In this study, PD-1 knockout mice and MPTP model of Parkinson’s disease (PD) were used to observe effects of PD-1-deficency on motor dysfunction of animals, glial cell reaction, and neuroinflammation in the substantia nigra. It revealed that PD-1/Iba1 double-immunopositive microglias reduced in WT-MPTP mice compared with that of WT-CON ones. Compared with WT-MPTP group, KO-MPTP mice showed increases in the motor dysfunction, decreased expression of TH protein and TH-positive neuronal protrusions, which was accompanied by microglial and astroglial activation, increased expression of proinflammatory cytokine iNOS, TNF-α, IL-1β and IL-6. Further signaling detection showed that PD-1-KO mice induced elevated expression and phosphorylation activation of AKT and ERK1/2. Results of this study together indicated that PD-1 deficiency could aggravate motor dysfunction of PD animal model, possibly by promoting microglial cell reaction, increasing proinflammatory cytokine generation and triggering AKT and ERK1/2 signaling activation in the midbrains, suggesting that PD-1 signaling abnormality might be most possibly involved in microglial activation and neuroinflammation in PD pathogenesis and 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 (M1 state), anti-inflammatory (M2 state) 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 (34, 35).
It was known that as an important inhibitory immune checkpoint, PD-1 is the regulating molecule of immune cell function (36). Studies showed that PD-1 and PD-L1 expression levels changes along with healthy or pathological states in the CNS, for example, 20% of microglial cells expressed PD-L1 in uninfected healthy 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 (37). 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 (38, 39). In addition, many studies focusing on brain tumors showed that tumor cells secreted high level of PD-L1, which thereafter induced T-lymphocytes to produce high level of PD-1 molecule and exact biological effects. 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 PD, which in turn affected course of disease, was major concerning question of this study. Present results showed that PD-1 deficiency state leaded to more obvious glial activation in MPTP mouse model, increased the expression level of inflammatory factors, and aggravated motor dysfunction of animals, 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 (40). It was not clear what causes difference of studies, which might be related to differences in application of 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 (41, 42). It remained a question whether of PD-1 signaling activation might produce a "double-edged sword" effects in different CNS diseases or distinct stages of disease. It was also evidenced that PD-1 showed a protective role by limiting inflammatory strength through glial inhibition of T-lymphocyte function (43). It was notice that, in addition, we still detected positive-band of PD-1 by western blot, indicating that PD-1 antibody showed cross-reaction in PD-1 KO mice used, and PD-1-KO information showed knockout of extracellular domains in this strain of PD-1 KO mice and this might only make PD-1 molecule lose functional binding of ligands instead of whole deletion of PD-1 expression. Nevertheless, further studies on mechanism of PD-1/PD-L1 signaling in abundant glial reaction and neuroinflammation in the CNS shall be helpfully expected to identify new intervention targets for manipulation of neuroinflammatory balance and to develop new strategies against PD pathogenesis and progression (2, 4, 44).