4. Discussion
HMGB1 is significantly up-regulated in PD model animals and patients, and its’ up-regulation is closely related to PD. The use of HMGB1 monoclonal antibody and a variety of compounds that inhibit HMGB1 have observed protective effects on TH+ neurons in PD model animals(Huh et al. , 2011; Santoro et al. , 2016; Sasakiet al. , 2016). Deficiency of HMGB1 downstream receptor TLR4 also protected TH+ neurons in a mouse model of MPTP (Campolo et al. , 2019). TLR4 are mainly expressed on the surface of microglia in the CNS and mediate the pro-inflammatory effects of glial cells (Rahimifard et al. , 2017). Among the three redox forms of HMGB1, only the dsHMGB1 has the ability to bind to TLRs (Venereau et al. , 2012; Yang et al. , 2010; Yang et al. , 2015). The evidence seems to suggest that in PD, dsHMGB1 is the HMGB1 that plays a deleterious role. However, due to frHMGB1 will always oxidize into dsHMGB1 in inflammatory state(Palmblad et al. , 2015), laboratory evidence for the role of frHMGB1 in PD is still few. Previously, it was shown that injection of frHMGB1 into the brain of healthy rats was sufficient to induce blood-brain barrier disruption and IL1-β production(Aucott et al. , 2018). In PD, the passive release of frHMGB1 in the nucleus of persistently injured neurons likely plays an important role in the negative effects of HMGB1. Given that knockdown of neuronal HMGB1 affects neuronal development in vivo , ourin vitro knockdown of midbrain neuron HMGB1 confirmed this.
Recently, Th17 cell subsets have revealed their deleterious effects in PD in both animal models and patient samples(Chandra et al. , 2016; Dutta et al. , 2019; Elgueta et al. , 2019; Reynoldset al. , 2010). We previously found that HMGB1 A Box has a surprising inhibitory effect on T cell infiltration and Th17 differentiation in addition to protecting TH+ neurons and inhibiting glial activation(Tian et al. , 2020). Although the stereotactic injection of exogenous frHMGB1 into the rat brain can’t increase the expression of phagocyte MHC Ⅱ like the rats injected with dsHMGB1, it is also enough to lead to the destruction of blood-brain barrier and the increase of CD68+ phagocytes(Aucottet al. , 2018). frHMGB1, passively released from the nucleus during dopaminergic neuron damage, may play a key role in the recruitment of peripheral immune cells.
Our results showed that CD45+ F4/80+peripherally derived macrophages were infiltrated in the SN of the MPTP mouse model and were significantly reduced after HMGB1 A Box application. Previously, significant nuclear-cytoplasmic HMGB1 translocations from neurons, astrocytes and microglia were observed in the PD mouse MPTP model(Santoro et al. , 2016). However, the roles of these different cell-derived HMGB1 on immune cells in the SN of PD model animals lacked precise definition. We found that in the MPP+ treated primary cell co-culture system, knockdown of HMGB1 in midbrain neurons had the most significant inhibitory effect on macrophage. This suggests that HMGB1 released by sustained injury of neurons plays a crucial role in the recruitment of peripheral monocyte/macrophages. The direct inhibitory effect of HMGB1 A Box on macrophage and T cell was confirmed by experiments with HMGB1 A Box-treated macrophages transwell experiment and transfusion of HMGB1 A Box-treated GFP+CD3+ T cells into wild type mice. Co-immunoprecipitation and immunofluorescence confirmed that HMGB1 A Box was able to bind to CXCR4 on the surface of these cells. Mice reinfused with CXCR4-neutralizing antibody-treated GFP+CD3+ T cells had significantly fewer GFP+ cells in the SN after 7 day-MPTP induction, although treatment with neutralizing antibody in vitro did not inhibit the CXCR4 expression of proliferated cells of GFP+CD3+ T cells in vivo . But it was also sufficient to confirm that the migration and infiltration of T cells into the SN was at least partially dependent on CXCR4. Ans in our previous study, we found that after the application of HMGB1 A Box, the levels of HMGB1 and CXCL12 were both reduced, and the infiltration of CD3+ T cells in the SN was almost disappear(Tianet al. , 2020). This also suggests that the HMGB1/CXCL12-CXCR4 axis plays an important role in T cell infiltration in PD.
Our previous study revealed the important role of microglia in the differentiation of Th17. In a pure in vitro co-culture system that excludes the influence of peripheral immune cells, naïve CD4+ T cells will not be able to differentiate into Th17 without the presence of microglia. This time, we used an in vivo model to confirm that peripheral monocytes also have a significant effect on Th17 differentiation in the SN in MPTP mouse model. Severalin vitro researches reported that the combination of CXCR4/CXCL12 induces the migration of macrophages, DCs and even microglia (Campanaet al. , 2009; Schiraldi et al. , 2012; Tanabe et al. , 1997). In contrast, CXCL12 needs to form a complex with HMGB1 to exert its strong migration-inducing effect(Schiraldi et al. , 2012). In this study, for the first time, we detected the presence of HMGB1/CXCL12 in an in vitro model of MPP+induced mouse midbrain cells, and also found evidence that HMGB1 A Box binds to the complex receptor CXCR4.
Previous studies reported an overall increase in CXCR4 and CXCL12 levels in the SN during PD(Shimoji et al. , 2009). In this study, we detected increased HMGB1 and CXCL12 in the serum of PD patients. Excitingly, we also found that the HMGB1/CXCL12 complex also exists in the serum of PD patients, and observed binding of CXCL12 and HMGB1 to CXCR4 on patients’ peripheral blood CD14+ monocytes and CD3+ T cells. Before, it is reported that CXCR4 was up-regulated in peripheral blood mononuclear cells of PD patients(Bagheri et al. , 2018). This proved that the HMGB1/CXCL12-CXCR4 axis related mechanism is also involved in human PD. In addition, in vitro experiments have long confirmed that CXCL12 induces microglial migration(Tanabe et al. , 1997), and recently, studies have confirmed that CXCR4 on the surface of microglia in the SN of A53T mice is also up-regulated(Li et al. , 2019). This suggests that when dopaminergic neurons are damaged, surrounding microglia may also aggregate to the injury site through the HMGB1/CXCL12-CXCR4 axis, but this hypothesis still needs more experimental evidence to confirm. Interestingly, we found that CXCL12 was significantly negatively correlated with the age of PD patients, suggesting that targeting the HMGB1/CXCL12-CXCR4 axis in PD could be more suitable for less old patients.
In conclusions, our results suggest that neuron-derived HMGB1 plays an important role in the recruitment of peripheral monocyte-macrophages and T cells in PD model mice, and peripheral monocytes/macrophages infiltration in the SN also affects Th17 infiltration and differentiation. The application of HMGB1 A Box significantly inhibited the infiltration of peripheral monocytes/macrophages and T cells in the SN by binding CXCR4 (Fig. 7). This effect may make this protein of great clinical value at least in the treatment of some PD classification, which is mainly characterized by neuroinflammation.