Positive feedback between PGE2 and COX2 redirects the differentiation of human dendritic cells toward stable myeloid-derived suppressor cells

Abstract

Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) show opposing roles in the immune system. In the present study, we report that the establishment of a positive feedback loop between prostaglandin E(2) (PGE(2)) and cyclooxygenase 2 (COX2), the key regulator of PGE(2) synthesis, represents the determining factor in redirecting the development of CD1a(+) DCs to CD14(+)CD33(+)CD34(+) monocytic MDSCs. Exogenous PGE(2) and such diverse COX2 activators as lipopolysaccharide, IL-1β, and IFNγ all induce monocyte expression of COX2, blocking their differentiation into CD1a(+) DCs and inducing endogenous PGE(2), IDO1, IL-4Rα, NOS2, and IL-10, typical MDSC-associated suppressive factors. The addition of PGE(2) to GM-CSF/IL-4-supplemented monocyte cultures is sufficient to induce the MDSC phenotype and cytotoxic T lymphocyte (CTL)-suppressive function. In accordance with the key role of PGE(2) in the physiologic induction of human MDSCs, the frequencies of CD11b(+)CD33(+) MDSCs in ovarian cancer are closely correlated with local PGE(2) production, whereas the cancer-promoted induction of MDSCs is strictly COX2 dependent. The disruption of COX2-PGE(2) feedback using COX2 inhibitors or EP2 and EP4 antagonists suppresses the production of MDSC-associated suppressive factors and the CTL-inhibitory function of fully developed MDSCs from cancer patients. The central role of COX2-PGE(2) feedback in the induction and persistence of MDSCs highlights the potential for its manipulation to enhance or suppress immune responses in cancer, autoimmunity, or transplantation.

Figure 1

Inhibition of DC differentiation by PGE₂, proinflammatory cytokines, and TLR ligands is associated with the induction of endogenous COX2- and MDSC-associated suppressive factors. (A) Expression of COX2 mRNA (panel A left) and protein (panel A right) levels is induced by synthetic PGE₂, initiating a positive feedback loop in iMCs. Regulation of COX1 and COX2 expression by synthetic PGE₂ was analyzed after 6-10 hours. (B) Induction of immunosuppressive factors IL-10, NOS2, IDO1, and IL-4Rα by synthetic PGE₂. (C-D) Induction of PGE₂ secretion (C) and COX2 mRNA (D left) and protein (D right) by the COX2 activators LPS (TLR4 ligand), IL-1β, and IFN-γ. (E) Celecoxib suppresses the induction of immunosuppressive factors (IDO1, IL-10, NOS2, and IL-4Rα) by COX2 activators. All data (panels A-E) were confirmed in at least 3 independent experiments. Histograms present data from a single representative experiment with different donors as mean ± SD. *P < .05; **P < .01; and ***P < .001.

Figure 2

PGE₂ redirects DC differentiation and induces CD14⁺CD33⁺CD34⁺ cells with the phenotype and function of monocytic MDSCs isolated from cancer patients. (A) Phenotype of PGE₂-induced CD1a⁻CD14⁺CD80⁻CD83⁻ MDSCs expressing inhibitory molecules ILT2, ILT3, ILT4, PDL-1, but not PDL-2. PGE₂-induced MDSCs express E-prostanoid receptors (labeled with α-EP1–, α-EP3–, sec.Alexa488, α-EP2–, and α-EP4–PE). (B) Expression of immunosuppressive factors IL-10, IDO1, IL-4Rα, and COX2 in PGE₂-induced MDSCs (see supplemental Figure 1A for corresponding protein levels of IDO and IL-10). (C) Immunosuppressive effects of PGE₂-induced MDSCs on allogeneic naive CFSE-labeled CD8⁺ T cells primed by CD3/CD28 and stained for granzyme B. Left panel: Percentages indicate the fraction of proliferating granzyme B⁺ (marker of CTL status) CD8⁺ cells. Right panel: Percentage of proliferating CD8⁺ T cells in the presence of PGE₂-induced MDSCs (PGE₂-d0) and PGE₂-conditioned DCs (PGE₂-d6). (D-F) MDSC phenotype and function of CD11b⁺ cells isolated from cancer ascites. (D) Characterization of cells from cancer ascites either before (left panel) or after (right panel box) isolation of CD11b⁺ cells. Note the high percentage of CD11b⁺ cells (8.9%-50.0%, mean 24.2%, n = 7) within the cancer-infiltrating primary cell population. (E) mRNA levels of IL-10, IDO1, IL-4Rα, and COX2 in CD11b⁺ cells isolated from cancer (see supplemental Figure 1B for corresponding protein levels of IDO and COX2) compared with CD11b⁺ cells isolated from blood (ascites-isolated, n = 7; control blood-isolated, n = 5). (F) Suppression of CFSE-labeled allogeneic naive CD8⁺ T-cell proliferation (CD3/CD28 stimulation) in the presence or absence of primary cells or CD11b⁺ cells isolated from cancer (ie, MDSCs isolated from cancer; n = 7). Percentages indicate the fraction of proliferating granzyme B⁺CD8⁺ cells. The gray squares represent the lymphocyte-specific gates used to exclude (CFSE-unlabeled) MDSCs. All data (panels A-F) were confirmed in at least 3 independent experiments. Histograms present data of a single representative experiment with different donors as means ± SD. *P < .05; **P < .01; and ***P < .001.

Figure 3

PGE₂ mediates the enhanced development of MDSCs in human cancer environment. (A) Correlation between the PGE₂ production and the frequencies of cancer-infiltrating CD11b⁺CD33⁺ cells from different patients. The percentage of cancer-infiltrating CD11b⁺CD33⁺ cells was determined by flow cytometry (n = 5 patients). The regression line and corresponding R² value are shown. (B) MDSC phenotype induced in GM-CSF⁺IL-4–cultured monocytes by membrane-permeable soluble factor(s) produced by cancer-infiltrating cells. Similar data were obtained using the CM from cancer-infiltrating cells and in a Transwell system (see supplemental Figure 3 for experimental design). Left panel: Suppression of DC differentiation by CM from cancer-infiltrating cells (manifested by loss of the DC marker CD1a). Right panel: Induction of the CD1a⁻CD14⁺DCSIGN⁻CD80⁻CD83⁻ MDSC phenotype. (C) mRNA levels of IL-10, arginase 1 (ARG1), IDO1, IL-4Rα, and COX2 in cancer-induced MDSCs. (D) Suppressed CD3/CD28–induced proliferation of granzyme B⁺ CTL (percentages) in the presence of cancer-induced MDSCs. Left panel: Percentages indicate the fraction of proliferating granzyme B⁺CD8⁺ cells. Right panel: Percentage of proliferating CD8⁺ T cells in the presence of cancer-induced MDSCs (cancer d0) and cancer-conditioned DCs (cancer d6). (E) Induction of immunosuppressive factors by cancer-associated PGE_2. (F) Induction of CD14⁺ MDSCs by CM from cancer-infiltrating cells is suppressed by COX2 inhibition and restored by synthetic PGE₂. (G) Regulation of COX1 and COX2 expression by CM from cancer-infiltrating cells generated in the presence or absence of celecoxib and/or synthetic PGE₂ and analyzed after 6-10 hours. (H) Immunosuppressive effects of cancer-induced MDSCs on naive CFSE-labeled CD8⁺ T cells primed by CD3/CD28 and stained for granzyme B. Cancer-infiltrating primary cell CM was generated in the presence or absence of the COX2 inhibitor celecoxib. (I) Induction of immunosuppressive factors by PGE₂, the EP4 agonist CAY10598, the EP2 agonist butaprost, but not the EP3/1 agonist sulprostone_. All data (panels A-I) were confirmed in 3-7 independent experiments. Histograms present data from a single representative experiment with different donors as mean ± SD.

Figure 4

Functional stability of MDSCs isolated from cancer patients requires continuous PGE₂-COX2 feedback involving EP2 and EP4 signaling. (A) Expression of immunosuppressive factors in cancer-isolated CD11b⁺ cells pretreated (24 hours) or not with celecoxib, the EP4 antagonist AH23848, the EP2/1 antagonist AH6809, and the EP3 antagonist L798106. (B) PGE₂ production and COX2 expression in primary cells (white bars) and CD11b⁺ cells (black bars) isolated from cancer, compared with control CD11b⁺ cells isolated from blood treated or not with celecoxib. Measurements were performed in triplicates. (C) Celecoxib pretreatment of MDSCs isolated from cancer abolishes their suppressive impact on CD3/CD28–activated naive CD8⁺ T cells. (D) Inhibition of COX2, arginase-1, IL-10, or IDO1 counteracts the suppressive functions of MDSCs isolated from cancer (n = 3). (E) Celecoxib, the EP4 antagonist AH23848, and the EP2/1 antagonist AH6809, but not the EP3 antagonist L798106, similarly reverse the suppressive functions of MDSCs. The addition of synthetic PGE₂ to celecoxib-pretreated MDSCs isolated from cancer restores immunosuppressive functions (n = 3). Neither celecoxib nor the EP antagonists showed any cytotoxic effects at the concentrations used. (F) CD3/CD28 activation of CD8⁺ T cells (absence of MDSCs) ± 1μM PGE₂ or different concentrations of the commercially available agonists butaprost (EP2) or sulprostone (EP3/1 agonist, negative control). All data (panels A-F) were confirmed in at least 3 independent experiments. Histograms present data from a single representative experiment with different donors as means ± SD.