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1.
PLoS One ; 11(9): e0161730, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27603520

RESUMO

Myeloid dendritic cells (mDCs) contribute to both HIV pathogenesis and elicitation of antiviral immunity. Understanding how mDC responses to stimuli shape HIV infection outcomes will inform HIV prevention and treatment strategies. The long double-stranded RNA (dsRNA) viral mimic, polyinosinic polycytidylic acid (polyIC, PIC) potently stimulates DCs to focus Th1 responses, triggers direct antiviral activity in vitro, and boosts anti-HIV responses in vivo. Stabilized polyICLC (PICLC) is being developed for vaccine adjuvant applications in humans, making it critical to understand how mDC sensing of PICLC influences HIV infection. Using the monocyte-derived DC (moDC) model, we sought to describe how PICLC (vs. other dsRNAs) impacts HIV infection within DCs and DC-T cell mixtures. We extended this work to in vivo macaque rectal transmission studies by administering PICLC with or before rectal SIVmac239 (SIVwt) or SIVmac239ΔNef (SIVΔNef) challenge. Like PIC, PICLC activated DCs and T cells, increased expression of α4ß7 and CD169, and induced type I IFN responses in vitro. The type of dsRNA and timing of dsRNA exposure differentially impacted in vitro DC-driven HIV infection. Rectal PICLC treatment similarly induced DC and T cell activation and pro- and anti-HIV factors locally and systemically. Importantly, this did not enhance SIV transmission in vivo. Instead, SIV acquisition was marginally reduced after a single high dose challenge. Interestingly, in the PICLC-treated, SIVΔNef-infected animals, SIVΔNef viremia was higher, in line with the importance of DC and T cell activation in SIVΔNef replication. In the right combination anti-HIV strategy, PICLC has the potential to limit HIV infection and boost HIV immunity.


Assuntos
Carboximetilcelulose Sódica/análogos & derivados , Infecções por HIV/terapia , Ativação Linfocitária/imunologia , Poli I-C/genética , Polilisina/análogos & derivados , RNA de Cadeia Dupla/genética , Animais , Carboximetilcelulose Sódica/administração & dosagem , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/virologia , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/transmissão , HIV-1/genética , Humanos , Interferon Tipo I/genética , Ativação Linfocitária/genética , Macaca/imunologia , Macaca/virologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Células Mieloides/virologia , Poli I-C/administração & dosagem , Polilisina/administração & dosagem , Polilisina/genética , RNA de Cadeia Dupla/administração & dosagem , Vírus da Imunodeficiência Símia/genética , Células Th1/efeitos dos fármacos , Células Th1/imunologia
2.
J Leukoc Biol ; 97(4): 737-49, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25673294

RESUMO

DCs are the first immune cells to be exposed to allergens, including chemical sensitizers, such as nickel, a human TLR4 agonist that induces DC maturation. In ACD, DCs can interact with PMNs that are recruited and activated, leading, in particular, to ectosome release. The objective of this work was to characterize the effects of PMN-Ect on DC functions in an ACD context. We first developed a standardized protocol to produce, characterize, and quantify ectosomes by use of human PLB-985 cells, differentiated into mature PMN (PLB-Ect). We then studied the in vitro effects of these purified ectosomes on human moDC functions in response to NiSO4 and to LPS, another TLR4 agonist. Confocal fluorescence microscopy showed that PLB-Ect was internalized by moDCs and localized in the lysosomal compartment. We then showed that PLB-Ect down-regulated NiSO4-induced moDC maturation, as witnessed by decreased expression of CD40, CD80, CD83, CD86, PDL-1, and HLA-DR and by decreased levels of IL-1ß, IL-6, TNF-α, and IL-12p40 mRNAs. These effects were related to p38MAPK and NF-κB down-regulation. However, no increase in pan-regulatory DC marker genes (GILZ, CATC, C1QA) was observed; rather, levels of effector DC markers (Mx1, NMES1) were increased. Finally, when these PLB-Ect + NiSO4-treated moDCs were cocultured with CD4(+) T cells, a Th2 cytokine profile seemed to be induced, as shown, in particular, by enhanced IL-13 production. Together, these results suggest that the PMN-Ect can modulate DC maturation in response to nickel, a common chemical sensitizer responsible for ADC.


Assuntos
Alérgenos/imunologia , Antígenos CD/biossíntese , Micropartículas Derivadas de Células/fisiologia , Células Dendríticas/imunologia , Dermatite Alérgica de Contato/imunologia , Regulação da Expressão Gênica/imunologia , Linfocinas/biossíntese , Células Mieloides/imunologia , Neutrófilos/imunologia , Níquel/imunologia , Células Th2/citologia , Alérgenos/farmacologia , Antígenos CD/genética , Antígeno B7-H1/biossíntese , Antígeno B7-H1/genética , Diferenciação Celular , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Dermatite Alérgica de Contato/etiologia , Antígenos HLA-DR/biossíntese , Antígenos HLA-DR/genética , Humanos , Leucemia Mieloide Aguda/patologia , Lipossomos , Linfocinas/genética , Monócitos/citologia , Células Mieloides/ultraestrutura , Neutrófilos/ultraestrutura , Níquel/farmacologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia
3.
Clin Sci (Lond) ; 128(4): 257-67, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25074471

RESUMO

Homing of inflammatory cells to the liver is key in the progression of non-alcoholic steatohepatitis (NASH). An abnormal response of CD4+ T-cells from obese mice to the chemotactic effect of CXCL12 has been reported but the mechanism involved in this process and relevance in patients are unknown. We aimed to explore the mechanism involved in the abnormal chemotaxis of CXC chemokine ligand 12 (CXCL12) in several mouse models of NASH and the relevance in the context of human non-alcoholic fatty liver disease (NAFLD). We assessed chemotactic responsiveness of CD4+ T-cells to CXCL12, the effect of AMD3100, a CXC chemokine receptor 4 (CXCR4) antagonist, in mice and lymphocytes from patients with NAFLD, and the affinity of CXCL12 for CXCR4. CXCL12-promoted migration of CD4+ T-cells from three different mouse models of NASH was increased and dependent of CXCR4. CD4+ T-cells from patients with NASH, but not from patients with pure steatosis, responded more strongly to the chemotactic effect of CXCL12, and this response was inhibited by AMD3100. Treatment with AMD3100 decreased the number of CD4+ T-cells to the liver in ob/ob mice. CXCL12 expression in the liver, CXCR4 and CXCR7 expression in CD4+ T-cells were not increased in three different mouse models of NASH. However, the affinity of CXCL12 for CXCR4 was increased in CD4+ T-cells of ob/ob mice. In conclusion, the CXCL12/CXCR4 pathway contributes in both mice and patients to the enhanced recruitment of CD4+ T-cells in NASH. An increased affinity of CXCL12 to CXCR4 rather than a higher expression of the chemokine or its receptors is involved in this process.


Assuntos
Hepatopatia Gordurosa não Alcoólica/metabolismo , Receptores CXCR4/metabolismo , Adulto , Animais , Benzilaminas , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Ciclamos , Modelos Animais de Doenças , Feminino , Compostos Heterocíclicos/farmacologia , Humanos , Contagem de Linfócitos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Obesos , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/patologia , Receptores CXCR/metabolismo , Regulação para Cima/efeitos dos fármacos
4.
J Immunol ; 192(3): 886-96, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24367031

RESUMO

Systemic lupus erythematosus is a chronic inflammatory autoimmune disease, the development of which is characterized by a progressive loss of renal function. Such dysfunction is associated with leukocyte infiltration in the glomerular and tubulointerstitial compartments in both human and experimental lupus nephritis. In this study, we investigated the role of the Ccr1 chemokine receptor in this infiltration process during the progression of nephritis in the lupus-prone New Zealand Black/New Zealand White (NZB/W) mouse model. We found that peripheral T cells, mononuclear phagocytes, and neutrophils, but not B cells, from nephritic NZB/W mice were more responsive to Ccr1 ligands than the leukocytes from younger prenephritic NZB/W mice. Short-term treatment of nephritic NZB/W mice with the orally available Ccr1 antagonist BL5923 decreased renal infiltration by T cells and macrophages. Longer Ccr1 blockade decreased kidney accumulation of effector/memory CD4(+) T cells, Ly6C(+) monocytes, and both M1 and M2 macrophages; reduced tubulointerstitial and glomerular injuries; delayed fatal proteinuria; and prolonged animal lifespan. In contrast, renal humoral immunity was unaffected in BL5923-treated mice, which reflected the unchanged numbers of infiltrated B cells in the kidneys. Altogether, these findings define a pivotal role for Ccr1 in the recruitment of T and mononuclear phagocyte cells to inflamed kidneys of NZB/W mice, which in turn contribute to the progression of renal injury.


Assuntos
Nefrite Lúpica/terapia , Células Mieloides/imunologia , Infiltração de Neutrófilos , Receptores CCR1/antagonistas & inibidores , Subpopulações de Linfócitos T/imunologia , Fatores Etários , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Quimiocina CCL3/biossíntese , Quimiocina CCL3/deficiência , Quimiocina CCL3/genética , Quimiocina CCL3/fisiologia , Quimiocina CCL5/biossíntese , Quimiocina CCL5/genética , Quimiocina CCL5/fisiologia , Quimiotaxia de Leucócito , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Humanos , Rim/imunologia , Rim/patologia , Ligantes , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Camundongos , Camundongos Endogâmicos NZB , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Células Mieloides/patologia , Infiltração de Neutrófilos/efeitos dos fármacos , RNA Mensageiro/biossíntese , Distribuição Aleatória , Receptores CCR1/biossíntese , Receptores CCR1/genética , Receptores CCR1/fisiologia , Baço/imunologia , Baço/patologia , Esplenomegalia/etiologia , Esplenomegalia/imunologia , Esplenomegalia/patologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia
5.
Autophagy ; 9(5): 714-29, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23519090

RESUMO

Breast cancer tissue contains a small population of cells that have the ability to self-renew; these cells are known as cancer stem-like cells (CSCs). We have recently shown that autophagy is essential for the tumorigenicity of these CSCs. Salinomycin (Sal), a K (+) /H (+) ionophore, has recently been shown to be at least 100 times more effective than paclitaxel in reducing the proportion of breast CSCs. However, its mechanisms of action are still unclear. We show here that Sal blocked both autophagy flux and lysosomal proteolytic activity in both CSCs and non-CSCs derived from breast cancer cells. GFP-LC3 staining combined with fluorescent dextran uptake and LysoTracker-Red staining showed that autophagosome/lysosome fusion was not altered by Sal treatment. Acridine orange staining provided evidence that lysosomes display the characteristics of acidic compartments in Sal-treated cells. However, tandem mCherry-GFP-LC3 assay indicated that the degradation of mCherry-GFP-LC3 is blocked by Sal. Furthermore, the protein degradation activity of lysosomes was inhibited, as demonstrated by the rate of long-lived protein degradation, DQ-BSA assay and measurement of cathepsin activity. Our data indicated that Sal has a relatively greater suppressant effect on autophagic flux in the ALDH (+) population in HMLER cells than in the ALDH (-) population; moreover, this differential effect on autophagic flux correlated with an increase in apoptosis in the ALDH (+) population. ATG7 depletion accelerated the proapoptotic capacity of Sal in the ALDH (+) population. Our findings provide new insights into how the autophagy-lysosomal pathway contributes to the ability of Sal to target CSCs in vitro.


Assuntos
Autofagia/efeitos dos fármacos , Neoplasias da Mama/patologia , Células-Tronco Neoplásicas/patologia , Piranos/farmacologia , Laranja de Acridina/metabolismo , Aldeído Desidrogenase/metabolismo , Apoptose/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Células MCF-7 , Fusão de Membrana/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem , Enzimas Ativadoras de Ubiquitina/metabolismo
6.
PLoS Pathog ; 7(6): e1002109, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21738472

RESUMO

Herpes simplex virus type 2 (HSV-2) increases the risk of HIV-1 infection and, although several reports describe the interaction between these two viruses, the exact mechanism for this increased susceptibility remains unclear. Dendritic cells (DCs) at the site of entry of HSV-2 and HIV-1 contribute to viral spread in the mucosa. Specialized DCs present in the gut-associated lymphoid tissues produce retinoic acid (RA), an important immunomodulator, able to influence HIV-1 replication and a key mediator of integrin α4ß7 on lymphocytes. α4ß7 can be engaged by HIV-1 on the cell-surface and CD4⁺ T cells expressing high levels of this integrin (α4ß7 (high)) are particularly susceptible to HIV-1 infection. Herein we provide in-vivo data in macaques showing an increased percentage of α4ß7 (high) CD4⁺ T cells in rectal mucosa, iliac lymph nodes and blood within 6 days of rectal exposure to live (n = 11), but not UV-treated (n = 8), HSV-2. We found that CD11c⁺ DCs are a major target of HSV-2 infection in in-vitro exposed PBMCs. We determined that immature monocyte-derived DCs (moDCs) express aldehyde dehydrogenase ALDH1A1, an enzyme essential for RA production, which increases upon HSV-2 infection. Moreover, HSV-2-infected moDCs significantly increase α4ß7 expression on CD4⁺ T lymphocytes and HIV-1 infection in DC-T cell mixtures in a RA-dependent manner. Thus, we propose that HSV-2 modulates its microenviroment, influencing DC function, increasing RA production capability and amplifying a α4ß7 (high)CD4⁺ T cells. These factors may play a role in increasing the susceptibility to HIV-1.


Assuntos
Linfócitos T CD4-Positivos/virologia , Coinfecção/virologia , Células Dendríticas/patologia , Infecções por HIV/virologia , HIV-1 , Herpes Genital/virologia , Herpesvirus Humano 2 , Animais , Antígeno CD11c/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Células Dendríticas/virologia , Suscetibilidade a Doenças/patologia , Infecções por HIV/complicações , Herpes Genital/complicações , Integrinas/metabolismo , Linfonodos , Macaca , Mucosa/patologia , Mucosa/virologia , Reto , Tretinoína/metabolismo
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