Klf10 inhibits IL-12p40 production in macrophage colony-stimulating factor-induced mouse bone marrow-derived macrophages

Abstract

Bone marrow-derived macrophages (BMMs) treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), differentiate into GM-CSF-induced mouse bone marrow-derived macrophages (GM-BMMs) or M-CSF-induced mouse bone marrow-derived macrophages (M-BMMs), which have an M1 or M2 profile, respectively. GM-BMMs produce large amounts of proinflammatory cytokines and mediate resistance to pathogens, whereas M-BMMs produce antiinflammatory cytokines that contribute to tissue repair and remodeling. M-BMMs stimulated with lipopolysaccharide (LPS) are in an antiinflammatory state, with an IL-12(low) IL-10(high) phenotype. However, the regulation of this process remains unclear. Klf10 belongs to the family of Krüppel-like transcription factors and was initially described as a TGF-β inducible early gene 1. IL-12p40 is upregulated in LPS-stimulated M-BMMs from Klf10-deficient mice, but downregulated during Klf10 overexpression. Klf11, another member of the Krüppel-like factor family, can also repress the production of IL-12p40. Furthermore, Klf10 binds to the CACCC element of the IL-12p40 promoter and inhibits its transcription. We have therefore identified Klf10 as a transcription factor that regulates the expression of IL-12p40 in M-BMMs.

Figure 1

Downregulation of Klf10 expression upon TLR4 activation by LPS in M-BMMs. (A) The mRNA levels of KLFs in M-BMMs. M-BMMs on day 5 were harvested and the gene expression levels of KLFs were assessed by qPCR. The actin mRNA level in these cells was set as 1. (B) Response of KLFs to LPS stimulation in M-BMMs. M-BMMs on day 5 were treated with 1 μg/mL LPS for the indicated time, then gene expression levels were assessed by qPCR and normalized to those in untreated cells. (C) Western blot analysis of Klf10 in M-BMMs treated with 1 μg/mL LPS for the indicated time. β-Actin was used as a loading control. Data shown are representative of three experiments performed. (D) qPCR analysis of Klf10 in M-BMMs treated with graded concentrations of LPS. (E) The role of the NF-κB pathway in the downregulation of Klf10 in LPS activated M-BMMs. M-BMMs on day 5 were pretreated with 5 μM BAY 11–7082 or without pretreatment, and then stimulated with 100 ng/mL LPS for the indicated time. Cells were harvested for qPCR analysis. (A–E) Data are shown as mean ± SD of three samples pooled from three independent experiments. **p < 0.01, determined by Student’s t-test.

Figure 2

Klf10 overexpression facilitates a reduction in the LPS-induced production of IL-12p40 and IL-6 in M-BMMs. (A and B) M-BMMs on day 5 were transfected with pcDNA3-Klf10 or empty vector by using the Amaxa mouse macrophage Nucleofector kit. The cells were cultured for 24 h, stimulated with 1 μg/mL LPS, and then harvested at the indicated times. Relative mRNA expression of (A) IL-12p40, IL-12p35, IL-6, and (B) TNF-α, and IL-10 was measured by qPCR and normalized to levels in untreated cells (left). Supernatants from the cultures were collected, and the levels of (A) IL-12p40, IL-12p70, IL-6, and (B) TNF-α and IL-10 were evaluated by ELISA (right). Data are shown as mean ± SD of three samples pooled from three independent experiments. *p < 0.05, **p < 0.01; determined by Student’s t-test.

Figure 3

Loss of Klf10 promotes the production of IL-12p40, IL-6, and NO in M-BMMs. (A and B) M-BMMs on day 5 from WT or Klf10-deficient mouse were stimulated with 1 μg/mL LPS. Cells were harvested at the indicated times. Relative mRNA expression of (A) IL-12p40, IL-12p35, IL-6, and (B) TNF-α and IL-10 were measured by qPCR and normalized to levels in untreated cells (left). Supernatants from the cultures were collected, and the levels of (A) IL-12p40, IL-12p70, IL-6, and (B) TNF-α and IL-10 were evaluated by ELISA (right). (C) NO levels in the supernatants were determined via the Griess reaction. Data are shown as mean ± SD of three samples pooled from three independent experiments. *p < 0.05, **p < 0.01; determined by Student’s t-test.

Figure 4

Different functions of Klf10 in M-BMMs and GM-BMMs. (A) Different expression patterns of IL-12p40, IL-6, and IL-10 in GM-BMMs and M-BMMs. Bone marrow cells from WT or Klf10-deficient mouse were cultured with either 20 ng/mL GM-CSF or 10 ng/mL M-CSF for macrophage polarization. On day 5, cells were stimulated with 1 μg/mL LPS. Cells were harvested at the indicated time, and the relative mRNA expression levels of IL-12p40, IL-6, and IL-10 were measured by qPCR and normalized to levels in untreated cells. (B) GM-BMMs on day 5 were pretreated with 2.5 × 10⁻⁷ M dexamethasone, 2 ng/mL TGF-β, or 10 ng/mL IL-10 for 16 h for deactivation, and then stimulated with 1 μg/mL LPS. Cells were harvested at the indicated time, and the relative mRNA expression levels of IL-12p40, IL-6, and IL-10 were measured by qPCR and normalized to those in untreated cells. (C) The expression of Klf10 in GM-BMMs from WT mouse in (B) was measured by qPCR. Data are shown as mean + SD of three samples pooled from three independent experiments. *p < 0.05, **p < 0.01; determined by Student’s t-test.

Figure 5

Similar function of Klf11 and Klf10 in the regulation of IL-12p40 expression.(A) Overexpression of Klf10 or Klf11 reduced the LPS-induced production of IL-12 p40 and IL-6 in M-BMMs from WT and Klf10-deficient mice. M-BMMs on day 5 were transfected with pcDNA3-Klf10, pcDNA3-Klf11, or empty vector (Mock), cultured for 24 h and then stimulated with 1 μg/mL LPS. Cells were harvested at the indicated times, and relative mRNA expressions of IL-12p40 and IL-6 was measured by qPCR and normalized to levels in untreated cells (left). Supernatants from the cultures were collected and the levels of IL-12p40 and IL-6 were evaluated by ELISA (right). (B) Silencing of Klf11 expression promoted the production of LPS-induced IL-12p40 in M-BMMs from WT and Klf10-deficient mice. On day 5, M-BMMs were transfected with Klf11 SiRNA or control SiRNA, cultured for 36 h, and then stimulated with 1 μg/mL LPS. Cells were harvested at the indicated times, and relative mRNA expression of IL-12p40 and IL-6 was measured by qPCR and normalized to levels in untreated cells (left). Supernatants from the cultures were collected, and the levels of IL-12p40 and IL-6 were evaluated by ELISA (right). Data are shown as mean ± SD of three samples pooled from three independent experiments. *p < 0.05, **p < 0.01, determined by Student’s t-test.

Figure 6

Klf10 binds to the promoter of IL12p40 and inhibits its transcriptional activity. (A) Putative and verified transcription factor-binding sites in the mouse IL-12p40 promoter region. The core-binding sequences are shown in bold. The positions are relative to the transcription initiation site (TIS) that is defined as +1. (B) High conservation of the CACCC-binding site in IL-12p40 promoter in mammals. (C) Klf10 inhibits the luciferase activity of the IL-12p40 promoter. HEK 293 cells were infected for 48 h with a WT IL-12p40 luciferase reporter (top), an IL-12p40 luciferase reporter with 2 bp mutations in the CACCC site at positions −233 to −229 (middle), or an IL-12p40 luciferase reporter that lack the CACCC-binding domain (bottom), plus a construct that encodes Klf10 or empty vector (mock), and then cells were harvested and analyzed for luciferase activity. (D–E) PCR and qPCR assays of the recruitment of Klf10 proteins to the IL-12p40 promoter. On day 5, M-BMMs were prepared and subjected to ChIP with antibody to IgG or Klf10. The immunoprecipitated DNA were analyzed by PCR and qPCR. The qPCR results were presented relative to those obtained with genomic DNA (input). Results shown are (D) representative of three independent experiments or (C, E) presented as mean + SD of three samples pooled from three independent experiments. *p < 0.05; determined by Student’s t-test.