RESUMO
The level of CD40 expression on dendritic cells (DCs) plays a decisive role in disease protection during Leishmania donovani (LD) infection. However, current understanding of the molecular regulation of CD40 expression remains elusive. Using molecular, cellular and functional approaches, we identified a role for Runx1 and Runx3 transcription factors in the regulation of CD40 expression in DCs. In response to lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα) or antileishmanial drug sodium antimony gluconate (SAG), both Runx1 and Runx3 translocated to the nucleus, bound to the CD40 promoter and upregulated CD40 expression on DCs. These activities of Runx proteins were mediated by the upstream phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Notably, LD infection attenuated LPS- or TNFα-induced CD40 expression in DCs by inhibiting PI3K-Akt-Runx axis via protein tyrosine phosphatase SHP-1. In contrast, CD40 expression induced by SAG was unaffected by LD infection, as SAG by blocking LD-induced SHP-1 activation potentiated PI3K-Akt signaling to drive Runx-mediated CD40 upregulation. Adoptive transfer experiments further showed that Runx1 and Runx3 play a pivotal role in eliciting antileishmanial immune response of SAG-treated DCs in vivo by promoting CD40-mediated type-1 T cell responses. Importantly, antimony-resistant LD suppressed SAG-induced CD40 upregulation on DCs by blocking the PI3K-Akt-Runx pathway through sustained SHP-1 activation. These findings unveil an immunoregulatory role for Runx proteins during LD infection.
Assuntos
Antígenos CD40/imunologia , Subunidades alfa de Fatores de Ligação ao Core/imunologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Leishmaniose Visceral/imunologia , Animais , Antígenos CD40/biossíntese , Cricetinae , Humanos , Leishmania donovani/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Trafficking of dendritic cells (DCs) from peripheral tissues to draining lymph nodes is a prerequisite for induction of adaptive immunity. An immunosuppressive cytokine transforming growth factor (TGF)-ß1, however, inhibits migration of DCs by downregulating the expression of chemokine receptor CCR-7. Whether TGF-ß1 engages any other receptor to mediate this inhibitory effect is currently unknown. In this article, we report that TGF-ß1 attenuated the lymph node homing ability of mouse DCs by reducing C-type lectin receptor-2 (CLEC-2) expression. Notably, TGF-ß1 inhibited CLEC-2 expression in DCs via c-Src. DCs silenced for c-Src were resistant to TGF-ß1-induced inhibition of CLEC-2 expression. Furthermore, silencing of c-Src substantially improved the lymph node homing capacity of TGF-ß1-treated DCs by restoring CLEC-2 expression. These results document a critical role for c-Src and CLEC-2 in TGF-ß1-mediated impairment of DC migration and define a previously unknown mechanism by which TGF-ß1 attenuates the lymph node homing ability of DCs.
Assuntos
Células Dendríticas/metabolismo , Regulação para Baixo/fisiologia , Lectinas Tipo C/metabolismo , Linfonodos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Domínios de Homologia de src/fisiologiaRESUMO
The enhanced expression of T cell Ig and mucin protein-3 (TIM-3) on tumor-associated dendritic cells (DCs) attenuates antitumor effects of DNA vaccines. To identify a potential target (or targets) for reducing TIM-3 expression on tumor-associated DCs, we explored the molecular mechanisms regulating TIM-3 expression. In this study, we have identified a novel signaling pathway (c-SrcâBruton's tyrosine kinaseâtranscription factors Ets1, Ets2, USF1, and USF2) necessary for TIM-3 upregulation on DCs. Both IL-10 and TGF-ß, which are produced in the tumor microenvironment, upregulated TIM-3 expression on DCs via this pathway. Suppressed expression of c-Src or downstream Bruton's tyrosine kinase, Ets1, Ets2, USF1, or USF2 blocked IL-10- and TGF-ß-induced TIM-3 upregulation on DCs. Notably, in vivo knockdown of c-Src in mice reduced TIM-3 expression on tumor-associated DCs. Furthermore, adoptive transfer of c-Src-silenced DCs in mouse tumors enhanced the in vivo antitumor effects of immunostimulatory CpG DNA; however, TIM-3 overexpression in c-Src-silenced DCs blocked this effect. Collectively, our data reveal the molecular mechanism regulating TIM-3 expression in DCs and identify c-Src as a target for improving the efficacy of nucleic acid-mediated anticancer therapy.
Assuntos
Células Dendríticas/imunologia , Genes src , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Neoplasias/imunologia , Linfócitos T/imunologia , Quinases da Família src/metabolismo , Transferência Adotiva , Tirosina Quinase da Agamaglobulinemia , Animais , Proteína Tirosina Quinase CSK , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Receptor Celular 2 do Vírus da Hepatite A/genética , Interleucina-10/imunologia , Interleucina-10/metabolismo , Camundongos , Neoplasias/metabolismo , Oligodesoxirribonucleotídeos/imunologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
The receptor T cell Ig and mucin protein-3 (TIM-3) has emerged as an important regulator of innate immune responses. However, whether TIM-3-induced signaling promotes or inhibits the activation and maturation of dendritic cells (DCs) still remains uncertain. In addition, the TIM-3 signaling events involved in this immunoregulatory function are yet to be established. In this article, we report that TIM-3 crosslinking by anti-TIM-3 Ab inhibited DC activation and maturation by blocking the NF-κB pathway. After Ab-mediated crosslinking, TIM-3 became tyrosine phosphorylated, which then sequentially bound and activated the nonreceptor tyrosine kinases Bruton's tyrosine kinase (Btk) and c-Src. Activation of Btk-c-Src signaling in turn triggered the secretion of some inhibitory factor (or factors) from DCs that inhibited the NF-κB pathway and subsequent activation and maturation of DCs. Silencing of Btk or c-Src abrogated the inhibitory effects of TIM-3 on DCs. These results demonstrate an essential role for Btk-c-Src signaling in TIM-3-induced DC suppression. Thus, in addition to demonstrating an inhibitory role for TIM-3 signaling in DC activation, we define the molecular mechanism by which TIM-3 mediates this effect.
Assuntos
Células Dendríticas/imunologia , Proteínas Tirosina Quinases/imunologia , Receptores Virais/imunologia , Transdução de Sinais/imunologia , Quinases da Família src/imunologia , Tirosina Quinase da Agamaglobulinemia , Animais , Proteína Tirosina Quinase CSK , Células Dendríticas/citologia , Inativação Gênica , Receptor Celular 2 do Vírus da Hepatite A , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/genética , NF-kappa B/imunologia , Proteínas Tirosina Quinases/genética , Receptores Virais/genética , Transdução de Sinais/genética , Quinases da Família src/genéticaRESUMO
The outcome of the disease visceral leishmaniasis (VL), caused by Leishmania donovani (LD), largely relies on the relative dominance of host-protective type-1 T helper (Th1) cell response versus disease-promoting type-2 T helper (Th2) cell response. The Th1 and Th2 responses, in turn, are believed to be elicited by type-1 conventional dendritic cells (cDC1) and type-2 conventional DCs (cDC2), respectively. However, it is still unknown which DC subtype (cDC1 or cDC2) predominates during chronic LD infection and the molecular mechanism governing such occurrence. Here we report that in chronically infected mice, the splenic cDC1-cDC2 balance shifted toward the cDC2 subtype and that the receptor T cell immunoglobulin and mucin protein-3 (TIM-3) expressed by DCs played a key role in mediating this effect. Transfer of TIM-3-silenced DCs in fact prevented the predominance of the cDC2 subtype in mice with chronic LD infection. We also found that LD actually upregulated TIM-3 expression on DCs by triggering a TIM-3-mediated signaling pathway STAT3 (signal transducer and activator of transcription 3)âinterleukin (IL)-10âc-Srcâtranscription factors Ets1, Ets2, USF1, and USF2. Notably, TIM-3 promoted STAT3 activation via a non-receptor tyrosine kinase Btk. Adoptive transfer experiments further demonstrated a critical role for STAT3-driven TIM-3 upregulation on DCs in increasing cDC2 abundance in chronically infected mice, which ultimately aided disease pathogenesis by augmenting Th2 responses. These findings document a new immunoregulatory mechanism contributing to disease pathology during LD infection and define TIM-3 as a key mediator of this process.
Assuntos
Receptor Celular 2 do Vírus da Hepatite A , Leishmania donovani , Animais , Camundongos , Células Dendríticas/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
To initiate an antileishmanial adaptive immune response, dendritic cells (DCs) must carry Leishmania antigens from peripheral tissues to local draining lymph nodes. However, the migratory capacity of DCs is largely compromised during Leishmania donovani infection. The molecular mechanism underlying this defective DC migration is not yet fully understood. Here, we demonstrate that L. donovani infection impaired the lymph node homing ability of DCs by decreasing C-type lectin receptor 2 (CLEC-2) expression. L. donovani exerted this inhibitory effect by inducing transforming growth factor-ß (TGF-ß) secretion from DCs. Indeed, TGF-ß produced in this manner inhibited nuclear factor-κB (NF-κB)-mediated CLEC-2 expression on DCs by activating c-Src. Notably, suppression of c-Src expression significantly improved the arrival of DCs in draining lymph nodes by preventing L. donovani-induced CLEC-2 downregulation on DCs. These findings reveal a unique mechanism by which L. donovani inhibits DC migration to lymph nodes and suggest a key role for TGF-ß, c-Src, and CLEC-2 in regulating this process. IMPORTANCE Dendritic cells (DCs) play a key role in initiating T cell-mediated protective immunity against visceral leishmaniasis (VL), the second most lethal parasitic disease in the world. However, the T cell-inducing ability of DCs critically depends on the extent of DC migration to regional lymph nodes. Notably, the migration of DCs is reported to be impaired during VL. The cause of this impaired DC migration, however, remains ill-defined. Here, we provide the first evidence that L. donovani, the causative agent of VL, attenuates the lymph node homing capacity of DCs by decreasing C-type lectin receptor 2 (CLEC-2) expression on DCs. Additionally, we have demonstrated how L. donovani mediates this inhibitory effect. Overall, our work has revealed a unique mechanism underlying L. donovani-induced impairment of DC migration and suggests a potential strategy to improve antileishmanial T cell activity by increasing DC arrival in lymph nodes.
Assuntos
Antiprotozoários , Leishmania donovani , Leishmaniose Visceral , Humanos , Leishmania donovani/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Linfonodos/metabolismo , Células Dendríticas , Fatores de Crescimento Transformadores/metabolismoRESUMO
The limited success of dendritic cell (DC)-based immunotherapy in multiple myeloma is partly due to hepatocyte growth factor (HGF)-induced DC dysfunction. From a therapeutic standpoint, it is important to understand the molecular events involved in inhibition of DC activation/maturation by HGF. Because Bruton's tyrosine kinase (Btk) negatively regulates maturation and immunostimulatory function of DCs, a role for Btk in HGF-induced inhibition of both murine and human DCs was investigated. We demonstrate that Btk is a novel proximal component of HGF-induced c-MET (HGF receptor) signaling. Following HGF treatment, Btk binds to c-MET and becomes activated. Btk activation in turn blocks the NF-κB pathway and subsequent DC activation via the c-Src-PI3K-AKT-mammalian target of rapamycin (mTOR) pathway. Notably, Btk activation is necessary for HGF-induced association of c-Src and PI3K with c-MET. Furthermore, we provide the first evidence that HGF inhibits DC activation by inducing autocrine interleukin (IL)-10 secretion, which requires activation of Btk. Blocking activation of Btk and its downstream the c-Src-PI3K-AKT-mTOR pathway prevents HGF-induced IL-10 secretion by DCs. In addition, neutralization of IL-10 secretion from DCs impaired the inhibitory effect of HGF on DCs. Thus, our study identifies a novel role for Btk in HGF-induced DC inhibition.
Assuntos
Células Dendríticas/imunologia , Fator de Crescimento de Hepatócito/imunologia , Proteínas Tirosina Quinases/imunologia , Tirosina Quinase da Agamaglobulinemia , Animais , Proteína Tirosina Quinase CSK , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Ativação Enzimática/imunologia , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Interleucina-10/imunologia , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/imunologia , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-met/imunologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Serina-Treonina Quinases TOR/imunologia , Serina-Treonina Quinases TOR/metabolismo , Quinases da Família srcRESUMO
The inability of sodium antimony gluconate (SAG)-unresponsive kala-azar patients to clear Leishmania donovani (LD) infection despite SAG therapy is partly due to an ill-defined immune-dysfunction. Since dendritic cells (DCs) typically initiate anti-leishmanial immunity, a role for DCs in aberrant LD clearance was investigated. Accordingly, regulation of SAG-induced activation of murine DCs following infection with LD isolates exhibiting two distinct phenotypes such as antimony-resistant (Sb(R)LD) and antimony-sensitive (Sb(S)LD) was compared in vitro. Unlike Sb(S)LD, infection of DCs with Sb(R)LD induced more IL-10 production and inhibited SAG-induced secretion of proinflammatory cytokines, up-regulation of co-stimulatory molecules and leishmanicidal effects. Sb(R)LD inhibited these effects of SAG by blocking activation of PI3K/AKT and NF-kappaB pathways. In contrast, Sb(S)LD failed to block activation of SAG (20 microg/ml)-induced PI3K/AKT pathway; which continued to stimulate NF-kappaB signaling, induce leishmanicidal effects and promote DC activation. Notably, prolonged incubation of DCs with Sb(S)LD also inhibited SAG (20 microg/ml)-induced activation of PI3K/AKT and NF-kappaB pathways and leishmanicidal effects, which was restored by increasing the dose of SAG to 40 microg/ml. In contrast, Sb(R)LD inhibited these SAG-induced events regardless of duration of DC exposure to Sb(R)LD or dose of SAG. Interestingly, the inhibitory effects of isogenic Sb(S)LD expressing ATP-binding cassette (ABC) transporter MRPA on SAG-induced leishmanicidal effects mimicked that of Sb(R)LD to some extent, although antimony resistance in clinical LD isolates is known to be multifactorial. Furthermore, NF-kappaB was found to transcriptionally regulate expression of murine gammaglutamylcysteine synthetase heavy-chain (mgammaGCS(hc)) gene, presumably an important regulator of antimony resistance. Importantly, Sb(R)LD but not Sb(S)LD blocked SAG-induced mgammaGCS expression in DCs by preventing NF-kappaB binding to the mgammaGCS(hc) promoter. Our findings demonstrate that Sb(R)LD but not Sb(S)LD prevents SAG-induced DC activation by suppressing a PI3K-dependent NF-kappaB pathway and provide the evidence for differential host-pathogen interaction mediated by Sb(R)LD and Sb(S)LD.
Assuntos
Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/farmacologia , Células Dendríticas , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral , Animais , Cricetinae , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/parasitologia , Resistência a Medicamentos , Feminino , Expressão Gênica/efeitos dos fármacos , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Interações Hospedeiro-Parasita/efeitos dos fármacos , Interleucina-10/metabolismo , Leishmania donovani/crescimento & desenvolvimento , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/imunologiaRESUMO
An immunological hallmark of visceral leishmaniasis (VL), caused by Leishmania donovani, is profound immunosuppression. However, the molecular basis for this immune dysfunction has remained ill defined. Since dendritic cells (DCs) normally initiate antileishmanial immune responses, we investigated whether DCs are dysregulated during L. donovani infection and assessed its role in immunosuppression. Accordingly, we determined the regulatory effect of L. donovani on DCs. Notably, it is still unclear whether L. donovani activates or suppresses DCs. In addition, the molecular mechanism and the relevant receptor (or receptors) mediating the immunoregulatory effect of L. donovani on DCs are largely undefined. Here, we report that L. donovani inhibited DC activation/maturation by transmitting inhibitory signals through the T cell immunoglobulin and mucin protein-3 (TIM-3) receptor and thereby suppressed antileishmanial immune responses. L. donovani in fact triggered TIM-3 phosphorylation in DCs, which in turn recruited and activated a nonreceptor tyrosine kinase, Btk. Btk then inhibited DC activation/maturation by suppressing the NF-κB pathway in an interleukin-10 (IL-10)-dependent manner. Treatment with TIM-3-specific blocking antibody or suppressed expression of TIM-3 or downstream effector Btk made DCs resistant to the inhibitory effects of L. donovani. Adoptive transfer experiments further demonstrated that TIM-3-mediated L. donovani-induced inhibition of DCs plays a crucial role in the suppression of the antileishmanial immune response in vivo. These findings identify TIM-3 as a new regulator of the antileishmanial immune response and demonstrate a unique mechanism for host immunosuppression associated with L. donovani infection. IMPORTANCE Visceral leishmaniasis (VL), a poverty-related disease caused by Leishmania donovani, is ranked by the World Health Organization as the second largest killer parasitic disease in the world. The protective immune response against VL is primarily regulated by dendritic cells (DCs), which upon activation/maturation initiate an antileishmanial immune response. However, it remains obscure whether L. donovani promotes or inhibits DC activation. In addition, the receptor through which L. donovani exerts immunoregulatory effect on DCs is ill defined. Here, we for the first time report that L. donovani inhibits DC activation and maturation via the T cell immunoglobulin and mucin protein-3 (TIM-3) receptor and thereby attenuates the capacity of DCs to trigger antileishmanial immune responses in vivo. In fact, we demonstrate here that suppression of TIM-3 expression in DCs augments antileishmanial immunity. Our study uncovers a unique mechanism by which L. donovani subverts host immune responses and suggests TIM-3 as a potential new target for immunotherapy against VL.
Assuntos
Antiprotozoários , Leishmania donovani , Leishmaniose Visceral , Animais , Células Dendríticas , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Imunoglobulinas/metabolismo , Imunoglobulinas/uso terapêutico , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Mucinas/metabolismoRESUMO
Bilirubin is regarded as a toxic waste, produced from heme degradation and also acts as a potentially important antioxidant. Bilirubin causes arrest in cell cycle and lead to lesser occurrence of malignancies in individuals, having normal or slender increase in levels of serum bilirubin. Prompted by the dynamic interaction between bilirubin and bovine serum albumin (BSA), bilirubin-BSA complex was explored as a biocompatible cap system for protease responsive delivery device of anticancer drug against colon cancer. Bilirubin, conjugated to the amine terminated and doxorubicin loaded mesoporous silica nanoparticles were employed as a novel formulation against colon carcinoma cells. Compared to doxorubicin only, bilirubin in combination with doxorubicin-loaded mesoporous silica nanoparticle significantly inhibits tumor cell growth as assessed in MC-38 (murine) and HCT-116 (human) colon cancer cells. Bilirubin-doxorubicin combination potently inhibits proliferation of tumor cells and acted as cytotoxic and pro-apoptotic agent in vitro. Our result demonstrates that this novel cap system could play a precise role in defense against colon cancer by interrupting the pro-cancerogenic survival pathways during carcinogenesis.
Assuntos
Quimiocinas CC/metabolismo , Leishmania/metabolismo , Animais , Cálcio/metabolismo , Quimiocina CCL2/metabolismo , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5 , Quimiotaxia , Interações Hospedeiro-Parasita , Humanos , Leishmania/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Mimetismo Molecular , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Hepatocyte growth factor modulates activation and antigen-presenting cell function of dendritic cells. However, the molecular basis for immunoregulation of dendritic cells by hepatocyte growth factor is undefined. In the current study, we demonstrate that hepatocyte growth factor exhibits inhibitory effect on dendritic cell activation by blocking IκB kinase activity and subsequent nuclear factor-κB activation. Inhibition of IκB kinase is mediated by hepatocyte growth factor-induced activation of c-Src. Proximal signaling events induced in dendritic cells by hepatocyte growth factor include a physical association of c-Src with the hepatocyte growth factor receptor c-MET and concomitant activation of c-Src. Activation of c-Src in turn establishes a complex consisting of phosphatidylinositol 3-kinase and c-MET, and promotes downstream activation of the phosphatidylinositol 3-kinase/AKT pathway and mammalian target of rapamycin. Blocking activation of c-Src, phosphatidylinositol 3-kinase and mammalian target of rapamycin prevents hepatocyte growth factor-induced inhibition of IκB kinase, nuclear factor-κB and dendritic cell activation. Notably, hepatocyte growth factor-stimulated c-Src activation results in induction of phosphatidylinositol 3-kinase complexes p85α/p110α and p85α/p110δ, which is required for activation of mammalian target of rapamycin, and consequent inhibition of IκB kinase and nuclear factor-κB activation. Our findings, for the first time, have identified the c-Src-phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway that plays a pivotal role in mediating the inhibitory effects of hepatocyte growth factor on dendritic cell activation by blocking nuclear factor-κB signaling.
Assuntos
Células Dendríticas/enzimologia , Fator de Crescimento de Hepatócito/farmacologia , Quinase I-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Proteína Tirosina Quinase CSK , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Transfecção , Quinases da Família srcRESUMO
In the recent past the standard treatment of kala-azar involved the use of pentavalent antimonials Sb(V). Because of progressive rise in treatment failure to Sb(V) was limited its use in the treatment program in the Indian subcontinent. Until now the mechanism of action of Sb(V) is not very clear. Recent studies indicated that both parasite and hosts contribute to the antimony efflux mechanism. Interestingly, antimonials show strong immunostimulatory abilities as evident from the upregulation of transplantation antigens and enhanced T cell stimulating ability of normal antigen presenting cells when treated with Sb(V) in vitro. Recently, it has been shown that some of the peroxovanadium compounds have Sb(V)-resistance modifying ability in experimental infection with Sb(V) resistant Leishmania donovani isolates in murine model. Thus, vanadium compounds may be used in combination with Sb(V) in the treatment of Sb(V) resistance cases of kala-azar.
RESUMO
Self-antigens expressed by apoptotic cells (ACs) may become targets for autoimmunity. Tolerance to these antigens is partly established by an ill-defined capacity of ACs to inhibit antigen-presenting cells such as dendritic cells (DCs). We present evidence that the receptor tyrosine kinase Mer (MerTK) has a key role in mediating AC-induced inhibition of DC activation/maturation. Pretreatment of DCs prepared from nonobese diabetic (NOD) mice with AC blocked secretion of proinflammatory cytokines, up-regulation of costimulatory molecule expression, and T cell activation. The effect of ACs on DCs was dependent on Gas6, which is a MerTK ligand. NOD DCs lacking MerTK expression (NOD.MerTK(KD/KD)) were resistant to AC-induced inhibition. Notably, autoimmune diabetes was exacerbated in NOD.MerTK(KD/KD) versus NOD mice expressing the transgenic BDC T cell receptor. In addition, beta cell-specific CD4(+) T cells adoptively transferred into NOD.MerTK(KD/KD) mice in which beta cell apoptosis was induced with streptozotocin exhibited increased expansion and differentiation into type 1 T cell effectors. In both models, the lack of MerTK expression was associated with an increased frequency of activated pancreatic CD11c(+)CD8alpha(+) DCs, which exhibited an enhanced T cell stimulatory capacity. These findings demonstrate that MerTK plays a critical role in regulating self-tolerance mediated between ACs, DCs, and T cells.
Assuntos
Apoptose , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Animais , Antígeno CD11c/biossíntese , Antígenos CD8/biossíntese , Separação Celular , Células Dendríticas , Diabetes Mellitus Experimental , Citometria de Fluxo , Tolerância Imunológica , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Modelos Biológicos , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Transgenes , Cicatrização , c-Mer Tirosina QuinaseRESUMO
Dendritic cells (DCs) play a key role in immune homeostasis and maintenance of self-tolerance. Tolerogenic DCs can be established by an encounter with apoptotic cells (ACs) and subsequent inhibition of maturation and effector functions. The receptor(s) and signaling pathway(s) involved in AC-induced inhibition of DCs have yet to be defined. We demonstrate that pretreatment with apoptotic but not necrotic cells inhibits activation of IkappaB kinase (IKK) and downstream NF-kappaB. Notably, receptor tyrosine kinase Mer (MerTK) binding of ACs is required for mediating this effect. Monocyte-derived DCs lacking MerTK expression (MerTKKD) or treated with blocking MerTK-specific antibodies (Abs) are resistant to AC-induced inhibition and continue to activate NF-kappaB and secrete proinflammatory cytokines. Blocking MerTK activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway prevents AC-induced inhibition. These results demonstrate an essential role for MerTK-mediated regulation of the PI3K/AKT and NF-kappaB pathways in AC-induced inhibition of monocyte-derived DCs.
Assuntos
Apoptose/imunologia , Células Dendríticas/imunologia , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Animais , Anticorpos/imunologia , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Fosfatidilinositol 3-Quinases/imunologia , Transdução de Sinais/imunologia , c-Mer Tirosina QuinaseRESUMO
EBV is associated with systemic lupus erythematosus (SLE), but how it might contribute to the etiology is not clear. Since EBV-encoded latent membrane protein 2A (LMP2A) interferes with normal B cell differentiation and function, we sought to determine its effect on B cell tolerance. Mice transgenic for both LMP2A and the Ig transgene 2-12H specific for the ribonucleoprotein Smith (Sm), a target of the immune system in SLE, develop a spontaneous anti-Sm response. LMP2A allows anti-Sm B cells to overcome the regulatory checkpoint at the early preplasma cell stage by a self-Ag-dependent mechanism. LMP2A induces a heightened sensitivity to TLR ligand stimulation, resulting in increased proliferation or Ab-secreting cell differentiation or both. Thus, we propose a model whereby LMP2A induces hypersensitivity to TLR stimulation, leading to activation of anti-Sm B cells through the BCR/TLR pathway. These data further implicate TLRs in the etiology of SLE and suggest a mechanistic link between EBV infection and SLE.
Assuntos
Linfócitos B/imunologia , Hipersensibilidade/imunologia , Ativação Linfocitária/imunologia , Receptores Toll-Like/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular , Movimento Celular , Proliferação de Células , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Fenótipo , Ribonucleoproteínas/imunologia , Baço/citologia , Baço/imunologiaRESUMO
Pentavalent antimony complexes, such as sodium stibogluconate and sodium antimony gluconate (SAG), are still the first choice for chemotherapy against various forms of leishmaniasis, including visceral leishmaniasis, or kala-azar. Although the requirement of a somewhat functional immune system for the antileishmanial action of antimony was reported previously, the cellular and molecular mechanism of action of SAG was not clear. Herein, we show that SAG induces extracellular signal-regulated kinase 1 (ERK-1) and ERK-2 phosphorylation through phosphoinositide 3-kinase (PI3K), protein kinase C, and Ras activation and p38 mitogen-activated protein kinase (MAPK) phosphorylation through PI3K and Akt activation. ERK-1 and ERK-2 activation results in an increase in the production of reactive oxygen species (ROS) 3 to 6 h after SAG treatment, while p38 MAPK activation and subsequent tumor necrosis factor alpha release result in the production of nitric oxide (NO) 24 h after SAG treatment. Thus, this study has provided the first evidence that SAG treatment induces activation of some important components of the intracellular signaling pathway, which results in an early wave of ROS-dependent parasite killing and a stronger late wave of NO-dependent parasite killing. This opens up the possibility of this metalloid chelate being used in the treatment of various diseases either alone or in combination with other drugs and vaccines.
Assuntos
Gluconato de Antimônio e Sódio/farmacologia , Leishmania donovani/imunologia , Macrófagos Peritoneais/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase Tipo II/análise , Fosfatidilinositol 3-Quinases/metabolismo , Espécies Reativas de Oxigênio/análise , Animais , Citocinas/análise , Ativação Enzimática/efeitos dos fármacos , Homozigoto , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The paradigm of tolerogenic/immature versus inflammatory/mature dendritic cells has dominated the recent literature regarding the role of these antigen-presenting cells in mediating immune homeostasis or self-tolerance and response to pathogens, respectively. This issue is further complicated by the identification of distinct subtypes of dendritic cells that exhibit different antigen-presenting cell effector functions. The discovery of pathogen-associated molecular patterns and toll-like receptors provides the mechanistic basis for dendritic cell recognition of specific pathogens and induction of appropriate innate and adaptive immune responses. Only recently has insight been gained into how dendritic cells contribute to establishing and/or maintaining immunological tolerance to self. Soluble and cellular mediators have been reported to effectively regulate the function of dendritic cells by inducing several outcomes ranging from non-inflammatory dendritic cells that lack the ability to induce T lymphocyte activation to dendritic cells that actively suppress T lymphocyte responses. A thorough discussion of these stimuli and their outcomes is essential to understanding the potential for modulating dendritic cell function in the treatment of inflammatory disease conditions.
Assuntos
Células Dendríticas/imunologia , Imunidade Celular/imunologia , Animais , Apoptose/imunologia , Células Dendríticas/citologia , Humanos , Fatores Imunológicos/fisiologia , Linfócitos T/imunologiaRESUMO
We have recently demonstrated that dendritic cells (DC) prepared from nonobese diabetic (NOD) mice, a spontaneous model for insulin-dependent diabetes mellitus, exhibit elevated levels of NF-kappaB activation upon stimulation. In the current study, we investigated the influence of dysregulation of NF-kappaB activation on the APC function of bone marrow-derived DC prepared from NOD vs BALB/c and nonobese diabetes-resistant mice. NOD DC pulsed with either peptide or virus were found to be more efficient than BALB/c DC at stimulating in vitro naive Ag-specific CD8+ T cells. The T cell stimulatory capacity of NOD DC was suppressed by gene transfer of a modified form of IkappaBalpha, indicating a direct role for NF-kappaB in this process. Furthermore, neutralization of IL-12(p70) to block autocrine-mediated activation of DC also significantly reduced the capacity of NOD DC to stimulate T cells. Despite a reduction in low molecular mass polypeptide-2 expression relative to BALB/c DC, no effect on proteasome-dependent events associated with the NF-kappaB signaling pathway or Ag processing was detected in NOD DC. Finally, DC from nonobese diabetes-resistant mice, a strain genotypically similar to NOD yet disease resistant, resembled BALB/c and not NOD DC in terms of the level of NF-kappaB activation, secretion of IL-12(p70) and TNF-alpha, and the capacity to stimulate T cells. Therefore, elevated NF-kappaB activation and enhanced APC function are specific for the NOD genotype and correlate with the progression of insulin-dependent diabetes mellitus. These results also provide further evidence indicating a key role for NF-kappaB in regulating the APC function of DC.
Assuntos
Cisteína Endopeptidases , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Proteínas I-kappa B , NF-kappa B/metabolismo , Animais , Anticorpos/farmacologia , Apresentação de Antígeno , Antígenos Virais/imunologia , Células Cultivadas , Citocinas/biossíntese , Proteínas de Ligação a DNA/metabolismo , Feminino , Interleucina-12/antagonistas & inibidores , Interleucina-12/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Inibidor de NF-kappaB alfa , Orthomyxoviridae/imunologia , Peptídeos/imunologia , Biossíntese de Proteínas , Especificidade da Espécie , Linfócitos T Citotóxicos/imunologiaRESUMO
Interleukin-10 (IL-10) has potent immunoregulatory effects on the maturation and the antigen-presenting cell (APC) function of dendritic cells (DCs). The molecular basis underlying these effects in DCs, however, is ill defined. It is well established that the transcription factor NF-kappaB is a key regulator of DC development, maturation, and APC function. This study was initiated to determine the effects of IL-10 on the NF-kappaB signaling pathway in immature DCs. IL-10 pretreatment of myeloid DCs cultured from bone marrow resulted in reduced DNA binding and nuclear translocation of NF-kappaB after anti-CD40 antibody or lipopolysaccharide (LPS) stimulation. Furthermore, inhibited NF-kappaB activation was characterized by reduced degradation, phosphorylation, or both of IkappaBalpha and IkappaBepsilon but not IkappaBbeta and by reduced phosphorylation of Ser536, located in the trans-activation domain of p65. Notably, IL-10-mediated inhibition of NF-kappaB coincided with suppressed IkappaB kinase (IKK) activity in vitro. Furthermore, IL-10 blocked inducible Akt phosphorylation, and inhibitors of phosphatidylinositol 3-kinase (PI3K) effectively suppressed the activation of Akt, IKK, and NF-kappaB. These findings demonstrate that IL-10 targets IKK activation in immature DCs and that suppressing the PI3K pathway in part mediates blockade of the pathway.