In this study, PD-1 knockout mice and MPTP model of Parkinson’s disease, motor behavior detection, immunohistochemistry and western blot were used to observe and analyze role of PD-1 signaling in brain glial activation, inflammation response and movement dysfunction of Parkinson’ animals. It revealed that PD-1/Iba1 double-positive microglial cells numerously distributed in control, whereas they were significantly reduced in the MPTP model. Compared with WT-MPTP, KO-MPTP group mice showed significant increases in motor dysfunction, decreased expression level of TH protein and decrease in TH-positive neuronal protrusions, accompanied by activation of microglial cells and astrocytes, increased expressional levels of proinflammatory cytokine iNOS, TNF-α, IL-1β and IL-6. Further detection showed that PD-1 knockout induced elevated expression and phosphorylation activation of AKT and ERK1/2. The results indicated that PD-1 knockout aggravated animal motor dysfunction of PD model, possibly by promoting microglial cell activation, increasing expression of proinflammatory cytokines and triggering AKT and ERK1/2 signaling in the substantia nigra, suggesting that PD-1 signaling abnormality may be involved in micrioglial activation and neuroinflammation response in Parkinson's disease progression.
Many studies showed that abnormal activation of microglial cells and astrocytes in the CNS dominated neuroinflammatory reaction, which might be a critical factor leading to the development of various neurodegenerative diseases such as AD, MS and PD (4–8). In the state of inflammatory reaction, microglial cells functioned as CNS inherent immune cells, activated microglial cells initiated differentiation in two opposite directions, namely, inflammatory state (M1), anti-inflammatory state (M2) functional polarization (29). On the one hand, microglial cells in M2 polarization could remove pathogens or cell fragments and protect from damage to the brains. On the other hand, microglial cell with M1 polarization was in an inflammatory state and could produce a series of cytokines, and thereby further activating astrocytes, being collaborative inflammatory response. The astrocytes regulated the immune response and react to pathological changes by hypertrophy, presented as functional activated state, the activated astrocytes also occurred in similar functional differentiation, that is, inflammatory state (A1) and anti-inflammatory state (A2) polarization, activated A2 astrocytes promoted tissue repair and help maintain function of the central neurons. The activated A1 astrocytes secreted a large number reactive oxygen and pro-inflammatory cytokines, affecting neurons and other glial function, triggering a vicious circle, exacerbating the biological process of amplification of inflammatory reactions and neuronal damage in the CNS (30, 31).
It was known that as an important inhibitory immune checkpoint, PD-1 is the regulating molecule of immune cell function (32). Data showed that PD-1 and PD-L1 expression levels changes with healthy and pathological state in the CNS, for example, 20% of microglial cells expressed PD-L1 in uninfected normal mice, while more than 90% of microglial cells showed induced PD-L1 expression 1week after infection (24, 25). Stimulation of interferon-gama (IFN-γ), autoimmune diseases, brain tumors and stroke state induced central T-cell activation and PD-1 production. Increased PD-L1 binding and activation of PD-1 signaling pathway regulated tumor microenvironment and inflammatory response, and thus affected progression of above diseases (33). Some studies indicated that PD1/PD-L1 signaling activation promoted differentiation of microglial cells into anti-inflammatory states (M2) and reduced secondary brain damage in the cerebral hemorrhage (34, 35). In addition, many studies focusing on brain tumors showed that tumor cells secreted high level of PD-L1, thereafter induced T cells to produce high level of PD-1 molecule. Combination of PD-1/PD-L1 or PD-1 signaling activation caused decrease or "failure" state of T-cell function, resulting in the migration and diffusion of tumor cells. As a targeted anti-tumor strategy, therefore, PD-1-based inhibitors (PD1 antibody) have attracted more and more attention and successfully used in clinical.
Whether PD-1 signaling pathway was involved in glial cell activation and inflammation of Parkinson's disease, which in turn affected course of the disease, was major concern of this study. Present results showed that the absence state of PD-1 leaded to more obvious glial activation in the MPTP model, increased the expression level of inflammatory factors, and aggravated motor dysfunction, which indicated that PD-1 had a certain restrictive effect on neuroinflammation dominated by activated glial cells, and played a neuroprotective role by regulating or limiting inflammatory response. Our result was supported by studies of Yao and other researchers. Yao and other applications used PD-1 knockout and spinal cord injury model, reveal that PD-1 knockout promoted direction of microglial cells and macrophage M1 polarization, and aggravated inflammatory response and neuronal damage (29). Contrary to these observations, Bodhankar used stroke models and found that PD-L1 deficiency improved infarction volume, reduced inflamed cells and inflammatory responses, and improved nerve function (36). It was not clear what causes this difference of study, and it might be related to differences in application of distinct animal models. Other scholars also reported that PD-1 signaling showed a protective role in persistent viral encephalitis. PD-1 signaling activation limited severity of inflammation during acute infection while it maintained a moderate inflammatory response during persistent infection and conduced to resistance to viral re-infection (37, 38). It remained a question that if the PD-1 signaling pathway exhibited a "double-edged sword" effect in the different CNS diseases or different state of disease. In addition, it was noticed that PD-1 activation played a protective role by limiting inflammatory response by glial inhibition of T-cell function (39), which was worthy of in-depth study. Further clarification of mechanism of PD-1/PD-L1 signaling pathway in glial cell activation and neuroinflammation shall be helpful to identify new drug intervention targets or establish new Parkinson's disease interventional treatment strategies.