) and CD4(+) T cells producing IFNγ in response to ex vivo peptide re-stimulation isolated from the spleen and lymph nodes were also increased in LCMV WE infected fgl2(-/-) mice. Increased frequencies of CD8(+) T cells specific for LCMV tetramers GP33 and NP396 were detected within the liver of fgl2(-/-) mice. Plasma from fgl2(-/-) mice contained higher titers of total and neutralizing anti-LCMV antibody. Enhanced anti-viral immunity in fgl2(-/-) mice was associated with increased levels of serum alanine transaminase (ALT), hepatic necrosis and inflammation following LCMV WE infection. These data demonstrate that targeting FGL2 leads to early increased viral replication but enhanced anti-viral adaptive T & B cell responses. Targeting FGL2 may enhance the efficacy of current anti-viral therapies for hepatotropic viruses.
Assuntos
Imunidade Adaptativa , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Fibrinogênio/imunologia , Hepatite/genética , Vírus da Coriomeningite Linfocítica/imunologia , Alanina Transaminase/sangue , Alanina Transaminase/genética , Animais , Anticorpos Neutralizantes/sangue , Linfócitos B/imunologia , Linfócitos B/virologia , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Células Dendríticas/virologia , Feminino , Fibrinogênio/genética , Deleção de Genes , Regulação da Expressão Gênica , Hepatite/imunologia , Hepatite/patologia , Hepatite/virologia , Interações Hospedeiro-Patógeno , Interferon gama/biossíntese , Interferon gama/imunologia , Camundongos , Camundongos Knockout , Transdução de Sinais , Baço/imunologia , Baço/virologia , Carga Viral , Replicação ViralRESUMO
Fibrinogen-like protein 2 (FGL2), a novel effector molecule of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), mediates its suppressive activity through binding to low affinity Fcγ receptors expressed on antigen presenting cells (APCs). FGL2 has been implicated in the pathogenesis of viral hepatitis, xeno- and allotransplant rejection, and rheumatoid arthritis. Here we fully analyzed the structure-function relationships of recombinant murine FGL2 generated in COS-7 cells and identified the receptor binding domains. Native FGL2 exists as an oligomer with a molecular weight of approximately 260 kDa, while under reducing conditions, FGL2 has a molecular weight of 65 kDa suggesting that native FGL2 is composed of four monomers. By site-directed mutation, cysteines at positions 94, 97, 184 and 187, found in the coiled-coil domain were shown to be crucial for FGL2 oligomerization. Monomeric FGL2 had a lower affinity binding to APCs, but increased immunosuppressive activity compared to oligomeric FGL2. Deglycosylation demonstrated that sugar moieties are critical for maintaining solubility of FGL2. SWISS-MODEL analysis suggested that FGL2 has a similar tertiary structure with other members of the fibrinogen family such as fibrinogen and tachylectin. Mutational analysis of cysteine residues and Western blots suggested an asymmetric bouquet-shaped quaternary structure for oligomeric FGL2, resembling many pattern-recognition molecules in the lectin pathway of innate immunity. The functional motifs of FGL2 were mapped to the C terminal globular domain, using a peptide blockade assay. These results collectively define the biochemical and immunological determinants of FGL2, an important immunosuppressive molecule of Treg providing important insights for designing FGL2-related therapeutics.
Assuntos
Fibrinogênio/farmacologia , Imunossupressores/farmacologia , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células COS , Proliferação de Células , Chlorocebus aethiops , Cisteína/química , Cisteína/genética , Feminino , Fibrinogênio/química , Fibrinogênio/fisiologia , Glicosilação , Imunossupressores/química , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Solubilidade , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/fisiologiaAssuntos
Vírus da Hepatite Murina/fisiologia , Síndrome do Desconforto Respiratório/virologia , Síndrome Respiratória Aguda Grave/virologia , Animais , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/virologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Sistema Imunitário , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Síndrome do Desconforto Respiratório/mortalidade , Síndrome Respiratória Aguda Grave/mortalidade , Distribuição TecidualRESUMO
Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide and remains the most common indication for liver transplantation. The current standard of care leads to a sustained viral response of roughly 50% of treated patients at best. Furthermore, anti-viral therapy is expensive, prolonged, and associated with serious side-effects. Evidence suggests that a poor response to treatment may be the result of a suppressed anti-viral immunity due to the presence of increased numbers and activity of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg cells). We and others have recently identified fibrinogen-like protein 2 (FGL2) as a putative effector of Treg cells, which accounts for their suppressive function through binding to Fc gamma receptors (FcγR). In an experimental model of fulminant viral hepatitis, our laboratory showed that increased plasma levels of FGL2 pre- and post-viral infection were predictive of susceptibility and severity of disease. Moreover, treatment with antibody to FGL2 fully protected susceptible animals from the lethality of the virus, and adoptive transfer of wild-type Treg cells into resistant fgl2-deficient animals accelerated their mortality post-infection. In patients with HCV infection, plasma levels of FGL2 and expression of FGL2 in the liver correlated with the course and severity of the disease. Collectively, these studies suggest that FGL2 may be used as a biomarker to predict disease progression in HCV patients and be a logical target for the development of novel therapeutic approaches for the treatment of patients with HCV infection.
RESUMO
Fibrinogen-like protein 2 (FGL2) is a multifunctional protein, which has been implicated in the pathogenesis of allograft and xenograft rejection. Previously, FGL2 was shown to inhibit maturation of BM-derived DC and T-cell proliferation. The mechanism of the immunosuppressive activity of FGL2 remains poorly elucidated. Here, we focus on identification of FGL2-specific receptor(s) and their ability to modulate APC activity and allograft survival. Using flow cytometry and surface plasmon resonance analysis, we show that FGL2 binds specifically to Fc gamma receptor (FcgammaR)IIB and FcgammaRIII receptors, which are expressed on the surface of APC, including B lymphocytes, macrophages and DC. Antibody to FcgammaRIIB and FcgammaRIII, or deficiency of these receptors, abrogated FGL2 binding. FGL2 inhibited the maturation of BMDC from FcgammaRIIB+/+ mice but not from FcgammaRIIB(-/-) mice and induced apoptosis in the FcgammaRIIB+ mouse B-cell line (A20) but not the A20IIA1.6 cell line that does not express FcgammaRIIB. Recombinant FGL2 infused into FcgammaRIIB+/+ (C57BL/6J, H-2b) mice but not FcgammaRIIB(-/-) mice inhibited rejection of fully mismatched BALB/cJ (H-2d) skin allografts. The identification of specific receptor binding has important implications for the pathogenesis of immune-mediated disease and suggests a potential for targeted FGL2 therapy.
Assuntos
Fibrinogênio/fisiologia , Tolerância Imunológica , Receptores de IgG/fisiologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Dendríticas/fisiologia , Feminino , Sobrevivência de Enxerto , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Pele/imunologia , Transplante HomólogoRESUMO
Mice with targeted deletion of fibrinogen-like protein 2 (fgl2) spontaneously developed autoimmune glomerulonephritis with increasing age, as did wild-type recipients reconstituted with fgl2-/- bone marrow. These data implicate FGL2 as an important immunoregulatory molecule and led us to identify the underlying mechanisms. Deficiency of FGL2, produced by CD4+CD25+ regulatory T cells (Treg), resulted in increased T cell proliferation to lectins and alloantigens, Th 1 polarization, and increased numbers of Ab-producing B cells following immunization with T-independent Ags. Dendritic cells were more abundant in fgl2-/- mice and had increased expression of CD80 and MHCII following LPS stimulation. Treg cells were also more abundant in fgl2-/- mice, but their suppressive activity was significantly impaired. Ab to FGL2 completely inhibited Treg cell activity in vitro. FGL2 inhibited dendritic cell maturation and induced apoptosis of B cells through binding to the low-affinity FcgammaRIIB receptor. Collectively, these data suggest that FGL2 contributes to Treg cell activity and inhibits the development of autoimmune disease.
Assuntos
Doenças Autoimunes/genética , Fibrinogênio/metabolismo , Glomerulonefrite/genética , Linfócitos T Reguladores/imunologia , Animais , Anticorpos/farmacologia , Antígenos CD/metabolismo , Doenças Autoimunes/patologia , Linfócitos B/imunologia , Peso Corporal/genética , Células Dendríticas/imunologia , Fibrinogênio/antagonistas & inibidores , Fibrinogênio/genética , Deleção de Genes , Glomerulonefrite/patologia , Linfonodos/imunologia , Camundongos , Camundongos Mutantes , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores de IgG/metabolismo , Baço/imunologia , Timo/imunologiaRESUMO
Previous studies from our laboratory have shown that fulminant hepatitis caused by the mouse hepatitis virus, MHV-3, is dependent on production of the novel immune coagulant fgl2/fibroleukin. In this study, we investigate the role of IFN-gamma and TNF-alpha in the induction of fgl2 expression and fgl2-dependent hepatic apoptosis. Infusion of IFN-gamma in combination with TNF-alpha through the portal vein of fgl2+/+ mice led to widespread hepatic apoptosis and fibrin deposition. Livers from fgl2-/- mice were normal, although strong expression of the fgl2 knockout reporter gene Lac Z was seen in both resident hepatic macrophages and endothelial cells. In vitro, IFN-gamma and TNF-alpha induced fgl2 expression in a macrophage and endothelial cell-specific manner. In macrophages (peritoneal and RAW 264.7 cells), IFN-gamma, but not IFN-alpha, LPS, TNF-alpha, or IL-1 induced fgl2 mRNA transcription and protein expression, while in endothelial cells TNF-alpha, but not IFN-gamma, induced fgl2 transcription. In addition, while TNF-alpha enhanced IFN-gamma-induced macrophage fgl2 transcription, IFN-gamma also enhanced TNF-alpha-induced endothelial cell fgl2 transcription. The induction of fgl2 by IFN-gamma in macrophages involved a STAT1-dependent pathway, involving the composite cis elements Sp1/Sp3 and GAS/PU.1. The latter interacted with IFN-gamma-dependent Sp1/Sp3, STAT1, and the ETS family of transcription factors member PU.1. The interaction of PU.1 with the IFN-gamma-activated sequence/ETS family of transcription factors site determined the macrophage-specific induction of fgl2 by IFN-gamma. Overall, this study demonstrates that IFN-gamma and TNF-alpha induce hepatocyte apoptosis in vivo, which is dependent on induction of fgl2, and defines the molecular basis of transcription of fgl2 in vitro.
Assuntos
Apoptose/imunologia , Fibrinogênio/fisiologia , Hepatócitos/citologia , Interferon gama/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Fator de Transcrição STAT1/fisiologia , Fator de Transcrição Sp1/fisiologia , Fator de Transcrição Sp3/fisiologia , Transativadores/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Apoptose/genética , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Linhagem Celular , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Fibrinogênio/biossíntese , Fibrinogênio/genética , Hepatócitos/imunologia , Hepatócitos/metabolismo , Interferon gama/administração & dosagem , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Transdução de Sinais/genética , Transdução de Sinais/imunologia , SuínosRESUMO
Severe acute respiratory syndrome (SARS) is a life-threatening infectious disease which has been difficult to study and treat because of the lack of a readily available animal model. Intranasal infection of A/J mice with the coronavirus murine hepatitis virus strain 1 (MHV-1) produced pulmonary pathological features of SARS. All MHV-1-infected A/J mice developed progressive interstitial pneumonitis, including dense macrophage infiltrates, giant cells, and hyaline membranes, resulting in death of all animals. In contrast, other mouse strains developed only mild transitory disease. Infected A/J mice had significantly higher cytokine levels, particularly macrophage chemoattractant protein 1 (MCP-1/CCL-2), gamma interferon, and tumor necrosis factor alpha. Furthermore, FGL2/fibroleukin mRNA transcripts and protein and fibrin deposits were markedly increased in the lungs of infected A/J mice. These animals developed a less robust type I interferon response to MHV-1 infection than resistant C57BL/6J mice, and treatment with recombinant beta interferon improved survival. This study describes a potentially useful small animal model of human SARS, defines its pathogenesis, and suggests treatment strategies.