Differential induction of PD-1/PD-L1 in Neuroimmune cells by drug of abuse

Abstract

Interaction of programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) plays a critical role in regulating the delicate balance between protective immunity and tolerance. Human neuroimmune cells express very low or undetectable levels of PD-1/PD-L1 in normal physiological condition.We seek to examine if exposure of these cells to drug of abuse such as methamphetamine (METH) alters the profile of PD-1/PD-L1 levels, thereby dampens the innate immune response of the host cells. Thus, we assessed the changes in the levels of PD-1/PD-L1 in primary human macrophages, brain endothelial cells (hBECs), astrocytes, microglia, and neurons after exposure to METH. We observed that stimulation of these neuroimmune cells by METH responded differentially to PD-1/PD-L1 expression. Stimulation of macrophage culture with 50 μM of METH exhibited immediate gradual upregulation of PD-L1, while increase in PD-1 took 2-4 hours later than PD-L1. The response of hBECs to PD-1/PD-L1 induction occurred at 24 hours, while increase of PD-1/PD-L1 levels in neurons and microglia was immediate following METH exposure. We found that astrocytes expressed moderate levels of endogenous PD-1/PD-L1, which was diminished by METH exposure. Our findings show a differential expression of PD-1/PD-L1 in neuroimmune cells in response to METH stimulation, suggesting that PD-1/PD-L1 interplay in these cell types could orchestrate the intercellular interactive communication for neuronal death or protection in the brain environment.

Keywords: Methamphetamine; neurodegeneration; neuroimmune cells; neuroinflammation; programmed cell death 1.

Figure 1

METH exposure up-regulated PD-L1 and PD-1 levels in human macrophages. Representative immunocytochemical staining of (A) PD-L1 and (B) PD-1, in human macrophages with/without METH at different time points; Ranks-Kruskal-Wallis test for quantitative densitometric analysis of (C) PD-L1 and (D) PD-1 levels. *P≤0.05, **P≤0.01, ***P≤0.001 and ****P≤0.0001 are the statistical significance compared with controls.

Figure 2

A delayed increase of PD-L1 and PD-1 in hBECs. Representative immunocytochemical staining of (A) PD-L1 and (B) PD-1, in hBECs with/without METH at 24 hrs and 48 hrs; Ranks-Kruskal-Wallis test for quantitative densitometric analysis of (C) PD-L1 and (D) PD-1 levels. **P≤0.01 is the statistical significance compared with controls.

Figure 3

METH down-regulated PD-L1 and PD-1 levels in human astrocytes. Representative immunocytochemical staining of (A) PD-L1 and (B) PD-1, in human astrocytes with/without METH at 24 hrs and 48 hrs; Ranks-Kruskal-Wallis test for quantitative densitometric analysis of (C) PD-L1 and (D) PD-1 levels. **P≤0.01 and ***P≤0.001 are the statistical significance compared with controls.

Figure 4

METH exposure elevated the expression of PD-L1 and PD-1 in human microglia. Representative immunocytochemical staining of (A) PD-L1 and (B) PD-1, in human microglia with/without METH at 24 hrs and 48 hrs; Ranks-Kruskal-Wallis test for quantitative densitometric analysis of (C) PD-L1 and (D) PD-1 levels. **P≤0.01 and ****P≤0.0001 are the statistical significance compared with controls.

Figure 5

METH up-regulated the expression of PD-L1 and PD-1 in human neurons. Representative immunocytochemical staining of (A) PD-L1 and (B) PD-1, in human neurons with/without METH at 24 hrs and 48 hrs; Ranks-Kruskal-Wallis test for quantitative densitometric analysis of (C) PD-L1 and (D) PD-1 levels. **P≤0.01 and ****P≤0.0001 are the statistical significance compared with controls.