RESUMO
As emerging pollutants in the environment, nanoplastics (NPs) can cross biological barriers and be enriched in organisms, posing a greatest threat to the health of livestock and humans. However, the size-dependent toxic effects of NPs in higher mammals remain largely unknown. To determine the size-dependent potential toxicities of NPs, we exposed mouse (AML-12) and human (L02) liver cell lines in vitro, and 6-week-old C57BL/6 mice (well-known preclinical model) in vivo to five different sizes of polystyrene NPs (PS-NPs) (20, 50, 100, 200 and 500 nm). We found that ultra-small NPs (20 nm) induced the highest cytotoxicity in mouse and human liver cell lines, causing oxidative stress and mitochondrial membrane potential loss on AML-12 cells. Unexpectedly in vivo, after long-term oral exposure to PS-NPs (75 mg/kg), medium NPs (200 nm) and large NPs (500 nm) induced significant hepatotoxicity, evidenced by increased oxidative stress, liver dysfunction, and lipid metabolism disorders. Most importantly, medium or large NPs generated local immunotoxic effects via recruiting and activating more numbers of neutrophils and monocytes in the liver or intestine, which potentially resulted in increased proinflammatory cytokine secretion and the tissue damage. The discrepancy in in vitro-in vivo toxic results might be attributed to the different properties of biodistribution and tissue accumulation of different sized NPs in vivo. Our study provides new insights regarding the hepatotoxicity and immunotoxicity of NPs on human and livestock health, warranting us to take immense measures to prevent these NPs-associated health damage.
Assuntos
Antineoplásicos , Doença Hepática Induzida por Substâncias e Drogas , Leucemia Mieloide Aguda , Nanopartículas , Poluentes Químicos da Água , Humanos , Animais , Camundongos , Camundongos Endogâmicos C57BL , Microplásticos/toxicidade , Poliestirenos/toxicidade , Distribuição Tecidual , Gado , MamíferosRESUMO
Nanovaccine-based immunotherapy (NBI) has received greater attention recently for its potential to prime tumor-specific immunity and establish a long-term immune memory that prevents tumor recurrence. Despite encouraging results in the recent studies, there are still numerous challenges to be tackled for eliciting potent antitumor immunity using NBI strategies. Based on the principles that govern immune response, here it is proposed that these challenges need to be addressed at the five critical cascading events: Loading tumor-specific antigens by nanoscale drug delivery systems (L); Draining tumor antigens to lymph nodes (D); Internalization by dendritic cells (DCs) (I); Maturation of DCs by costimulatory signaling (M); and Presenting tumor-peptide-major histocompatibility complexes to T cells (P) (LDIMP cascade in short). This review provides a detailed and objective overview of emerging NBI strategies to improve the efficacy of nanovaccines in each step of the LDIMP cascade. It is concluded that the balance between each step must be optimized by delicate designing and modification of nanovaccines and by combining with complementary approaches to provide a synergistic immunity in the fight against cancer.
Assuntos
Vacinas Anticâncer , Neoplasias , Antígenos de Neoplasias , Células Dendríticas , Humanos , Imunoterapia , Neoplasias/terapia , VacinaçãoRESUMO
The extracellular signal-regulated kinase (ERK) pathway has been shown to regulate pathogenesis of many viral infections, but its role during rabies virus (RV) infection in vivo is not clear. In the present study, we investigated the potential role of MEK-ERK1/2 signalling pathway in the pathogenesis of rabies in mouse model and its regulatory effects on pro-inflammatory cytokines and other mediators of immunity, and kinetics of immune cells. Mice were infected with 25 LD50 of challenge virus standard (CVS) strain of RV by intracerebral (i.c.) inoculation and were treated i.c. with U0126 (specific inhibitor of MEK1/2) at 10 µM/mouse at 0, 2, 4 and 6 days post-infection. Treatment with U0126 resulted in delayed disease development and clinical signs, increased survival time with lesser mortality than untreated mice. The better survival of inhibitor-treated and RV infected mice was positively correlated with reduced viral load and reduced viral spread in the brain as quantified by real-time PCR, direct fluorescent antibody test and immunohistochemistry. CVS-infected/mock-treated mice developed severe histopathological lesions with increased Fluoro-Jade B positive degenerating neurons in brain, which were associated with higher levels of serum nitric oxide, iNOS, TNF-α, and CXCL10 mRNA. Also CVS-infected/U0126-treated mice revealed significant decrease in caspase 3 but increase in Bcl-2 mRNA levels and less TUNEL positive apoptotic cells. CVS-infected/U0126-treated group also showed significant increase in CD4+, CD8+ T lymphocytes and NK cells in blood and spleen possibly due to less apoptosis of these cells. In conclusion, these data suggest that MEK-ERK1/2 signalling pathway play critical role in the pathogenesis of RV infection in vivo and opens up new avenues of therapeutics.
Assuntos
Butadienos/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Nitrilas/antagonistas & inibidores , Vírus da Raiva/efeitos dos fármacos , Vírus da Raiva/patogenicidade , Raiva/tratamento farmacológico , Animais , Apoptose , Encéfalo/patologia , Encéfalo/virologia , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Caspase 3/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/virologia , Quimiocina CXCL10/sangue , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Imuno-Histoquímica , Imunofenotipagem , Células Matadoras Naturais , Cinética , Masculino , Camundongos , Degeneração Neural , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo II/sangue , RNA Mensageiro/sangue , Raiva/mortalidade , Raiva/virologia , Vírus da Raiva/genética , Transcriptoma , Fator de Necrose Tumoral alfa/sangue , Carga ViralRESUMO
Microgravity has been known to induce cell death. However, its underlying mechanism is less studied. In this study, BL6-10 melanoma cells were cultured in flasks under simulated microgravity (SMG). We examined cell apoptosis, and assessed expression of genes associated with apoptosis and genes regulating apoptosis in cells under SMG. We demonstrate that SMG induces cell morphological changes and microtubule alterations by confocal microscopy, and enhances apoptosis by flow cytometry, which was associated with up- and down-regulation of pro-apoptotic and anti-apoptotic genes, respectively. Moreover, up- and down-regulation of pro-apoptotic (Caspases 3, 7, 8) and anti-apoptotic (Bcl2 and Bnip3) molecules was confirmed by Western blotting analysis. Western blot analysis also indicates that SMG causes inhibition of an apoptosis suppressor, pNF-κB-p65, which is complemented by the predominant localization of NF-κB-p65 in the cytoplasm. SMG also reduces expression of molecules regulating the NF-κB pathway including Uev1A, TICAM, TRAF2, and TRAF6. Interestingly, 10 DNA repair genes are down-regulated in cells exposed to SMG, among which down-regulation of Parp, Ercc8, Rad23, Rad51, and Ku70 was confirmed by Western blotting analysis. In addition, we demonstrate a significant inhibition of molecules involved in the DNA-damage response, such as p53, PCNA, ATM/ATR, and Chk1/2. Taken together, our work reveals that SMG promotes the apoptotic response through a combined modulation of the Uev1A/TICAM/TRAF/NF-κB-regulated apoptosis and the p53/PCNA- and ATM/ATR-Chk1/2-controlled DNA-damage response pathways. Thus, our investigation provides novel information, which may help us to determine the cause of negative alterations in human physiology occurring at spaceflight environment. J. Cell. Biochem. 117: 2138-2148, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Apoptose , Quinase 1 do Ponto de Checagem/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ausência de Peso , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase do Ponto de Checagem 2/genética , Camundongos , Antígeno Nuclear de Célula em Proliferação/genética , Fator 2 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/genética , Fator de Transcrição RelA/genética , Proteína Supressora de Tumor p53/genética , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Introduction: Bluetongue (BT) poses a significant threat to the livestock industry, affecting various animal species and resulting in substantial economic losses. The existence of numerous BT virus (BTV) serotypes has hindered control efforts, highlighting the need for broad-spectrum vaccines. Methodology: In this study, we evaluated the conserved amino acid sequences within key non-structural (NS) proteins of BTV and identified numerous highly conserved murine- and bovine-specific MHC class I-restricted (MHC-I) CD8+ and MHC-II-restricted CD4+ epitopes. We then screened these conserved epitopes for antigenicity, allergenicity, toxicity, and solubility. Using these epitopes, we developed in silico-based broad-spectrum multiepitope vaccines with Toll-like receptor (TLR-4) agonists. The predicted proinflammatory cytokine response was assessed in silico using the C-IMMSIM server. Structural modeling and refinement were achieved using Robetta and GalaxyWEB servers. Finally, we assessed the stability of the docking complexes through extensive 100-nanosecond molecular dynamics simulations before considering the vaccines for codon optimization and in silico cloning. Results: We found many epitopes that meet these criteria within NS1 and NS2 proteins and developed in silico broad-spectrum vaccines. The immune simulation studies revealed that these vaccines induce high levels of IFN-γ and IL-2 in the vaccinated groups. Protein-protein docking analysis demonstrated promising epitopes with strong binding affinities to TLR-4. The docked complexes were stable, with minimal Root Mean Square Deviation and Root Mean Square Fluctuation values. Finally, the in silico-cloned plasmids have high % of GC content with > 0.8 codon adaptation index, suggesting they are suitable for expressing the protein vaccines in prokaryotic system. Discussion: These next-generation vaccine designs are promising and warrant further investigation in wet lab experiments to assess their immunogenicity, safety, and efficacy for practical application in livestock. Our findings offer a robust framework for developing a comprehensive, broad-spectrum vaccine, potentially revolutionizing BT control and prevention strategies in the livestock industry.
Assuntos
Vírus Bluetongue , Biologia Computacional , Epitopos de Linfócito T , Proteínas não Estruturais Virais , Vacinas Virais , Animais , Vírus Bluetongue/imunologia , Epitopos de Linfócito T/imunologia , Vacinas Virais/imunologia , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/genética , Camundongos , Biologia Computacional/métodos , Sorogrupo , Bovinos , Bluetongue/prevenção & controle , Bluetongue/imunologia , Bluetongue/virologia , Sequência ConservadaRESUMO
Cardiac fibrosis can be mitigated by limiting fibroblast-to-myofibroblast differentiation and proliferation. Human antigen R (HuR) modulates messenger RNA stability and expression of multiple genes. However, the direct role of cardiac myofibroblast HuR is unknown. Myofibroblast-specific deletion of HuR limited cardiac fibrosis and preserved cardiac functions in pressure overload injury. Knockdown of HuR in transforming growth factor-ß1-treated cardiac fibroblasts suppressed myofibroblast differentiation and proliferation. HuR deletion abrogated the expression and messenger RNA stability of cyclins D1 and A2, suggesting a potential mechanism by which HuR promotes myofibroblast proliferation. Overall, these data suggest that inhibition of HuR could be a potential therapeutic approach to limit cardiac fibrosis.
RESUMO
Invariant NKT (iNKT) cells comprise a heterogeneous group of non-circulating, tissue-resident T lymphocytes that recognize glycolipids, including alpha-galactosylceramide (αGalCer), in the context of CD1d, but whether peripheral iNKT cell subsets are terminally differentiated remains unclear. Here we show that mouse and human liver-resident αGalCer/CD1d-binding iNKTs largely correspond to a novel Zbtb16+Tbx21+Gata3+MaflowRorc- subset that exhibits profound transcriptional, phenotypic and functional plasticity. Repetitive in vivo encounters of these liver iNKT (LiNKT) cells with intravenously delivered αGalCer/CD1d-coated nanoparticles (NP) trigger their differentiation into immunoregulatory, IL-10+IL-21-producing Zbtb16highMafhighTbx21+Gata3+Rorc- cells, termed LiNKTR1, expressing a T regulatory type 1 (TR1)-like transcriptional signature. This response is LiNKT-specific, since neither lung nor splenic tissue-resident iNKT cells from αGalCer/CD1d-NP-treated mice produce IL-10 or IL-21. Additionally, these LiNKTR1 cells suppress autoantigen presentation, and recognize CD1d expressed on conventional B cells to induce IL-10+IL-35-producing regulatory B (Breg) cells, leading to the suppression of liver and pancreas autoimmunity. Our results thus suggest that LiNKT cells are plastic for further functional diversification, with such plasticity potentially targetable for suppressing tissue-specific inflammatory phenomena.
Assuntos
Linfócitos B Reguladores , Células T Matadoras Naturais , Animais , Antígenos CD1d/metabolismo , Autoimunidade , Linfócitos B Reguladores/metabolismo , Galactosilceramidas , Interleucina-10/metabolismo , Fígado/metabolismo , CamundongosRESUMO
T cell-T cell Ag presentation is increasingly attracting attention. We previously showed that the in vitro OVA-pulsed dendritic cell (DC(OVA))-activated CD4(+) Th cells acquired OVA peptide/MHC (pMHC) class I and costimulatory molecules such as CD54 and CD80 from DC(OVA) and acted as CD4(+) Th-APC capable of stimulating OVA-specific CD8(+) CTL responses. In this study, we further applied the OVA-specific TCR-transgenic OT I and OT II mice with deficiency of various cytokines or costimulatory molecule genes useful for studying the molecular mechanisms underlying in Th-APC's stimulatory effect. We demonstrated that DC(OVA)-stimulated OT II CD4(+) Th-APC also acquired costimulatory molecules such as CD40, OX40L, and 4-1BBL and the functional pMHC II complexes by DC(OVA) activation. CD4(+) Th-APC with acquired pMHC II and I were capable of stimulating CD4(+) Th1 and central memory CD8(+)44(+)CD62L(high)IL-7R(+) T cell responses leading to antitumor immunity against OVA-expressing mouse B16 melanoma. Their stimulatory effect on CD8(+) CTL responses and antitumor immunity is mediated by IL-2 secretion, CD40L, and CD80 signaling and is specifically targeted to CD8(+) T cells in vivo via acquired pMHC I. In addition, CD4(+) Th-APC expressing OVA-specific TCR, FasL, and perforin were able to kill DC(OVA) and neighboring Th-APC expressing endogenous and acquired pMHC II. Taken together, we show that CD4(+) Th-APC can modulate immune responses by stimulating CD4(+) Th1 and central memory CD8(+) T cell responses and eliminating DC(OVA) and neighboring Th-APC. Therefore, our findings may have great impacts in not only the antitumor immunity, but also the regulatory T cell-dependent immune tolerance in vivo.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Antígenos de Histocompatibilidade Classe I/fisiologia , Memória Imunológica , Ativação Linfocitária/imunologia , Fragmentos de Peptídeos/fisiologia , Células Th1/imunologia , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Testes Imunológicos de Citotoxicidade , Receptores de Hialuronatos/biossíntese , Selectina L/biossíntese , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Receptores de Interleucina-7/biossíntese , Células Th1/metabolismoRESUMO
Neutrophils with immunoregulatory properties, also referred to as type-2 neutrophils (N2), myeloid-derived suppressor cells (MDSCs), or tumor-associated neutrophils (TANs), comprise a heterogeneous subset of cells that arise from unknown precursors in response to poorly understood cues. Here, we find that, in several models of liver autoimmunity, pharmacologically induced, autoantigen-specific T regulatory type-1 (TR1) cells and TR1-cell-induced B regulatory (Breg) cells use five immunoregulatory cytokines to coordinately recruit neutrophils into the liver and program their transcriptome to generate regulatory neutrophils. The liver-associated neutrophils from the treated mice, unlike their circulating counterparts or the liver neutrophils of sick mice lacking antigen-specific TR1 cells, are proliferative, can transfer disease protection to immunocompromised hosts engrafted with pathogenic effectors, and blunt antigen-presentation and local autoimmune responses via cathelin-related anti-microbial peptide (CRAMP), a cathelicidin, in a CRAMP-receptor-dependent manner. These results, thus, identify antigen-specific regulatory T cells as drivers of tissue-restricted regulatory neutrophil formation and CRAMP as an effector of regulatory neutrophil-mediated immunoregulation.
Assuntos
Autoimunidade , Catelicidinas/metabolismo , Fígado/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos/metabolismo , Linfócitos B Reguladores/imunologia , Polaridade Celular/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Inflamação/patologia , Células de Kupffer/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Mitose/genética , Células Supressoras Mieloides/imunologia , Infiltração de Neutrófilos , Neutrófilos , Especificidade de Órgãos , Fenótipo , Transcrição GênicaRESUMO
Peptide MHC class II-based (pMHCII-based) nanomedicines trigger the formation of multicellular regulatory networks by reprogramming autoantigen-experienced CD4+ T cells into autoimmune disease-suppressing T regulatory type 1 (TR1) cells. We have shown that pMHCII-based nanomedicines displaying liver autoimmune disease-relevant yet ubiquitously expressed antigens can blunt various liver autoimmune disorders in a non-disease-specific manner without suppressing local or systemic immunity against infectious agents or cancer. Here, we show that such ubiquitous autoantigen-specific T cells are also awakened by extrahepatic tissue damage and that the corresponding TR1 progeny can suppress experimental autoimmune encephalomyelitis (EAE) and pancreatic ß cell autoreactivity. In mice having EAE, nanomedicines displaying either ubiquitous or CNS-specific epitopes triggered the formation and expansion of cognate TR1 cells and their recruitment to the CNS-draining lymph nodes, sparing their liver-draining counterparts. Surprisingly, in mice having both liver autoimmunity and EAE, liver inflammation sequestered these ubiquitous or even CNS-specific TR1 cells away from the CNS, abrogating their antiencephalitogenic activity. In these mice, only the ubiquitous antigen-specific TR1 cells suppressed liver autoimmunity. Thus, the scope of antigen spreading in autoimmune disorders is larger than previously anticipated, involving specificities expected to be silenced by mechanisms of tolerance; the regulatory activity, but not the retention of autoreactive TR1 cells, requires local autoantigen expression.
Assuntos
Autoimunidade , Encefalomielite Autoimune Experimental/imunologia , Hepatite Autoimune/imunologia , Fígado/imunologia , Linfócitos T Reguladores/imunologia , Animais , Autoantígenos/imunologia , Encefalomielite Autoimune Experimental/patologia , Hepatite Autoimune/patologia , Antígenos de Histocompatibilidade Classe II/imunologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos NOD , Linfócitos T Reguladores/patologiaRESUMO
Application of cancer vaccines is limited due to their systemic immunotoxicity and inability to satisfy all the steps, including loading of tumor antigens, draining of antigens to lymph nodes (LNs), internalization of antigens by dendritic cells (DCs), DC maturation, and cross-presentation of antigens for T cell activation. Here, we present a combinatorial therapy, based on a α-cyclodextrin (CD)-based gel system, DOX/ICG/CpG-P-ss-M/CD, fabricated by encapsulating doxorubicin (DOX) and the photothermal agent indocyanine green (ICG). Upon irradiation, the gel system exhibited heat-responsive release of DOX and vaccine-like nanoparticles, CpG-P-ss-M, along with chemotherapy- and phototherapy-generated abundant tumor-specific antigen storage in situ. The released CpG-P-ss-M acted as a carrier adsorbed and delivered antigens to LNs, promoting the uptake of antigens by DCs and DC maturation. Notably, combined with PD-L1 blocking, the therapy effectively inhibited primary tumor growth and induced tumor-specific immune response against tumor recurrence and metastasis.
RESUMO
To achieve safe and effective antitumor immunity, we constructed the M1-macrophage-membrane-coated nanoparticles [(C/I)BP@B-A(D)&M1m] having laser-responsive, size-changeable, on-demand drug release and prolonged circulation retention properties. (C/I)BP@B-A(D)&M1m delayed clearance by the phagocytic system and homed to tumor efficiently. Upon 650 nm laser irradiation, the hydrophobic core of the PEGylated bilirubin nanoparticles (BP) got disrupted, releasing small-sized deep-penetrating B-A(D) particles, photosensitive chlorin e6 (C), and tolerance-inducing indoleamine 2,3-dioxygenase inhibitor, indoximode (I). Treatment-induced immunogenic cell death and antitumor immunity, suppressing primary tumor growth in both 4T1 and B16F10 models without causing any adverse effects. Most importantly, it inhibited primary tumor recurrence as well as metastasis. Thus, this study provides a promising combinatorial strategy to trigger antitumor immunity in malignancies.
Assuntos
Nanopartículas , Neoplasias , Animais , Linhagem Celular Tumoral , Imunoterapia , Lasers , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/tratamento farmacológico , FagócitosRESUMO
Peptide-major histocompatibility complex class II (pMHCII)-based nanomedicines displaying tissue-specific autoantigenic epitopes can blunt specific autoimmune conditions by re-programming cognate antigen-experienced CD4+ T-cells into disease-suppressing T-regulatory type 1 (TR1) cells. Here, we show that single pMHCII-based nanomedicines displaying epitopes from mitochondrial, endoplasmic reticulum or cytoplasmic antigens associated with primary biliary cholangitis (PBC) or autoimmune hepatitis (AIH) can broadly blunt PBC, AIH and Primary Sclerosing Cholangitis in various murine models in an organ- rather than disease-specific manner, without suppressing general or local immunity against infection or metastatic tumors. Therapeutic activity is associated with cognate TR1 cell formation and expansion, TR1 cell recruitment to the liver and draining lymph nodes, local B-regulatory cell formation and profound suppression of the pro-inflammatory capacity of liver and liver-proximal myeloid dendritic cells and Kupffer cells. Thus, autoreactivity against liver-enriched autoantigens in liver autoimmunity is not disease-specific and can be harnessed to treat various liver autoimmune diseases broadly.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Antígenos de Histocompatibilidade Classe II/imunologia , Hepatopatias/tratamento farmacológico , Nanopartículas/administração & dosagem , Peptídeos/administração & dosagem , Idoso , Animais , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Linhagem Celular , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/química , Humanos , Fígado/efeitos dos fármacos , Fígado/imunologia , Hepatopatias/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Nanomedicina/métodos , Nanopartículas/química , Peptídeos/química , Peptídeos/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologiaRESUMO
Nanomedicine is at the forefront of targeted drug delivery for cancer therapy. An improved combinatorial approach is highlighted for breast cancer treatment by Hu et al. in this issue of ACS Applied Materials and Interfaces. The authors demonstrated that, by combining multistage-responsive nanoparticles carrying a therapeutic drug, doxorubicin, a photothermal agent, indocyanine green, and a nitric oxide donor with photothermal therapy and intravenous injection of a tumor-homing iRGD peptide, one could achieve efficient therapeutics distribution deep inside the tumor and nearly eradicate primary tumor growth. An in-depth understanding of this approach in combination with other strategies such as the use of immunomodulators would facilitate treating metastasis in distant organs, and clinical translation of this platform, benefiting cancer patients by providing long-lasting efficacy.
Assuntos
Neoplasias da Mama , Nanopartículas , Linhagem Celular Tumoral , Doxorrubicina , Sistemas de Liberação de Medicamentos , HumanosRESUMO
We have shown that nanoparticles (NPs) can be used as ligand-multimerization platforms to activate specific cellular receptors in vivo. Nanoparticles coated with autoimmune disease-relevant peptide-major histocompatibility complexes (pMHC) blunted autoimmune responses by triggering the differentiation and expansion of antigen-specific regulatory T cells in vivo. Here, we define the engineering principles impacting biological activity, detail a synthesis process yielding safe and stable compounds, and visualize how these nanomedicines interact with cognate T cells. We find that the triggering properties of pMHC-NPs are a function of pMHC intermolecular distance and involve the sustained assembly of large antigen receptor microclusters on murine and human cognate T cells. These compounds show no off-target toxicity in zebrafish embryos, do not cause haematological, biochemical or histological abnormalities, and are rapidly captured by phagocytes or processed by the hepatobiliary system. This work lays the groundwork for the design of ligand-based NP formulations to re-program in vivo cellular responses using nanotechnology.
Assuntos
Autoimunidade , Antígenos de Histocompatibilidade , Nanomedicina/métodos , Nanopartículas/química , Peptídeos , Linfócitos T Reguladores/imunologia , Animais , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/imunologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Peptídeos/química , Peptídeos/imunologia , Linfócitos T Reguladores/patologiaAssuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Animais , Diferenciação Celular/imunologia , HumanosRESUMO
The greatest hurdle in cancer treatment is the metastasis of primary tumors to distant organs. Our knowledge on how different immune cells, in the absence of exogenous stimulation, prevent tumor metastasis in distant organs is poorly understood. Using a highly metastatic murine lung B16 melanoma cell line BL6-10, we employed naive mice that genetically lack CD4(+) or CD8(+) T cells, or are depleted of dendritic cells (DCs) or natural killer (NK) cells to understand the relative importance of these cells in metastasis prevention. Irrespective of the presence of naïve CD4(+) T, CD8(+) T, DCs, or NK cells, lungs, which act as primary site of predilection for B16 melanoma, readily developed numerous lung BL6-10 melanoma colonies. However, their absence led to B16 melanoma metastasis in variable proportions to distant organs, particularly livers, kidneys, adrenals, ovaries, and hearts. NK cells mediate prevention of BL6-10 metastasis to various organs, especially to livers. Mechanistically, CD40L signaling, a critical factor required for DC licensing and CD8(+) cytotoxic T lymphocyte (CTL) responses, was required for CD4(+) T cell-mediated prevention of systemic BL6-10 metastasis. These results suggest that the composition and functions of different immune cells in distant tissue microenvironments (distant organs other than primary sites of predilection) robustly mediate natural resistance against melanoma metastasis. Thus, harnessing these immune cells' responses in immunotherapeutics would considerably limit organ metastasis.
Assuntos
Imunidade Adaptativa/imunologia , Imunidade Inata/imunologia , Neoplasias Pulmonares/imunologia , Melanoma Experimental/imunologia , Metástase Neoplásica/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/patologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/imunologiaRESUMO
Involvement of CD4(+) helper T (Th) cells is crucial for CD8(+) cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4(+) Th's signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4(+) Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4(+) T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K(b)/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4(+) Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4(+) Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4(+) Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.
Assuntos
Ligante de CD40/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Memória Imunológica , Interleucina-2/metabolismo , Transdução de Sinais , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Apoptose/genética , Apoptose/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/imunologia , Análise por Conglomerados , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Perfilação da Expressão Gênica , Memória Imunológica/genética , Camundongos , Camundongos Knockout , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
The limitations of highly active anti-retroviral therapy have necessitated the development of alternative therapeutics for human immunodeficiency virus type-1 (HIV-1)-infected patients with dysfunctional dendritic cells (DCs) and CD4(+) T cell deficiency. We previously demonstrated that HIV-1 Gp120-specific T cell-based Gp120-Texo vaccine by using ConA-stimulated C57BL/6 (B6) mouse CD8(+) T (ConA-T) cells with uptake of pcDNA(Gp120)-transfected B6 mouse DC line DC2.4 (DC2.4(Gp120))-released exosomes (EXO(Gp120)) was capable of stimulating DC and CD4(+) T cell-independent CD8(+) cytotoxic T lymphocyte (CTL) responses detected in wild-type B6 mice using non-specific PE-anti-CD44 and anti-IFN-γ antibody staining by flow cytometry. To assess effectiveness of Gp120-Texo vaccine in transgenic (Tg) HLA-A2 mice mimicking the human situation, we constructed adenoviral vector AdV(Gp120) expressing HIV-1 GP120 by recombinant DNA technology, and generated Gp120-Texo vaccine by using Tg HLA-A2 mouse CD8(+) ConA-T cells with uptake of AdV(Gp120)-transfected HLA-A2 mouse bone marrow DC (DC(Gp120))-released EXO(Gp120). We then performed animal studies to assess Gp120-Texo-induced stimulation of Gp120-specific CTL responses and antitumor immunity in Tg HLA-A2 mice. We demonstrate that Gp120-Texo vaccine stimulates Gp120-specific CTL responses detected in Tg HLA-A2 mice using Gp120-specific PE-HLA-A2/Gp120 peptide (KLTPLCVTL) tetramer staining by flow cytometry. These Gp120-specific CTLs are capable of further differentiating into functional effectors with killing activity to Gp120 peptide-pulsed splenocytes in vivo. In addition, Gp120-Texo vaccine also induces Gp120-specific preventive, therapeutic (for 6 day tumor lung metastasis) and CD4(+) T cell-independent long-term immunity against B16 melanoma BL6-10(Gp120/A2Kb) expressing both Gp120 and A2Kb (α1 and α2 domains of HLA-A2 and α3 domain of H-2K(b)) in Tg HLA-A2 mice. Taken together, the novel CD8(+) Gp120-Texo vaccine capable of stimulating efficient CD4(+) T cell-independent Gp120-specific CD8(+) CTL responses leading to therapeutic and long-term immunity in Tg HLA-A2 mice may represent a new immunotherapeutic vaccine for treatment of HIV-1 patients with CD4(+) T cell deficiency.