RESUMO
BACKGROUND: Allergic contact dermatitis (CD) is a chronic inflammatory skin disease caused by type 1 biased adaptive immunity for which there is an unmet need for antigen (Ag)-specific immunotherapies. Exposure to skin sensitizers stimulates secretion of the proinflammatory neuropeptides substance P and hemokinin 1, which signal via the neurokinin-1 receptor (NK1R) to promote the innate and adaptive immune responses of CD. Accordingly, mice lacking the NK1R develop impaired CD. Nonetheless, the role and therapeutic opportunities of targeting the NK1R in CD remain to be elucidated. OBJECTIVE: We sought to develop an Ag-specific immunosuppressive approach to treat CD by skin codelivery of hapten and NK1R antagonists integrated in dissolvable microneedle arrays (MNA). METHODS: In vivo mouse models of contact hypersensitivity and ex vivo models of human skin were used to delineate the effects and mechanisms of NK1R signaling and the immunosuppressive effects of the contact sensitizer NK1R antagonist MNA in CD. RESULTS: We demonstrated in mice that CD requires NK1R signaling by substance P and hemokinin 1. Specific deletion of the NK1R in keratinocytes and dendritic cells, but not in mast cells, prevented CD. Skin codelivery of hapten or Ag MNA inhibited neuropeptide-mediated skin inflammation in mouse and human skin, promoted deletion of Ag-specific effector T cells, and increased regulatory T cells, which prevented CD onset and relapses locally and systemically in an Ag-specific manner. CONCLUSIONS: Immunoregulation by engineering localized skin neuroimmune networks can be used to treat cutaneous diseases that like CD are caused by type 1 immunity.
Assuntos
Dermatite Alérgica de Contato , Antagonistas dos Receptores de Neurocinina-1 , Animais , Dermatite Alérgica de Contato/tratamento farmacológico , Haptenos , Camundongos , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Receptores da Neurocinina-1 , Substância PRESUMO
There is increasing evidence of the relevant connection and regulation between the gut and skin immune axis. In fact, oral administration of lipoteichoic acid (LTA) from Lactobacillus rhamnosus GG (LGG) prevents the development of UV-induced skin tumors in chronically exposed mice. Here we aim to evaluate whether this LTA is able to revert UV-induced immunosuppression as a mechanism involved in its anti-tumor effect and whether it has an immunotherapeutic effect against cutaneous squamous cell carcinoma. Using a mouse model of contact hypersensitivity, we demonstrate that LTA overcomes UV-induced skin immunosuppression. This effect was in part achieved by modulating the phenotype of lymph node resident dendritic cells (DC) and the homing of skin migratory DC. Importantly, oral LTA reduced significantly the growth of established skin tumors once UV radiation was discontinued, demonstrating that it has a therapeutic, besides the already demonstrated preventive antitumor effect. The data presented here strongly indicates that oral administration of LTA represents a promising immunotherapeutic approach for different conditions in which the skin immune system is compromised.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Lacticaseibacillus rhamnosus/química , Lipopolissacarídeos/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Ácidos Teicoicos/farmacologia , Raios Ultravioleta/efeitos adversos , Administração Oral , Animais , Antineoplásicos/isolamento & purificação , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Movimento Celular/efeitos da radiação , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/efeitos da radiação , Dermatite de Contato/genética , Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Feminino , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/patologia , Trato Gastrointestinal/efeitos da radiação , Lipopolissacarídeos/isolamento & purificação , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/patologia , Linfonodos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologia , Pele/efeitos da radiação , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Ácidos Teicoicos/isolamento & purificaçãoRESUMO
BACKGROUND: Efficient development of atopic diseases requires interactions between allergen and adjuvant to initiate and amplify the underlying inflammatory responses. Substance P (SP) and hemokinin-1 (HK-1) are neuropeptides that signal through the neurokinin-1 receptor (NK1R) to promote inflammation. Mast cells initiate the symptoms and tissue effects of atopic disorders, secreting TNF and IL-6 after FcεRI cross-linking by antigen-IgE complexes (FcεRI-activated mast cells [FcεRI-MCs]). Additionally, MCs express the NK1R, suggesting an adjuvant role for NK1R agonists in FcεRI-MC-mediated pathologies; however, in-depth research addressing this relevant aspect of MC biology is lacking. OBJECTIVE: We sought to investigate the effect of NK1R signaling and the individual roles of SP and HK-1 as potential adjuvants for FcεRI-MC-mediated allergic disorders. METHODS: Bone marrow-derived mast cells (BMMCs) from C57BL/6 wild-type (WT) or NK1R(-/-) mice were used to investigate the effects of NK1R signaling on FcεRI-MCs. BMMCs generated from Tac1(-/-) mice or after culture with Tac4 small interfering RNA were used to address the adjuvancy of SP and HK-1. WT, NK1R(-/-), and c-Kit(W-sh/W-sh) mice reconstituted with WT or NK1R(-/-) BMMCs were used to evaluate NK1R signaling on FcεRI-MC-mediated passive local and systemic anaphylaxis and on airway inflammation. RESULTS: FcεRI-activated MCs upregulated NK1R and HK-1 transcripts and protein synthesis, without modifying SP expression. In a positive signaling loop HK-1 promoted TNF and IL-6 secretion by MC degranulation and protein synthesis, the latter through the phosphoinositide 3-kinase/Akt/nuclear factor κB pathways. In vivo NK1R signaling was necessary for the development of passive local and systemic anaphylaxis and airway inflammation. CONCLUSIONS: FcεRI stimulation of MCs promotes autocrine secretion of HK-1, which signals through NK1R to provide adjuvancy for efficient development of FcεRI-MC-mediated disorders.
Assuntos
Comunicação Autócrina , Imunoglobulina E/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Taquicininas/metabolismo , Anafilaxia/imunologia , Anafilaxia/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Interleucina-6/biossíntese , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Knockout , Receptores de IgE/metabolismo , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais , Fatores de Necrose Tumoral/biossínteseRESUMO
Substance-P and hemokinin-1 are proinflammatory neuropeptides with potential to promote type 1 immunity through agonistic binding to neurokinin-1 receptor (NK1R). Dendritic cells (DCs) are professional antigen-presenting cells that initiate and regulate the outcome of innate and adaptive immune responses. Immunostimulatory DCs are highly desired for the development of positive immunization techniques. DCs express functional NK1R; however, regardless of their potential DC-stimulatory function, the ability of NK1R agonists to promote immunostimulatory DCs remains unexplored. Here, we demonstrate that NK1R signaling activates therapeutic DCs capable of biasing type 1 immunity by inhibition of interleukin-10 (IL-10) synthesis and secretion, without affecting their low levels of IL-12 production. The potent type 1 effector immune response observed following cutaneous administration of NK1R-signaled DCs required their homing in skin-draining lymph nodes (sDLNs) where they induced inflammation and licensed endogenous-conventional sDLN-resident and -recruited inflammatory DCs to secrete IL-12. Our data demonstrate that NK1R signaling promotes immunostimulatory DCs, and provide relevant insight into the mechanisms used by neuromediators to regulate innate and adaptive immune responses.
Assuntos
Células Dendríticas/imunologia , Imunidade Celular/imunologia , Interleucina-12/imunologia , Receptores da Neurocinina-1/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Citometria de Fluxo , Imunização/métodos , Imunofenotipagem , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , Receptores da Neurocinina-1/agonistas , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/imunologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.
Assuntos
Comunicação Celular , Células Dendríticas/metabolismo , Endossomos/metabolismo , Exossomos/genética , MicroRNAs/fisiologia , Animais , Apresentação de Antígeno , Biomarcadores/metabolismo , Citosol/metabolismo , Células Dendríticas/citologia , Exossomos/metabolismo , Perfilação da Expressão Gênica , Fusão de Membrana , Camundongos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The prevailing idea regarding the mechanism(s) by which therapeutic immunosuppressive dendritic cells (DCs) restrain alloimmunity is based on the concept that they interact directly with antidonor T cells, inducing anergy, deletion, and/or regulation. However, this idea has not been tested in vivo. Using prototypic in vitro-generated maturation-resistant (MR) DCs, we demonstrate that once MR-DCs carrying donor antigen (Ag) are administered intravenously, they decrease the direct and indirect pathway T-cell responses and prolong heart allograft survival but fail to directly regulate T cells in vivo. Rather, injected MR-DCs are short-lived and reprocessed by recipient DCs for presentation to indirect pathway CD4(+) T cells, resulting in abortive activation and deletion without detrimental effect on the number of indirect CD4(+) FoxP3(+) T cells, thus increasing the regulatory to effector T cell relative percentage. The effect on the antidonor response was independent of the method used to generate therapeutic DCs or their viability; and in accordance with the idea that recipient Ag-presenting cells mediate the effects of therapeutic DCs in transplantation, prolongation of allograft survival was achieved using donor apoptotic MR-DCs or those lacking surface major histocompatibility complex molecules. We therefore conclude that therapeutic DCs function as Ag-transporting cells rather than Ag-presenting cells to prolong allograft survival.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/transplante , Animais , Apresentação de Antígeno , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Primers do DNA/genética , Células Dendríticas/citologia , Terapia de Imunossupressão , Injeções Intravenosas , Isoantígenos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Doadores de Tecidos , Transplante HomólogoRESUMO
Dendritic cells (DCs) are the preferred targets for immunotherapy protocols focused on stimulation of cellular immune responses. However, regardless of initial promising results, ex vivo generated DCs do not always promote immune-stimulatory responses. The outcome of DC-dependent immunity is regulated by proinflammatory cytokines and neuropeptides. Proinflammatory neuropeptides of the tachykinin family, including substance P (SP) and hemokinin-1 (HK-1), bind the neurokinin 1 receptor (NK1R) and promote stimulatory immune responses. Nevertheless, the ability of pro-inflammatory tachykinins to affect the immune functions of DCs remains elusive. In the present work, we demonstrate that mouse bone marrow-derived DCs (BMDCs) generated in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), express functional NK1R. Signaling via NK1R with SP, HK-1, or the synthetic agonist [Sar(9)Met(O(2))(11)]-SP rescues DCs from apoptosis induced by deprivation of GM-CSF and IL-4. Mechanistic analysis demonstrates that NK1R agonistic binding promotes DC survival via PI3K-Akt signaling cascade. In adoptive transfer experiments, NK1R-signaled BMDCs loaded with Ag exhibit increased longevity in draining lymph nodes, resulting in enhanced and prolonged effector cellular immunity. Our results contribute to the understanding of the interactions between the immune and nervous systems that control DC function and present a novel approach for ex vivo-generation of potent immune-stimulatory DCs.
Assuntos
Células Dendríticas/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Mediadores da Inflamação/farmacologia , Receptores da Neurocinina-1/fisiologia , Taquicininas/farmacologia , Transferência Adotiva , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Células da Medula Óssea/metabolismo , Antígenos CD40/metabolismo , Antígenos CD40/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Dendríticas/metabolismo , Células Dendríticas/fisiologia , Células Dendríticas/transplante , Ativação Enzimática/efeitos dos fármacos , Imunidade Celular/genética , Imunidade Celular/fisiologia , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores da Neurocinina-1/agonistas , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Taquicininas/metabolismoRESUMO
Human skin-migratory dendritic cells (DCs) have the ability to prime and bias Th1 and Th2 CD4+ T lymphocytes. However, whether human cutaneous DCs are capable of initiating proinflammatory Th17 responses remains undetermined. We report that skin-migratory DCs stimulate allogeneic naive CD4+ T cells that differentiate simultaneously into two distinct effector Th17 and Th1 populations capable of homing to the skin, where they induce severe cutaneous damage. Skin-migratory Langerhans cells (smiLCs) were the main cutaneous DC subset capable of inducing Th17 responses dependent on the combined effects of IL-15 and stabilized IL-6, which resulted in IL-6 trans-signaling of naive CD4+ T cells. Different from smiLCs, purified skin-migratory dermal DCs did not synthesize IL-15 and were unable to bias Th17 responses. Nevertheless, these dermal DCs were capable of differentiating Th17 cells in mixed leukocyte cultures supplemented with IL-15 and stabilized IL-6. Overall, our data demonstrate that human epidermal smiLCs induce Th17 responses by mechanisms different from those previously described and highlight the need to target clinical treatments based on these variations.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Mediadores da Inflamação/fisiologia , Interleucina-17/fisiologia , Pele/imunologia , Pele/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Técnicas de Cocultura , Células Dendríticas/patologia , Humanos , Interleucina-15/biossíntese , Interleucina-15/fisiologia , Interleucina-6/metabolismo , Interleucina-6/fisiologia , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Teste de Cultura Mista de Linfócitos , Técnicas de Cultura de Órgãos , Transdução de Sinais/imunologia , Pele/patologia , Linfócitos T Auxiliares-Indutores/patologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/patologiaRESUMO
Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that targets the beta-cells of the pancreas. We investigated the ability of soluble galectin-1 (gal-1), an endogenous lectin that promotes T cell apoptosis, to down-regulate the T cell response that destroys the pancreatic beta-cells. We demonstrated that in nonobese diabetic (NOD) mice, gal-1 therapy reduces significantly the amount of Th1 cells, augments the number of T cells secreting IL-4 or IL-10 specific for islet cell Ag, and causes peripheral deletion of beta-cell-reactive T cells. Administration of gal-1 prevented the onset of hyperglycemia in NOD mice at early and subclinical stages of T1D. Preventive gal-1 therapy shifted the composition of the insulitis into an infiltrate that did not invade the islets and that contained a significantly reduced number of Th1 cells and a higher percentage of CD4(+) T cells with content of IL-4, IL-5, or IL-10. The beneficial effects of gal-1 correlated with the ability of the lectin to trigger apoptosis of the T cell subsets that cause beta-cell damage while sparing naive T cells, Th2 lymphocytes, and regulatory T cells in NOD mice. Importantly, gal-1 reversed beta-cell autoimmunity and hyperglycemia in NOD mice with ongoing T1D. Because gal-1 therapy did not cause major side effects or beta-cell toxicity in NOD mice, the use of gal-1 to control beta-cell autoimmunity represents a novel alternative for treatment of subclinical or ongoing T1D.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Galectina 1/fisiologia , Hipoglicemiantes/administração & dosagem , Animais , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Doenças Autoimunes/prevenção & controle , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/prevenção & controle , Feminino , Galectina 1/administração & dosagem , Humanos , Injeções Intraperitoneais , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos TransgênicosRESUMO
Despite the role of donor-specific antibodies (DSAs) in recognizing major histocompatibility complex (MHC) antigens and mediating transplant rejection, how and where recipient B cells in lymphoid tissues encounter donor MHC antigens remains unclear. Contrary to the dogma, we demonstrated here that migration of donor leukocytes out of skin or heart allografts is not necessary for B or T cell allosensitization in mice. We found that mouse skin and cardiac allografts and human skin grafts release cell-free donor MHC antigens via extracellular vesicles (EVs) that are captured by subcapsular sinus (SCS) macrophages in lymph nodes or analog macrophages in the spleen. Donor EVs were transported across the SCS macrophages, and donor MHC molecules on the EVs were recognized by alloreactive B cells. This triggered B cell activation and DSA production, which were both prevented by SCS macrophage depletion. These results reveal an unexpected role for graft-derived EVs and open venues to interfere with EV biogenesis, trafficking, or function to restrain priming or reactivation of alloreactive B cells.
Assuntos
Vesículas Extracelulares , Transplante de Coração , Animais , Linfócitos B , Rejeição de Enxerto , Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Plasmacytoid dendritic cells (pDCs) play a key role in the innate immune response to viral infection, due largely to their ability to produce large quantities of type I IFNs. These cells are also notable for their ability to differentiate into conventional dendritic cells after appropriate stimulation. Here, we show that a splenic population of murine CD11c(+) cells expressing pDC markers Gr-1, B220, and PDCA-1 is preferentially parasitized after infection with the virulent RH strain of Toxoplasma gondii. Although these markers are closely associated with pDCs, the population we identified was unusual because the cells express CD11b and higher than expected levels of CD11c. By adoptive transfer of CD45.1-positive cells into CD45.2 congenic mice, we show that CD11c(+)Gr-1(+) cells migrate from the peritoneal cavity to the spleen. During infection, these cells accumulate in the marginal zone region. Recruitment of infected CD11c(+)Gr-1(+) cells to the spleen is partially dependent upon signaling through chemokine receptor CCR2. Intracellular cytokine staining demonstrates that infected, but not noninfected, splenic CD11c(+)Gr-1(+) dendritic cells are suppressed in their ability to respond to ex vivo TLR stimulation. We hypothesize that Toxoplasma exploits pDCs as Trojan horses, targeting them for early infection, suppressing their cytokine effector function, and using them for dissemination within the host.
Assuntos
Antígenos de Superfície/biossíntese , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Toxoplasmose/imunologia , Toxoplasmose/parasitologia , Animais , Antígenos de Superfície/genética , Biomarcadores/metabolismo , Antígeno CD11c/biossíntese , Antígeno CD11c/genética , Células Cultivadas , Células Dendríticas/metabolismo , Feminino , Imunofenotipagem , Interleucina-12/antagonistas & inibidores , Interleucina-12/biossíntese , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peritônio/imunologia , Peritônio/parasitologia , Peritônio/patologia , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/genética , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/imunologia , Toxoplasmose/metabolismoRESUMO
Efficient Ca2+ flux induced during cognate T cell activation requires signaling the T cell receptor (TCR) and unidentified G-protein-coupled receptors (GPCRs). T cells express the neurokinin-1 receptor (NK1R), a GPCR that mediates Ca2+ flux in excitable and non-excitable cells. However, the role of the NK1R in TCR signaling remains unknown. We show that the NK1R and its agonists, the neuropeptides substance P and hemokinin-1, co-localize within the immune synapse during cognate activation of T cells. Simultaneous TCR and NK1R stimulation is necessary for efficient Ca2+ flux and Ca2+-dependent signaling that sustains the survival of activated T cells and helper 1 (Th1) and Th17 bias. In a model of contact dermatitis, mice with T cells deficient in NK1R or its agonists exhibit impaired cellular immunity, due to high mortality of activated T cells. We demonstrate an effect of the NK1R in T cells that is relevant for immunotherapies based on pro-inflammatory neuropeptides and its receptors.
Assuntos
Cálcio/metabolismo , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Animais , Comunicação Autócrina/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Polaridade Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinapses Imunológicas/efeitos dos fármacos , Sinapses Imunológicas/metabolismo , Interleucina-2/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Receptores da Neurocinina-1/agonistas , Transdução de Sinais/efeitos dos fármacos , Substância P/farmacologia , Linfócitos T/efeitos dos fármacos , Taquicininas/farmacologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologiaRESUMO
Hematopoietic growth factors (HGF) mobilize potential tolerogenic cells in transplant donors. Fms-like tyrosine kinase 3 ligand (Flt3L) mobilizes stem cells and dendritic cells (DCs) in human and nonhuman primate blood. Blood and renal and liver biopsies were obtained from untreated and Flt3L-mobilized rhesus macaques. Flt3L increased the number of myeloid CD11c(hi) and plasmacytoid CD123(hi) precursors in blood and both myeloid CD11c(+) HLA-DR(+) fascin(+) (CD45RA(-)) DCs and putative plasmacytoid CD11c(lo) CD45RA(hi) DC precursors in liver and kidneys, without affecting organ function. DC in Flt3L-treated monkeys were concentrated in the glomeruli and interstitium of kidneys, and in the portal triads and parenchyma of liver. These DCs exhibited the phenotype of immature antigen-presenting cells (APCs; CD83(-) CD86(lo) CCR5(+) CCR7(-)). HGF-induced changes reversed significantly within 7 days of Flt3L withdrawal. Therapeutic protocols that mobilize donor hematopoietic cells should consider the influence of HGF on the APC constituency of prospective organ allografts.
Assuntos
Células Dendríticas/imunologia , Substâncias de Crescimento/farmacologia , Transplante de Rim/imunologia , Transplante de Fígado/imunologia , Adjuvantes Imunológicos/uso terapêutico , Animais , Terapia Biológica , Células Dendríticas/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Transplante de Rim/patologia , Transplante de Fígado/patologia , Macaca mulatta , Proteínas de Membrana/uso terapêutico , Modelos AnimaisRESUMO
The immune response against transplanted allografts is one of the most potent reactions mounted by the immune system. The acute rejection response has been attributed to donor dendritic cells (DCs), which migrate to recipient lymphoid tissues and directly activate alloreactive T cells against donor MHC molecules. Here, using a murine heart transplant model, we determined that only a small number of donor DCs reach lymphoid tissues and investigated how this limited population of donor DCs efficiently initiates the alloreactive T cell response that causes acute rejection. In our mouse model, efficient passage of donor MHC molecules to recipient conventional DCs (cDCs) was dependent on the transfer of extracellular vesicles (EVs) from donor DCs that migrated from the graft to lymphoid tissues. These EVs shared characteristics with exosomes and were internalized or remained attached to the recipient cDCs. Recipient cDCs that acquired exosomes became activated and triggered full activation of alloreactive T cells. Depletion of recipient cDCs after cardiac transplantation drastically decreased presentation of donor MHC molecules to directly alloreactive T cells and delayed graft rejection in mice. These findings support a key role for transfer of donor EVs in the generation of allograft-targeting immune responses and suggest that interrupting this process has potential to dampen the immune response to allografts.
Assuntos
Aloenxertos/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Exossomos/metabolismo , Tolerância Imunológica/imunologia , Animais , Movimento Celular , Rejeição de Enxerto , Sobrevivência de Enxerto , Transplante de Coração , Complexo Principal de Histocompatibilidade/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Transplante de Pele , Baço/metabolismo , Linfócitos T/citologia , Transplante HomólogoRESUMO
Successful engraftment of organ transplants has traditionally relied on preventing the activation of recipient (host) T cells. Once T-cell activation has occurred, however, stalling the rejection process becomes increasingly difficult, leading to graft failure. Here we demonstrate that graft-infiltrating, recipient (host) dendritic cells (DCs) play a key role in driving the rejection of transplanted organs by activated (effector) T cells. We show that donor DCs that accompany heart or kidney grafts are rapidly replaced by recipient DCs. The DCs originate from non-classical monocytes and form stable, cognate interactions with effector T cells in the graft. Eliminating recipient DCs reduces the proliferation and survival of graft-infiltrating T cells and abrogates ongoing rejection or rejection mediated by transferred effector T cells. Therefore, host DCs that infiltrate transplanted organs sustain the alloimmune response after T-cell activation has already occurred. Targeting these cells provides a means for preventing or treating rejection.
Assuntos
Células Dendríticas/imunologia , Rejeição de Enxerto/imunologia , Linfócitos T/imunologia , Animais , Transplante de Coração , Transplante de Rim , Ativação Linfocitária , Camundongos , TransplantesRESUMO
BACKGROUND: Migratory antigen-presenting cells resident in kidneys may have tolerogenic potential. Difficulties inherent in their isolation have limited their characterization. The authors examined the phenotype and function of murine kidney dendritic cells (DC) mobilized in vivo by systemic administration of fms-like tyrosine 3 kinase ligand (Flt3L). METHODS: Monoclonal antibody staining was used to characterize DC subsets in situ, immediately after their isolation, and after lipopolysaccharide stimulation. Cytokine and CC chemokine receptor (CCR) gene expression was analyzed by RNase protection assay. Mixed leukocyte reactions were performed to assess DC allostimulatory ability and also the function of putative T-regulatory cells. In vivo DC trafficking was monitored by fluorescence imaging of dye-labeled cells and the influence of renal DC on vascularized heart allograft survival was determined. RESULTS: Flt3L induced a marked increase both in CD11cCD8alpha and in CD11cCD8alpha DC within the renal cortex and medulla. Rarer, CD11cB220 (precursor plasmacytoid) DC were also detected. Bulk freshly isolated DC exhibited no interleukin (IL)-12p35 mRNA, low surface co-stimulatory molecule expression, and CCR transcripts, consistent with immaturity. They elicited only weak allogeneic T-cell proliferative responses, and repeated stimulation induced CD4CD25 IL-10 T cells. In vivo, the freshly isolated DC failed to prime T cells of naive allogeneic hosts for anti-donor cytotoxic T-cell responses. When infused systemically, 1 week before organ transplantation, they prolonged graft survival without immunosuppressive therapy. CONCLUSIONS: Hematopoietin-mobilized renal DC are functionally immature and exhibit tolerogenic potential. Mobilization of DC within kidneys is likely to affect their antigen-handling capacity, immunogenicity, and tolerogenic ability.
Assuntos
Células Dendríticas/fisiologia , Sobrevivência de Enxerto , Rim/imunologia , Linfócitos T/imunologia , Animais , Movimento Celular , Imunofenotipagem , Interleucina-12/genética , Subunidade p35 da Interleucina-12 , Subunidade p40 da Interleucina-12 , Lipopolissacarídeos/farmacologia , Masculino , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos , Subunidades Proteicas/genética , Transcrição Gênica , Transplante HomólogoRESUMO
Chronic allograft vasculopathy (CAV) is an atheromatous-like lesion that affects vessels of transplanted organs. It is a component of chronic rejection that conventional immuno-suppression fails to prevent, and is a major cause of graft loss. Indirect allo-recognition through T cells and allo-Abs are critical during CAV pathogenesis. We tested whether the indirect allo-response and its impact on CAV is down-regulated by in situ-delivery of donor Ags to recipient's dendritic cells (DCs) in lymphoid organs in a pro-tolerogenic fashion, through administration of donor splenocytes undergoing early apoptosis. Following systemic injection, donor apoptotic cells were internalized by splenic CD11c(hi) CD8alpha(+) and CD8(-) DCs, but not by CD11c(int) plasmacytoid DCs. Those DCs that phagocytosed apoptotic cells in vivo remained quiescent, resisted ex vivo-maturation, and presented allo-Ag for up to 3 days. Administration of donor apoptotic splenocytes, unlike cells alive, (i) promoted deletion, FoxP3 expression and IL-10 secretion, and decreased IFN-gamma-release in indirect pathway CD4 T cells; and (ii) reduced cross-priming of anti-donor CD8 T cells in vivo. Targeting recipient's DCs with donor apoptotic cells reduced significantly CAV in a fully-mismatched aortic allograft model. The effect was donor specific, dependent on the physical characteristics of the apoptotic cells, and was associated to down-regulation of the indirect type-1 T cell allo-response and secretion of allo-Abs, when compared to recipients treated with donor cells alive or necrotic. Down-regulation of indirect allo-recognition through in situ-delivery of donor-Ag to recipient's quiescent DCs constitutes a promising strategy to prevent/ameliorate indirect allorecognition and CAV.
Assuntos
Apoptose , Transplante de Células/métodos , Células Dendríticas/imunologia , Rejeição de Enxerto/prevenção & controle , Tolerância ao Transplante/imunologia , Animais , Apresentação Cruzada/imunologia , Camundongos , Transplante de Órgãos , Baço/citologia , Linfócitos T/imunologia , Transplante HomólogoRESUMO
Exosomes are nanovesicles released by different cell types including dendritic cells (DCs). The fact that exosomes express surface MHC-peptide complexes suggests that they could function as Ag-presenting vesicles or as vehicles to spread allogeneic Ags for priming of anti-donor T cells during elicitation of graft rejection or induction/maintenance of transplant tolerance. We demonstrate that circulating exosomes transporting alloantigens are captured by splenic DCs of different lineages. Internalization of host-derived exosomes transporting allopeptides by splenic DCs leads to activation of anti-donor CD4 T cells by the indirect pathway of allorecognition, a phenomenon that requires DC-derived, instead of exosome-derived, MHC class II molecules. By contrast, allogeneic exosomes are unable to stimulate direct-pathway T cells in vivo. We demonstrate in mice that although graft-infiltrating leukocytes release exosomes ex vivo, they do not secrete enough concentrations of exosomes into circulation to stimulate donor-reactive T cells in secondary lymphoid organs. Instead, our findings indicate that migrating DCs (generated in vitro or isolated from allografts), once they home in the spleen, they transfer exosomes expressing the reporter marker GFP to spleen-resident DCs. Our results suggest that exchange of exosomes between DCs in lymphoid organs might constitute a potential mechanism by which passenger leukocytes transfer alloantigens to recipient's APCs and amplify generation of donor-reactive T cells following transplantation.
Assuntos
Apresentação de Antígeno , Vesículas Citoplasmáticas/imunologia , Vesículas Citoplasmáticas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Isoantígenos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Transplante de Coração/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Transplante de Pele/imunologiaRESUMO
Exosomes are 50- to 100-nm vesicles that are formed within the late endocytic compartment and released from a variety of cell types. Previously, we demonstrated that exosomes derived from dendritic cells transduced with adenoviral vectors expressing IL-10, IL-4, or Fas ligand (FasL) produce anti-inflammatory exosomes able to reduce inflammation in a murine paw delayed-type hypersensitivity model, suppress the onset on murine collagen-induced arthritis, and reduce the severity of established collagen-induce arthritis. In this study, we examined the ability of endogenous, blood-borne exosomes to regulate the immune response. Exosomes isolated from plasma of mice immunized to keyhole limpet hemocyanin, but not from naive or OVA-immunized mice, were able to suppress the keyhole limpet hemocyanin-specific delayed-type hypersensitivity inflammatory response. The anti-inflammatory effect was mediated by MHC class II(+) plasma exosomes that were also FasL(+) and CD11b(+), but CD11c(-). Moreover, the anti-inflammatory effect of the MHC class II(+) plasma-derived exosomes was, in part, dependent upon the presence of FasL in the exosomes and Fas in the recipient mouse. These results suggest that exosomes in the plasma, produced by MHC class II(+) and CD11b(+) cells, have the ability to suppress the immune response in an Ag-specific manner in part through a Fas/FasL-dependent manner.
Assuntos
Antígenos/imunologia , Proteína Ligante Fas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Tolerância Imunológica , Plasma/imunologia , Vesículas Transportadoras/imunologia , Receptor fas/imunologia , Adenoviridae , Animais , Antígenos CD1/genética , Antígenos CD1/imunologia , Artrite Experimental/genética , Artrite Experimental/imunologia , Células Dendríticas/imunologia , Hemocianinas/imunologia , Hemocianinas/farmacologia , Hipersensibilidade Tardia/imunologia , Tolerância Imunológica/genética , Inflamação/genética , Inflamação/imunologia , Interleucina-10/imunologia , Interleucina-4/imunologia , Masculino , Camundongos , Camundongos Endogâmicos MRL lpr , Ovalbumina/imunologia , Ovalbumina/farmacologia , Transdução Genética , Vesículas Transportadoras/genéticaRESUMO
In this study, we demonstrate that genetically modified bone marrow-derived dendritic cells (DC) and exosomes derived from the DC, expressing either secreted IL-4 or membrane-bound IL-4, can reduce the severity and the incidence of established collagen-induced arthritis and inhibit inflammation of delayed-type hypersensitivity (DTH) in mice. The ability of the DC and DC-derived exosomes to suppress the DTH response was MHC class II and, in part, Fas ligand/Fas dependent. The DC-derived exosomes were internalized by CD11c(+) DC in the dermis at the site of injection and in the draining lymph node as well as by CD11c(+) DC and F4/80(+) macrophages in the spleen. Moreover, adoptive transfer of CD11c(+) or CD3(+) splenic cells from mice treated with exosomes showed significant reduction of footpad swelling in the DTH model. These results demonstrate that administration of DC/IL-4 or exosomes derived from DC/IL-4 are able to modulate the activity of APC and T cells in vivo through a MHC class II and partly Fas ligand/Fas-dependent mechanism, resulting in effective treatment of established collagen-induced arthritis and suppression of the DTH inflammatory response. Thus, APC-derived exosomes could be used therapeutically for the treatment of autoimmune disease and inflammatory disorders.