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Abstract
PD-L1 is expressed in a variety of antigen-presenting cells and provides T cell tolerance via ligation with its receptor PD-1 and B7-1 on T cells. Stimulation with lipopolysaccharide (LPS) can increase the level of PD-L1 expression in B cells and macrophages, which suggests that this molecule plays a role in the immunosuppression observed in severe sepsis. The aim of this study was to identify which of the downstream pathways of TLR4 are involved in the up-regulation of PD-L1 by LPS in macrophages. Flow cytometry was used to examine the expression of PD-L1 in RAW 264.7 macrophages stimulated with LPS. The following chemical inhibitors were used to evaluate the role of each pathway: LY294002 for PI3K/Akt, SB202190 for p38 MAPK, and U0126 for MEK. LPS induced the expression of PD-L1 in a time- and dose-dependent manner. Transfection of siRNA for TLR4 suppressed the induction of PD-L1. Pretreatment with LY294002 and SB202190 decreased the level of PD-L1 expression but U0126 did not. Overall, the PI3K/Akt and p38 MAPK pathways are involved in the up-regulation of PD-L1 expression in RAW 264.7 macrophages stimulated with LPS.
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Figure 1
PD-L1 is up-regulated by LPS in mouse macrophage cell line. RAW 264.7 cells were treated with LPS (1.0 µg/ml) for 24 hrs. Surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI (mean fluorescence intensity). Number: mean fluorscence intensity (MFI). Gray: isotype control, Black: PD-L1.
Figure 2
PD-L1 is up-regulated by LPS in time-dependent & concentration-dependent manner. RAW 264.7 cells were treated with LPS (1.0 µg/ml) for the indicated times (A) and with the indicated concentrations of LPS for 24 hrs (B). Surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI.
Figure 3
Blocking of TLR4 with siRNA suppress LPS-induced PD-L1 expression. RAW 264.7 cells were transfected with control siRNA and siRNA for TLR4. The levels of TLR4 in cellular extracts were detected by Western blot analysis (A). After cells were treated with LPS (1.0 µg/ml) for 24 hrs, surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI (B).
Figure 4
PI3K/Akt pathway is involved in up-regulation of PD-L1 by LPS. RAW 264.7 cells were pretreated with LY294002 (50 µM) for 2 hrs and then stimulated with LPS for 24 hrs. Surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI. Cell viability by MTT assay: > 90% of vehicle control.
Figure 5
MEK/ERK pathway has no role in up-regulation of PD-L1 by LPS. RAW 264.7 cells were pretreated with U0126 (10 µM) for 2 hrs and then stimulated with LPS for 24 hrs. Surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI. Cell viability by MTT assay: > 90% of vehicle control.
Figure 6
p38 MAPK pathway is involved in up-regulation of PD-L1 by LPS. RAW 264.7 cells were pretreated with SB202190 (10 µM) for 2 hrs and then stimulated with LPS for 24 hrs. Surface expressions of PD-L1 were measured by flow cytometry and expressed via MFI. Cell viability by MTT assay: > 90% of vehicle control.
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