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1.
Immunohorizons ; 3(12): 573-584, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31836639

RESUMO

Neutrophils and inflammatory monocytes are innate immune cells essential for protection during Listeria monocytogenes infection. Although certain functions have been generally assigned to each of the cells, similarities and differences in functions necessary for bacterial clearance have not previously been investigated. In the current study, phagocytosis, phagosomal containment, bacterial killing, and cytokine production by neutrophils and monocytes during L. monocytogenes infection were studied. Data obtained via in vitro studies show that neutrophils are more effective at L. monocytogenes uptake, phagosomal containment, and killing than monocytes. However, monocytes were found to be more effective at cytokine production during L. monocytogenes infection, in vivo. Additionally, the data demonstrated that neutrophils and monocytes are also capable of producing IL-1α, a cytokine that does not yet have a clearly defined role during infection with L. monocytogenes Furthermore, we were able to demonstrate a population of monocytes producing both TNF-α and IL-α, concurrently. This study highlights the multifunctional capabilities of neutrophils and monocytes, further adding to our knowledge of these innate immune cells during L. monocytogenes infection.


Assuntos
Listeria monocytogenes/imunologia , Listeriose/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Fagocitose/imunologia , Fagossomos/imunologia , Animais , Imunidade Inata , Interleucina-1alfa/metabolismo , Listeriose/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/metabolismo
2.
PLoS One ; 14(12): e0226778, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31877174

RESUMO

Francisella tularensis, a category-A bioterrorism agent causes tularemia. F. tularensis suppresses the immune response of host cells and intracellularly proliferates. However, the detailed mechanisms of immune suppression and intracellular growth are largely unknown. Here we developed a transposon mutant library to identify novel pathogenic factors of F. tularensis. Among 750 transposon mutants of F. tularensis subsp. novicida (F. novicida), 11 were isolated as less cytotoxic strains, and the genes responsible for cytotoxicity were identified. Among them, the function of slt, which encodes soluble lytic transglycosylase (SLT) was investigated in detail. An slt deletion mutant (Δslt) was less toxic to the human monocyte cell line THP-1 vs the wild-type strain. Although the wild-type strain proliferated in THP-1 cells, the number of intracellular Δslt mutant decreased in comparison. The Δslt mutant escaped from phagosomes during the early stages of infection, but the mutant was detected within the autophagosome, followed by degradation in lysosomes. Moreover, the Δslt mutant induced host cells to produce high levels of cytokines such as tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß, compared with the wild-type strain. These results suggest that the SLT of F. novicida is required for immune suppression and escape from autophagy to allow its survival in host cells.


Assuntos
Proteínas de Bactérias/imunologia , Francisella tularensis/imunologia , Glicosiltransferases/imunologia , Tularemia/imunologia , Animais , Linhagem Celular , Francisella tularensis/crescimento & desenvolvimento , Humanos , Evasão da Resposta Imune , Lisossomos/imunologia , Lisossomos/microbiologia , Camundongos , Monócitos/imunologia , Monócitos/microbiologia , Fagossomos/imunologia , Fagossomos/microbiologia , Tularemia/microbiologia
3.
Proc Natl Acad Sci U S A ; 116(50): 25106-25114, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31754039

RESUMO

Receptors of innate immune cells function synergistically to detect pathogens and elicit appropriate immune responses. Many receptor pairs also appear "colocalized" on the membranes of phagosomes, the intracellular compartments for pathogen ingestion. However, the nature of the seemingly receptor colocalization and the role it plays in immune regulation are unclear, due to the inaccessibility of intracellular phagocytic receptors. Here, we report a geometric manipulation technique to directly probe the role of phagocytic receptor "colocalization" in innate immune regulation. Using particles with spatially patterned ligands as phagocytic targets, we can decouple the receptor pair, Dectin-1 and Toll-like receptor (TLR)2, to opposite sides on a single phagosome or bring them into nanoscale proximity without changing the overall membrane composition. We show that Dectin-1 enhances immune responses triggered predominantly by TLR2 when their centroid-to-centroid proximity is <500 nm, but this signaling synergy diminishes upon receptor segregation beyond this threshold distance. Our results demonstrate that nanoscale proximity, not necessarily colocalization, between Dectin-1 and TLR2 is required for their synergistic regulation of macrophage immune responses. This study elucidates the relationship between the spatial organization of phagocytic receptors and innate immune responses. It showcases a technique that allows spatial manipulation of receptors and their signal cross-talk on phagosomes inside living cells.


Assuntos
Lectinas Tipo C , Fagossomos , Transdução de Sinais/imunologia , Receptor 2 Toll-Like , Animais , Membrana Celular/química , Membrana Celular/imunologia , Membrana Celular/metabolismo , Imunidade Inata/imunologia , Lectinas Tipo C/química , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Camundongos , Fagocitose/imunologia , Fagossomos/química , Fagossomos/imunologia , Fagossomos/metabolismo , Células RAW 264.7 , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo
4.
Science ; 366(6464): 460-467, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31649195

RESUMO

The nucleotide oligomerization domain (NOD)-like receptors 1 and 2 (NOD1/2) are intracellular pattern-recognition proteins that activate immune signaling pathways in response to peptidoglycans associated with microorganisms. Recruitment to bacteria-containing endosomes and other intracellular membranes is required for NOD1/2 signaling, and NOD1/2 mutations that disrupt membrane localization are associated with inflammatory bowel disease and other inflammatory conditions. However, little is known about this recruitment process. We found that NOD1/2 S-palmitoylation is required for membrane recruitment and immune signaling. ZDHHC5 was identified as the palmitoyltransferase responsible for this critical posttranslational modification, and several disease-associated mutations in NOD2 were found to be associated with defective S-palmitoylation. Thus, ZDHHC5-mediated S-palmitoylation of NOD1/2 is critical for their ability to respond to peptidoglycans and to mount an effective immune response.


Assuntos
Aciltransferases/metabolismo , Lipoilação , Proteína Adaptadora de Sinalização NOD1/química , Proteína Adaptadora de Sinalização NOD2/química , Transdução de Sinais , Animais , Cisteína/química , Células HCT116 , Células HEK293 , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptidoglicano , Fagossomos/imunologia , Fagossomos/microbiologia , Processamento de Proteína Pós-Traducional , Células RAW 264.7 , Salmonella typhimurium
5.
Nat Cell Biol ; 21(11): 1357-1369, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31659275

RESUMO

αMß2 integrin (complement receptor 3) is a major receptor for phagocytosis in macrophages. In other contexts, integrins' activities and functions are mechanically linked to actin dynamics through focal adhesions. We asked whether mechanical coupling of αMß2 integrin to the actin cytoskeleton mediates phagocytosis. We found that particle internalization was driven by formation of Arp2/3 and formin-dependent actin protrusions that wrapped around the particle. Focal complex-like adhesions formed in the phagocytic cup that contained ß2 integrins, focal adhesion proteins and tyrosine kinases. Perturbation of talin and Syk demonstrated that a talin-dependent link between integrin and actin and Syk-mediated recruitment of vinculin enable force transmission to target particles and promote phagocytosis. Altering target mechanical properties demonstrated more efficient phagocytosis of stiffer targets. Thus, macrophages use tyrosine kinase signalling to build a mechanosensitive, talin- and vinculin-mediated, focal adhesion-like molecular clutch, which couples integrins to cytoskeletal forces to drive particle engulfment.


Assuntos
Macrófagos/imunologia , Mecanotransdução Celular , Fagocitose/imunologia , Quinase Syk/genética , Talina/genética , Vinculina/genética , Citoesqueleto de Actina/imunologia , Citoesqueleto de Actina/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Actinas/genética , Actinas/imunologia , Animais , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Adesões Focais/imunologia , Adesões Focais/ultraestrutura , Forminas/genética , Forminas/imunologia , Regulação da Expressão Gênica , Humanos , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/imunologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Fagossomos/imunologia , Fagossomos/ultraestrutura , Poliestirenos , Cultura Primária de Células , Células RAW 264.7 , Quinase Syk/imunologia , Células THP-1 , Talina/imunologia , Vinculina/imunologia
6.
Med Sci (Paris) ; 35(8-9): 635-642, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31532375

RESUMO

Phagocytosis and macroautophagy, named here autophagy, are two essential mechanisms of lysosomal degradation of diverse cargos into membrane structures. Both mechanisms are involved in immune regulation and cell survival. However, phagocytosis triggers degradation of extracellular material whereas autophagy engulfs only cytoplasmic elements. Furthermore, activation and maturation of these two processes are different. LAP (LC3-associated phagocytosis) is a form of phagocytosis that uses components of the autophagy pathway. It can eliminate (i) pathogens, (ii) immune complexes, (iii) threatening neighbouring cells, dead or alive, and (iv) cell debris, such as POS (photoreceptor outer segment) and the midbody released at the end of mitosis. Cells have thus optimized their means of elimination of dangerous components by sharing some fundamental elements coming from the two main lysosomal degradation pathways.


Assuntos
Autofagia/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Fagocitose/fisiologia , Animais , Humanos , Evasão da Resposta Imune/fisiologia , Infecções/imunologia , Infecções/metabolismo , Infecções/patologia , Macrófagos/imunologia , Fagossomos/imunologia
7.
Biol Cell ; 111(10): 262-270, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31400233

RESUMO

BACKGROUND INFORMATION: After macrophage recognises and phagocytoses the microorganism, their phagosome undergoes a maturation process, which creates a hostile environment for the bacterium. The lumen is acidified, and proteolysis occurs to kill and degrade pathogen for further antigen presentation. It is important to understand the association between the macrophage intracellular activities and the outcome of infection. Different methods have been developed to measure the phagosome dynamics of macrophages, but there are still limitations. RESULTS: We used Mycobacterium tuberculosis (Mtb) antigens, the causative agent of tuberculosis (TB), as a model of infectious disease. Adopting a fluorescent bead-based assay, we developed beads coated with trehalose 6,6'dimycolate (TDM) from Mtb cell wall and ß-glucan from yeast cell wall to measure the macrophage phagosomal activities using a microplate reader. We examined the consistency of the assay using J774 cells and validated it using human monocyte-derived macrophages (hMDM) from healthy volunteers and TB patients. There was a decreased pH and increased proteolysis in the lumen of J774 cells after phagocytosing the ligand-coated beads. J774 macrophage showed no difference in the acidification and proteolysis in response to control IgG beads, TDM and ß-glucan beads. hMDM from healthy volunteers or TB patients showed heterogeneity in the intracellular activities when treated with ligand-coated beads. CONCLUSIONS AND SIGNIFICANCE: The beads coated with specific ligands from Mtb worked well in both macrophage cell line and human primary macrophages, which can be exploited to further study the phagosomal function of macrophage in TB. Our bead model can be applied to different ligands from other pathogens, which could extend the understanding of the associations between macrophage antimicrobial functions and outcomes of infectious diseases and the possible cellular mechanisms involved.


Assuntos
Antígenos de Bactérias/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Fagossomos/imunologia , Animais , Linhagem Celular , Humanos , Modelos Biológicos , Nanopartículas/química , beta-Glucanas/química
8.
EMBO J ; 38(16): e99266, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31271236

RESUMO

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human ß2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.


Assuntos
Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/química , Complexo de Endopeptidases do Proteassoma/imunologia , Microglobulina beta-2/metabolismo , Animais , Apresentação do Antígeno , Células Cultivadas , Apresentação Cruzada , Células Dendríticas/citologia , Endocitose , Humanos , Camundongos , Fagossomos/imunologia , Proteólise , Microglobulina beta-2/genética
9.
PLoS Pathog ; 15(6): e1007879, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199852

RESUMO

Burkholderia pseudomallei is a gram-negative, facultative intracellular bacterium, which causes a disease known as melioidosis. Professional phagocytes represent a crucial first line of innate defense against invading pathogens. Uptake of pathogens by these cells involves the formation of a phagosome that matures by fusing with early and late endocytic vesicles, resulting in killing of ingested microbes. Host Rab GTPases are central regulators of vesicular trafficking following pathogen phagocytosis. However, it is unclear how Rab GTPases interact with B. pseudomallei to regulate the transport and maturation of bacterial-containing phagosomes. Here, we showed that the host Rab32 plays an important role in mediating antimicrobial activity by promoting phagosome maturation at an early phase of infection with B. pseudomallei. And we demonstrated that the expression level of Rab32 is increased through the downregulation of the synthesis of miR-30b/30c in B. pseudomallei infected macrophages. Subsequently, we showed that B. pseudomallei resides temporarily in Rab32-positive compartments with late endocytic features. And Rab32 enhances phagosome acidification and promotes the fusion of B. pseudomallei-containing phagosomes with lysosomes to activate cathepsin D, resulting in restricted intracellular growth of B. pseudomallei. Additionally, Rab32 mediates phagosome maturation depending on its guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding state. Finally, we report the previously unrecognized role of miR-30b/30c in regulating B. pseudomallei-containing phagosome maturation by targeting Rab32 in macrophages. Altogether, we provide a novel insight into the host immune-regulated cellular pathway against B. pseudomallei infection is partially dependent on Rab32 trafficking pathway, which regulates phagosome maturation and enhances the killing of this bacterium in macrophages.


Assuntos
Burkholderia pseudomallei/imunologia , Melioidose/imunologia , MicroRNAs/imunologia , Fagossomos/imunologia , Proteínas rab de Ligação ao GTP/imunologia , Animais , Burkholderia pseudomallei/patogenicidade , Melioidose/patologia , Camundongos , Viabilidade Microbiana/imunologia , Fagossomos/microbiologia , Fagossomos/patologia , Células RAW 264.7
10.
Int Rev Immunol ; 38(2): 57-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31117900

RESUMO

Phagosome-lysosome (P-L) fusion is one of the central immune-effector responses of host. It is known that phagosome maturation process is associated with numerous signaling cascades and among these, important role of calcium (Ca2+) signaling has been realized recently. Ca2+ plays key roles in actin rearrangement, activation of NADPH oxidase and protein kinase C (PKC). Involvement of Ca2+ in these cellular processes directs phagosomal maturation process. Some of the intracellular pathogens have acquired the strategies to modulate Ca2+ associated pathways to block P-L fusion process. In this review we have described the mechanism of Ca2+ signals that influence P-L fusion by controlling ROS, actin and PKC signaling cascades. We have also discussed the strategies implemented by the intracellular pathogens to manipulate Ca2+ signaling to consequently subvert P-L fusion. A detail study of factors associated in manipulating Ca2+ signaling may provide new insights for the development of therapeutic tools for more effective treatment options against infectious diseases.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Fagocitose , Fagossomos/metabolismo , Actinas/metabolismo , Animais , Citotoxicidade Imunológica , Humanos , Espaço Intracelular , Lisossomos/metabolismo , Macrófagos/metabolismo , Fagocitose/imunologia , Fagossomos/imunologia , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
11.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31036602

RESUMO

The fungal pathogen Histoplasma capsulatum causes a spectrum of disease, ranging from local pulmonary infection to disseminated disease. The organism seeks residence in macrophages, which are permissive for its survival. Hypoxia-inducible factor 1α (HIF-1α), a principal regulator of innate immunity to pathogens, is necessary for macrophage-mediated immunity to H. capsulatum in mice. In the present study, we analyzed the effect of HIF-1α in human macrophages infected with this fungus. HIF-1α stabilization was detected in peripheral blood monocyte-derived macrophages at 2 to 24 h after infection with viable yeast cells. Further, host mitochondrial respiration and glycolysis were enhanced. In contrast, heat-killed yeasts induced early, but not later, stabilization of HIF-1α. Since the absence of HIF-1α is detrimental to host control of infection, we asked if large amounts of HIF-1α protein, exceeding those induced by H. capsulatum, altered macrophage responses to this pathogen. Exposure of infected macrophages to an HIF-1α stabilizer significantly reduced recovery of H. capsulatum from macrophages and produced a decrement in mitochondrial respiration and glycolysis compared to those of controls. We observed recruitment of the autophagy-related protein LC3-II to the phagosome, whereas enhancing HIF-1α reduced phagosomal decoration. This finding suggested that H. capsulatum exploited an autophagic process to survive. In support of this assertion, inhibition of autophagy activated macrophages to limit intracellular growth of H. capsulatum Thus, enhancement of HIF-1α creates a hostile environment for yeast cells in human macrophages by interrupting the ability of the pathogen to provoke host cell autophagy.


Assuntos
Histoplasma/imunologia , Histoplasmose/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Macrófagos/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Animais , Autofagia , Histoplasmose/genética , Histoplasmose/microbiologia , Histoplasmose/fisiopatologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Macrófagos/microbiologia , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Fagossomos/imunologia , Fagossomos/microbiologia
12.
J Clin Invest ; 129(5): 1926-1939, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30939123

RESUMO

IL-26 is an antimicrobial protein secreted by Th17 cells that has the ability to directly kill extracellular bacteria. To ascertain whether IL-26 contributes to host defense against intracellular bacteria, we studied leprosy, caused by the obligate intracellular pathogen Mycobacterium leprae, as a model. Analysis of leprosy skin lesions by gene expression profiling and immunohistology revealed that IL-26 was more strongly expressed in lesions from the self-limited tuberculoid compared with expression in progressive lepromatous patients. IL-26 directly bound to M. leprae in axenic culture and reduced bacteria viability. Furthermore, IL-26, when added to human monocyte-derived macrophages infected with M. leprae, entered the infected cell, colocalized with the bacterium, and reduced bacteria viability. In addition, IL-26 induced autophagy via the cytoplasmic DNA receptor stimulator of IFN genes (STING), as well as fusion of phagosomes containing bacilli with lysosomal compartments. Altogether, our data suggest that the Th17 cytokine IL-26 contributes to host defense against intracellular bacteria.


Assuntos
Interleucinas/imunologia , Hanseníase Virchowiana/microbiologia , Hanseníase Tuberculoide/microbiologia , Células Th17/imunologia , Autofagia , Citocinas/imunologia , Perfilação da Expressão Gênica , Humanos , Lisossomos/imunologia , Lisossomos/microbiologia , Macrófagos/imunologia , Monócitos/citologia , Mycobacterium leprae , Mycobacterium tuberculosis , Fagossomos/imunologia , Proteínas Recombinantes/imunologia , Transdução de Sinais
13.
Am J Physiol Regul Integr Comp Physiol ; 316(6): R819-R831, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30943046

RESUMO

Voltage-gated proton channel (Hv1) has been studied in various immune cells, including neutrophils. However, most studies have taken an in vitro approach using isolated cells or primary cultured cells of mammals; therefore, limited evidence is available on the function of Hv1 in a physiological context. In this study, we have developed the in vivo system that enables real-time functional analysis of Hv1 using zebrafish embryos (Danio rerio). Hvcn1-deficiency (hvcn1-/-) in zebrafish completely abolished voltage-gated proton current, which is typically observed in wild-type neutrophils. Importantly, hvcn1-deficiency significantly reduced reactive oxygen species production and calcium response of zebrafish neutrophils, comparable to the results observed in mammalian models. These findings verify zebrafish Hv1 (DrHv1) as the primary contributor for native Hv1-derived proton current in neutrophils and suggest the conserved function of Hv1 in the immune cells across vertebrate animals. Taking advantage of Hv1 zebrafish model, we compared real-time behaviors of neutrophils between wild-type and hvcn1-/- zebrafish in response to tissue injury and acute bacterial infection. Notably, we observed a significant increase in the number of phagosomes in hvcn1-/- neutrophils, raising a possible link between Hv1 and phagosomal maturation. Furthermore, survival analysis of zebrafish larvae potentially supports a protective role of Hv1 in the innate immune response against systemic bacterial infection. This study represents the influence of Hv1 on neutrophil behaviors and highlights the benefits of in vivo approach toward the understanding of Hv1 in a physiological context.


Assuntos
Canais Iônicos/metabolismo , Neutrófilos/metabolismo , Fagossomos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio , Imunidade Inata , Ativação do Canal Iônico , Canais Iônicos/deficiência , Canais Iônicos/genética , Potenciais da Membrana , Neutrófilos/imunologia , Fagocitose , Fagossomos/imunologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
14.
J Immunol ; 202(10): 2971-2981, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30944160

RESUMO

Phagocytosis is an essential element of the immune response, assuring the elimination of pathogens, cellular debris, and apoptotic and tumoral cells. Activation of phagocytosis by the FcγR stimulates phospholipase D (PLD) activity and triggers the production of phosphatidic acid (PA) at the plasma membrane of macrophages, but the regulatory mechanisms involved are still not clearly understood. In this study, we examined the role of the small GTPase Arf6 in the activation of the PLD isoforms during FcγR-mediated phagocytosis. In RAW 264.7 macrophage cells, expressed Arf6-GFP partially colocalized with PLD1-hemagglutinin on intracellular membrane-bound vesicles and with PLD2-hemagglutinin at the plasma membrane. Both PLD isoforms were found to interact with Arf6 during FcγR-mediated phagocytosis as seen by immunoprecipitation experiments. In macrophages stimulated for phagocytosis, Arf6 was observed to be associated with nascent phagosomes. RNA interference knockdown of Arf6 reduced the amount of active Arf6 associated with phagosomes, revealed by the MT2-GFP probe that specifically binds to Arf6-GTP. Arf6 silencing concomitantly decreased PLD activity as well as the levels of PA found on phagosomes and phagocytic sites as shown with the PA probe Spo20p-GFP. Altogether, our results indicate that Arf6 is involved in the regulation of PLD activity and PA synthesis required for efficient phagocytosis.


Assuntos
Fatores de Ribosilação do ADP/imunologia , Macrófagos/imunologia , Fagocitose , Fosfolipase D/imunologia , Receptores de IgG/imunologia , Fatores de Ribosilação do ADP/genética , Animais , Isoenzimas/genética , Isoenzimas/imunologia , Macrófagos/citologia , Camundongos , Fagossomos/genética , Fagossomos/imunologia , Ácidos Fosfatídicos/genética , Ácidos Fosfatídicos/imunologia , Fosfolipase D/genética , Células RAW 264.7 , Receptores de IgG/genética
15.
Insect Biochem Mol Biol ; 109: 1-12, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30953686

RESUMO

Phagocytosis is an evolutionarily conserved mechanism that plays a key role in both host defence and tissue homeostasis in multicellular organisms. A range of surface receptors expressed on different cell types allow discriminating between self and non-self (or altered) material, thus enabling phagocytosis of pathogens and apoptotic cells. The phagocytosis process can be divided into four main steps: 1) binding of the phagocyte to the target particle, 2) particle internalization and phagosome formation, through remodelling of the plasma membrane, 3) phagosome maturation, and 4) particle destruction in the phagolysosome. In this review, we describe our present knowledge on phagocytosis in the fruit fly Drosophila melanogaster, assessing each of the key steps involved in engulfment of both apoptotic cells and bacteria. We also assess the physiological role of phagocytosis in host defence, development and tissue homeostasis.


Assuntos
Drosophila melanogaster/imunologia , Imunidade Inata , Fagocitose/imunologia , Fagossomos/imunologia , Animais , Drosophila melanogaster/crescimento & desenvolvimento
16.
J Infect Dis ; 220(3): 514-523, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-30923818

RESUMO

As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF-exposed Mtb had reduced control and fewer phagosome-lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.


Assuntos
Pulmão/imunologia , Pulmão/microbiologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Tuberculose/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imunidade Inata/imunologia , Inflamação/imunologia , Inflamação/microbiologia , Lisossomos/imunologia , Lisossomos/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Fagossomos/imunologia , Fagossomos/microbiologia , Proteína A Associada a Surfactante Pulmonar/imunologia , Proteína D Associada a Surfactante Pulmonar/imunologia , Tuberculose/microbiologia , Adulto Jovem
17.
J Immunol Methods ; 468: 55-60, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30880262

RESUMO

Antibody-based therapeutics are powerful tools to treat disease. While their mechanism of action (MOA) always involves binding to a specific target via the Fab region of the antibody, the induction of effector functions through the Fc region of the antibody is equally important for antibody therapeutics designed to deplete tumor cells. By binding of the Fc region to Fc gamma receptors (FcγRs) on the surface of immune cells or complement factors, antibody therapeutics exert effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), both of which induce target cell death and aid in the efficacy of treatment. Another major Fc effector function is antibody-dependent cellular phagocytosis (ADCP). ADCP is the mechanism by which antibody-opsonized target cells activate the FcγRs on the surface of macrophages to induce phagocytosis, resulting in internalization and degradation of the target cell through phagosome acidification. ADCP has been implicated as a major MOA of several biologics, but this activity is difficult to measure in in vitro. Most assays measure the association of target cells and macrophages; however, co-localization can represent cell attachment rather than internalization. Here, we describe the development of a novel method to accurately measure ADCP activity. By labeling target cells with a pH sensitive dye that only fluoresces in mature phagosomes, the ADCP activity of antibody therapeutics can be accurately quantitated via flow cytometry.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Macrófagos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Fagocitose/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Rituximab/farmacologia , Anticorpos Monoclonais Humanizados/metabolismo , Linhagem Celular Tumoral , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Fagossomos/imunologia , Fagossomos/metabolismo , Fagossomos/patologia , Receptores de IgG/metabolismo , Rituximab/metabolismo
18.
Cell Physiol Biochem ; 52(2): 280-301, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30816675

RESUMO

Acid sphingomyelinase hydrolyzes sphingomyelin to ceramide and phosphorylcholine. Ceramide molecules spontaneously interact with each other and generate ceramide-enriched membrane domains. These ceramide-enriched domains further fuse, forming large ceramideenriched platforms that participate in the organization of receptors and in the amplification of signaling molecules. Recent studies have suggested several bacteria and bacterial toxins that stimulate the activation and the translocation of acid sphingomyelinase, which leads to the release of ceramide. The acid sphingomyelinase/ceramide system also regulates the internalization of bacteria into the host cell, the subsequent cytokine release, inflammatory response, and initiation of host cell apoptosis. In addition, ceramide has been implicated in the fusion of phagosomes and lysosomes upon bacterial infection. Thus, this system modulates the reorganization of cell membrane receptors and intracellular signaling molecules during bacteria-host interactions. The acid sphingomyelinase and ceramide system may thus serve as a novel therapeutic target for treating infections.


Assuntos
Infecções Bacterianas/imunologia , Toxinas Bacterianas/imunologia , Ceramidas/imunologia , Transdução de Sinais/imunologia , Esfingomielina Fosfodiesterase/imunologia , Animais , Infecções Bacterianas/patologia , Ativação Enzimática/imunologia , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Lisossomos/imunologia , Lisossomos/microbiologia , Fagossomos/imunologia , Fagossomos/microbiologia
19.
Am J Med Sci ; 357(3): 184-194, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30797501

RESUMO

Phagocytosis is necessary for antigen degradation and presentation, the activation of the adaptive immune response and the elimination of pathogenic micro-organisms. The phagosomal vacuole formed during phagocytosis requires a process of maturation that involves fusion with lysosomes, a decrease in luminal pH and the activation of the enzymes that eventually will destroy phagocytized micro-organisms. Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. This agent has developed various strategies to prevent phagosome maturation and persist indefinitely in latency mode. Herein, we review these strategies in the light of available experimental evidence. A better understanding of them may be essential in the development of more effective therapies against tuberculosis.


Assuntos
Imunidade Adaptativa/fisiologia , Evasão da Resposta Imune , Mycobacterium tuberculosis/fisiologia , Fagocitose/fisiologia , Humanos , Lisossomos/imunologia , Fagossomos/imunologia
20.
PLoS One ; 14(1): e0210979, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30689633

RESUMO

Interaction between surfactant protein-A (SP-A) and toll-like receptor (TLR)4 plays a critical role in host defense. In this work, we studied the host defense function of SPA4 peptide (amino acids GDFRYSDGTPVNYTNWYRGE), derived from the TLR4-interacting region of SP-A, against Pseudomonas aeruginosa. We determined the binding of SPA4 peptide to live bacteria, and its direct antibacterial activity against P. aeruginosa. Pro-phagocytic and anti-inflammatory effects were investigated in JAWS II dendritic cells and primary alveolar macrophages. The biological relevance of SPA4 peptide was evaluated in a mouse model of acute lung infection induced by intratracheal challenge with P. aeruginosa. Our results demonstrate that the SPA4 peptide does not interact with or kill P. aeruginosa when cultured outside the host. The SPA4 peptide treatment induces the uptake and localization of bacteria in the phagolysosomes of immune cells. At the same time, the secreted amounts of TNF-α are significantly reduced in cell-free supernatants of SPA4 peptide-treated cells. In cells overexpressing TLR4, the TLR4-induced phagocytic response is maintained, but the levels of TLR4-stimulated TNF-α are reduced. Furthermore, our results demonstrate that the therapeutic administration of SPA4 peptide reduces bacterial burden, inflammatory cytokines and chemokines, intracellular signaling, and lactate levels, and alleviates lung edema and tissue damage in P. aeruginosa-infected mice. Together, our results suggest that the treatment with SPA4 peptide can help control the bacterial burden, inflammation, and tissue injury in a P. aeruginosa lung infection model.


Assuntos
Fragmentos de Peptídeos/uso terapêutico , Pneumonia Bacteriana/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa , Proteína A Associada a Surfactante Pulmonar/uso terapêutico , Receptor 4 Toll-Like/metabolismo , Animais , Carga Bacteriana , Células Cultivadas , Modelos Animais de Doenças , Feminino , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fagocitose/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Fagossomos/imunologia , Fagossomos/microbiologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Ligação Proteica , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Proteína A Associada a Surfactante Pulmonar/imunologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/imunologia
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