RESUMO
Receptor CD300b is implicated in regulating the immune response to bacterial infection by an unknown mechanism. Here, we identified CD300b as a lipopolysaccharide (LPS)-binding receptor and determined the mechanism underlying CD300b augmentation of septic shock. In vivo depletion and adoptive transfer studies identified CD300b-expressing macrophages as the key cell type augmenting sepsis. We showed that CD300b, and its adaptor DAP12, associated with Toll-like receptor 4 (TLR4) upon LPS binding, thereby enhancing TLR4-adaptor MyD88- and TRIF-dependent signaling that resulted in an elevated pro-inflammatory cytokine storm. LPS engagement of the CD300b-TLR4 complex led to the recruitment and activation of spleen tyrosine kinase (Syk) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). This resulted in an inhibition of the ERK1/2 protein kinase- and NF-κB transcription factor-mediated signaling pathways, which subsequently led to a reduced interleukin-10 (IL-10) production. Collectively, our data describe a mechanism of TLR4 signaling regulated by CD300b in myeloid cells in response to LPS.
Assuntos
Interleucina-10/metabolismo , Macrófagos/imunologia , Peritonite/imunologia , Receptores Imunológicos/metabolismo , Sepse/imunologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Células HEK293 , Humanos , Interleucina-10/genética , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Receptores Imunológicos/genética , Transdução de Sinais , Quinase Syk/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.
Assuntos
Actinas/metabolismo , Bactérias/imunologia , Caspases/metabolismo , Lisossomos/metabolismo , Fagossomos/metabolismo , Multimerização Proteica , Fatores de Despolimerização de Actina/metabolismo , Animais , Bactérias/crescimento & desenvolvimento , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Caspase 1/deficiência , Caspase 1/genética , Caspase 1/metabolismo , Caspases/deficiência , Caspases/genética , Caspases Iniciadoras , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagossomos/microbiologia , FosforilaçãoRESUMO
The increasing prevalence of inflammatory diseases and the adverse effects associated with the long-term use of current anti-inflammatory therapies prompt the identification of alternative approaches to reestablish immune balance. Apigenin, an abundant dietary flavonoid, is emerging as a potential regulator of inflammation. Here, we show that apigenin has immune-regulatory activity in vivo. Apigenin conferred survival to mice treated with a lethal dose of Lipopolysaccharide (LPS) restoring normal cardiac function and heart mitochondrial Complex I activity. Despite the adverse effects associated with high levels of splenocyte apoptosis in septic models, apigenin had no effect on reducing cell death. However, we found that apigenin decreased LPS-induced apoptosis in lungs, infiltration of inflammatory cells and chemotactic factors' accumulation, re-establishing normal lung architecture. Using NF-κB luciferase transgenic mice, we found that apigenin effectively modulated NF-κB activity in the lungs, suggesting the ability of dietary compounds to exert immune-regulatory activity in an organ-specific manner. Collectively, these findings provide novel insights into the underlying immune-regulatory mechanisms of dietary nutraceuticals in vivo.
Assuntos
Anti-Inflamatórios/farmacologia , Apigenina/farmacologia , Infiltração Leucêmica/tratamento farmacológico , NF-kappa B/metabolismo , Sepse/tratamento farmacológico , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Apigenina/administração & dosagem , Apigenina/uso terapêutico , Apoptose , Suplementos Nutricionais , Infiltração Leucêmica/imunologia , Lipopolissacarídeos/toxicidade , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Sepse/imunologia , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologiaRESUMO
Monocytes, key components of the immune system, are a heterogeneous population comprised of classical monocytes (CD16(-) ) and non-classical monocytes (CD16(+) ). Monocytes are short lived and undergo spontaneous apoptosis, unless stimulated. Dysregulation of monocyte numbers contribute to the pathophysiology of inflammatory diseases, yet the contribution of each subset remains poorly characterized. Protein kinase C (PKC) family members are central to monocyte biology; however, their role in regulating lifespan and immune function of CD16(-) and CD16(+) monocytes has not been studied. Here, we evaluated the contribution of PKCδ and PKCε in the lifespan and immune response of both monocyte subsets. We showed that CD16(+) monocytes are more susceptible to spontaneous apoptosis because of the increased caspase-3, -8 and -9 activities accompanied by higher kinase activity of PKCδ. Silencing of PKCδ reduced apoptosis in both CD16(+) and CD16(-) monocytes. CD16(+) monocytes express significantly higher levels of PKCε and produce more tumour necrosis factor-α in CD16(+) compared with CD16(-) monocytes. Silencing of PKCε affected the survival and tumour necrosis factor-α production. These findings demonstrate a complex network with similar topography, yet unique regulatory characteristics controlling lifespan and immune response in each monocyte subset, helping define subset-specific coordination programmes controlling monocyte function.
Assuntos
Monócitos/enzimologia , Monócitos/imunologia , Proteína Quinase C-delta/imunologia , Proteína Quinase C-delta/metabolismo , Proteína Quinase C-épsilon/imunologia , Proteína Quinase C-épsilon/metabolismo , Apoptose , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Sobrevivência Celular , Células Cultivadas , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/imunologia , Humanos , Monócitos/classificação , Monócitos/patologia , Proteína Quinase C-delta/genética , Proteína Quinase C-épsilon/genética , Interferência de RNA , Receptores de IgG/deficiência , Receptores de IgG/imunologia , Transdução de Sinais , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/sangueRESUMO
Some arthropod-borne obligate intracellular rickettsiae are among the most virulent human pathogens. Upon entry, Rickettsia species modulate immune (e.g., macrophages; MΦ) and non-immune cell (e.g., endothelial cells) responses to create a habitable environment for host colonization. In particular, MΦ play a crucial role in either terminating an infection at an early stage or succumbing to bacterial replication and colonization. However, our understanding on how Rickettsia species modulate crucial cellular processes within MΦ, including phagocytosis, and host cell defenses, to establish an intracytosolic replication niche, remain poorly defined. In this study, we describe a previously unappreciated mechanism, in which pathogenic rickettsiae infection is mediated by the phosphatidylserine (PS)-binding receptor, CD300f. We found that CD300f -/- mice but not wild-type (WT) C57BL/6J mice were protected against R. typhi - or R. rickettsii [ Shelia Smith ]-induced fatal rickettsiosis. Adoptative transfer studies further revealed that CD300f-expressing bone marrow-derived macrophages (BMDMΦ) are important mediators to control rickettsiosis in WT mice. Mechanistical analysis, using WT or CD300f -/- BMDMΦ, showed that CD300f facilitates the engulfment of both pathogenic R. typhi and R. rickettsii species, likely via a PS-mediated mechanism. Furthermore, CD300f was involved in the intracytosolic replication of both pathogenic rickettsiae by differentially modulating the anti-inflammatory Interleukin (IL)-10 and anti-rickettsial IL-1α and IL-1ß cytokine responses. Collectively, our findings describe a previously unappreciated role for the efferocytic receptor, CD300f, to facilitate engulfment and the intracellular survival of pathogenic rickettsiae within the host. Significance Statement: Vector-borne diseases, which are transmitted by hematophagous arthropods, like ticks and fleas, present a perilous threat to public health. In fact, tick- and flea-borne rickettsial diseases are on the rise globally and our current inadequate understanding on how Rickettsia interacts with their mammalian host has significantly impaired the development of effective interventions against pathogenic rickettsial infections. Here, we identified the phosphatidylserine (PS)-receptor, CD300f, as an important mediator of pathogenic rickettsiae infection in vivo and in vitro . Specifically, we showed that CD300f-expressing macrophages facilitate rickettsial infection by differentially modulating anti-inflammatory Interleukin (IL)-10 and anti-rickettsial IL-1α and IL-1ß cytokine responses. In sum, our data described CD300f as an important regulator of rickettsial infection and may present a target for therapeutic intervention.
RESUMO
IMPORTANCE: Rickettsia spp. are intracellular bacterial parasites of a wide range of arthropod and vertebrate hosts. Some rickettsiae are responsible for several severe human diseases globally. One interesting feature of these pathogens is their ability to exploit host cytosolic defense responses to their benefits. However, the precise mechanism by which pathogenic Rickettsia spp. elude host defense responses remains unclear. Here, we observed that pathogenic Rickettsia typhi and Rickettsia rickettsii (Sheila Smith [SS]), but not non-pathogenic Rickettsia montanensis, become ubiquitinated and induce autophagy upon entry into macrophages. Moreover, unlike R. montanensis, R. typhi and R. rickettsii (SS) colocalized with LC3B but not with Lamp2 upon host cell entry. Finally, we observed that both R. typhi and R. rickettsii (SS), but not R. montanensis, reduce pro-inflammatory interleukin-1 (IL-1) responses, likely via an autophagy-mediated mechanism. In summary, we identified a previously unappreciated pathway by which both pathogenic R. typhi and R. rickettsii (SS) become ubiquitinated, induce autophagy, avoid autolysosomal destruction, and reduce microbicidal IL-1 cytokine responses to establish an intracytosolic niche in macrophages.
Assuntos
Interleucina-1 , Rickettsia , Humanos , Citocinas , Rickettsia/fisiologia , Macrófagos/microbiologia , AutofagiaRESUMO
Differences in CD8(+)CD57(-) and CD8(+)CD57(+) lymphocyte lifespan have been documented. Lower numbers and shorter lifespan are characteristic of CD8(+)CD57(+) in normal individuals. However, CD8(+)CD57(+) are expanded in certain disease states including T cell large granular leukemia and other hematologic malignancies. The mechanisms responsible for the differences in CD8(+)CD57(-) and CD8(+)CD57(+) lifespan remain elusive. In this study, we demonstrate that the small heat shock protein (Hsp) 27 is a key regulator of CD8(+)CD57(+) lymphocyte lifespan. We found that Hsp27 expression is significantly lower in CD8(+)CD57(+) than in CD8(+)CD57(-) lymphocytes. In contrast, Hsp60 and Hsp70 are expressed at comparable levels. Unlike other antiapoptotic Bcl-2-like molecules, the expression of Hsp27 tightly correlates with CD8(+)CD57(+) and CD8(+)CD57(-) lifespan. We demonstrate that Hsp27 overexpression in CD8(+)CD57(+) lymphocytes to levels found normally in CD8(+)CD57(-) lymphocytes decreased apoptosis. Accordingly, silencing of Hsp27 in CD8(+)CD57(-) lymphocytes increased apoptosis. Collectively these results demonstrate that Hsp27 is a critical regulator of normal CD8(+)CD57(+) lifespan supporting its use as a marker of lifespan in this lineage, and suggest a mechanism responsible for the decreased apoptosis and clonal expansion characteristic of certain disease states.
Assuntos
Antígenos CD57/biossíntese , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteínas de Choque Térmico HSP27/fisiologia , Apoptose/genética , Apoptose/imunologia , Antígenos CD57/genética , Antígenos CD57/metabolismo , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Células Clonais , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Regulação da Expressão Gênica/imunologia , Marcadores Genéticos/genética , Proteínas de Choque Térmico HSP27/biossíntese , Proteínas de Choque Térmico HSP27/genética , Humanos , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Bacterial infection is a highly complex biological process involving a dynamic interaction between the invading microorganism and the host. Specifically, intracellular pathogens seize control over the host cellular processes including membrane dynamics, actin cytoskeleton, phosphoinositide metabolism, intracellular trafficking and immune defense mechanisms to promote their host colonization. To accomplish such challenging tasks, virulent bacteria deploy unique species-specific secreted effectors to evade and/or subvert cellular defense surveillance mechanisms to establish a replication niche. However, despite superficially similar infection strategies, diverse Rickettsia species utilize different effector repertoires to promote host colonization. This review will discuss our current understandings on how different Rickettsia species deploy their effector arsenal to manipulate host cellular processes to promote their intracytosolic life within the mammalian host.
Assuntos
Vetores Artrópodes/microbiologia , Interações Hospedeiro-Patógeno , Infecções por Rickettsia/microbiologia , Rickettsia/classificação , Rickettsia/patogenicidade , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/microbiologia , Animais , Especificidade de Hospedeiro , Humanos , Redes e Vias Metabólicas , Ácaros/microbiologia , Fosfatidilinositóis/metabolismo , Ftirápteros/microbiologia , Filogenia , Rickettsia/crescimento & desenvolvimento , Rickettsia/metabolismo , Infecções por Rickettsia/genética , Infecções por Rickettsia/patologia , Sifonápteros/microbiologia , Especificidade da Espécie , Carrapatos/microbiologiaRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selective killing of cancer cells underlines its anticancer potential. However, poor tolerability and resistance underscores the need to identify cancer-selective TRAIL-sensitizing agents. Apigenin, a dietary flavonoid, sensitizes lung cancer cell lines to TRAIL. It remains unknown, however, whether apigenin sensitizes primary lung cancer cells to TRAIL and its underlying mechanisms. Here we show that apigenin reprograms alternative splicing of key TRAIL/death-inducing-signaling-complex (DISC) components: TRAIL Death Receptor 5 (DR5) and cellular-FLICE-inhibitory-protein (c-FLIP) by interacting with the RNA-binding proteins hnRNPA2 and MSI2, resulting in increased DR5 and decreased c-FLIPS protein levels, enhancing TRAIL-induced apoptosis of primary lung cancer cells. In addition, apigenin directly bound heat shock protein 70 (Hsp70), promoting TRAIL/DISC assembly and triggering apoptosis. Our findings reveal that apigenin directs alternative splicing and inhibits Hsp70 enhancing TRAIL anticancer activity. These findings underscore impactful synergies between diet and cancer treatments opening new avenues for improved cancer treatments.
Assuntos
Neoplasias Pulmonares/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Apoptose , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/patologia , Transdução de SinaisRESUMO
Rickettsia species (spp.) are strict obligate intracellular bacteria, some of which are pathogenic in their mammalian host, including humans. One critical feature of these stealthy group of pathogens is their ability to manipulate hostile cytosolic environments to their benefits. Although our understanding of Rickettsia cell biology and pathogenesis is evolving, the mechanisms by which pathogenic Rickettsia spp. evade host innate immune detection remain elusive. Here, we show that disease severity in wild-type (WT) C57BL/6J mice infected with Rickettsia typhi (the etiologic agent of murine typhus) and Rickettsia rickettsii (the etiologic agent of Rocky Mountain spotted fever), but not with the nonpathogenic species Rickettsia montanensis, correlated with levels of bacterial burden as detected in the spleens of mice, as well as the serum concentrations of proinflammatory cytokine interleukin-1α (IL-1α) and, to a lesser extent, IL-1ß. Antibody-mediated neutralization of IL-1α confirmed a key role in controlling mortality rates and bacterial burdens of rickettsia-infected WT mice. As macrophages are a primary source of both IL-1α and IL-1ß cytokines, we determined the mechanism of the antirickettsial activities using bone marrow-derived macrophages. We found that pathogenic R. typhi and R. rickettsii, but not nonpathogenic R. montanensis, eluded pro-IL-1α induction and benefited predominantly from the reduced IL-1α secretion, via a caspase-11-gasdermin D (Gsdmd)-dependent pathway, to facilitate intracytosolic replication. Adoptive transfer experiments identified that IL-1α secretion by macrophages was critical for controlling rickettsiosis in WT mice. In sum, we identified a previously unappreciated pathway by which pathogenic, unlike nonpathogenic, rickettsiae preferentially target the caspase-11-Gsdmd-IL-1α signaling axis in macrophages, thus supporting their replication within the host. IMPORTANCE Currently, no vaccines are available to prevent rickettsioses, while vector-borne rickettsial infections in humans are on the rise globally. In fact, the insufficient understanding of how pathogenic Rickettsia species circumvent host immune defense mechanisms has significantly hindered the development of more effective therapeutics. Here, we identified a previously unappreciated role for the caspase-11-Gsdmd-IL-1α signaling axis in limiting the replication of pathogenic R. rickettsia and R. typhi species in murine macrophages and wild-type (WT) C57BL/6J mice. Adoptive transfer studies further identified IL-1α-secreting macrophages as critical mediators in controlling rickettsial infection in WT mice. Collectively, these findings provide insight into the potential mechanism of how pathogenic, but not nonpathogenic, Rickettsia spp. benefit from a reduction in the caspase-11-Gsdmd-mediated release of IL-1α to support host colonization.
Assuntos
Inflamassomos , Rickettsia , Humanos , Animais , Camundongos , Inflamassomos/metabolismo , Interleucina-1alfa , Camundongos Endogâmicos C57BL , Caspases , Mamíferos/metabolismoRESUMO
To establish a habitable intracellular niche, various pathogenic bacteria secrete effectors that target intracellular trafficking and modulate phosphoinositide (PI) metabolism. Murine typhus, caused by the obligate intracellular bacterium Rickettsia typhi, remains a severe disease in humans. However, the mechanisms by which R. typhi effector molecules contribute to internalization by induced phagocytosis and subsequent phagosomal escape into the cytosol to facilitate the intracellular growth of the bacteria remain ill-defined. Here, we characterize a new molecule, Risk1, as a phosphatidylinositol 3-kinase (PI3K) secreted effector and the first bacterial secretory kinase with both class I and III PI3K activities. Inactivation of Risk1 PI3K activities reduced the phosphorylation of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate within the host, which consequently diminished host colonization by R. typhi During infection, Risk1 targets the Rab5-EEA1-phosphatidylinositol 3-phosphate [PI(3)P] signaling axis to promote bacterial phagosomal escape. Subsequently, R. typhi undergoes ubiquitination and induces host autophagy; however, maturation to autolysosomes is subverted to support intracellular growth. Intriguingly, only enzymatically active Risk1 binds the Beclin-1 core complex and contributes to R. typhi-induced autophagosome formation. In sum, our data suggest that Risk1, with dual class I and class III PI3K activities, alters host PI metabolism and consequently subverts intracellular trafficking to facilitate intracellular growth of R. typhiIMPORTANCERickettsia species are Gram-negative obligate intracellular bacteria that infect a wide range of eukaryotes and vertebrates. In particular, human body louse-borne Rickettsia prowazekii and flea-borne Rickettsia typhi have historically plagued humankind and continue to reemerge globally. The unavailability of vaccines and limited effectiveness of antibiotics late in infection place lethality rates up to 30%, highlighting the need to elucidate the mechanisms of Rickettsia pathogenicity in greater detail. Here, we characterize a new effector, Risk1, as a secreted phosphatidylinositol 3-kinase (PI3K) with unique dual class I and class III activities. Risk1 is required for host colonization, and its vacuolar phosphatidylinositol 3-phosphate generation modulates endosomal trafficking to arrest autophagosomal maturation. Collectively, Risk1 facilitates R. typhi growth by altering phosphoinositide metabolism and subverting intracellular trafficking.
Assuntos
Proteínas de Bactérias/genética , Espaço Intracelular/microbiologia , Fagossomos/microbiologia , Fosfatidilinositol 3-Quinase/genética , Rickettsia typhi/genética , Rickettsia typhi/patogenicidade , Animais , Proteínas de Bactérias/metabolismo , Chlorocebus aethiops , Endossomos/metabolismo , Células HeLa , Humanos , Camundongos , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositóis/metabolismo , Transdução de Sinais , Células VeroRESUMO
Efficient phagocytosis of apoptotic cells (efferocytosis) is essential for immune homeostasis. Phospholipids exposed on the surface of apoptotic cells, such as phosphatidylserine, supply important "eat-me" signals. Liposomes are lipid bilayer vesicles that can be generated from one or several types of phospholipids of interest. Thus, these vesicles offer versatility, flexibility, and, importantly, a three-dimensional structure for studying the interaction between lipids and their receptors as well as the lipid-receptor interaction-mediated signaling events controlling efferocytosis by cells like professional phagocytes. Here, we describe methods to prepare liposomes, perform liposome-based lipid-receptor binding assays, use liposomes to block efferocytosis, and utilize liposome-coated beads as apoptotic cell surrogates for phagocytosis. © 2018 by John Wiley & Sons, Inc.
Assuntos
Lipossomos , Fagocitose , Animais , Apoptose , Camundongos Endogâmicos C57BL , Fosfolipídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Ressonância de Plasmônio de Superfície , Timócitos/fisiologiaRESUMO
Apigenin, a flavone abundantly found in fruits and vegetables, exhibits antiproliferative, anti-inflammatory, and antimetastatic activities through poorly defined mechanisms. In the present study, the treatment of different cell lines with apigenin resulted in selective antiproliferative and apoptotic effect in monocytic and lymphocytic leukemias. Apigenin-induced-apoptosis was mediated by the activation of caspase-9 and caspase-3. Apigenin was found intracellularly and localized to the mitochondria. Treatment of monocytic cells with apigenin was accompanied by an increase in reactive oxygen species (ROS) and phosphorylation of the MAPKs, p38 and ERK. However, the inhibition of ROS, p38 or ERK failed to block apoptosis, suggesting that these cellular responses induced by apigenin are not essential for the induction of apoptosis. In addition, apigenin induced the activation of PKCdelta. Pharmacological inhibition of PKCdelta, the expression of dominant-negative PKCdelta and silencing of PKCdelta in leukemia cells showed that apigenin-induced-apoptosis requires PKCdelta activity. Together, these results indicate that this flavonoid provides selective activity to promote caspase-dependent-apoptosis of leukemia cells and uncover an essential role of PKCdelta during the induction of apoptosis by apigenin.
Assuntos
Apigenina/farmacologia , Apoptose/efeitos dos fármacos , Caspases/fisiologia , Proteína Quinase C-delta/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células 3T3 , Animais , Apoptose/fisiologia , Caspase 3 , Caspase 9 , Caspases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HL-60 , Humanos , Células Jurkat , Células K562 , Leucemia/patologia , Camundongos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas , Células U937RESUMO
Engulfment of apoptotic cells is predominantly executed by phagocytes via the recognition of "eat me" signals like phosphatidylserine (PS). Various PS-specific receptors exist on phagocytes, including Tyro3, Axl, and MerTK receptor tyrosine kinases (TAMs), T-cell immunoglobulin and mucin domain containing 1 and 4 (TIM1/4), and the newly identified CD300 family. The aim of the present auto-commentary is to highlight recent findings regarding the Cd300lf and Cd300lb receptors and their emerging roles in the development of autoimmune disease.
RESUMO
Oxidative stress is the main etiological factor behind the pathogenesis of various diseases including inflammation, cancer, cardiovascular and neurodegenerative disorders. Due to the spin trapping abilities and various pharmacological properties of nitrones, their application as therapeutic agent has been gaining attention. Though the antioxidant properties of the nitrones are well known, the mechanism by which they modulate the cellular defense machinery against oxidative stress is not well investigated and requires further elucidation. Here, we have investigated the mechanisms of cytoprotection of the nitrone spin traps against oxidative stress in bovine aortic endothelial cells (BAEC). Cytoprotective properties of both the cyclic nitrone 5,5-dimethyl-pyrroline N-oxide (DMPO) and linear nitrone α-phenyl N-tert-butyl nitrone (PBN) against H2O2-induced cytotoxicity were investigated. Preincubation of BAEC with PBN or DMPO resulted in the inhibition of H2O2-mediated cytotoxicity and apoptosis. Nitrone-treatment resulted in the induction and restoration of phase II antioxidant enzymes via nuclear translocation of NF-E2-related factor 2 (Nrf-2) in oxidatively-challenged cells. Furthermore, the nitrones were found to inhibit the mitochondrial depolarization and subsequent activation of caspase-3 induced by H2O2. Significant down-regulation of the pro-apoptotic proteins p53 and Bax, and up-regulation of the anti-apoptotic proteins Bcl-2 and p-Bad were observed when the cells were preincubated with the nitrones prior to H2O2-treatment. It was also observed that Nrf-2 silencing completely abolished the protective effects of nitrones. Hence, these findings suggest that nitrones confer protection to the endothelial cells against oxidative stress by modulating phase II antioxidant enzymes and subsequently inhibiting mitochondria-dependent apoptotic cascade.
Assuntos
Apoptose/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Células Endoteliais/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredutases/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Animais , Aorta/citologia , Caspase 3/metabolismo , Bovinos , Núcleo Celular/metabolismo , Células Cultivadas , Citoproteção , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Ativação Enzimática , Glutationa Peroxidase/biossíntese , Glutationa Redutase/biossíntese , Heme Oxigenase-1/biossíntese , Peróxido de Hidrogênio/farmacologia , NAD(P)H Desidrogenase (Quinona)/biossíntese , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Transporte Proteico , Transdução de Sinais , Detecção de SpinRESUMO
Caspase-3 is an essential executioner of apoptosis responsible for regulating many important cellular processes, among them the number of circulating monocytes, central players in the innate immune response. The activation of caspase-3 requires its processing from an inactive precursor. Here we show that the small heat shock protein 27 (Hsp27) associates with caspase-3 and protein-protein interaction experiments in vivo and with purified proteins demonstrate a direct interaction between Hsp27 and the amino-terminal prodomain of caspase-3. Using an in vitro caspase-3 activation assay, our results further establish that the interaction of Hsp27 with the caspase-3 prodomain inhibits the second proteolytic cleavage necessary for caspase-3 activation, revealing a novel mechanism for the regulation of this effector caspase. Hsp27 expression in monocytes is constitutive. Consistent with a central role of Hsp27 in blocking caspase-3 activation, Hsp27 down-regulation by double-stranded RNA interference induces apoptosis of macrophages, whereas Hsp27 overexpression increases the life span of monocytes by inhibiting apoptosis. Highlighting the importance of cell partitioning in the regulation of apoptosis, immunofluorescence, and subcellular fractionation studies revealed that whereas both caspase-3 and Hsp27 are cytoplasmic in fresh monocytes (i.e. not undergoing apoptosis), Hsp27 moves to the nucleus during apoptosis, a relocalization that can be blocked by promoting the differentiation of monocytes to macrophages or by inhibiting cell death. These results reveal a novel mechanism of caspase-3 regulation and underscore a novel and fundamental role of Hsp27 in the regulation of monocyte life span.
Assuntos
Caspase 3/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico/metabolismo , Monócitos/patologia , Apoptose , Diferenciação Celular , Ativação Enzimática , Humanos , Receptores de Lipopolissacarídeos/biossíntese , Modelos Biológicos , Monócitos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Frações Subcelulares/metabolismoRESUMO
LPS stimulates monocytes/macrophages through the activation of signaling events that modulate the production of inflammatory cytokines. Apigenin, a flavonoid abundantly found in fruits and vegetables, exhibits anti-proliferative and anti-inflammatory activities through poorly defined mechanisms. In this study, we demonstrate that apigenin inhibits the production of proinflammatory cytokines IL-1beta, IL-8, and TNF in LPS-stimulated human monocytes and mouse macrophages. The inhibitory effect on proinflammatory cytokine production persists even when apigenin is administered after LPS stimulation. Transient transfection experiments using NF-kappaB reporter constructs indicated that apigenin inhibits the transcriptional activity of NF-kappaB in LPS-stimulated mouse macrophages. The classical proteasome-dependent degradation of the NF-kappaB inhibitor IkappaBalpha was observed in apigenin LPS-stimulated human monocytes. Using EMSA, we found that apigenin does not alter NF-kappaB-DNA binding activity in human monocytes. Instead we show that apigenin, as part of a non-canonical pathway, regulates NF-kappaB activity through hypophosphorylation of Ser536 in the p65 subunit and the inactivation of the IKK complex stimulated by LPS. The decreased phosphorylation on Ser536 observed in LPS-stimulated mouse macrophages treated with apigenin was overcome by the over-expression of IKKbeta. In addition, our studies indicate that apigenin inhibits in vivo LPS-induced TNF and the mortality induced by lethal doses of LPS. Collectively, these findings suggest a molecular mechanism by which apigenin suppresses inflammation and modulates the immune response in vivo.
Assuntos
Apigenina/farmacologia , Citocinas/imunologia , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Monócitos/imunologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Fator de Transcrição RelA/imunologia , Animais , Linhagem Celular , Citocinas/biossíntese , Humanos , Quinase I-kappa B/imunologia , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/imunologia , Proteínas I-kappa B/metabolismo , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Monócitos/metabolismo , Inibidor de NF-kappaB alfa , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/imunologia , Fator de Transcrição RelA/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologiaRESUMO
Monocytes are central components of the innate immune response and normally circulate for a short period of time before undergoing spontaneous apoptosis. During inflammation, differentiation, or oncogenic transformation, the life span of monocytes is prolonged by preventing the activation of the apoptotic program. Here we showed that caspase-3, a cysteine protease required for monocyte apoptosis, is a phosphoprotein. We identified protein kinase Cdelta (PKCdelta) as a member of the protein kinase C family that associates with and phosphorylates caspase-3. The PKCdelta-dependent phosphorylation of caspase-3 resulted in an increase in the activity of caspase-3. This effect of PKCdelta is specific to caspase-3, as evidenced by the absence of similar effects on caspase-9. The activity of PKCdelta precedes the activation of caspase-3 during spontaneous monocyte apoptosis and in monocyte-induced apoptosis. We found that the overexpression of PKCdelta resulted in an increase of apoptosis, whereas its inhibition blocked caspase-3 activity and decreased apoptosis. Our results provided evidence that the PKCdelta-dependent phosphorylation of caspase-3 provided a novel pro-apoptotic mechanism involved in the regulation of monocyte life span.