Abstract
Programmed death ligand-1 (PD-L1) is a critical regulator of T cell function contributing to peripheral immune tolerance. Although it has been shown that posttranscriptional regulatory mechanisms control PD-L1 expression in cancer, it remains unknown whether such regulatory loops operate also in non-transformed cells. Here we studied PD-L1 expression in human dermal lymphatic endothelial cells (HDLECs), which play key roles in immunity and cancer. Treatment of HDLECs with the pro-inflammatory cytokines IFN-γ and TNF-α synergistically up-regulated PD-L1 expression. IFN-γ and TNF-α also affected expression of several microRNAs (miRNAs) that have the potential to suppress PD-L1 expression. The most highly up-regulated miRNA following IFN-γ and TNF-α treatment in HDLECs was miR-155, which has a central role in the immune system and cancer. Induction of miR-155 was driven by TNF-α, the effect of which was significantly enhanced by IFN-γ. The PD-L1 3′-UTR contains two functional miR-155-binding sites. Endogenous miR-155 controlled the kinetics and maximal levels of PD-L1 induction upon IFN-γ and TNF-α treatments. We obtained similar findings in dermal fibroblasts, demonstrating that the IFN-γ/TNF-α/miR-155/PD-L1 pathway is not restricted to HDLECs. These results reveal miR-155 as a critical component of an inflammation-induced regulatory loop controlling PD-L1 expression in primary cells.
Highlights
Programmed death ligand-1 (PD-L1) is a critical regulator of T cell function contributing to peripheral immune tolerance
Inhibition of miR-155 led to an increase of PD-L1 expression (Fig. 6G). miR-155 inhibition did not result in a statistically significant effect on PD-L1 mRNA under these conditions (Fig. 6H). These results indicate that miR-155-mediated suppression of PD-L1 is not specific to human dermal lymphatic endothelial cells (HDLECs) and could be observed in other dermal primary cells responding to inflammatory stimuli
We reveal that in addition to promoting PD-L1 expression TNF-␣ and IFN-␥ concurrently lead to induction of PD-L1targeting miRNAs during a physiological immune response (Fig. 7)
Summary
PD-L1 is expressed in LECs and can be synergistically induced by IFN-␥ and TNF-␣. Human macrovascular endothelial cells become activated by pro-inflammatory cytokines IFN-␥ and TNF-␣ and display increased PD-L1 expression [5, 22]. Small RNA sequencing of IFN-␥ and TNF-␣-stimulated LECs reveal inflammation-responsive miRNAs. Having shown that PD-L1 is inducible in HDLECs responding to inflammatory stimuli, we reasoned that this was an appropriate cellular model for identifying posttranscriptional PD-L1 regulators during inflammatory responses of primary human cells. Expression of STAT1 was increased in IFN-␥- and TNF-␣-stimulated cells and decreased in untreated cells but resulted overall in no change in phosphorylation of STAT1 relative to total STAT1 (Fig. 5A and supplemental Fig. S4A). Expression of PD-L1 at all three time points was increased following inhibition of miR-155 compared with control This demonstrated that lack of miR-155 could affect the onset and maximum levels of PD-L1 expression upon IFN-␥ and TNF-␣ treatments. HDLECs and could be observed in other dermal primary cells responding to inflammatory stimuli
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