IL-10 Production in Macrophages Is Regulated by a TLR-Driven CREB-Mediated Mechanism That Is Linked to Genes Involved in Cell Metabolism

David E. Sanin, Catriona T. Prendergast and Adrian P. Mountford
J Immunol August 1, 2015, 195 (3) 1218-1232; DOI: https://doi.org/10.4049/jimmunol.1500146

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    BMMs internalize cercarial E/S products and produce IL-10 in a TLR-dependent manner. (A) Representative confocal image (12 × 20 μm) of a BMM exposed to 50 μg/ml 0–3hRPAF633 (red) for 60 min, labeled with anti–EEA-1 Abs (cyan), and counterstained with DAPI (blue). Arrowheads indicate the location of 0–3hRPAF633 within EE-A+ (white) or EEA-1− (yellow) endosomes. Cell membrane is shown in white. Representative overlaid flow cytometry histograms (B) and mean MFI ± SEM (C) of BMMs exposed to 50 μg/ml 0–3hRPAF633 (closed circles) or media control (open circles). Symbols are means of three technical replicates. Fold changes in transcript RNA (D and E) and protein levels of IL-10 and IL-12p40 (F and G) in cultures of WT BMMs exposed to 50 μg/ml 0–3hRP (closed circles) or corresponding media controls (open circles). Symbols represent mean from four biological replicates. (H and I) IL-10 and IL-12p40 produced by overnight cultures of WT, MyD88−/−, TLR2−/−, or TLR4−/− BMMs exposed to 50 μg/ml 0–3hRP or media. Histogram bars are means for selected groups of WT and transgenic BMMs (four biological replicates). ANOVA and multiple comparisons tests (Bonferroni, Dunnett, and Sidak) were performed to examine statistically significant differences (C–G) between 0–3hRP–treated BMMs and corresponding media control at each time point or (H and I) between treated BMMs from different strains of mice. Dotted lines represent no fold changes in RNA levels (D and E) or lower detection limit of ELISA tests (F–I). Results are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, p > 0.05.

  • FIGURE 2.
    FIGURE 2.

    Cercarial E/S products trigger MAPK activation in BMMs in a TLR-dependent manner. Phosphorylation of MAPKs shown as representative overlaid flow cytometry histograms (A–C) and mean MFI ± SEM (D–I) for BMMs exposed to 50 μg/ml 0–3hRP (closed circles) or media control (open circles). Results are shown at 30 min (A–C and J–L) or 0–100 min (D–I). MFI values are shown for phosphorylation of ERK1/2, p38, and CREB (A–F and J–L) and for total MAPKs (G–I). Symbols and bars are means of four biological replicates and representative of three independent experiments. ANOVA and multiple comparisons tests (Sidak and Bonferroni) were performed to examine statistically significant differences between 0–3hRP–treated BMMs and corresponding media control at each time point (D–I) or between the means of selected groups of WT and transgenic BMMs (J and K) (four biological replicates). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, p > 0.05.

  • FIGURE 3.
    FIGURE 3.

    CREB is downstream from p38 and ERK1/2. Representative overlaid flow cytometry histograms (A and B) and mean MFI + SEM (C–E) of BMMs treated with p38 inhibitor (SB203580, 1 μmol) or MEK inhibitor (U0126, 10 μmol) for 2 h and then exposed for 30 min to 50 μg/ml 0–3hRP (closed bars) or media control (open bars). Cells were labeled with Abs against p-CREB (C), p-ERK1/2 (D), or p-p38 (E). Bars represent means of three biological replicates. ANOVA and Tukey multiple comparisons test were performed to examine statistically significant differences between the means of selected groups. Results are representative of four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. ns, p > 0.05.

  • FIGURE 4.
    FIGURE 4.

    MAPK activation induces IL-10 production, while limiting IL-12. BMMs from WT (A–F) and IL-10−/− (G and H) mice were treated with p38 inhibitor (SB203580, 0.3–3 μmol) (A–C and G) or MEK inhibitor (U0126, 3–30 μmol) (D–F and H) for 2 h and then exposed overnight to 50 μg/ml 0–3hRP (closed circles) or media control (open circles). Culture supernatants were tested for the presence of IL-10 (A and D), IL-12p40 (B, E, G, and H), and IL-12p70 (C and F). Symbols and bars are means of four biological replicates. ANOVA and multiple comparisons tests (Dunnett and Sidak) were performed to examine statistically significant differences (A–F) between 0–3hRP–treated BMMs and corresponding media control at each dose of the inhibitor or between the means of WT and IL-10−/− BMMs (G and H) (four biological replicates). Dotted lines represent lower detection limit in ELISA tests. Results are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, p > 0.05.

  • FIGURE 5.
    FIGURE 5.

    Uptake of 0–3hRP and PI3K signaling is required for full MAPK activation and IL-10 production by BMMs. Representative overlaid flow cytometry histograms (A) and mean MFI ± SEM (B) of BMMs pretreated for 2 h with PI3K inhibitor (LY294002; 25 μmol; gray line/squares) or left untreated (circles) and then exposed to 50 μg/ml 0–3hRPAF633 (closed circles) or media control (dashed line, open circles) for 0–300 min. Symbols represent means of three biological replicates. (C–G) BMMs pretreated for 2 h with PI3K inhibitors LY294002 or IC87114 or left untreated and then exposed to 0–3hRP for 30 min (closed bars) or media control (open bars). Cells were labeled with Abs against p-ERK1/2 (C), p-p38 (D), or p-CREB (E). Bars represent mean MFI + SEM of three biological replicates. Supernatants from BMM cultures treated with PI3K inhibitors LY294002 and IC87114 (F and G) and stimulated with 0–3hRP (closed bars) or media control (open bars) were tested for the presence of IL-10 and IL-12p40. Bars represent means of three biological replicates. ANOVA and multiple comparisons tests (Sidak and Dunnett) were performed to examine statistically significant differences (B–G) between BMMs exposed to 0–3hRP only versus cells exposed 0–3hRP + inhibitor. Dotted lines are the lower detection limit in ELISA tests. Results are representative of four independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, p > 0.05.

  • FIGURE 6.
    FIGURE 6.

    CREB is recruited to a specific regulatory element of the Il10 promoter. (A) Scan strategy used to determine binding site in the Il10 promoter for CREB using sets of primers to amplify four DNA fragments between 100 and 300 bp (dotted boxes). Transcription and translation start sites are indicated above diagram. Agarose gel of amplified DNA fragments from Il10 promoter using all designed primers (B) and enrichment of promoter regions based on densitometry analysis of PCR results (C). Sonicated chromatin from BMMs exposed to 0–3hRP for 30 min was precipitated using Abs against p-CREB (black bar), total CREB (checked bar), plus [in (D)] control Abs for RNA polymerase II (Pol II) and CD36, alongside no Ab control. Results are representative of four independent experiments. AU, arbitrary units.

  • FIGURE 7.
    FIGURE 7.

    TLR-activated CREB regulates an extensive network of genes in BMMs stimulated with cercarial E/S products. GO term enrichment network divided into three clusters from enriched peaks + peak scores [−10*log10(p value)] from sequenced fragments of sonicated chromatin from BMMs exposed 0–3hRP precipitated using an Ab against CREB calculated against input control. (A) Significantly enriched GO terms (p < 0.05) colored based on significance (according to intensity of color gradient). Node size is representative of the number of genes mapping to each term. Significantly enriched genes within “localization” (B), “biological regulation” (C), and “metabolism” (D) clusters, presented according to their GO term and KEGG pathway. (E) Mean fold change + SEM for selected RNA transcript levels obtained from BMMs pretreated with MEK inhibitor or left untreated and then exposed to 0–3hRP or media control. Bars represent means of three technical replicates. Statistically significant differences between the means of selected groups were determined using ANOVA and Tukey multiple comparisons test. Dotted lines represent no-fold change in the levels of mRNA. ***p < 0.001, ****p < 0.0001. ns, p > 0.05.

  • FIGURE 8.
    FIGURE 8.

    0–3hRP stimulation increases oxidative phosphorylation over anaerobic glycolysis in BMMs downstream of MEK1/2. Culture supernatants from BMMs exposed for 18 h to 50 μg/ml 0–3hRP or left unstimulated (media) were tested for the amount of remaining glucose (A) and lactate production (B). (C and D) BMMs were treated for 2 h with MEK1/2 inhibitor U0126 (10 μmol) and then exposed for 18 h to 50 μg/ml 0–3hRP or left unstimulated (media). Culture supernatants were tested for the amount of remaining glucose (C) and lactate production (D). Additionally, stimulated BMMs were lysed and hexokinase activity measured (E). Bars represent the mean difference + SEM of the amount of each metabolite (n = 3) (A–D) or mean fold changes + SEM (E) in hexokinase activity (n = 3). In all cases, media was used as a reference point arbitrarily set to 0. (A and B) Unpaired two-tailed t tests were performed to examine differences between means of 0–3hRP–treated cells compared with media. (C and D) ANOVA and Sidak multiple comparisons test were performed to examine statistically significant differences between control BMMs stimulated with 0–3hRP (black bars) compared with BMMs treated with MEK1/2 inhibitor then stimulated with 0–3hRP (hatched bars). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, p > 0.05.

  • FIGURE 9.
    FIGURE 9.

    IL-10 is produced in the skin by two different monocyte populations following S. mansoni cercariae infection. Representative flow cytometry dot plots (A and C) and mean percentage ± SEM (B, D, and E) of DEC according to expression of MHC-II and F4/80. Gates show R1 (F4/80−MHC-IIhigh), R2 (F4/80+MHC-IIhigh), and R3 (F4/80+MHC-IImid) monocytes. MHC-II+ DEC (A and B) and IL-10/GFP+ MHC-II+ DEC (C–E), recovered from naive animals or specified days postinfection of WT or IL-10+/GFP mice. (E) IL-10/GFP+ MHC-II+ DEC separated according to expression of F4/80 as in (A). Symbols are values for cells obtained from individual naive/infected mice. Horizontal bars are the means ± SEM. n = 4–10 pinnae. ANOVA and Tukey multiple comparisons test show statistically significant differences between the means of indicated groups. *p < 0.05, ***p < 0.001, ****p < 0.0001. ns, p > 0.05.

  • FIGURE 10.
    FIGURE 10.

    Hypothetical model of the molecular events leading to the production of IL-10 in macrophages exposed to S. mansoni cercarial E/S products. S. mansoni cercarial E/S products are likely to engage TLR2 and TLR4 in macrophages, after which TIRAP, and consequently MyD88, would be recruited to the activated receptors in a PI3K-dependent manner. These events trigger the phosphorylation (+Ph) of p38 and NF-κB proteins p65 and p105. The latter releases TPL2, thus activating the MEK/ERK/RSK cascade. The two axes of this signaling pathway (enclosed in a dotted line) converge on the phosphorylation of CREB, which is recruited to the Il10 promoter, leading to the production of IL-10, and also modulates the expression of genes associated with metabolism and several transcription factors. Phosphorylated p65 and p50, the degradation product of p105, can form a heterodimer, which modulates the transcription of IL-12, a process negatively impacted by the mechanism that leads to IL-10 production (enclosed in dotted lines). Peak phosphorylation times in minutes are given for several kinases, as well as the initial detection times for the mRNA and protein of cytokines.

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