Coxsackievirus B3 (CVB3) is a globally prevalent enterovirus of the Picornaviridae family that is frequently associated with viral myocarditis (VM). Neutrophils, as first responders, may be key cells ...in determining viral disease outcomes; however, neutrophils have been poorly studied with respect to viral infection. Although neutrophils have been ascribed a relevant role in early cardiac inflammation, their precise role in CVB3 infection has not yet been evaluated. In this study, we aimed to determine if the interaction between human neutrophils and CVB3 could lead to viral replication and/or modulation of neutrophil survival and biological functions, and whether neutrophil depletion in a murine model has a beneficial or harmful effect on CVB3 infection. Our results show that CVB3 interacted with but did not replicate in human neutrophils. Neutrophils recognized CVB3 mainly through endosomal TLR-8, and infection triggered NFκB activation. Virus internalization resulted in increased cell survival, up-regulation of CD11b, enhanced adhesion to fibrinogen and fibronectin, and the secretion of IL-6, IL-1β, TNF-α, and IL-8. Supernatants from infected neutrophils exerted chemotactic activity partly mediated by IL-8. The infected neutrophils released myeloperoxidase and triggered neutrophil extracellular trap formation in the presence of TNF-α. In mice infected with CVB3, viral RNA was detected in neutrophils as well as in mononuclear cells. After neutrophil depletion, mice showed reduced VM reflected by a reduction in viral titers, cell exudates, and CCL-2 mRNA levels, as well as the abrogation of reactive cardiomyocyte hypertrophy. Our results indicate that neutrophils have relevant direct and indirect roles in the pathogenesis of CVB3-induced VM.
•-Coxsackievirus B3 (CVB3) interacts with, but does not replicate in human neutrophils.•-CVB3 increases neutrophil survival, CD11b expression, cytokine and IL-8 release.•-CVB3 triggers neutrophil release of myeloperoxidase and NETs.•-Neutrophils sense CVB3 mainly through TLR-8 and the activation of NFκB.•-Neutrophil-depleted CVB3-infected mice exhibit reduced viral myocarditis.
Glucocorticoids play an important role in adipogenesis through the glucocorticoid receptor (GR) that forms a heterocomplex with Hsp90•Hsp70 and one high molecular weight immunophilin, either FKBP51 ...or FKBP52. When 3T3-L1 preadipocytes are induced to differentiate, FKBP51 expression progressively increases, whereas FKBP52 decreases, and Hsp90, Hsp70, p23 and Cyp40 remain unchanged. Interestingly, FKBP51 rapidly translocates from mitochondria to the nucleus where it is retained upon its interaction with chromatin and the nuclear matrix. FKBP51 nuclear localization is transient, and after 48 hours it cycles back to mitochondria. Importantly, this dynamic FKBP51 mitochondrial-nuclear shuttling depends on PKA signaling, because its inhibition by PKI or knockdown of PKA-cα by siRNA, prevented FKBP51 nuclear translocation induced by IBMX. In addition, the electrophoretic pattern of migration of FKBP51 is altered by treatment of cells with PKI or knockdown of PKA-cα, suggesting that FKBP51 is a PKA substrate. In preadipocytes, FKBP51 colocalizes with PKA-cα in mitochondria. When adipogenesis is triggered, PKA-cα also moves to the nucleus colocalizing with FKBP51 mainly in the nuclear lamina. Moreover, FKBP51 and GR interaction increases when preadipocytes are induced to differentiate. GR transcriptional capacity is reduced when cells are incubated in the presence of IBMX, forskolin or dibutyryl-cAMP, compounds that induced FKBP51 nuclear translocation, but not by a specific activator of EPAC. FKBP51 knockdown facilitates adipogenesis, whereas ectopic expression of FKBP51 blocks adipogenesis. These findings indicate that the dynamic mitochondrial-nuclear shuttling of FKBP51 regulated by PKA may be key in fine-tuning the transcriptional control of GR target genes required for the acquisition of adipocyte phenotype.
Confocal and electron microscopy images, and WB analysis of cellular fractions revealed that HP1γ is in the nucleus but also in the cytoplasm of C2C12 myoblasts, myotubes, skeletal and cardiac ...muscles, N2a, HeLa and HEK293T cells. Signal specificity was tested with different antibodies and by HP1γ knockdown. Leptomycin B treatment of myoblasts increased nuclear HP1γ, suggesting that its nuclear export is Crm-1-dependent. HP1γ exhibited a filamentous pattern of staining partially co-localizing with actin in the cytoplasm of myotubes and myofibrils. Immunoelectron microscopic analysis showed high-density immunogold particles that correspond to HP1γ localized to the Z-disk and A-band of the sarcomere of skeletal muscle. HP1γ partially co-localized with actin in C2C12 myotubes and murine myofibrils. Importantly, actin co-immunoprecipitated with HP1γ in the nuclear and cytosolic fractions of myoblasts. Actin co-immunoprecipitated with HP1γ in myoblasts incubated in the absence or presence of the actin depolymerizing agent cytochalasin D, suggesting that HP1γ may interact with G-and F-actin. In the cytoplasm, HP1γ was associated to the perinuclear actin cap that controls nuclear shape and position. In the nucleus, re-ChIP assays showed that HP1γ-actin associates to the promoter and transcribed regions of the house keeping gene GAPDH, suggesting that HP1γ may function as a scaffold protein for the recruitment of actin to control gene expression. When HP1γ was knocked-down, myoblasts were unable to differentiate or originated thin myotubes. In summary, HP1γ is present in the nucleus and the cytoplasm interacting with actin, a protein complex that may exert different functions depending on its subcellular localization.
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•HP1γ, a protein so far considered exclusively nuclear, is present in the cytoplasm of C2C12, N2a, HeLa and HEK293T cells.•Immunoelectron microscopic analysis of skeletal muscle shows HP1γ present in the sarcomere.•HP1γ is exported from the nucleus in a Crm-1 dependent manner.•In the cytoplasm, HP1γ is in the perinuclear actin cap. In the nucleus, HP1γ-actin associates to actively transcribed genes.•When HP1γ was knocked-down, myoblasts were unable to differentiate or originated thin myotubes.
Archaebacterias are considered a unique source of novel biomaterials of interest for nanomedicine. In this perspective, the effects of nanoarchaeosomes (ARC), which are nanovesicles prepared from ...polar lipids extracted from the extreme halophilic
, on human umbilical vein endothelial cells (HUVEC) were investigated in physiological and under inflammatory static conditions. Upon incubation, ARC (170 nm mean size, -41 mV ζ) did not affect viability, cell proliferation, and expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin under basal conditions, but reduced expression of both molecules and secretion of IL-6 induced by lypopolysaccharide (LPS), Pam3CSK4 or
. Such effects were not observed with TNF-α or IL-1β stimulation. Interestingly, ARC significantly decreased basal levels of von Willebrand factor (vWF) and levels induced by all stimuli. None of these parameters was altered by liposomes of hydrogenated phosphatidylcholine and cholesterol of comparable size and concentration. Only ARC were endocytosed by HUVEC and reduced mRNA expression of ICAM-1 and vWF via NF-ĸB and ERK1/2 in LPS-stimulated cells. This is the first report of the anti-inflammatory effect of ARC on endothelial cells and our data suggest that its future use in vascular disease may hopefully be of particular interest.
Obesity is a serious health problem worldwide since it is a major risk factor for chronic diseases such as type II diabetes. Obesity is the result of hyperplasia (associated with increased ...adipogenesis) and hypertrophy (associated with decreased adipogenesis) of the adipose tissue. Therefore, understanding the molecular mechanisms underlying the process of adipocyte differentiation is relevant to delineate new therapeutic strategies for treatment of obesity. As in all differentiation processes, temporal patterns of transcription are exquisitely controlled, allowing the acquisition and maintenance of the adipocyte phenotype. The genome is spatially organized; therefore decoding local features of the chromatin language alone does not suffice to understand how cell type-specific gene expression patterns are generated. Elucidating how nuclear architecture is built during the process of adipogenesis is thus an indispensable step to gain insight in how gene expression is regulated to achieve the adipocyte phenotype. Here we will summarize the recent advances in our understanding of the organization of nuclear architecture as progenitor cells differentiate in adipocytes, and the questions that still remained to be answered.
CD34+ cells, megakaryocytes (MKs), and platelets express toll-like receptors (TLRs) that enable these cells to amplify the host innate immune response. However, the role of TLR7/TLR8 activation in ...megakaryopoiesis has not yet been investigated.
We evaluated the effect of coxsackievirus B3 (CVB3) and synthetic TLR7/TLR8 agonists on the development of human MKs and production of platelets.
CD34+ cells from human umbilical cord were inoculated with CVB3 or stimulated with synthetic TLR7/TLR8 agonists and then cultured in the presence of thrombopoietin.
CD34+ cells, MK progenitor cells, and mature MKs expressed TLR7 and TLR8, and exposure to CVB3 resulted in productive infection, as determined by the presence of viral infectious particles in culture supernatants. Cell expansion, differentiation into MKs, MK maturation, and platelet biogenesis were significantly reduced in CD34+-infected cultures. The reduction in MK growth was not due to an alteration in cellular proliferation but was accompanied by an increase in cellular apoptosis and pyroptosis. Impairment of MK generation and maturation of viable cells were also associated with decreased expression of transcription factors involved in these processes. These effects were completely abrogated by TLR7 but not TLR8 antagonists and mimicked by TLR7 but not TLR8 agonists. CVB3 infection of CD34+ cells increased the immunophenotype of MKs characterized as CD148+/CD48+ or CD41+/CD53+ cells.
These data suggest a novel role of TLR7 in megakaryo/thrombopoiesis that may contribute to a better understanding of the molecular basis underlying thrombocytopenia and the immunologic role of MKs in viral infection processes.
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•The role of receptors that sense viruses in megakaryocyte and platelet production is unknown.•Human stem cells were used to study platelet biogenesis in a viral infection model.•Virus recognition by toll-like receptor 7 (TLR7) impairs megakaryocyte and platelet development.•Viral infection and TLR7 activation increase megakaryocytes with immune properties.
Adipose tissue plays a central role in the control of energy balance as well as in the maintenance of metabolic homeostasis. It was not until recently that the first evidences of the role of heat ...shock protein (Hsp) 90 and high molecular weight immunophilin FKBP51 have been described in the process of adipocyte differentiation. Recent reports describe their role in the regulation of PPARγ, a key transcription factor in the control of adipogenesis and the maintenance of the adipocyte phenotype. In addition, novel roles have been uncovered for FKBP51 in the organization of the architecture of the nucleus through its participation in the reorganization of the nuclear lamina. Therefore, the aim of this review is to integrate and discuss the recent advances in the field, with special emphasis on the roles of Hsp90 and FKBP51 in the process of adipocyte differentiation.
How the co-ordinated events of gene activation and silencing during cellular differentiation are influenced by spatial organization of the cell nucleus is still poorly understood. Little is known ...about the molecular mechanisms controlling subnuclear distribution of transcription factors, and their interplay with nuclear proteins that shape chromatin structure. Here we show that C/EBPβ not only associates with pericentromeric heterochromatin but also interacts with the nucleoskeleton upon induction of adipocyte differentiation of 3T3-L1 cells. Different C/EBPβ dimers localize in different nuclear domains. Using BiFC in living cells, we show that LAP (
Liver
Activating
Protein) homodimers localize in euchromatin and heterochromatin. In contrast, LIP (
Liver
Inhibitory
Protein) homodimers localize exclusively in heterochromatin. Importantly, their differential subnuclear distribution mirrors the site for interaction with HP1α. HP1α inhibits LAP transcriptional capacity and occupies the promoter of the C/EBPβ-dependent gene
c/ebpα in 3T3-L1 preadipocytes. When adipogenesis is induced, HP1α binding decreases from
c/ebpα promoter, allowing transcription. Thus, the equilibrium among different pools of C/EBPβ associated with chromatin or nucleoskeleton, and dynamic changes in their interaction with HP1α, play key roles in the regulation of C/EBP target genes during adipogenesis.
Snake venoms are intricate mixtures of enzymes and bioactive factors that induce a range of detrimental effects in afflicted hosts. Certain Viperids, including Bothrops jararacussu, harbor C-type ...lectins (CTLs) known for their modulation of a variety of host cellular responses. In this study, we isolated and purified BjcuL, a CTL from B. jararacussu venom and investigated its impact on endothelial cell behavior, contrasting it with human galectin-1 (Gal-1), a prototype member of the galectin family with shared β-galactoside-binding activity. We found that BjcuL binds to human dermal microvascular endothelial cells (HMECs) in a concentration- and carbohydrate-dependent fashion and reprograms the function of these cells, favoring a pro-inflammatory and pro-coagulant endothelial phenotype. In light of the quest for universal antagonists capable of mitigating the harmful consequences of snake venoms, BjcuL emerges as a promising target to be blocked in order to regulate pathological endothelial cell responses.Snake venoms are intricate mixtures of enzymes and bioactive factors that induce a range of detrimental effects in afflicted hosts. Certain Viperids, including Bothrops jararacussu, harbor C-type lectins (CTLs) known for their modulation of a variety of host cellular responses. In this study, we isolated and purified BjcuL, a CTL from B. jararacussu venom and investigated its impact on endothelial cell behavior, contrasting it with human galectin-1 (Gal-1), a prototype member of the galectin family with shared β-galactoside-binding activity. We found that BjcuL binds to human dermal microvascular endothelial cells (HMECs) in a concentration- and carbohydrate-dependent fashion and reprograms the function of these cells, favoring a pro-inflammatory and pro-coagulant endothelial phenotype. In light of the quest for universal antagonists capable of mitigating the harmful consequences of snake venoms, BjcuL emerges as a promising target to be blocked in order to regulate pathological endothelial cell responses.
Multiple blood cell abnormalities participate in the development of inflammation in systemic lupus erythematosus (SLE). Although platelets have been suggested as one of these contributors through the ...release of their content during activation, there are limited specific data about their role as immune players in SLE.
Thirteen SLE patients were included. Flow cytometry was used to measure Toll-like receptors (TLR) 2, 4, and 9 in resting platelets, platelet-activation markers (PAC-1 binding, P-selectin, CD63, and CD40 ligand -L) and platelet-leukocyte aggregates before and after specific TLR stimulation. Soluble CD40L and von Willebrand factor (vWf) release from stimulated platelets was measured using ELISA.
In resting conditions, SLE platelets showed normal expression levels of TLR 2, 4 and 9. Platelet surface activation markers, soluble CD40L, and vWf release were normal at baseline and after TLR stimulation. Platelet-monocyte aggregates were elevated in resting conditions in SLE samples and showed only a marginal increase after TLR stimulation, while baseline and stimulated platelet-neutrophil and platelet-lymphocyte aggregates were normal. C-reactive protein levels positively correlated with platelet-monocyte aggregates both at baseline and after stimulation with the TLR-2 agonist PAM3CSK4, suggesting these complexes could reflect the inflammatory activity in SLE. In our cohort, 12 of 13 patients received treatment with hydroxychloroquine (HCQ), a known inhibitor of endosomal activity and a potential inhibitor of platelet activation. The fact that SLE platelets showed an adequate response to TLR agonists suggests that, despite this treatment, they retain the ability to respond to the increased levels of damage-associated molecular patterns (DAMPs), which represent known TLR ligands, present in the circulation of SLE patients. Interestingly, elevated plasma levels of high mobility group box 1 (HMGB1), a classical DAMP, correlated with vWf release from TLR-stimulated platelets, suggesting that HMGB1 may also be released by platelets, thereby creating a positive feedback loop for platelet activation that contributes to inflammation.
Our study demonstrates normal platelet TLR expression and function together with increased circulating platelet-monocyte aggregates. In addition, a direct correlation was observed between plasma HMGB1 levels and platelet vWf release following TLR2 stimulation. This platelet behavior in a group of patients undergoing HCQ treatment suggests that platelets could play a role in the inflammatory state of SLE.
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