Lipopolysaccharide-mediated IL-10 transcriptional regulation requires sequential induction of type I IFNs and IL-27 in macrophages

Abstract

IL-10 is a potent anti-inflammatory molecule that regulates excessive production of inflammatory cytokines during an infection or tissue damage. Dysregulation of IL-10 is associated with a number of autoimmune diseases, and so, understanding the mechanisms by which IL-10 gene expression is regulated remains an important area of study. Macrophages represent a major source of IL-10, which is generated in response to TLR signaling as a feedback mechanism to curtail inflammatory response. In this study, we identify a signaling pathway in murine bone marrow-derived macrophages in which activation of TLR4 by LPS induces the expression of IL-10 through the sequential induction of type I IFNs followed by induction and signaling through IL-27. We demonstrate that IL-27 signaling is required for robust IL-10 induction by LPS and type I IFNs. IL-27 leads directly to transcription of IL-10 through the activation of two required transcription factors, STAT1 and STAT3, which are recruited to the IL-10 promoter. Finally, through systematic functional promoter-reporter analysis, we identify three cis elements within the proximal IL-10 promoter that play an important role in regulating transcription of IL-10 in response to IL-27.

FIGURE 1

Induction of IL-10 protein and mRNA in BMDMs. A, Total of 0.5 × 10⁶ WT, IFN-αR KO, and IL-27R KO BMDMs were stimulated with LPS (50 ng/ml), and supernatant was collected at the indicated time points. IL-10 protein production was quantified via ELISA. B, Total of 0.5 × 10⁶ BMDMs were unstimulated or stimulated with LPS (50 ng/ml), IFN-α (250 U/ml), or IL-27 (10 ng/ml), supernatant was collected, and IL-10 protein production was quantified via ELISA. C, Total of 0.5 × 10⁶ BMDMs were unstimulated or stimulated with LPS (50 ng/ml), IFN-α (250U/ml), or IL-27 (10 ng/ml) for 4 h, RNA was harvested, and relative IL-10 transcript was detected via qPCR normalized to L32.

FIGURE 2

LPS-mediated induction of IL-10 is type I IFN and IL-27 signaling dependent. A, Total of 0.5 × 10⁶ WT were unstimulated or stimulated with LPS (50 ng/ml), rIFN-α (250 U/ml), or rIL-27 (10 ng/ml) for 2 h. CM were collected and transferred to 0.5 × 10⁶ WT or IFN-αR^−/− previously serum-starved (1% FBS) BMDMs in the absence of any stimulation. Cells were incubated with the indicated CM for 3 h, RNA was collected, and IL-10 transcript level was detected via qPCR normalized to L32. B, Total of 0.5 × 10⁶ WT BMDMs were unstimulated or stimulated with LPS (50 ng/ml) or IFN-α (250 U/ml) for 2 h. CM were collected and transferred with 0.5 × 10⁶ WT or IL27R^−/− previously serum-starved BMDMs in the absence of any stimulation. Cells were incubated with the indicated CM for 3 h, and IL-10 expression was assessed as in A. Data from supernatant transfers (A, B) represent two independent experiments. C, Total of 0.5 × 10⁶ WT or IFN-αR^−/− BMDMs were unstimulated or stimulated with LPS (50 ng/ml), IFN-α (250 U/ml), or IL-27 (10 ng/ml) for 3 h. RNA was collected, and IL-10 transcript level was detected via qPCR normalized to L32. D, WT or IL27R^−/− BMDMs were unstimulated or stimulated with LPS (50 ng/ml), IFN-α (250 U/ml), or IL-27 (10 ng/ml) for 3 h. RNA was collected, and IL-10 transcript level was detected via qPCR normalized to L32. E, Total of 0.5 × 10⁶ WT BMDMs were pretreated with DMSO or CHX (2 µg/ml) and then stimulated with LPS (50 ng/ml), IFN-α (250 U/ml), or IL-27 (10 ng/ml) for 3 h. RNAwas collected, and IL-10 transcript level was detected via qPCR normalized to L32. Data from C and D represent mean and SD from three independent experiments. Student t test was performed. *p < 1 × 10⁻⁴.

FIGURE 3

LPS induction of IL-10 requires sequential induction of type I IFNs and IL-27, respectively. A, Total of 0.5 × 10⁶ WT BMDMs were were stimulated with LPS (50 ng/ml) for the indicated time points. RNAwas collected, and IFN-β, IFN-α4, and IL-27p28 transcript level was detected via qPCR normalized to L32. B, Total of 0.5 × 10⁶ WT were unstimulated or stimulated with LPS for 2 h. CM were collected and transferred to 0.5 × 10⁶ WT or IFN-αR^−/− previously serum-starved (1% FBS) BMDMs in the absence of any stimulation. Cells were incubated with the indicated CM for 3 h, RNA was collected, and IL-27p28 transcript level was detected via qPCR normalized to L32. C, Total of 0.5 × 10⁶ WT or IFN-αR^−/− BMDMs were unstimulated or stimulated with LPS (50 ng/ml) or IFN-α (250 U/ml) for 3 h. RNA was collected, and IL-27p28 or EBI3 transcript level was detected via qPCR normalized to L32. D, Total of 0.5 × 10⁶ WT or IL-27R^−/− BMDMs were unstimulated or stimulated with LPS (50 ng/ml), IFN-α (250 U/ml), or rIL-27 (10 ng/ml) for 3 h. RNA was collected, and IFN-α and IL-27p28 transcript level was detected via qPCR normalized to L32. Data represent mean and SD from three independent experiments. Student t test was performed. *p < 0.01.

FIGURE 4

Identification of LPS, IFN-α, and IL-27 response elements in IL-10 proximal promoter. A, RAW264.7 were transiently transfected with −1954/+64 IL-10 promoter ligated to firefly luciferase reporter (2.5 µg) and were unstimulated or titrated with stimulated with LPS, IFN-α, or IL-27. Stimulation with LPS (1 µg/ml), rIFN-α (1000 U/ml), or IL-27 (80 ng/ml) consistently gave equivalent firefly luciferase activities (normalized to renilla luciferase activity). Empty pGL4.20 or IL-10 promoter 5′ deletion mutants (as indicated) sharing +64 transcription start site were transfected into RAW264.7 cells and left untreated or stimulated with LPS (1 µg/ml) (B), rIFN-α (1000 U/ml) (C), or rIL-27 (80 ng/ml) (D). Luciferase activity displayed as percent of full-length (−1954/+64) IL-10 promoter activity normalized to renilla luciferase activity representing two independent experiments. Student t test was performed. Arrows indicate p < 0.05.

FIGURE 5

Mapping of critical LPS, IFN-α, and IL-27 response elements in IL-10 promoter by substitution mutant analysis. A, 6-nt mutants were constructed spanning across entirety of the regions −1541/−1232 and −595/−455 within the context of the −1954/+64 IL-10 promoter reporter construct. Constructs were transfected in RAW 264.7 cells and assessed for luciferase activity as described in B. Sequences of representative wild-type or substitution mutants are indicated. B, Empty pGL4.20, −1954/+64 IL10 promoter, and mutant IL-10 promoter constructs (−1954/+64 backbone) were transfected into RAW264.7 cells and left unstimulated or stimulated with LPS (1 µg/ml) (B), rIFN-α (1000 U/ml) (C), and rIL-27 (80 ng/ml) (D). Luciferase activity is displayed as percent of full-length wild-type (−1954/+64) IL-10 promoter activity normalized to renilla activity representing three independent experiments. Student t test was performed. *p < 0.05.

FIGURE 6

LPS, IFN-α, and IL-27 induction of IL-10 is STAT1 and STAT3 dependent. A, Total of 0.5 × 10⁶ WT BMDMs were unstimulated or stimulated with LPS (50 ng/ml), IFN-α (250 U/ml), or IL-27 (10 ng/ml) for indicated time points. Cell lysate was collected and immune-blotted for STAT1 and STAT3 expression and STAT1 and STAT3 phosphorylation using specific Abs. B, Total of 0.5 × 10⁶ WT and STAT1^−/− BMDMs were unstimulated or stimulated with LPS (50 ng/ml), IFN-α (250 U/ml), or IL- 27 (10 ng/ml) for 3 h. RNA was harvested, and relative IL-10 transcript was detected via qPCR normalized to L32. Student t test was performed. *p < 1 × 10³. C, Total of 0.5 × 10⁶ macrophage-specific STAT3^+/+ and STAT3^−/− BMDMs were unstimulated or stimulated with LPS (50 ng/ml), IFN-α (250 U/ml), or IL-27 (10 ng/ml) for 3 h. RNA was harvested, and relative IL-10 transcript was detected via qPCR normalized to L32. Data represent two independent experiments. *p < 0.05. D, ChIP using Abs against STAT1, STAT2, STAT3, c-Maf, and Sp1 using primers specific to the IL-10 promoter. Transcription factor enrichment presented as percent of input representing two independent experiments. E, Model of LPS-mediated induction of IL-10 gene expression involves sequential induction and signaling type I IFN followed induction and signaling by IL-27 signaling in macrophages.