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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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Related in: MedlinePlus

Neutralisation of IL-10 releases the impairment of DC activation in response to TLR stimulation in the presence of OC-associated ascites.(A, B) Monocyte derived DC were cultured in the presence of 3μg/ml R848, 10% ascites and neutralizing antibodies (5μg/ml) as indicated. Neutralizing antibodies were added to cultures alone or in combination. (A) CD86 expression levels were measured by flow-cytometry, and (B) cytokine levels were measured in cell culture supernatants by flow cytomix analysis (IL-6, TNFα) or sandwich ELISA (IL-12p40). n = 9–13 (13 independent experiments; monocyte-derived DC from 8 different healthy donors, cultured with 1 (n = 4), 2 (n = 3) or 3 (n = 1) different ascites samples. IL-6 neutralizing antibody was only used in 9 out of 13 experiments). One-way ANOVA (Friedman test with Dunn post test): *** = p<0.001. Please note that the quantification of IL-6 in samples containing IL-6 neutralizing antibody (αIL-6 and all AB) is compromised. (C, D) For allogeneic MLR, monocyte-derived DC from healthy donors were cultured overnight in complete medium only, or stimulated with 3μg/ml R848 in the presence or absence of 10% ascites fluid (AF) and/or IL-10 neutralizing antibody (5μg/ml) as indicated. Subsequently, such pre-treated DC were washed and co-cultured with CFSE-stained CD4+ naïve T cells from another healthy donor at a 1:200 ratio for 6 days. Naïve T cells from the same donor were used consistently for all experiments. The proliferation of T cells was determined by CFSE dilution after 6 days. (C) A representative example of flow-cytometry analysis is shown. (D) Cumulative data from 4 independent experiments showing proliferation in percent of CD3+ cells that have undergone at least one round of division. n = 4 (4 independent experiments; monocyte-derived DC from 3 different healthy donors, cultured with ascites from two (n = 1) or one (n = 2) OC patient).
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pone.0175712.g004: Neutralisation of IL-10 releases the impairment of DC activation in response to TLR stimulation in the presence of OC-associated ascites.(A, B) Monocyte derived DC were cultured in the presence of 3μg/ml R848, 10% ascites and neutralizing antibodies (5μg/ml) as indicated. Neutralizing antibodies were added to cultures alone or in combination. (A) CD86 expression levels were measured by flow-cytometry, and (B) cytokine levels were measured in cell culture supernatants by flow cytomix analysis (IL-6, TNFα) or sandwich ELISA (IL-12p40). n = 9–13 (13 independent experiments; monocyte-derived DC from 8 different healthy donors, cultured with 1 (n = 4), 2 (n = 3) or 3 (n = 1) different ascites samples. IL-6 neutralizing antibody was only used in 9 out of 13 experiments). One-way ANOVA (Friedman test with Dunn post test): *** = p<0.001. Please note that the quantification of IL-6 in samples containing IL-6 neutralizing antibody (αIL-6 and all AB) is compromised. (C, D) For allogeneic MLR, monocyte-derived DC from healthy donors were cultured overnight in complete medium only, or stimulated with 3μg/ml R848 in the presence or absence of 10% ascites fluid (AF) and/or IL-10 neutralizing antibody (5μg/ml) as indicated. Subsequently, such pre-treated DC were washed and co-cultured with CFSE-stained CD4+ naïve T cells from another healthy donor at a 1:200 ratio for 6 days. Naïve T cells from the same donor were used consistently for all experiments. The proliferation of T cells was determined by CFSE dilution after 6 days. (C) A representative example of flow-cytometry analysis is shown. (D) Cumulative data from 4 independent experiments showing proliferation in percent of CD3+ cells that have undergone at least one round of division. n = 4 (4 independent experiments; monocyte-derived DC from 3 different healthy donors, cultured with ascites from two (n = 1) or one (n = 2) OC patient).

Mentions: In order to clarify whether IL-10 is mediating the inhibitory effect on TLR-mediated DC activation partially or in full, we added neutralizing antibodies against IL-10 to monocyte-derived DC cultures containing R848 and 10% OC-associated ascites. We chose R848 as TLR agonist for this set of experiments, since it induced the highest levels of cytokine production by DC in the absence of ascites and robust and reproducible suppression of R848-induced cytokine production was observed in the presence of 10% ascites. In parallel, we also tested neutralizing antibodies against IL-6, TGFβ, LIF and VEGFα to identify any contributions to the observed immunosuppressive effect by these factors. IL-6, LIF and VEGFα were present in the ascites samples investigated in this study (Fig 3A). We also included TGFβ, which has been ascribed immunosuppressive roles in different physiological scenarios but the concentration of which we had not determined in the ascites. Upon addition of IL-10 neutralizing antibody, we observed restored CD86 up-regulation and production of IL-6, IL-12p40 and TNFα to or beyond the activation levels of DC stimulated with R848 alone without OC-associated ascites (Fig 4A and 4B) indicating that the observed immunosuppressive effect is indeed IL-10 dependent. None of the other tested neutralizing antibodies had the same effect nor did the combination of neutralizing antibodies show an additive or synergistic effect ruling out a contribution by the other tested immunosuppressive factors in our system (Fig 4A and 4B).


Interleukin-10 and prostaglandin E 2 have complementary but distinct suppressive effects on Toll-like receptor-mediated dendritic cell activation in ovarian carcinoma
Neutralisation of IL-10 releases the impairment of DC activation in response to TLR stimulation in the presence of OC-associated ascites.(A, B) Monocyte derived DC were cultured in the presence of 3μg/ml R848, 10% ascites and neutralizing antibodies (5μg/ml) as indicated. Neutralizing antibodies were added to cultures alone or in combination. (A) CD86 expression levels were measured by flow-cytometry, and (B) cytokine levels were measured in cell culture supernatants by flow cytomix analysis (IL-6, TNFα) or sandwich ELISA (IL-12p40). n = 9–13 (13 independent experiments; monocyte-derived DC from 8 different healthy donors, cultured with 1 (n = 4), 2 (n = 3) or 3 (n = 1) different ascites samples. IL-6 neutralizing antibody was only used in 9 out of 13 experiments). One-way ANOVA (Friedman test with Dunn post test): *** = p<0.001. Please note that the quantification of IL-6 in samples containing IL-6 neutralizing antibody (αIL-6 and all AB) is compromised. (C, D) For allogeneic MLR, monocyte-derived DC from healthy donors were cultured overnight in complete medium only, or stimulated with 3μg/ml R848 in the presence or absence of 10% ascites fluid (AF) and/or IL-10 neutralizing antibody (5μg/ml) as indicated. Subsequently, such pre-treated DC were washed and co-cultured with CFSE-stained CD4+ naïve T cells from another healthy donor at a 1:200 ratio for 6 days. Naïve T cells from the same donor were used consistently for all experiments. The proliferation of T cells was determined by CFSE dilution after 6 days. (C) A representative example of flow-cytometry analysis is shown. (D) Cumulative data from 4 independent experiments showing proliferation in percent of CD3+ cells that have undergone at least one round of division. n = 4 (4 independent experiments; monocyte-derived DC from 3 different healthy donors, cultured with ascites from two (n = 1) or one (n = 2) OC patient).
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pone.0175712.g004: Neutralisation of IL-10 releases the impairment of DC activation in response to TLR stimulation in the presence of OC-associated ascites.(A, B) Monocyte derived DC were cultured in the presence of 3μg/ml R848, 10% ascites and neutralizing antibodies (5μg/ml) as indicated. Neutralizing antibodies were added to cultures alone or in combination. (A) CD86 expression levels were measured by flow-cytometry, and (B) cytokine levels were measured in cell culture supernatants by flow cytomix analysis (IL-6, TNFα) or sandwich ELISA (IL-12p40). n = 9–13 (13 independent experiments; monocyte-derived DC from 8 different healthy donors, cultured with 1 (n = 4), 2 (n = 3) or 3 (n = 1) different ascites samples. IL-6 neutralizing antibody was only used in 9 out of 13 experiments). One-way ANOVA (Friedman test with Dunn post test): *** = p<0.001. Please note that the quantification of IL-6 in samples containing IL-6 neutralizing antibody (αIL-6 and all AB) is compromised. (C, D) For allogeneic MLR, monocyte-derived DC from healthy donors were cultured overnight in complete medium only, or stimulated with 3μg/ml R848 in the presence or absence of 10% ascites fluid (AF) and/or IL-10 neutralizing antibody (5μg/ml) as indicated. Subsequently, such pre-treated DC were washed and co-cultured with CFSE-stained CD4+ naïve T cells from another healthy donor at a 1:200 ratio for 6 days. Naïve T cells from the same donor were used consistently for all experiments. The proliferation of T cells was determined by CFSE dilution after 6 days. (C) A representative example of flow-cytometry analysis is shown. (D) Cumulative data from 4 independent experiments showing proliferation in percent of CD3+ cells that have undergone at least one round of division. n = 4 (4 independent experiments; monocyte-derived DC from 3 different healthy donors, cultured with ascites from two (n = 1) or one (n = 2) OC patient).
Mentions: In order to clarify whether IL-10 is mediating the inhibitory effect on TLR-mediated DC activation partially or in full, we added neutralizing antibodies against IL-10 to monocyte-derived DC cultures containing R848 and 10% OC-associated ascites. We chose R848 as TLR agonist for this set of experiments, since it induced the highest levels of cytokine production by DC in the absence of ascites and robust and reproducible suppression of R848-induced cytokine production was observed in the presence of 10% ascites. In parallel, we also tested neutralizing antibodies against IL-6, TGFβ, LIF and VEGFα to identify any contributions to the observed immunosuppressive effect by these factors. IL-6, LIF and VEGFα were present in the ascites samples investigated in this study (Fig 3A). We also included TGFβ, which has been ascribed immunosuppressive roles in different physiological scenarios but the concentration of which we had not determined in the ascites. Upon addition of IL-10 neutralizing antibody, we observed restored CD86 up-regulation and production of IL-6, IL-12p40 and TNFα to or beyond the activation levels of DC stimulated with R848 alone without OC-associated ascites (Fig 4A and 4B) indicating that the observed immunosuppressive effect is indeed IL-10 dependent. None of the other tested neutralizing antibodies had the same effect nor did the combination of neutralizing antibodies show an additive or synergistic effect ruling out a contribution by the other tested immunosuppressive factors in our system (Fig 4A and 4B).

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