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Interleukin-10 and prostaglandin E 2 have complementary but distinct suppressive effects on Toll-like receptor-mediated dendritic cell activation in ovarian carcinoma

View Article: PubMed Central - PubMed

ABSTRACT

Dendritic cells (DC) have the potential to instigate a tumour-specific immune response, but their ability to prime naïve lymphocytes depends on their activation status. Thus, for tumour immunotherapy to be effective, the provision of appropriate DC activation stimuli such as Toll-like receptor (TLR) agonists is crucial in order to overcome immunosuppression associated with the tumour microenvironment. To address this, we investigated how ovarian carcinoma (OC)-associated ascites impedes activation of DC by TLR agonists. Our results show that ascites reduces the TLR-mediated up-regulation of CD86 and partially inhibits the production of the pro-inflammatory cytokines interleukin 6 (IL-6), IL-12 and tumour necrosis factor α (TNFα) in monocyte-derived DC from healthy controls. We further observe an impaired T cell stimulatory capacity of DC upon activation with TLR agonists in the presence of ascites, indicating that their functionality is affected by the immunosuppressive factors. We identify IL-10 and prostaglandin E2 (PGE2) as the pivotal immunosuppressive components in OC-associated ascites compromising TLR-mediated DC activation. Interestingly, IL-10 is present in both ascites from patients with malignant OC and in peritoneal fluid from patients with benign ovarian conditions and both fluids have similar ability to reduce TLR-mediated DC activation. However, depletion of IL-10 from ascites revealed that the presence of paracrine IL-10 is not crucial for ascites-mediated suppression of DC activation in response to TLR activation. Unlike IL-10, PGE2 is absent from peritoneal fluid of patients with benign conditions and selectively reduces TNFα induction in response to TLR-mediated activation in the presence of OC-associated ascites. Our study highlights PGE2 as an immunosuppressive component of the malignant OC microenvironment rendering PGE2 a potentially important target for immunotherapy in OC.

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Up-regulations of CD86 and induction of cytokines in response to TLR stimulation in the presence or absence of OC-associated ascites.(A) Monocyte-derived DC were stimulated overnight with 3μg/ml R848, 1μg/ml LPS, 100μg/ml polyI:C in the presence of 0%, 10% or 25% of ascites from patients with malignant OC. The mean fluorescence intensity (MFI) of the surface marker CD86 was assessed by flow cytometry. (B) Monocyte-derived DC were cultured overnight as described above and cytokines were measured in culture supernatants by flow cytomix analysis or sandwich ELISA (IL-12p40). In total, 12 independent experiments were performed (n = 12) with DC from individual healthy volunteers cultured with ascites from 4, 3, 2 or 1 OC patients (n = 1, 1, 1 and 3 healthy volunteers, respectively). One-way ANOVA was used for statistical analysis (Friedman test with Dunn post test): * = p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant.
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pone.0175712.g001: Up-regulations of CD86 and induction of cytokines in response to TLR stimulation in the presence or absence of OC-associated ascites.(A) Monocyte-derived DC were stimulated overnight with 3μg/ml R848, 1μg/ml LPS, 100μg/ml polyI:C in the presence of 0%, 10% or 25% of ascites from patients with malignant OC. The mean fluorescence intensity (MFI) of the surface marker CD86 was assessed by flow cytometry. (B) Monocyte-derived DC were cultured overnight as described above and cytokines were measured in culture supernatants by flow cytomix analysis or sandwich ELISA (IL-12p40). In total, 12 independent experiments were performed (n = 12) with DC from individual healthy volunteers cultured with ascites from 4, 3, 2 or 1 OC patients (n = 1, 1, 1 and 3 healthy volunteers, respectively). One-way ANOVA was used for statistical analysis (Friedman test with Dunn post test): * = p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant.

Mentions: In order to investigate the immunosuppressive influence of soluble factors associated with the OC microenvironment, we examined the effect of ascites collected from patients suffering from advanced-stage serous epithelial OC on activation of monocyte-derived DC by TLR agonists. The cellular fraction was removed from the ascites samples by centrifugation and only the cell-free ascites was investigated for its immunosuppressive properties. Monocyte-derived DC express TLR3, TLR4 and TLR8 and as such, polyI:C, LPS and R848 were chosen for stimulation of these TLR, respectively. The TLR agonists were used at concentrations optimised for the induction of pro-inflammatory cytokines such as IL-6 and TNFα in overnight cultures. Compared to R848 and LPS, polyI:C induced rather low levels of IL-6 and TNFα and no IL-12p40 (S1 Fig). For all three TLR agonists, the up-regulation of the co-stimulatory molecule CD86 was reduced in the presence of 25% of ascites (Fig 1A). In the presence of 10% ascites, CD86 up-regulation was significantly reduced in response to R848, but not in response to LPS or polyI:C (Fig 1A). This was the case both when monocyte-derived DC from different healthy donors were cultured with ascites from the same OC patient as well as when culturing the same donor’s monocyte-derived DC with ascites from up to four different patients. CD86 levels on monocyte-derived DC cultured without TLR agonists were not affected by the presence of ascites (S2A Fig). In addition to CD86, we monitored changes in the up-regulation of other surface markers such as CD40 and HLA-DR, as well as the immunoregulatory molecules PD-L1 (CD274) and PD-L2 (CD273). While all of these molecules were up-regulated in response to TLR stimulation, their expression at the cell surface was not consistently affected by the presence of ascites (data not shown).


Interleukin-10 and prostaglandin E 2 have complementary but distinct suppressive effects on Toll-like receptor-mediated dendritic cell activation in ovarian carcinoma
Up-regulations of CD86 and induction of cytokines in response to TLR stimulation in the presence or absence of OC-associated ascites.(A) Monocyte-derived DC were stimulated overnight with 3μg/ml R848, 1μg/ml LPS, 100μg/ml polyI:C in the presence of 0%, 10% or 25% of ascites from patients with malignant OC. The mean fluorescence intensity (MFI) of the surface marker CD86 was assessed by flow cytometry. (B) Monocyte-derived DC were cultured overnight as described above and cytokines were measured in culture supernatants by flow cytomix analysis or sandwich ELISA (IL-12p40). In total, 12 independent experiments were performed (n = 12) with DC from individual healthy volunteers cultured with ascites from 4, 3, 2 or 1 OC patients (n = 1, 1, 1 and 3 healthy volunteers, respectively). One-way ANOVA was used for statistical analysis (Friedman test with Dunn post test): * = p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant.
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Related In: Results  -  Collection

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pone.0175712.g001: Up-regulations of CD86 and induction of cytokines in response to TLR stimulation in the presence or absence of OC-associated ascites.(A) Monocyte-derived DC were stimulated overnight with 3μg/ml R848, 1μg/ml LPS, 100μg/ml polyI:C in the presence of 0%, 10% or 25% of ascites from patients with malignant OC. The mean fluorescence intensity (MFI) of the surface marker CD86 was assessed by flow cytometry. (B) Monocyte-derived DC were cultured overnight as described above and cytokines were measured in culture supernatants by flow cytomix analysis or sandwich ELISA (IL-12p40). In total, 12 independent experiments were performed (n = 12) with DC from individual healthy volunteers cultured with ascites from 4, 3, 2 or 1 OC patients (n = 1, 1, 1 and 3 healthy volunteers, respectively). One-way ANOVA was used for statistical analysis (Friedman test with Dunn post test): * = p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant.
Mentions: In order to investigate the immunosuppressive influence of soluble factors associated with the OC microenvironment, we examined the effect of ascites collected from patients suffering from advanced-stage serous epithelial OC on activation of monocyte-derived DC by TLR agonists. The cellular fraction was removed from the ascites samples by centrifugation and only the cell-free ascites was investigated for its immunosuppressive properties. Monocyte-derived DC express TLR3, TLR4 and TLR8 and as such, polyI:C, LPS and R848 were chosen for stimulation of these TLR, respectively. The TLR agonists were used at concentrations optimised for the induction of pro-inflammatory cytokines such as IL-6 and TNFα in overnight cultures. Compared to R848 and LPS, polyI:C induced rather low levels of IL-6 and TNFα and no IL-12p40 (S1 Fig). For all three TLR agonists, the up-regulation of the co-stimulatory molecule CD86 was reduced in the presence of 25% of ascites (Fig 1A). In the presence of 10% ascites, CD86 up-regulation was significantly reduced in response to R848, but not in response to LPS or polyI:C (Fig 1A). This was the case both when monocyte-derived DC from different healthy donors were cultured with ascites from the same OC patient as well as when culturing the same donor’s monocyte-derived DC with ascites from up to four different patients. CD86 levels on monocyte-derived DC cultured without TLR agonists were not affected by the presence of ascites (S2A Fig). In addition to CD86, we monitored changes in the up-regulation of other surface markers such as CD40 and HLA-DR, as well as the immunoregulatory molecules PD-L1 (CD274) and PD-L2 (CD273). While all of these molecules were up-regulated in response to TLR stimulation, their expression at the cell surface was not consistently affected by the presence of ascites (data not shown).

View Article: PubMed Central - PubMed

ABSTRACT

Dendritic cells (DC) have the potential to instigate a tumour-specific immune response, but their ability to prime na&iuml;ve lymphocytes depends on their activation status. Thus, for tumour immunotherapy to be effective, the provision of appropriate DC activation stimuli such as Toll-like receptor (TLR) agonists is crucial in order to overcome immunosuppression associated with the tumour microenvironment. To address this, we investigated how ovarian carcinoma (OC)-associated ascites impedes activation of DC by TLR agonists. Our results show that ascites reduces the TLR-mediated up-regulation of CD86 and partially inhibits the production of the pro-inflammatory cytokines interleukin 6 (IL-6), IL-12 and tumour necrosis factor &alpha; (TNF&alpha;) in monocyte-derived DC from healthy controls. We further observe an impaired T cell stimulatory capacity of DC upon activation with TLR agonists in the presence of ascites, indicating that their functionality is affected by the immunosuppressive factors. We identify IL-10 and prostaglandin E2 (PGE2) as the pivotal immunosuppressive components in OC-associated ascites compromising TLR-mediated DC activation. Interestingly, IL-10 is present in both ascites from patients with malignant OC and in peritoneal fluid from patients with benign ovarian conditions and both fluids have similar ability to reduce TLR-mediated DC activation. However, depletion of IL-10 from ascites revealed that the presence of paracrine IL-10 is not crucial for ascites-mediated suppression of DC activation in response to TLR activation. Unlike IL-10, PGE2 is absent from peritoneal fluid of patients with benign conditions and selectively reduces TNF&alpha; induction in response to TLR-mediated activation in the presence of OC-associated ascites. Our study highlights PGE2 as an immunosuppressive component of the malignant OC microenvironment rendering PGE2 a potentially important target for immunotherapy in OC.

No MeSH data available.


Related in: MedlinePlus