Inhibition of Breast Cancer Resistance Protein (ABCG2) in Human Myeloid Dendritic Cells Induces Potent Tolerogenic Functions during LPS Stimulation

<div><p>Breast cancer resistance protein (ABCG2), a member of the ATP-binding cassette transporters has been identified as a major determinant of multidrug resistance (MDR) in cancer cells, but ABC transporter inhibition has limited therapeutic value <i>in vivo</i>. In this research, we demonstrated that inhibition of efflux transporters ABCG2 induced the generation of tolerogenic DCs from human peripheral blood myeloid DCs (mDCs). ABCG2 expression was present in mDCs and was further increased by LPS stimulation. Treatment of CD1c⁺ mDCs with an ABCG2 inhibitor, Ko143, during LPS stimulation caused increased production of IL-10 and decreased production of pro-inflammatory cytokines and decreased expression of CD83 and CD86. Moreover, inhibition of ABCG2 in monocyte-derived DCs (MDDCs) abrogated the up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in these cells in response to LPS. Furthermore, CD1c⁺ mDCs stimulated with LPS plus Ko143 inhibited the proliferation of allogeneic and superantigen-specific syngenic CD4⁺ T cells and promoted expansion of CD25⁺FOXP3⁺ regulatory T (Treg) cells in an IL-10-dependent fashion. These tolerogenic effects of ABCG2 inhibition could be abolished by ERK inhibition. Thus, we demonstrated that inhibition of ABCG2 in LPS-stimulated mDCs can potently induce tolerogenic potentials in these cells, providing crucial new information that could lead to development of better strategies to combat MDR cancer.</p></div

ERK pathway is essential for optimal induction of tolerogenic mDCs by LPS and Ko143.

<p>(A) MDDCs (1×10⁶) were pre-treated with or without Ko143 for 1 hour and then incubated with LPS for 10 minutes. The samples were then subjected to Western blotting using anti-phosphorylated p38 (p-p38), ERK (pERK), AKT (p-AKT) and IKK (p-IKK) Abs. (B) MDDCs were treated with Ko143, LPS or Ko143 plus LPS for indicated amount of time. Phosphorylation of MEK and ERK were subjected to Western blotting. (C) MDDCs were pre-incubated with PD98059 for 10 minutes and further cultured with Ko143, LPS or Ko143 plus LPS for 30 minutes. Phosphorylation of ERK was measured by Western blotting. (D) CD1c⁺ mDCs were stimulated with LPS together with Ko143 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104753#pone-0104753-g002" target="_blank">Figure 2</a> in the presence or absence of PD98059. Flow cytometry of CD83 expression in CD1c⁺ mDCs is shown. (E) Concentrations of IL-12p40, IL-12p70, TNF-α and IL-10 in the culture medium. (F) CD1c⁺ mDCs were co-cultured with allergenic CD4 T cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104753#pone-0104753-g004" target="_blank">Figure 4</a>. Expression of CD25 and CFSE dilution were analyzed in CD4⁺ T cells (left panel). Mean percentage of proliferating CD4⁺CD25⁻ cells (middle panel) or CD4⁺CD25⁺ cells (right panel) was shown. (G) CD4, CD25 and FOXP3 expression was analyzed by flow cytometry. Data in A–C are the representative of at least three independent experiments, and data in D–G are representative or the average of analyses of 4 samples from 4 donors for each group.</p

Generation of Treg cells induced by LPS plus Ko143-treated mDCs is dependent on IL-10.

<p>CD1c⁺ mDCs and CD4 T cells were co-cultured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104753#pone-0104753-g004" target="_blank">Figure 4</a> in the presence of anti-IL-10 or control IgG Abs. (A) Expression of CD25 and CFSE dilution were analyzed in CD4⁺ T cells. (B) CD4, CD25 and FOXP3 expression was analyzed by flow cytometry. (C) IFN-γ (left panel) and IL-10 (right panel) concentrations in the culture medium were measured by ELISA. Data are representative or the average of analyses of 3 samples from 3 donors.</p

Ko143 suppresses LPS-induced mDC maturation.

<p>(A) Purified CD1c⁺ mDCs were pre-treated with Ko143 for 1 hour and incubated with or without LPS for 24 hours. CD83 and CD86 expression were analyzed by flow cytometry (left panel). MFI of CD83 and CD86 was shown (right panel). Data are representative or the average of analyses of 3 samples from 3 donors. (B) Intracellular IL-12p40, TNF-α and IL-10 production in purified CD1c⁺ mDCs were analyzed by flow cytometry. Data are representative or the average of analyses of 3 samples from 3 donors. (C) Concentrations of IL-12p40, IL-12p70, TNF-α and IL-10 in the culture medium of CD1c⁺ mDCs as measured by ELISA. Data are representative or the average of analyses of 6 samples from 6 donors.</p

ABCG2 is required for LPS-induced DC maturation.

<p>MDDCs were transfected with ABCG2 siRNA or control siRNA for 24 hours and then stimulated with LPS for 24 hours. (A) mRNA levels of ABCG2 were measured by real time qPCR. (B) Protein levels of ABCG2 were subjected by western blotting using anti-ABCG2 Abs. (C) The expression levels of CD83 and CD86 were analyzed by flow cytometry. (D) Levels of IL-12p40, IL-12p70, TNF-α and IL-10 in the culture medium as measured by ELISA. All data are representative or the average of analyses of 4 samples from 4 donors for each group.</p

LPS induces over-expression of ABCG2 in blood mDCs.

<p>(A) PBDCs were purified from peripheral blood by PBDC isolation kit. Surface ABCG2 expression levels were measured in CD11c⁻CD123⁺ pDCs, CD11c⁺CD123^inter mDCs and CD11c⁻CD123⁻ cells by flow cytometry. (B) Real-time PCR analysis of ABCG2 gene expression, presented relative to that of β-actin, in purified pDCs, mDCs and CD11c⁻CD123⁻ cells (D/N). Data are representative or the average of analyses of 4 samples from 4 donors for each group. (C) PBDCs were stimulated with LPS for 24 hours. ABCG2 expression in gated pDCs, mDCs and CD11c⁻CD123⁻ cells were analyzed on a flow cytometry. Data are representative of analyses of 3 samples from 3 donors. (D) Mitoxantrone efflux in purified mDCs and LPS-stimulated mDCs in the presence or absence of Ko143 was analyzed by flow cytometry (left panel) and mean fluorescence intensity (MFI) was shown (right panel). Data are representative or the average of analyses of 3 samples from 3 donors.</p

LPS plus Ko143-treated mDCs promote expansion of Treg cells.

<p>Ko143-, LPS- or LPS plus Ko143-treated CD1c⁺ mDCs were co-cultured with CFSE-labeled allogeneic CD4⁺ T cells (1×10⁵) in a 1∶10 ratio for 4 days. (A) Surface expression of CD25 and CFSE dilution was analyzed in CD4⁺ T cells by flow cytometry. (B) Flow cytometry of CD4, CD25 and FOXP3 expression was shown. Data are representative of analyses of 3 samples from 3 donors. (C) Flow cytometry of intracellular IL-10 and IFN-γ production in gated CD4⁺ T cells was shown. (D) Intracellular IL-10 expression in CD4⁺FOXP3⁺ T cells (left panel) and mean percentage of IL-10⁺FOXP3⁺ or IL-10⁺FOXP3⁻ cells (right panel) were shown. Data are representative or the average of analyses of 3 samples from 3 donors. (E) CFSE-labeled PBMCs were incubated with soluble anti-CD3 and CD28 Abs for 4 days in the presence or absence of CD25⁺ Treg cells. CFSE dilution was analyzed in CD4⁺CD25⁻ and CD8⁺ T cells (left panel) and mean percentage of proliferating cells (right panel) was shown. Data are representative or the average of analyses of 4 samples from 4 donors. (F) CD1c⁺ mDCs were cultured with LPS and CFSE-labeled syngenic CD4 T cells, in the presence or absence of Staphylococcal Enterotoxin B (SEB) and Ko143 for 4 days. CFSE dilution was analyzed in CD4⁺TCRVβ3⁺ T cells by flow cytometry. Data are representative of analyses of 3 samples from 3 donors.</p

Functionalization of Fatty Acid Vesicles through Newly Synthesized Bolaamphiphile–DNA Conjugates

The surface functionalization of fatty acid vesicles will allow their use as nanoreactors for complex chemistry. In this report, the tethering of several DNA conjugates to decanoic acid vesicles for molecular recognition and synthetic purposes was explored. Due to the highly dynamic nature of these structures, only one novel bola-amphiphile DNA conjugate could interact efficiently with or spontaneously pierce into the vesicle bilayers without jeopardizing their self-assembly or stability. This molecule was synthesized via a Cu­(I)-catalyzed [3 + 2] azide–alkyne cycloaddition (click reaction), and consists of a single hydrocarbon chain of 20 carbons having on one end a triazole group linked to the 5′-phosphate of the nucleic acid and on the other side a hydroxyl-group. Its insertion was so effective that a fluorescent label on the DNA complementary to the conjugate could be used to visualize fatty acid structures

Non-covalent Monolayer-Piercing Anchoring of Lipophilic Nucleic Acids: Preparation, Characterization, and Sensing Applications

Functional interfaces of biomolecules and inorganic substrates like semiconductor materials are of utmost importance for the development of highly sensitive biosensors and microarray technology. However, there is still a lot of room for improving the techniques for immobilization of biomolecules, in particular nucleic acids and proteins. Conventional anchoring strategies rely on attaching biomacromolecules via complementary functional groups, appropriate bifunctional linker molecules, or non-covalent immobilization via electrostatic interactions. In this work, we demonstrate a facile, new, and general method for the reversible non-covalent attachment of amphiphilic DNA probes containing hydrophobic units attached to the nucleobases (lipid–DNA) onto SAM-modified gold electrodes, silicon semiconductor surfaces, and glass substrates. We show the anchoring of well-defined amounts of lipid–DNA onto the surface by insertion of their lipid tails into the hydrophobic monolayer structure. The surface coverage of DNA molecules can be conveniently controlled by modulating the initial concentration and incubation time. Further control over the DNA layer is afforded by the additional external stimulus of temperature. Heating the DNA-modified surfaces at temperatures >80 °C leads to the release of the lipid–DNA structures from the surface without harming the integrity of the hydrophobic SAMs. These supramolecular DNA layers can be further tuned by anchoring onto a mixed SAM containing hydrophobic molecules of different lengths, rather than a homogeneous SAM. Immobilization of lipid–DNA on such SAMs has revealed that the surface density of DNA probes is highly dependent on the composition of the surface layer and the structure of the lipid–DNA. The formation of the lipid–DNA sensing layers was monitored and characterized by numerous techniques including X-ray photoelectron spectroscopy, quartz crystal microbalance, ellipsometry, contact angle measurements, atomic force microscopy, and confocal fluorescence imaging. Finally, this new DNA modification strategy was applied for the sensing of target DNAs using silicon-nanowire field-effect transistor device arrays, showing a high degree of specificity toward the complementary DNA target, as well as single-base mismatch selectivity