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Inhibition of breast cancer resistance protein (ABCG2) in human myeloid dendritic cells induces potent tolerogenic functions during LPS stimulation.

Jin JO, Zhang W, Wong KW, Kwak M, van Driel IR, Yu Q - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Public Health Clinical Center, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT
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 in vivo. 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.

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LPS induces over-expression of ABCG2 in blood mDCs.(A) PBDCs were purified from peripheral blood by PBDC isolation kit. Surface ABCG2 expression levels were measured in CD11c−CD123+ pDCs, CD11c+CD123inter 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.
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pone-0104753-g001: LPS induces over-expression of ABCG2 in blood mDCs.(A) PBDCs were purified from peripheral blood by PBDC isolation kit. Surface ABCG2 expression levels were measured in CD11c−CD123+ pDCs, CD11c+CD123inter 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.

Mentions: Previous reports have shown that ex vivo human MDDCs and LCs express ABCG2 on their surface, but the expression of ABCG2 in human blood DC subsets has not been investigated. We therefore assessed the expression levels of ABCG2 in human peripheral blood CD11c+CD123inter mDCs and CD11c−CD123+ plasmacytoid DCs (pDCs). mDCs, but not pDCs, expressed detectable levels of ABCG2 on cell surface (Figure 1A). To confirm this observation, we sorted PBDCs into 3 populations: CD11c−CD123+ pDCs, CD11c+CD123inter mDCs and CD11c−CD123− cells, which are non-DCs, and measured mRNA levels of ABCG2 mRNA. As expected, only the mDC population expressed high levels of ABCG2 mRNA (Figure 1B). Since DCs can alter the expression of some surface proteins during activation and maturation, we next assessed whether LPS stimulation can alter ABCG2 expression in MDDCs and PBDCs. Interestingly, LPS stimulation induced up-regulation of ABCG2 expression in MDDCs (Figure S1A) and blood mDCs, but not in pDCs (Figure 1C). Consistent with the changes at protein levels, LPS treatment led to marked increases in ABCG2 mRNA levels in MDDCs (Figure S1B).


Inhibition of breast cancer resistance protein (ABCG2) in human myeloid dendritic cells induces potent tolerogenic functions during LPS stimulation.

Jin JO, Zhang W, Wong KW, Kwak M, van Driel IR, Yu Q - PLoS ONE (2014)

LPS induces over-expression of ABCG2 in blood mDCs.(A) PBDCs were purified from peripheral blood by PBDC isolation kit. Surface ABCG2 expression levels were measured in CD11c−CD123+ pDCs, CD11c+CD123inter 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.
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Related In: Results  -  Collection

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pone-0104753-g001: LPS induces over-expression of ABCG2 in blood mDCs.(A) PBDCs were purified from peripheral blood by PBDC isolation kit. Surface ABCG2 expression levels were measured in CD11c−CD123+ pDCs, CD11c+CD123inter 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.
Mentions: Previous reports have shown that ex vivo human MDDCs and LCs express ABCG2 on their surface, but the expression of ABCG2 in human blood DC subsets has not been investigated. We therefore assessed the expression levels of ABCG2 in human peripheral blood CD11c+CD123inter mDCs and CD11c−CD123+ plasmacytoid DCs (pDCs). mDCs, but not pDCs, expressed detectable levels of ABCG2 on cell surface (Figure 1A). To confirm this observation, we sorted PBDCs into 3 populations: CD11c−CD123+ pDCs, CD11c+CD123inter mDCs and CD11c−CD123− cells, which are non-DCs, and measured mRNA levels of ABCG2 mRNA. As expected, only the mDC population expressed high levels of ABCG2 mRNA (Figure 1B). Since DCs can alter the expression of some surface proteins during activation and maturation, we next assessed whether LPS stimulation can alter ABCG2 expression in MDDCs and PBDCs. Interestingly, LPS stimulation induced up-regulation of ABCG2 expression in MDDCs (Figure S1A) and blood mDCs, but not in pDCs (Figure 1C). Consistent with the changes at protein levels, LPS treatment led to marked increases in ABCG2 mRNA levels in MDDCs (Figure S1B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Public Health Clinical Center, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT
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 in vivo. 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.

Show MeSH
Related in: MedlinePlus