Syk negatively regulates TLR4-mediated IFNβ and IL-10 production and promotes inflammatory responses in dendritic cells

Abstract

Background: While Syk has been shown to associate with TLR4, the immune consequences of Syk-TLR interactions and related molecular mechanisms are unclear.

Methods: Gain- and loss-of-function approaches were utilized to determine the regulatory function of Syk and elucidate the related molecular mechanisms in TLR4-mediated inflammatory responses. Cytokine production was measured by ELISA and phosphorylation of signaling molecules determined by Western blotting.

Results: Syk deficiency in murine dendritic cells resulted in the enhancement of LPS-induced IFNβ and IL-10 but suppression of pro-inflammatory cytokines (TNFα, IL-6). Deficiency of Syk enhanced the activity of PI3K and elevated the phosphorylation of PI3K and Akt, which in turn, lead to the phospho-inactivation of the downstream, central gatekeeper of the innate response, GSK3β. Inhibition of PI3K or Akt abrogated the ability of Syk deficiency to enhance IFNβ and IL-10 in Syk deficient cells, confirmed by the overexpression of Akt (Myr-Akt) or constitutively active GSK3β (GSK3 S9A). Moreover, neither inhibition of PI3K-Akt signaling nor neutralization of de novo synthesized IFNβ could rescue TNFα and IL-6 production in LPS-stimulated Syk deficient cells. Syk deficiency resulted in decreased phosphorylation of IKKβ and the NF-κB p65 subunit, further suggesting a divergent influence of Syk on pro- and anti-inflammatory TLR responses.

Conclusions: Syk negatively regulates TLR4-mediated production of IFNβ and IL-10 and promotes inflammatory responses in dendritic cells through divergent regulation of downstream PI3K-Akt and NF-κB signaling pathways.

General significance: Syk may represent a novel target for manipulating the direction or intensity of the innate response, depending on clinical necessity.

Keywords: GSK3β; IL-10; Interferon-β; PI3K; Syk; Toll-like receptor 4.

Figure 1. Expression of Syk and its influences on the maturation of dendritic cells

(A)Western blot of the whole cell lysates from wild type and Cre-loxP-mediated Syk deficient dendritic cells. Blots were probed with antibodies to total-Syk and GAPDH as a loading control; (B) Densitometric quantification of the ratio of total-Syk to total-GAPDH; (C) WT and Syk deficient cells were stimulated with LPS (1 μg/ml) for 24 hours, and the expression of CD80 and CD86 was detected by flow cytometry; (D) Cell viability was determined by trypan blue exclusion.

Figure 2. Deficiency of Syk elevates the production of IFNβ and IL-10, but suppresses the levels of TNFα and IL-6 in LPS-stimulated dendritic cells

Cell-free supernatants were collected from wild type and Syk deficient dendritic cells stimulated with LPS for 24 h, and the production of (A) IFNβ, (C) IL-10, (D) TNFα, and (E) IL-6 was determined by ELISA. The mRNA levels of IFNβ (B) and other cytokines (F) from wild type and Syk deficient cells stimulated with LPS (1 μg/ml) for the time indicated were examined by RT- PCR; TLR3 (Poly I:C LMW; 1 μg/ml) and TLR9 (ODN1826; 10μg/ml) agonists were used to stimulate wild type and Syk deficient BMDCs for 24 h, and the production of IFNβ (G) and other cytokines (H) measured by ELISA, “*” and “***” indicates P<0.05 and P<0.001, respectively. Data represents the mean ± S.D. of three biological replicates.

Figure 3. Deficiency of Syk elevated IFNβ production and leads to the enhancement of STATs phosphorylation

Wild type and Syk deficiency cells were pretreated with IFNβ neutralizing antibody or isotype control for 2 h and then stimulated with LPS. Cell lysates were prepared at the indicated time points and then analyzed by Western blot. (A) Blots were probed with antibodies to phospho-STATs and GAPDH as a loading control; (B) Densitometric quantification of the ratio of phospho-STATs to total-GAPDH.

Figure 4. Syk deficiency enhances PI3-kinase activity and phosphorylation of downstream molecules, Akt and GSK3β in LPS-stimulated cells

(A) Western blots of whole cell lysates from LPS-stimulated wild type and Syk deficient cells. Blots were probed with antibodies to phospho-p85 and total GAPDH as a loading control; (B) Densitometric quantification of the ratio of phospho-p85 to total-GAPDH; (C) Wild type and Syk deficient cells were stimulated for 30 min and 1 h with 1 μg/ml LPS. Subsequently, PI3K was immunoprecipitated from cell lysates and enzymatic activity was assessed. Data are presented as the mean ±SD from three biological replicates. Syk deficiency resulted in the significant (***, p<0.001) enhancement of PIP3 generation where indicated. (D) Blots in (A) were also probed with antibodies to phospho-Akt, GSK3β, and total GAPDH as a loading control; (E) Densitometric quantification of the ratio of phospho- Akt, -GSK3β to total-GAPDH.

Figure 5. Inhibition of PI3K or Akt abrogates the regulatory effects of Syk deficiency on TLR4-mediated IFNβ and IL-10 production

(A, B) Syk deficiency cells were pre-treated with LY294002 (25 μM) or ZSTK474 (50 nM) and stimulated with LPS (1 μg/ml). Cell lysates were collected at the time points indicated and phosphorylation of Akt were detected by Western blot. (C, D) Densitometric quantification of the ratio of phospho-Akt to total-GAPDH. (E, F) Wild type and Syk deficient cells were pretreated with PI3K inhibitor, LY294002 or ZSTK474, or Akt inhibitor, for 2 h and then stimulated with LPS for 24 h. Cell free supernatants were collected and LPS-induced production of IFNβ (E) and IL-10 (F) was determined by ELISA. “***” indicates P<0.001. Data represent the mean ± S.D. of three biological replicates.

Figure 6. Syk deficiency-mediated elevated IFNβ and IL-10 production is dependent on the activity of Akt-GSK3β signaling

Syk-deficient cells were transfected with Myr-Akt or pcDNA3-GSK3β (S9A) plasmid which encode constitutively active Akt and GSK3β, respectively. Empty vectors were also transfected into wild type and Syk-deficient cells as a control. After 48 h transfection, cells were stimulated with LPS (1 μg/ml) for 6 h and cells lysates were collected for Western blot analysis. (A) Blot was probed with antibodies to total Akt and GAPDH to ensure equivalent sample loading. (B) After 48 h, transfected cells were stimulated with LPS for 24 h, and then the cell free supernatants were harvested to detect the production of IFNβ and IL-10. (C) Western blot of cell lysates from Syk deficient BMDCs pretreated with PI3K inhibitor, LY294002, for 2 h, then stimulated with LPS to 2 h, the blot was probed with antibodies to phospho-and total GSK3β, densitometric quantification of the ratio of phospho- to total-GSK3β was also calculated as shown. (D) For GSK3β transfection, phosphorylation levels of glycogen synthase (GS), a specific substrate of GSK3β, and the expression level of Hemagglutinin (HA) tag protein, encoded by the HA DNA sequence inserted at the C terminal of GSK3β mutant, were detected by Western blot to assess the transfection efficiency. (E) ELISA of LPS-induced IFNβ and IL-10 in cell-free supernatant in the absence and presence of SB216763 and constitutively active GSK3β. For B and E, data are representative of at least three separate experiments. “*” and “***” indicate p<0.05, and p<0.001 respectively. Data represent the mean ± S.D. of three biological replicates.

Figure 7. Syk regulation of TNFα and IL-6 was not affected by the activity of PI3K signaling and the de novo production of IFNβ

Syk-deficient BMDCs were pre-treated with LY294002, IFNβ or IL-10 neutralizing antibody, or isotype control for 2 h, then stimulated with LPS (1 μg/ml) for 24 h. Cell free supernatants were collected to determine the production of TNFα (A, C) and IL-6 (B, D) by ELISA. “*” represents p<0.05. Data are representative of at least three separate experiments.

Figure 8. Syk deficiency reduces phosphorylation of IKKβ and NF-κB p65 but not phosphorylation of MAPK signaling

Cell lysates were collected from LPS-stimulated wild type and Syk-deficient BMDCs at the time points indicated for Western blot analysis. Blots were probed with the antibodies to phospho-and total IKKβ, NF-κBp65 (A, B), P38, ERK (C, D), and GAPDH as a loading control. (B, D) Densitometric quantification of the ratio of phospho- to total proteins.