RESUMO
BACKGROUND: Cryptococcal meningitis is the most common fungal infection of the central nervous system (CNS) in HIV/AIDS. HIV-1 virotoxins (e.g., gp41) are able to induce disorders of the blood-brain barrier (BBB), which mainly consists of BMEC. Our recent study suggests that α7 nAChR is an essential regulator of inflammation, which contributes to regulation of NF-κB signaling, neuroinflammation and BBB disorders caused by microbial (e.g., HIV-1 gp120) and non-microbial [e.g., methamphetamine (METH)] factors. However, the underlying mechanisms for multiple comorbidities are unclear. METHODS: In this report, an aggravating role of α7 nAChR in host defense against CNS disorders caused by these comorbidities was demonstrated by chemical [inhibitor: methyllycaconitine (MLA)] and genetic (α7(-/-) mice) blockages of α7 nAChR. RESULTS: As shown in our in vivo studies, BBB injury was significantly reduced in α7(-/-) mice infected with C. neoformans. Stimulation by the gp41 ectodomain peptide (gp41-I90) and METH was abolished in the α7(-/-) animals. C. neoformans and gp41-I90 could activate NF-κB. Gp41-I90- and METH-induced monocyte transmigration and senescence were significantly inhibited by MLA and CAPE (caffeic acid phenethyl ester, an NF-κB inhibitor). CONCLUSIONS: Collectively, our data suggest that α7 nAChR plays a detrimental role in the host defense against C. neoformans- and HIV-1 associated comorbidity factors-induced BBB injury and CNS disorders.
Assuntos
Barreira Hematoencefálica/metabolismo , Cryptococcus neoformans , Meningite Criptocócica/genética , Receptor Nicotínico de Acetilcolina alfa7/genética , Aconitina/análogos & derivados , Aconitina/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , Coinfecção , Proteína gp41 do Envelope de HIV/farmacologia , Infecções por HIV/metabolismo , HIV-1/metabolismo , Inflamação , Metanfetamina/farmacologia , Camundongos , Camundongos Knockout , NF-kappa B/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidoresRESUMO
Cryptococcus neoformans is a pathogenic yeast that can invade the brain and cause meningoencephalitis. Our previous in vitro studies suggested that the interaction between C. neoformans hyaluronic acid and human brain endothelial CD44 could be the initial step of brain invasion. In this report, we used a CD44 knock-out (KO or CD44(-/-)) mouse model to explore the importance of CD44 in C. neoformans brain invasion. Our results showed that C. neoformans-infected CD44 KO mice survived longer than the infected wild-type mice. Consistent with our in vitro results, the brain and cerebrospinal fluid fungal burden was reduced in CD44-deficient mice. Histopathological studies showed smaller and fewer cystic lesions in the brains of CD44 KO mice. Interestingly, the cystic lesions contained C. neoformans cells embedded within their polysaccharide capsule and were surrounded by host glial cells. We also found that a secondary hyaluronic acid receptor, RHAMM (receptor of hyaluronan-mediated motility), was present in the CD44 KO mice. Importantly, our studies demonstrated an in vivo blocking effect of simvastatin. These results suggest that the CD44 and RHAMM receptors function on membrane lipid rafts during invasion and that simvastatin may have a potential therapeutic role in C. neoformans infections of the brain.
Assuntos
Encéfalo/metabolismo , Criptococose/metabolismo , Cryptococcus neoformans/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Receptores de Hialuronatos/metabolismo , Animais , Western Blotting , Encéfalo/efeitos dos fármacos , Encéfalo/microbiologia , Criptococose/líquido cefalorraquidiano , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/fisiologia , Proteínas da Matriz Extracelular/genética , Feminino , Interações Hospedeiro-Patógeno , Receptores de Hialuronatos/genética , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/microbiologia , Ligação Proteica , Interferência de RNA , Sinvastatina/farmacologia , VirulênciaRESUMO
Cryptococcus neoformans is a neurotropic fungal pathogen, which provokes the onset of devastating meningoencephalitis. We used human brain microvascular endothelial cells (HBMEC) as the in vitro model to investigate how C. neoformans traverses across the blood-brain barrier. In this study, we present several lines of evidence indicating that C. neoformans invasion is mediated through the endocytic pathway via lipid rafts. Human CD44 molecules from lipid rafts can directly interact with hyaluronic acid, the C. neoformans ligand. Bikunin, which perturbs CD44 function in the lipid raft, can block C. neoformans adhesion and invasion of HBMEC. The lipid raft marker, ganglioside GM1, co-localizes with CD44 on the plasma membrane, and C. neoformans cells can adhere to the host cell in areas where GM1 is enriched. These findings suggest that C. neoformans entry takes place on the lipid rafts. Upon C. neoformans engagement, GM1 is internalized through vesicular structures to the nuclear membrane. This endocytic redistribution process is abolished by cytochalasin D, nocodazole, or anti-DYRK3 (dual specificity tyrosine-phosphorylation-regulated kinase 3) siRNA. Concomitantly, the knockdown of DYRK3 significantly reduces C. neoformans invasion across the HBMEC monolayer in vitro. Our data demonstrate that the lipid raft-dependent endocytosis process mediates C. neoformans internalization into HBMEC and that the CD44 protein of the hosts, cytoskeleton, and intracellular kinase-DYRK3 are involved in this process.
Assuntos
Encéfalo/irrigação sanguínea , Encéfalo/microbiologia , Criptococose/microbiologia , Cryptococcus neoformans/metabolismo , Microdomínios da Membrana/microbiologia , Microcirculação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Membrana Celular/metabolismo , Citocalasina D/farmacologia , Gangliosídeo G(M1)/análogos & derivados , Gangliosídeo G(M1)/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência/métodos , Nocodazol/farmacologia , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Cryptococcus neoformans has a predilection for central nervous system infection. C. neoformans traversal of the blood brain barrier, composed of human brain microvascular endothelial cells (HBMEC), is the crucial step in brain infection. However, the molecular mechanism of the interaction between Cryptococcus neoformans and HBMEC, relevant to its brain invasion, is still largely unknown. METHODS: In this report, we explored several cellular and molecular events involving the membrane lipid rafts and caveolin-1 (Cav1) of HBMEC during C. neoformans infection. Immunofluorescence microscopy was used to examine the roles of Cav1. The knockdown of Cav1 by the siRNA treatment was performed. Phosphorylation of Cav1 relevant to its invasion functions was investigated. RESULTS: We found that the host receptor CD44 colocalized with Cav1 on the plasma membrane, and knockdown of Cav1 significantly reduced the fungal ability to invade HBMEC. Although the CD44 molecules were still present, HBMEC membrane organization was distorted by Cav1 knockdown. Concomitantly, knockdown of Cav1 significantly reduced the fungal crossing of the HBMEC monolayer in vitro. Upon C. neoformans engagement, host Cav1 was phosphorylated in a CD44-dependent manner. This phosphorylation was diminished by filipin, a disrupter of lipid raft structure. Furthermore, the phosphorylated Cav1 at the lipid raft migrated inward to the perinuclear localization. Interestingly, the phospho-Cav1 formed a thread-like structure and colocalized with actin filaments but not with the microtubule network. CONCLUSION: These data support that C. neoformans internalization into HBMEC is a lipid raft/caveolae-dependent endocytic process where the actin cytoskeleton is involved, and the Cav1 plays an essential role in C. neoformans traversal of the blood-brain barrier.
Assuntos
Caveolina 1/genética , Caveolina 1/metabolismo , Cryptococcus neoformans/patogenicidade , Células Endoteliais/metabolismo , Receptores de Hialuronatos/metabolismo , Microdomínios da Membrana/metabolismo , Citoesqueleto de Actina , Barreira Hematoencefálica/metabolismo , Cavéolas/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Infecções do Sistema Nervoso Central/microbiologia , Cryptococcus neoformans/metabolismo , Endocitose , Células Endoteliais/citologia , Filipina/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Fosforilação/efeitos dos fármacos , RNA Interferente PequenoRESUMO
Cryptococcus neoformans causes life-threatening meningoencephalitis, particularly prevalent in AIDS patients. The interrelationship between C. neoformans and HIV-1 is intriguing, as both pathogens elicit severe neuropathological complications. We have previously demonstrated that the HIV-1 gp41 ectodomain fragments gp41-I33 (amino acids 579-611) and gp41-I90 (amino acids 550-639) can enhance C. neoformans binding to HBMECs (human brain microvascular endothelial cells). Both peptides contain the loop region of gp41. In the present study, we used immunofluorescence microscopy and transmission and scanning electron microscopy to explore the underlying mechanisms. Our findings indicated that both C. neoformans and gp41-I90 up-regulated ICAM-1 (intercellular adhesion molecule 1) on the HBMECs and elicited membrane ruffling on the surface of HBMECs. The HIV-1 gp41 ectodomain could also induce CD44 and ß-actin redistribution to the membrane lipid rafts, but it could not enhance PKCα (protein kinase Cα) phosphorylation like C. neoformans. Instead, gp41-I90 was able to induce syncytium formation on HBMECs. The results of the present study suggest HIV-1 gp41-enhanced C. neoformans binding to HBMECs via gp41 core domain-induced membrane activities, revealing a potential mechanism of invasion for this pathogenic fungus into the brain tissues of HIV-1-infected patients.
Assuntos
Encéfalo/microbiologia , Membrana Celular/metabolismo , Cryptococcus neoformans/fisiologia , Células Endoteliais/microbiologia , Endotélio Vascular/microbiologia , Proteína gp41 do Envelope de HIV/química , Actinas/metabolismo , Sítios de Ligação , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Membrana Celular/microbiologia , Células Cultivadas , Criptococose/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mutação , Proteína Quinase C-alfa/metabolismoRESUMO
Although the role of the classic retinoic acid (RA)-induced genomic pathway in cancer cell differentiation is well recognized, the underlying mechanisms remain to be dissected. Retinoic acid receptor alpha (RARalpha) is a transcription factor activated by RA, and its serine 77 (RARalphaS77) is the main residue phosphorylated by the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex. We report here that in both human myeloid leukemia and mouse embryonic teratocarcinoma stem cells, either RA-suppressed CAK phosphorylation of RARalpha or mutation of RARalphaS77 to alanine (RARalphaS77A) coordinates CAK-dependent G(1) arrest with cancer cell differentiation by transactivating RA-target genes. Both hypophosphorylated RARalpha and RARalphaS77A reduce binding to retinoic acid-responsive elements (RARE) in the promoters of RA-target genes while stimulating gene transcription. The enhanced transactivation and reduced RARalpha-chromatin interaction are accompanied by RARalpha dissociation from the transcriptional repressor N-CoR and are association with the coactivator NCoA-3. Such effects of decreased CAK phosphorylation of RARalphaS77 on mediating RA-dependent transcriptional control of cancer cell differentiation are examined correspondingly in both RA-resistant myeloid leukemia and embryonic teratocarcinoma stem RARalpha(-/-) cells. These studies demonstrate, for the first time, that RA couples G(1) arrest to transcriptional control of cancer cell differentiation by suppressing CAK phosphorylation of RARalpha to release transcriptional repression.-Wang, A., Alimova, I. N., Luo, P. Jong, A., Triche, T. J., Wu, L. Loss of CAK phosphorylation of RARalpha mediates transcriptional control of retinoid-induced cancer cell differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Receptores do Ácido Retinoico/metabolismo , Tretinoína/farmacologia , Animais , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Camundongos , Fosforilação/fisiologia , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Receptor alfa de Ácido Retinoico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Pathogenic yeast Cryptococcus neoformans causes devastating cryptococcal meningoencephalitis. Our previous studies demonstrated that C. neoformans hyaluronic acid was required for invasion into human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. In this report, we demonstrate that C. neoformans hyaluronic acid interacts with CD44 on HBMEC. Our results suggest that HBMEC CD44 is a primary receptor during C. neoformans infection, based on the following observations. First, anti-CD44 neutralizing antibody treatment was able to significantly reduce C. neoformans association with HBMEC. Second, C. neoformans association was considerably impaired using either CD44-knock-down HBMEC or C. neoformans hyaluronic acid-deficient strains. Third, overexpression of CD44 in HBMEC increased their association activity towards C. neoformans. Fourth, confocal microscopic images showed that CD44 was enriched at and around the C. neoformans association sites. Fifth, upon C. neoformans and HBMEC engagement, a subpopulation of CD44 and actin translocated to the host membrane rafts. Our results highlight the interactions between C. neoformans hyaluronic acid and host CD44 and the dynamic results of these interactions, which may represent events during the adhesion and entry of C. neoformans at HBMEC membrane rafts.
Assuntos
Criptococose/microbiologia , Cryptococcus neoformans/fisiologia , Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologia , Receptores de Hialuronatos/metabolismo , Encéfalo/irrigação sanguínea , Membrana Celular/metabolismo , Células Cultivadas , Cryptococcus neoformans/química , Interações Hospedeiro-Patógeno , Humanos , Ácido Hialurônico/metabolismo , Meningoencefalite/microbiologiaRESUMO
Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans. Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant-negative form of PKCalpha in HBMEC. During C. neoformans infection, phosphorylation of PKCalpha was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKCalpha isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho-PKCalpha colocalized with beta-actin on the membrane of HBMEC. In addition, cytochalasin D (an F-filament-disrupting agent) inhibited fungus invasion into HBMEC in a dose-dependent manner. Furthermore, blockage of PKCalpha function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKCalpha and downstream actin filament activity during the fungal invasion into HBMEC.
Assuntos
Barreira Hematoencefálica/enzimologia , Cryptococcus neoformans/patogenicidade , Endotélio Vascular/enzimologia , Meningite Criptocócica/enzimologia , Proteína Quinase C-alfa/metabolismo , Citoesqueleto de Actina/metabolismo , Barreira Hematoencefálica/microbiologia , Barreira Hematoencefálica/patologia , Capilares/enzimologia , Capilares/microbiologia , Capilares/patologia , Células Cultivadas , Endotélio Vascular/microbiologia , Endotélio Vascular/patologia , Ativação Enzimática , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Meningite Criptocócica/microbiologia , Meningite Criptocócica/patologia , Fosforilação , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/genéticaRESUMO
IbeR is a regulator present in meningitic Escherichia coli strain E44 that carries a loss-of-function mutation in the stationary-phase (SP) regulatory gene rpoS. In order to determine whether IbeR is an SP regulator in E44, two-dimensional gel electrophoresis and LC-MS were used to compare the proteomes of a noninvasive ibeR deletion mutant BR2 and its parent strain E44 in the SP. Four up-regulated (TufB, GapA, OmpA, AhpC) and three down-regulated (LpdA, TnaA, OpmC) proteins in BR2 were identified when compared to E44. All these proteins contribute to energy metabolism or stress resistance, which is related to SP regulation. One of the down-regulated proteins, tryptophanase (TnaA), which is regulated by RpoS in other E. coli strains, is associated with SP regulation via production of a signal molecule indole. Our studies demonstrated that TnaA was required for E44 invasion, and that indole was able to restore the noninvasive phenotype of the tnaA mutant. The production of indole was significantly reduced in BR2, indicating that ibeR is required for the indole production via tnaA. Survival studies under different stress conditions indicated that IbeR contributed to bacteria stress resistance in the SP. Taken together, IbeR is a novel regulator contributing to the SP regulation.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fase S/genética , Fator sigma/metabolismo , Proteínas de Bactérias/genética , Células Cultivadas , Eletroforese em Gel Bidimensional , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Escherichia coli/fisiologia , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Indóis/metabolismo , Meningite devida a Escherichia coli/microbiologia , Mutação , Proteômica , Fator sigma/genética , Estresse Fisiológico , Triptofanase/metabolismoRESUMO
Extracellular vesicles (EVs) are secreted membrane vesicles, which play complex physiological and pathological functions in intercellular communication. Recently, we isolated natural killer (NK) cell-derived EVs (NK-EVs) from ex vivo expansion of NK cell cultures. The isolated NK-EVs contained cytotoxic proteins and several activated caspases, and they induced apoptosis in target cells. In this report, the protein levels of cytotoxic proteins from NK-EV isolates were analysed by ELISA. The mean values of perforin (PFN, 550 ng/mL), granzyme A (GzmA, 185 ng/mL), granzyme B (GzmB, 23.4 ng/mL), granulysin (GNLY, 56 ng/mL), and FasL (2.5 ng/mL) were obtained from >60 isolations using dot plots. The correlation between cytotoxicity and cytotoxic protein levels was examined by linear regression. PFN, GzmA, GzmB, GNLY all had a positive, moderate correlation with cytotoxicity, suggesting that there is not a single cytotoxic protein dominantly involved in killing and that all of these proteins may contribute to cytotoxicity. To further explore the possible killing mechanisms, cells were treated with NK-EVs, proteins extracted and lysates assessed by Western blotting. The levels of Gzm A substrates, SET and HMG2, were diminished in targeted cells, indicating that GzmA may induce a caspase-independent death pathway. Also, cytochrome C was released from mitochondria, a central hallmark of caspase-dependent death pathways. In addition, several ER-associated proteins were altered, suggesting that NK-EVs may induce ER stress resulting in cell death. Our results indicate that multiple killing mechanisms are activated by NK-derived EVs, including caspase-independent and -dependent cell death pathways, which can mediate cytotoxicity against cancer cells. Abbreviations: NK: natural killer cells; aNK: activated NK cells; EV: extracellular vesicles; ER: endoplasmic reticulum; ALL: acute lymphoblastic leukaemia; FBS: foetal bovine serum. GzmA: granzyme A; GzmB: granzyme B; GNLY: granulysin; PFN: perforin.
RESUMO
In neuroblastoma, the interplay between immune cells of the tumor microenvironment and cancer cells contributes to immune escape mechanisms and drug resistance. In this study, we show that natural killer (NK) cell-derived exosomes carrying the tumor suppressor microRNA (miR)-186 exhibit cytotoxicity against MYCN-amplified neuroblastoma cell lines. The cytotoxic potential of these exosomes was partly dependent upon expression of miR-186. miR-186 was downregulated in high-risk neuroblastoma patients, and its low expression represented a poor prognostic factor that directly correlated with NK activation markers (i.e., NKG2D and DNAM-1). Expression of MYCN, AURKA, TGFBR1, and TGFBR2 was directly inhibited by miR-186. Targeted delivery of miR-186 to MYCN-amplified neuroblastoma or NK cells resulted in inhibition of neuroblastoma tumorigenic potential and prevented the TGFß1-dependent inhibition of NK cells. Altogether, these data support the investigation of a miR-186-containing nanoparticle formulation to prevent tumor growth and TGFß1-dependent immune escape in high-risk neuroblastoma patients as well as the inclusion of ex vivo-derived NK exosomes as a potential therapeutic option alongside NK cell-based immunotherapy.Significance: These findings highlight the therapeutic potential of NK cell-derived exosomes containing the tumor suppressor miR-186 that inhibits growth, spreading, and TGFß-dependent immune escape mechanisms in neuroblastoma.
Assuntos
Exossomos/metabolismo , Células Matadoras Naturais/imunologia , MicroRNAs/genética , Neuroblastoma/prevenção & controle , Microambiente Tumoral/imunologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Exossomos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neuroblastoma/imunologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In order to dissect the pathogenesis of Cryptococcus neoformans meningoencephalitis, a genomic survey of the changes in gene expression of human brain microvascular endothelial cells infected by C. neoformans was carried out in a time-course study. Principal component analysis (PCA) revealed significant fluctuations in the expression levels of different groups of genes during the pathogen-host interaction. Self-organizing map (SOM) analysis revealed that most genes were up- or downregulated 2 folds or more at least at one time point during the pathogen-host engagement. The microarray data were validated by Western blot analysis of a group of genes, including beta-actin, Bcl-x, CD47, Bax, Bad, and Bcl-2. Hierarchical cluster profile showed that 61 out of 66 listed interferon genes were changed at least at one time point. Similarly, the active responses in expression of MHC genes were detected at all stages of the interaction. Taken together, our infectomic approaches suggest that the host cells significantly change the gene profiles and also actively participate in immunoregulations of the central nervous system (CNS) during C. neoformans infection.
Assuntos
Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidade , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Meningite Criptocócica/genética , Meningite Criptocócica/microbiologia , Microcirculação/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , HumanosRESUMO
Extracellular vesicles (EVs) deliver bioactive macromolecules (i.e. proteins, lipids and nucleic acids) for intercellular communication in multicellular organisms. EVs are secreted by all cell types including immune cells. Immune cell-derived EVs modulate diverse aspects of the immune system to either enhance or suppress immune activities. The extensive effects of immune cell-derived EVs have become the focus of great interest for various nano-biomedical applications, ranging from the medical use of nanoplatform-based diagnostic agents to the development of therapeutic interventions as well as vaccine applications, and thus may be ideal for 'immune-theranostic'. Here, we review the latest advances concerning the biological roles of immune cell-derived EVs in innate and acquired immunity. The intercellular communication amongst immune cells through their EVs is highlighted, showing that all immune cell-derived EVs have their unique function(s) in immunity through intricate interaction(s). Natural-killer (NK) cell-derived EVs, for example, contain potent cytotoxic proteins and induce apoptosis to targeted cancer cells. On the other hand, cancer cell-derived EVs bearing NK ligands may evade immune surveillance and responses. Finally, we discuss possible medical uses for the immune cell-derived EVs as a tool for immune-theranostic: as diagnostic biomarkers, for use in therapeutic interventions and for vaccination.
RESUMO
Extracellular vesicles (EVs) have been the focus of great interest, as they appear to be involved in numerous important cellular processes. They deliver bioactive macromolecules such as proteins, lipids, and nucleic acids, allowing intercellular communication in multicellular organisms. EVs are secreted by all cell types, including immune cells such as natural killer cells (NK), and they may play important roles in the immune system. Currently, a large-scale procedure to obtain functional NK EVs is lacking, limiting their use clinically. In this report, we present a simple, robust, and cost-effective method to isolate a large quantity of NK EVs. After propagating and activating NK cells ex vivo and then incubating them in exosome-free medium for 48 h, EVs were isolated using a polymer precipitation method. The isolated vesicles contain the tetraspanin CD63, an EV marker, and associated proteins (fibronectin), but are devoid of cytochrome C, a cytoplasmic marker. Nanoparticle tracking analysis showed a size distribution between 100 and 200 nm while transmission electron microscopy imaging displayed vesicles with an oval shape and comparable sizes, fulfilling the definition of EV. Importantly, isolated EV fractions were cytotoxic against cancer cells. Furthermore, our results demonstrate for the first time that isolated activated NK (aNK) cell EVs contain the cytotoxic proteins perforin, granulysin, and granzymes A and B, incorporated from the aNK cells. Activation of caspase -3, -7 and -9 was detected in cancer cells incubated with aNK EVs, and caspase inhibitors blocked aNK EV-induced cytotoxicity, suggesting that aNK EVs activate caspase pathways in target cells. The ability to isolate functional aNK EVs on a large scale may lead to new clinical applications. Abbreviations: NK: natural killer cells; activated NK (aNK) cells; EVs: extracellular vesicles; ALL: acute lymphoblastic leukaemia; aAPC: artificial antigen-presenting cell; TEM: transmission electron microscope; PBMC: peripheral blood mononuclear cells; FBS: foetal bovine serum.
RESUMO
One of the most challenging issues in HIV-associated neurocognitive disorders (HAND) caused by HIV-1 virotoxins and drug abuse is the lack of understanding the underlying mechanisms that are commonly associated with disorders of the blood-brain barrier (BBB), which mainly consists of brain microvascular endothelial cells (BMEC). Here, we hypothesized that Glycoprotein 120 (gp120), methamphetamine (METH) and nicotine (NT) can enhance amyloid-beta (Aß) accumulation in BMEC through Alpha7 nicotinic acetylcholine receptor (α7 nAChR). Both in vitro (human BMEC) (HBMEC) and in vivo (mice) models of BBB were used to dissect the role of α7 nAChR in up-regulation of Aß induced by gp120, METH and NT. Aß release from and transport across HBMEC were significantly increased by these factors. Methyllycaconitine (MLA), an antagonist of α7 nAChR, could efficiently block these pathogenic effects. Furthermore, our animal data showed that these factors could significantly increase the levels of Aß, Tau and Ubiquitin C-Terminal Hydrolase L1 (UCHL1) in mouse cerebrospinal fluid (CSF) and Aß in the mouse brains. These pathogenicities were significantly reduced by MLA, suggesting that α7 nAChR may play an important role in neuropathology caused by gp120, METH and NT, which are the major pathogenic factors contributing to the pathogenesis of HAND.
Assuntos
Amiloide/metabolismo , Encéfalo/patologia , Células Endoteliais/metabolismo , Proteína gp120 do Envelope de HIV/farmacologia , Metanfetamina/farmacologia , Nicotina/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Aconitina/análogos & derivados , Aconitina/farmacologia , Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Biomarcadores/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/lesões , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Movimento Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células HL-60 , Humanos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Transporte Proteico/efeitos dos fármacos , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteínas S100/metabolismo , Fatores de Tempo , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidoresRESUMO
Cip/Kip family protein p21, a cyclin-dependent kinase (CDK) inhibitor, is directly transactivated by retinoic acid receptor alpha (RARalpha) upon retinoic acid (RA):RARalpha binding. Yet the role of p21 upregulation by RA in lymphoma cells remains unknown. Here, we show that, in human pre-B lymphoma Nalm6 cells, RA-induced proliferation inhibition results from massive cell death characterized by apoptosis. Upregulated p21 by RA accompanies caspase-3 activation and precedes the occurrence of apoptosis. p21 induction leads to increased p21 complex formation with cyclin E/CDK2, which occurs when cyclin E and CDK2 levels remain constant. CDK2 can alternatively promote apoptosis, but the mechanisms remain unknown. Data presented here suggest a novel RA-signaling, by which RA-induced p21 induction and complex formation with cyclin E/CDK2 diverts CDK2 function from normally driving proliferation to alternatively promoting apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Tretinoína/farmacologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido RetinoicoRESUMO
BACKGROUND: Myocardial infarction (MI) elicits nerve sprouting. OBJECTIVES: The purpose of this study was to determine the spatial distribution of nerve sprouting and neurotrophic gene expression after MI. METHODS: We created MI in mice by coronary artery ligation. The hearts were removed 3 hours to 2 months after MI and examined for nerve fiber density and neurotrophic factor gene expression using Affymetrix microarray and mRNA analyses. RESULTS: The density of nerve fibers immunopositive for growth-associated protein (GAP)-43 was the highest 3 hours after MI both in the peri-infarct area and in the area remote to infarct, resulting in sympathetic (but not parasympathetic) hyperinnervation in the ventricles. The GAP-43-positive nerve fiber density of myocardium was greater in the outer transverse loop than in the inner vertical loop. The differences between these two myocardial loops peaked within 3 hours after MI and persisted for 2 months afterward. Gene expression of nerve growth factor, insulin-like growth factor, leukemia inhibitory factor, transforming growth factor-beta(3), and interleukin-1alpha was increased up to 2 months after MI compared with normal control. Expression of these growth factors was more pronounced and persistent in the peri-infarct area than in the remote area. CONCLUSION: MI induces sympathetic nerve sprouting in both peri-infarct and remote areas, more in the outer transverse loop. Selective up-regulation of nerve growth factor, insulin-like growth factor, leukemia inhibitory factor, transforming growth factor-beta(3), and interleukin-1alpha occurred in the peri-infarct area and, to a lesser extent, in the remote area.
Assuntos
Coração/inervação , Infarto do Miocárdio/fisiopatologia , Regeneração Nervosa , Sistema Nervoso Simpático/fisiologia , Animais , Eletrocardiografia , Proteína GAP-43/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interleucina-6/biossíntese , Interleucina-6/genética , Fator Inibidor de Leucemia , Camundongos , Modelos Animais , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Fibras Nervosas/metabolismo , Fator de Crescimento Neural/biossíntese , Fator de Crescimento Neural/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , Somatomedinas/biossíntese , Somatomedinas/genética , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/metabolismo , Fatores de TempoRESUMO
Eukaryotic CDC6 gene function is required for the initiation of DNA replication and is a key regulatory protein during cell cycle progression. The human CDC6 gene is not expressed in most normal tissues, in contrast with its marked expression in proliferating cancer cells. An effective way to explore the gene functions of CDC6 is to knock-down the CDC6 messenger RNA (mRNA) and examine the phenotypic consequences. In this chapter, we describe the construction of a lentivirus vector to express a CDC6 DNA segment. The transcript is able to fold by itself because the sense and antisense regions are complementary. There is a 9-nucleotide (nt) loop region allowing for the short hairpin RNA (shRNA) to form. Cellular ribonucleases process the shRNA into a functional short interfering RNA (siRNA). Down-regulation of Cdc6 protein is confirmed by Western blots.
Assuntos
Proteínas de Ciclo Celular/genética , Regulação para Baixo , Vetores Genéticos , MicroRNAs/genética , Proteínas Nucleares/genética , Interferência de RNA , Linhagem Celular , Linhagem Celular Tumoral , Exorribonucleases/genética , Células HeLa , Humanos , Lentivirus/genética , RNA Interferente Pequeno/metabolismo , Transdução GenéticaRESUMO
Bacterial meningitis remains the leading cause of disabilities worldwide. This life-threatening disease has a high mortality rate despite the availability of antibiotics and improved critical care. The interactions between bacterial surface components and host defense systems that initiate bacterial meningitis have been studied in molecular and cellular detail over the past several decades. Bacterial meningitis commonly exhibits triad hallmark features (THFs): pathogen penetration, nuclear factor-kappaB (NF-κB) activation in coordination with type 1 interferon (IFN) signaling and leukocyte transmigration that occur at the blood-brain barrier (BBB), which consists mainly of brain microvascular endothelial cells (BMEC). This review outlines the progression of these early inter-correlated events contributing to the central nervous system (CNS) inflammation and injury during the pathogenesis of bacterial meningitis. A better understanding of these issues is not only imperative to elucidating the pathogenic mechanism of bacterial meningitis, but may also provide the in-depth insight into the development of novel therapeutic interventions against this disease.
RESUMO
Cryptococcus neoformans is a life-threatening pathogenic yeast that causes devastating meningoencephalitis. The mechanism of cryptococcal brain invasion is largely unknown, and recent studies suggest that its extracellular microvesicles may be involved in the invasion process. The 14-3-3 protein is abundant in the extracellular microvesicles of C. neoformans, and the 14-3-3-GFP fusion has been used as the microvesicle's marker. However, the physiological role of 14-3-3 has not been explored. In this report, we have found that C. neoformans contains a single 14-3-3 gene that apparently is an essential gene. To explore the functions of 14-3-3, we substituted the promoter region of the 14-3-3 with the copper-controllable promoter CTR4. The CTR4 regulatory strain showed an enlarged cell size, drastic changes in morphology, and a decrease in the thickness of the capsule under copper-enriched conditions. Furthermore, the mutant cells produced a lower amount of total proteins in their extracellular microvesicles and reduced adhesion to human brain microvascular endothelial cells in vitro. Proteomic analyses of the protein components under 14-3-3-overexpressed and -suppressed conditions revealed that the 14-3-3 function(s) might be associated with the microvesicle biogenesis. Our results support that 14-3-3 has diverse pertinent roles in both physiology and pathogenesis in C. neoformans. Its gene functions are closely relevant to the pathogenesis of this fungus.