Prostaglandin E2 synergistically with interleukin-23 favors human Th17 expansion

Abstract

Microenvironment molecular cues direct T helper (Th) cell differentiation; however, Th17 fate determination is still imprecisely understood in humans. To assess the role of prostaglandin E(2) (PGE(2)) in Th expansion, we activated peripheral blood mononuclear cells by CD3 cross-linking. In the presence of exogenous PGE(2), peripheral blood mononuclear cells produced higher interleukin-17 (IL-17), C-C chemokine ligand 20 (CCL20)/macrophage inflammatory protein 3alpha (MIP-3alpha), CXC chemokine ligand 8 (CXCL8)/IL-8, and lower interferon-gamma and IL-22 levels than in control cultures. Exogenous PGE(2) and IL-23 synergized in inducing IL-17, whereas indomethacin and IL-23 blockade drastically reduced IL-17 but not interferon-gamma production. Furthermore, IL-1 but not tumor necrosis factor was absolutely required for IL-17 production. PGE(2) doubled the frequency of CD4+ T cells producing IL-17 and within the CD4+ subset enhanced C-C chemokine receptor 6 (CCR6) and CCR4 while decreasing CXC chemokine receptor 3 (CXCR3) expression. Furthermore, in CD4+ T-cell lines, the production of IL-17 segregated with the CCR6+ subset. In the presence of CCR6+ compared with CXCR3+ Th cells, monocytes/macrophages produced much higher levels of matrix metalloproteinase-1, -3, and -9 but similar levels of CXCL10 and IL-1beta. These results identify PGE(2) and IL-23 as participating in the expansion of CD4+ T cells endowed with high IL-17 production capacity, which in turn favors monocyte production of mediators important for host defense and tissue destruction.

Figure 1

PGE₂ and IL-23 specifically enhance the production of IL-17 and some of the related cytokines by human PBMCs. (A) IL-17; (B) IFN-γ; (C) IL-1β; (D) CCL20/MIP-3α; (E) IL-22; (F) CXCL8/IL-8. PBMCs (1 × 10⁶) were activated by CD3 cross-linking and cultured for 7 days before harvesting of supernatants. PGE₂ (50 ng/mL) and IL-23 (10 ng/mL) were added at the beginning, and IL-2 (20 U/mL) was added after 48 hours of culture. IL-17, IFN-γ, and IL-1β were assessed by multiplex immunoassay, whereas CCL20/MIP-3α, IL-22, and CXCL8/IL-8 were assessed by ELISA. Box plots represent the 10th, 25th, 50th, 75th, and 90th percentiles of 11 distinct individual donors (except CXCL8/IL-8, n = 7). Circles represent outliers. *Significant differences (P < .01) compared with the nil culture condition by paired Student t test. Note that the IL-17 scale is logarithmic.

Figure 2

Endogenous PGE₂ and IL-23 affect the production of IL-17 by human PBMCs. Culture conditions as in the legend of Figure 1. Indomethacin (10⁻⁵ M) and anti–IL-23 mAb (10 μg/mL) were added at the beginning of the culture. Cytokines were assessed by multiplex immunoassay. Bars represent the mean percentage plus or minus SD of the response of the nil culture condition of 9 distinct individual donors. In the nil culture condition, IL-17 was 198.9 (± 182.4) ng/mL, IFN-γ was 1693.9 (± 1365.5) ng/mL, and IL-1β was 617.0 (± 257.2) ng/mL. *Significant differences (P < .01) compared with nil by paired Student t test.

Figure 3

PGE₂ and IL-23 favor Th17 expansion. (A,B) Culture conditions as in the legend of Figure 1. Cells were harvested after 7 days of culture and processed for intracellular staining on PMA and ionomycin activation as described in “Flow cytometry.” (A) Representative example of a culture in the presence of IL-23 (10 ng/mL) with or without PGE₂ (50 ng/mL). (B) Box plots represent the 10th, 25th, 50th, 75th, and 90th percentiles of 7 distinct individual donors. Circles represent outliers. *Significant differences (P < .01) compared with the nil culture condition by paired Student's t test. Note that IL-17 positivity was restricted to CD4⁺ T cells. (C) Peripheral blood CD4⁺ T cells were sorted into CD45RO⁻ (naive) and CD45RO⁺ (memory) subsets, activated by CD3/CD28-coated beads, and cultured in the presence of IL-2 (20 U/mL), IL-23 (10 ng/mL), with or without PGE₂ (50 ng/mL). Experiment representative of similar results with cells from three distinct donors.

Figure 4

IL-1 but not TNF is required for the production of IL-17 by human PBMCs cultured in the presence of PGE₂. Culture conditions as in the legend of Figure 1. IL-1Ra (1 μg/mL), TNF-bp (10⁻⁸ M), IL-1β (500 ng/mL), and indomethacin (10⁻⁵ M) were added at the beginning of the culture in the absence of exogenous IL-23 except when mentioned. IL-17, IFN-γ, and IL-1β were assessed by multiplex immunoassay in 7-day culture supernatants. Bars represent the mean plus or minus SD of 6 (A-C) or 4 (D) distinct individual donors. *Significant differences (P < .01) compared with the nil culture condition by paired Student t test.

Figure 5

PGE₂ favors the expression of CCR6 and CCR4 in CD4⁺ T cells. PBMCs were cultured as described in the legend of Figure 1. The cells were harvested after 7 days and submitted to 4-color FACS analysis. (A) Representative histogram of CD4 expression in PBMCs cultured in the absence (thin line) or presence (thick line) of PGE₂ (50 ng/mL). (B) Representative histogram of CCR6, CCR4, and CXCR3 expression (thick line) in CD4⁺ T cells (thin line represents isotype control). (C) Representative dot blot of CCR4 and CXCR3 expression in CD4⁺CCR6⁺ T cells. (D) The bars represent the mean percentage plus or minus SD of CD4 and chemokine receptor expression of 11 distinct individual donors. *Significant differences (P < .01) compared with the nil culture condition by paired Student t test.

Figure 6

IL-17 production in Th17 cell generated under the influence of PGE₂ segregates with CCR6 expression. (A-C) CD4⁺ T cells were activated and expanded thereafter in the presence of irradiated allogeneic PBMCs and OKT-3 (0.1 μg/mL) in medium supplemented with IL-2, IL-23, and PGE₂. CCR6⁺ (B) and CXCR3⁺ (C) were obtained from the parental Th17 cell line (A) by sorting using FACSAria. (D) Naive CD4⁺ T cells were activated by CD3 cross-linking in the presence of IL-12 and anti–IL-4 to obtain Th1 and expanded thereafter in the presence of IL-2. (A-D) The cells were harvested 10 days after sorting and processed for chemokine receptor expression and intracellular staining on PMA and ionomycin activation as described in “Flow cytometry.” The cells were all from the same patient and cultured for the same amount of time. The results here shown are representative of results generated with T-cell lines from two healthy persons.

Figure 7

Th17 are more potent than Th1 cells to induce MMP production by monocytes/macrophages. (A) IL-17, IFN-γ, IL-4; (B) MMP-1, MMP-3, MMP-9; (C) IL-1β, CCL20/MIP-3α, CXCL10/IP-10. T-cell lines were generated as described in the legend of Figure 6. A total of 50 000 T cells were cocultured with equal numbers of allogeneic monocytes for 48 hours. IL-1β, IL-4, IL-17, IFN-γ, MMP-1, MMP-3, and MMP-9 were assessed by multiplex immunoassay, whereas CCL20/MIP-3α and CXCL10 were assessed by ELISA. Bars represent the mean of duplicate cultures; the SD was within 15% of the mean for all conditions. The results are representative of similar data obtained with T cells generated from 2 healthy donors.