ABSTRACT
Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor density across healthy and diseased cells. Engaging receptors above a threshold concentration would be desirable in devising selective diagnostics. Integrins are prime target candidates as they are readily available on the cell surface and have been reported to be overexpressed in diseases. Insights into their spatial organization would therefore be advantageous to design selective targeting agents. Here, we investigated the effect of activation method on integrin α5ß1 clustering by immunofluorescence and modeled the global neighbor distances with input from an immuno-staining assay and image processing of microscopy images. This data was used to engineer spatially-controlled DNA-scaffolded bivalent ligands, which we used to compare trends in spatial-selective binding observed across HUVEC, CHO and HeLa in resting versus activated conditions in confocal microscopy images. For HUVEC and CHO, the data demonstrated an improved selectivity and localisation of binding for smaller spacings ~7 nm and ~24 nm, in good agreement with the model. A deviation from the mode predictions for HeLa was observed, indicative of a clustered, instead of homogeneous, integrin organization. Our findings demonstrate how low-technology imaging methods can guide the design of spatially controlled ligands to selectively differentiate between cell type and integrin activation state.
Subject(s)
Integrin alpha5beta1 , Nanoparticles , DNA , Integrin alpha5beta1/metabolism , Integrins/metabolism , LigandsABSTRACT
DNA-based nanomaterials are gaining popularity as uniform and programmable bioengineering tools as a result of recent solutions to their weak stability under biological conditions. The DNA nanotechnology platform uniquely allows decoupling of engineering parameters to comprehensively study the effect of each upon cellular encounter. We here present a systematic analysis of the effect of surface parameters of DNA-based nanoparticles on uptake in three different cell models: tumor cells, macrophages, and dendritic cells. The influence of surface charge, stabilizing coating, fluorophore types, functionalization technique, and particle concentration employed is found to cause significant differences in material uptake among these cell types. We therefore provide new insights into the large variance in cell type-specific uptake, highlighting the necessity of proper engineering and careful assay development when DNA-based materials are used as tools in bioengineering and as future nanotherapeutic agents.
Subject(s)
Nanoparticles , Nanostructures , Biological Transport , DNA , NanotechnologyABSTRACT
First evidence of geometrical patterns and defined distances of biomolecules as fundamental parameters to regulate receptor binding and cell signaling have emerged recently. Here, we demonstrate the importance of controlled nanospacing of immunostimulatory agents for the activation of immune cells by exploiting DNA-based nanomaterials and pre-existing crystallography data. We created DNA origami nanoparticles that present CpG-motifs in rationally designed spatial patterns to activate Toll-like Receptor 9 in RAW 264.7 macrophages. We demonstrated that stronger immune activation is achieved when active molecules are positioned at the distance of 7 nm, matching the active dimer structure of the receptor. Moreover, we show how the introduction of linkers between particle and ligand can influence the spatial tolerance of binding. These findings are fundamental for a fine-tuned manipulation of the immune system, considering the importance of spatially controlled presentation of therapeutics to increase efficacy and specificity of immune-modulating nanomaterials where multivalent binding is involved.
Subject(s)
Nanostructures , Toll-Like Receptor 9 , DNA/chemistry , Ligands , Protein Binding , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/metabolismABSTRACT
Dendritic cells (DCs) are professional antigen-presenting cells involved in the initiation of immune responses. We generated a tolerogenic DC (tolDC) line that constitutively secretes interleukin-10 (IL10-DCs), expressed lower levels of co-stimulatory and MHCII molecules upon stimulation, and induced antigen-specific proliferation of T cells. Vaccination with IL10-DCs combined with another tolDC line that secretes IL-35, reduced antigen-specific local inflammation in a delayed-type hypersensitivity assay independently on regulatory T cell differentiation. In an autoimmune model of rheumatoid arthritis, vaccination with the combined tolDCs after the onset of the disease impaired disease development and promoted recovery of mice. After stable memory was established, the tolDCs promoted CD4 downregulation and induced lymphocyte activation gene 3 (LAG-3) expression in reactivated memory T cells, reducing T cell activation. Taken together, our findings indicate the benefits of combining anti-inflammatory cytokines in an antigen-specific context to treat excessive inflammation when memory is already established.
Subject(s)
Antigens, CD/genetics , Dendritic Cells/immunology , Dendritic Cells/metabolism , Immunologic Memory , Interleukin-10/biosynthesis , Interleukin-12 Subunit p35/biosynthesis , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Animals , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Arthritis, Rheumatoid/therapy , Cell Communication/immunology , Cell Line , Cytokines/metabolism , Female , Gene Expression , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Immune Tolerance , Immunomodulation , Immunotherapy/methods , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Knockout , Vaccines/administration & dosage , Vaccines/immunology , Lymphocyte Activation Gene 3 ProteinABSTRACT
The role of Epithelial to Mesenchymal Transition (EMT) factor Zeb1 is well defined in metastasis and cancer progression but it's importance in dendritic cells (DCs) is unexplored until now. For the first time we report here that Zeb1 controls immunogenic responses of CD8α+ conventional Type-I (cDC1) DCs. We found that ZEB1 expression increases significantly after TLR9 stimulation and its depletion impairs activation, co-stimulation and secretion of important cytokines like IL-6, IL-10 and IL-12 in cDC1 MutuDC line. We further confirmed our findings in primary cDC1 DCs derived from bone marrow. Co-culture of these Zeb1 knock down (KD) DCs with OT-II CD4+ T helper cells skewed their differentiation toward Th2 subtype. Moreover, adoptive transfer of activated Zeb1 KD DCs cleared intestinal worms in helminth infected mice by increasing Th2 responses in vivo. Integrative genomic analysis showed Zeb1 as an activator of immune response genes in cDC1 MutuDCs as compared to other pathway genes. In addition, differentially regulated genes in Zeb1 KD RNA-seq showed significant enrichment of Th2 activation pathways supporting our in vitro findings. Mechanistically, we showed that decreased IL-12 secreted by Zeb1 KD DCs is the plausible mechanism for increased Th2 differentiation. Collectively our data demonstrate that Zeb1 could be targeted in DCs to modulate T-cell mediated adaptive immune responses.
Subject(s)
Dendritic Cells/immunology , Epithelial-Mesenchymal Transition/immunology , Th1 Cells/immunology , Zinc Finger E-box-Binding Homeobox 1/immunology , Adaptive Immunity/immunology , Adoptive Transfer/methods , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Cells, Cultured , Coculture Techniques/methods , Female , HEK293 Cells , Humans , Interleukin-10/immunology , Interleukin-12/immunology , Interleukin-6/immunology , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , T-Lymphocytes, Helper-Inducer/immunology , Th2 Cells/immunology , Toll-Like Receptor 9/immunologyABSTRACT
We have previously shown that the Platelet-Activating Factor Receptor (PAFR) engagement in murine macrophages and dendritic cells (DCs) promotes a tolerogenic phenotype reversed by PAFR-antagonists treatment in vitro. Here, we investigated whether a PAFR antagonist would modulate the immune response in vivo. Mice were subcutaneously injected with OVA or OVA with PAFR-antagonist WEB2170 on days 0 and 7. On day 14, OVA-specific IgG2a and IgG1 were measured in the serum. The presence of WEB2170 during immunization significantly increased IgG2a without affecting IgG1 levels. When WEB2170 was added to OVA in complete Freund's adjuvant, enhanced IgG2a but not IgG1 production was also observed, and CD4+ FoxP3+ T cell frequency in the spleen was reduced compared to mice immunized without the antagonist. Similar results were observed in PAFR-deficient mice, along with increased Tbet mRNA expression in the spleen. Additionally, bone marrow-derived DCs loaded with OVA were transferred into naïve mice and their splenocytes were co-cultured with fresh OVA-loaded DCs. CD4+ T cell proliferation was higher in the group transferred with DCs treated with the PAFR-antagonist. We propose that the activation of PAFR by ligands present in the site of immunization is able to fine-tune the adaptive immune response.
Subject(s)
Adaptive Immunity , Azepines/administration & dosage , Immunoglobulin G/blood , Ovalbumin/administration & dosage , Platelet Aggregation Inhibitors/administration & dosage , Spleen/immunology , Triazoles/administration & dosage , Animals , Azepines/immunology , CD4-Positive T-Lymphocytes/metabolism , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/immunology , Freund's Adjuvant/administration & dosage , Freund's Adjuvant/immunology , Immunization , Mice , Ovalbumin/immunology , Platelet Aggregation Inhibitors/immunology , Platelet Membrane Glycoproteins/antagonists & inhibitors , Receptors, G-Protein-Coupled/antagonists & inhibitors , Spleen/cytology , Triazoles/immunologyABSTRACT
Activation of the platelet-activating factor receptor (PAFR) in macrophages is associated with suppressor phenotype. Here, we investigated the PAFR in murine dendritic cells (DC). Bone marrow-derived dendritic cells (BALB/c) were cultured with GM-CSF and maturation was induced by LPS. The PAFR antagonists (WEB2086, WEB2170, PCA4248) and the prostaglandin (PG) synthesis inhibitors (indomethacin, nimesulide and NS-398) were added before LPS. Mature and immature DCs expressed PAFR. LPS increased MHCII, CD40, CD80, CD86, CCR7 and induced IL-10, IL-12, COX-2 and PGE2 expression. IL-10, COX-2 and PGE2 levels were reduced by PAFR antagonists and increased by cPAF. The IL-10 production was independent of PGs. Mature DCs induced antigen-specific lymphocyte proliferation. PAFR antagonists or PG-synthesis inhibitors significantly increased lymphocyte proliferation. It is proposed that PAF has a central role in regulatory DC differentiation through potentiation of IL-10 and PGE2 production.
Subject(s)
Bone Marrow Cells/metabolism , Dendritic Cells/metabolism , Dinoprostone/metabolism , Platelet Membrane Glycoproteins/genetics , Receptors, G-Protein-Coupled/genetics , T-Lymphocytes/metabolism , Animals , Antigen Presentation , Antigens, CD/genetics , Antigens, CD/immunology , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/immunology , Cell Differentiation , Cyclooxygenase 2/genetics , Cyclooxygenase 2/immunology , Cyclooxygenase Inhibitors/pharmacology , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Gene Expression Regulation , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-12/genetics , Interleukin-12/immunology , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred BALB C , Platelet Aggregation Inhibitors/pharmacology , Platelet Membrane Glycoproteins/agonists , Platelet Membrane Glycoproteins/immunology , Primary Cell Culture , Receptors, CCR7/genetics , Receptors, CCR7/immunology , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/immunology , Signal Transduction , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Macrophage interaction with oxidized low-density lipoprotein (oxLDL) leads to its differentiation into foam cells and cytokine production, contributing to atherosclerosis development. In a previous study, we showed that CD36 and the receptor for platelet-activating factor (PAFR) are required for oxLDL to activate gene transcription for cytokines and CD36. Here, we investigated the localization and physical interaction of CD36 and PAFR in macrophages stimulated with oxLDL. We found that blocking CD36 or PAFR decreases oxLDL uptake and IL-10 production. OxLDL induces IL-10 mRNA expression only in HEK293T expressing both receptors (PAFR and CD36). OxLDL does not induce IL-12 production. The lipid rafts disruption by treatment with ßCD reduces the oxLDL uptake and IL-10 production. OxLDL induces co-immunoprecipitation of PAFR and CD36 with the constitutive raft protein flotillin-1, and colocalization with the lipid raft-marker GM1-ganglioside. Finally, we found colocalization of PAFR and CD36 in macrophages from human atherosclerotic plaques. Our results show that oxLDL induces the recruitment of PAFR and CD36 into the same lipid rafts, which is important for oxLDL uptake and IL-10 production. This study provided new insights into how oxLDL interact with macrophages and contributing to atherosclerosis development.
Subject(s)
CD36 Antigens/metabolism , Interleukin-10/biosynthesis , Lipoproteins, LDL/metabolism , Macrophages/cytology , Macrophages/metabolism , Membrane Microdomains/metabolism , Platelet Membrane Glycoproteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , G(M1) Ganglioside/metabolism , HEK293 Cells , Humans , Male , Membrane Proteins/metabolism , Mice, Inbred C57BL , Plaque, Atherosclerotic/metabolism , Plaque, Atherosclerotic/pathology , Protein TransportABSTRACT
OxLDL is recognized by macrophage scavenger receptors, including CD36; we have recently found that Platelet-Activating Factor Receptor (PAFR) is also involved. Since PAFR in macrophages is associated with suppressor function, we examined the effect of oxLDL on macrophage phenotype. It was found that the presence of oxLDL during macrophage differentiation induced high mRNA levels to IL-10, mannose receptor, PPAR γ and arginase-1 and low levels of IL-12 and iNOS. When human THP-1 macrophages were pre-treated with oxLDL then stimulated with LPS, the production of IL-10 and TGF- ß significantly increased, whereas that of IL-6 and IL-8 decreased. In murine TG-elicited macrophages, this protocol significantly reduced NO, iNOS and COX2 expression. Thus, oxLDL induced macrophage differentiation and activation towards the alternatively activated M2-phenotype. In murine macrophages, oxLDL induced TGF- ß , arginase-1 and IL-10 mRNA expression, which were significantly reduced by pre-treatment with PAFR antagonists (WEB and CV) or with antibodies to CD36. The mRNA expression of IL-12, RANTES and CXCL2 were not affected. We showed that this profile of macrophage activation is dependent on the engagement of both CD36 and PAFR. We conclude that oxLDL induces alternative macrophage activation by mechanisms involving CD36 and PAFR.
Subject(s)
CD36 Antigens/metabolism , Gene Expression Regulation , Lipoproteins, LDL/chemistry , Macrophages/cytology , Platelet Membrane Glycoproteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Arginase/metabolism , Cell Line , Cyclooxygenase 2/metabolism , Humans , Interleukin-10/metabolism , Interleukin-12/metabolism , Lectins, C-Type/metabolism , Lymphocyte Activation , Macrophages/metabolism , Male , Mannose Receptor , Mannose-Binding Lectins/metabolism , Mice , Mice, Inbred C57BL , Monocytes/cytology , Nitric Oxide/chemistry , Nitric Oxide Synthase Type II/metabolism , PPAR gamma/metabolism , Phenotype , Receptors, Cell Surface/metabolismABSTRACT
We recently reported that paracrine Fibroblast Growth Factor 2 (FGF2) triggers senescence in Ras-driven Y1 and 3T3(Ras) mouse malignant cell lines. Here, we show that although FGF2 activates mitogenic pathways in these Ras-dependent malignant cells, it can block cell proliferation and cause a G2/M arrest. These cytostatic effects of FGF2 are inhibited by PD173074, an FGF receptor (FGFR) inhibitor. To determine which downstream pathways are induced by FGF2, we tested specific inhibitors targeting mitogen-activated protein kinase (MEK), phosphatidylinositol 3 kinase (PI3K) and protein kinase C (PKC). We show that these classical mitogenic pathways do not mediate the cytostatic activity of FGF2. On the other hand, the inhibition of Src family kinases rescued Ras-dependent malignant cells from the G2/M irreversible arrest induced by FGF2. Taken together, these data indicate a growth factor-sensitive point in G2/M that likely involves FGFR/Ras/Src pathway activation in a MEK, PI3K and PKC independent manner.
Subject(s)
Cell Division/physiology , Fibroblast Growth Factor 2/physiology , G2 Phase/physiology , Proto-Oncogene Proteins p21(ras)/physiology , Proto-Oncogene Proteins pp60(c-src)/physiology , Animals , DNA Replication , Mice , Phosphatidylinositol 3-Kinases/metabolismABSTRACT
Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant Y1 adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated beta-galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Y1 adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either Y1 or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Ras-dependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RhoA-GTP. Surprisingly, attempts to select FGF2-resistant cells from the Y1 and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Ras-dependent malignant cells could rarely overcome.