Prostaglandin E2 inhibits Tr1 cell differentiation through suppression of c-Maf

<div><p>Prostaglandin E2 (PGE2), a major lipid mediator abundant at inflammatory sites, acts as a proinflammatory agent in models of inflammatory/autoimmune diseases by promoting CD4 Th1/Th17 differentiation. Regulatory T cells, including the IL-10 producing Tr1 cells counterbalance the proinflammatory activity of effector Th1/Th17 cells. Tr1 cell differentiation and function are induced by IL-27, and depend primarily on sustained expression of c-Maf in addition to AhR and Blimp-1. In agreement with the in vivo proinflammatory role of PGE2, here we report for the first time that PGE2 inhibits IL-27-induced differentiation and IL-10 production of murine CD4⁺CD49b⁺LAG-3⁺Foxp3^- Tr1 cells. The inhibitory effect of PGE2 was mediated through EP4 receptors and induction of cAMP, leading to a significant reduction in c-Maf expression. Although PGE2 reduced IL-21 production in differentiating Tr1 cells, its inhibitory effect on Tr1 differentiation and c-Maf expression also occurred independent of IL-21 signaling. PGE2 did not affect STAT1/3 activation, AhR expression and only marginally reduced Egr-2/Blimp-1 expression. The effect of PGE2 on CD4⁺CD49b⁺LAG-3⁺ Tr1 differentiation was not associated with either induction of Foxp3 or IL-17 production, suggesting a lack of transdifferentiation into Foxp3⁺ Treg or effector Th17 cells. We recently reported that PGE2 inhibits the expression and production of IL-27 from activated conventional dendritic cells (cDC) in vivo and in vitro. The present study indicates that PGE2 also reduces murine Tr1 differentiation and function directly by acting on IL-27-differentiating Tr1 cells. Together, the ability of PGE2 to inhibit IL-27 production by cDC, and the direct inhibitory effect on Tr1 differentiation mediated through reduction in c-Maf expression, represent a new mechanistic perspective for the proinflammatory activity of PGE2.</p></div

Inhibition of Tr1 differentiation by PGE2 is independent of STAT1/3.

<p>Naïve CD4⁺CD62L⁺ cells from wildtype 129S6 (WT) and <i>Stat1</i>^{<i>-/- </i>}mice were stimulated in the presence of IL-27 and PGE2. (A) Samples were collected on day 3 and cells were analyzed for Tr1 markers. (B) Cells were subjected to RNA extraction and subsequent analysis by RT-PCR for <i>Maf</i> expression. (C) Naïve CD4⁺CD62L⁺ cells from C57BL/6 mice were stimulated in the presence of IL-27, PGE2 and 10^-4M dbcAMP. Cells were collected 15, 30 60 minutes later and analyzed by FACS for intracellular phospho-STAT1 (Tyr701). (D) Naïve CD4⁺CD62L⁺ cells from C57BL/6 mice were stimulated as described in (C) for 15 and 60 minutes. Cells were analyzed by FACS for intracellular phospho-STAT3 (Tyr705). Data are cumulative from three independent experiments. Each sample was tested in duplicate and results represent means ± SD. Significance was determined using one-way ANOVA; ***<i>P<0</i>.<i>001</i>.</p

PGE2 inhibits Tr1 differentiation in vivo.

<p>(A) Schematic of timeline and experimental groups. <i>Il10</i>^<i>gfp</i> mice were injected i.p. with vehicle, 20 μg/ml anti-CD3 or anti-CD3 and 4μg dmPGE2 at 0hr and 48hr. Peyer’s patches, spleen, and intraepithelial lymphocyte populations were collected. (B) CD4⁺ populations were analyzed by FACS for Tr1 cell populations (CD49b⁺LAG-3⁺). (C-D) CD4⁺ populations were analyzed by FACS for IL-10 expression in both Tr1 populations and non-Tr1 populations (including CD49b⁺LAG-3^-, CD49b^-LAG-3⁺ and CD49b^-LAG-3^- populations). Data are cumulative of two independent experiments. Significance was evaluated by unpaired t-test.</p

PGE2 inhibits IL-27 induced differentiation of Tr1 cells.

<p>(A-C) Naïve CD4⁺CD62L⁺ cells from <i>il10</i>^<i>gfp</i> mice were stimulated with plate-bound anti-CD3 (3 μg/ml) and soluble anti-CD28 (1 μg/ml) in the presence of 50 ng/ml IL-27 and various concentrations of PGE2 for three days. Cells were collected on day 3 and analyzed by FACS for Tr1 markers, and IL-10 within CD49b⁺LAG-3⁺ populations. Representative samples show CD49b⁺LAG-3⁺ populations (upper panel) and histograms of IL-10 (lower panel). Data are representative of four independent experiments. Each sample was tested in duplicate and results represent means ± SD. Significance was determined using one-way ANOVA and * represents the P value for a sample versus IL-27 control. **<i>P<0</i>.<i>01</i>, ***<i>P<0</i>.<i>001</i>. (D) For cell proliferation experiments, naïve CD4⁺CD62L⁺ cells were stained with CFSE per manufacturer’s instructions prior to stimulation with anti-CD3, anti-CD28, IL-27 and PGE2. Incorporation of CFSE was analyzed by FACS on day 3. Data are representative of two independent experiments. Each sample was tested in triplicate and results represent means ± SD. Significance was determined using unpaired t-test. (E) Naïve CD4⁺CD62L⁺ cells were stimulated in the presence of 50 ng/ml IL-27 and 10^-6M PGE2. Supernatant was collected on day 3 and subjected to ELISA to determine IL-17 levels. Data are representative of three independent experiments. Each sample was tested in duplicate and results represent means ± SD. Significance was determined using one-way ANOVA.</p

PGE2 signals through EP4 to inhibit Tr1 cell differentiation and expression of IL-10.

<p>(A) Naïve CD4⁺CD62L⁺ cells were stimulated in the presence of IL-27 and 10^-6M PGE2 or 10^-5M of EP receptor agonists: Butaprost (EP2), Misoprostol (EP3, EP4>EP1) or Sulprostone (EP3>EP1). Cells were collected on day 3 and analyzed by FACS for Tr1 cell markers. (B,C) Naïve CD4⁺CD62L⁺ cells from <i>il10</i>^<i>gfp</i> mice were pretreated for 30 minutes with 10^-6M EP receptor antagonists, ONO-AE3-208 (ONO; EP4) or PF-04418948 (PF; EP2) prior to treatment with 50 ng/ml IL-27 and 10^-7M PGE2. Cells were collected on day 3 and analyzed by FACS for Tr1 markers and IL-10 expression. Data are representative of three independent experiments. Each sample was tested in duplicate and results represent means ± SD. Significance was evaluated by one-way ANOVA and * symbolizes significance of experimental condition versus IL-27 control; *<i>P<0</i>.<i>05</i>, **<i>P<0</i>.<i>01</i>, ***<i>P<0</i>.<i>001</i>.</p

PGE2 inhibits c-Maf in CD4 T cells differentiated in the presence of IL-27.

<p>(A) Naïve CD4⁺CD62L⁺ cells were stimulated in the presence of IL-27 and PGE2. RNA was collected at 24, 48 and 72 hr and analyzed by RT-PCR for <i>Maf</i> and <i>Ahr</i> expression. (B) Naïve CD4⁺CD62L⁺ cells were stimulated in the presence of IL-27 and PGE2 or dbcAMP. Cells were collected on day 3 and analyzed by RT-PCR for <i>Maf</i> expression. (C) Splenocytes from <i>il10</i>^<i>gfp</i> mice were stimulated with 3 μg/ml anti-CD3 and 1 μg LPS in the presence of IL-27 and PGE2. RNA was collected on day 3 and <i>Maf</i> expression was analyzed by RT-PCR. (D) Naïve CD4⁺CD62L⁺ cells were stimulated in the presence of IL-27 and PGE2 or dbcAMP. Cells were collected on day 3 and analyzed by FACS for intracellular c-Maf within the whole cell population (upper panel) and within CD49b⁺LAG-3⁺ populations (lower panel). Graphical representation of data is on the right. (E) Naïve CD4⁺CD62L⁺ cells were stimulated as in D. Cells were collected on day 3 and analyzed by FACS for intracellular Egr-2 within the whole cell population (upper panel) and within CD49b⁺LAG-3⁺ populations (lower panel). (F) Naïve CD4⁺CD62L⁺ cells were stimulated as in D. Cells were collected on day 3 and analyzed by FACS for intracellular Blimp-1 within the whole cell population and within CD49b⁺LAG-3⁺ populations. Data are representative of two to three independent experiments. Each sample was tested in duplicate and results represent means ± SD. Significance was evaluated by one-way ANOVA and * symbolizes significance of experimental condition versus IL-27 control (B, D, E); *<i>P<0</i>.<i>05</i>, **<i>P<0</i>.<i>01</i>, ***<i>P<0</i>.<i>001</i>.</p

Inhibition of c-Maf by PGE2 is independent of IL-21.

<p>(A) Naïve CD4⁺CD62L⁺ cells were stimulated in the presence of IL-27 and PGE2 or dbcAMP. Supernatant was collected on day 3 and analyzed by ELISA for IL-21. Data are cumulative from two independent experiments. (B) Naïve CD4⁺CD62L⁺ cells from <i>il10</i>^<i>gfp</i> mice were stimulated in the presence of IL-27, 100 ng/ml IL-21 and PGE2. Cells were collected on day 3 and analyzed by FACS to determine IL-10 production within CD49b⁺LAG-3⁺ CD4 T cells. Data are representative of two independent experiments. (C) Naïve CD4⁺CD62L⁺ cells from C57BL/6 mice were stimulated as above. Cells were collected on day 3 and analyzed by FACS to determine presence of intracellular c-Maf within CD4 T cells (top) and CD49b⁺LAG-3⁺ CD4 T cells (bottom). Histograms show representative samples, while graphs present data from one of three independent experiments. Each sample was tested in duplicate and results represent means ± SD. Significance was determine by one-way ANOVA; **<i>P<0</i>.<i>01</i>, ***<i>P<0</i>.<i>001</i>.</p

PGE2 inhibits Tr1 cell differentiation in splenocyte cultures.

<p>(A) <i>il10</i>^<i>gfp</i> splenocytes were stimulated with 3 μg/ml anti-CD3 and 1 μg LPS in the presence of neutralizing IL-27 antibodies (5 and 10 μg/ml) or IgG control (20 μg/ml). Cells were collected on day 3 and CD4⁺CD49b⁺LAG-3⁺ Tr1 cells were identified by FACS. Data are cumulative from two independent experiments. (B) Splenocytes were stimulated with anti-CD3 and LPS in the presence of PGE2 (10^-6M). Supernatant was collected on day 3 and IL-27 levels analyzed by ELISA. (C-D) Splenocytes were stimulated with anti-CD3 and LPS in the presence or absence of IL-27 (50 ng/ml) and PGE2 (10^-6M). Cells were collected on day 3 and CD4⁺CD49b⁺LAG-3⁺ Tr1 cells were identified by FACS. (C) Representative sample shows gating strategy to determine percentage of CD49b⁺LAG-3⁺ within CD4⁺ T cells and (D) graph presents representative data from three independent experiments. Each sample was tested in duplicate and results represent means ± SD. Significance was evaluated by one-way ANOVA; **<i>P<0</i>.<i>01</i>.</p

Different Microtubule Structures Assembled by Kinesin Motors

The microtubule–kinesin system is used to form microtubule-based structures via microtubule gliding motility. On the kinesin-coated surface, the microtubules can be easily assembled into stable micro- and nanostructures like circles and microtubule bundles using the streptavidin–biotin system. Furthermore, these microtubules structures can still retain performance with kinesin motor movement in spite of different velocities. Collisions bear responsibility for the majority of events leading to circle formation. By taking advantage of biological substances, some micro- or nanostructures, which are difficult to fabricate by artificial processes, can be easily obtained

Different Microtubule Structures Assembled by Kinesin Motors

The microtubule–kinesin system is used to form microtubule-based structures via microtubule gliding motility. On the kinesin-coated surface, the microtubules can be easily assembled into stable micro- and nanostructures like circles and microtubule bundles using the streptavidin–biotin system. Furthermore, these microtubules structures can still retain performance with kinesin motor movement in spite of different velocities. Collisions bear responsibility for the majority of events leading to circle formation. By taking advantage of biological substances, some micro- or nanostructures, which are difficult to fabricate by artificial processes, can be easily obtained