3.2. HMGB1 A Box reduces Th17 and protects TH+neurons by inhibiting monocytes/macrophages infiltration.
Clodronate liposome depleted peripheral monocyte/macrophage mice showed more TH+ neurons in the SN compared to MPTP induced mice, and at the protein level, this protective effect was similar to that of HMGB1 A Box (Fig. 2A, B). Subsequently, we performed flow cytometry on the above-mentioned mouse SN, and the gating strategy is shown in the figure (Fig. 2C). Injections of HMGB1 A Box and clodronate liposomes did not affect the proportion of CD45intCD11b+ microglia in the SN (Fig. 2D), but compared with the MPTP group, HMGB1 A Box and clodronate liposome injected mice showed fewer CD45hiCD11b+ cells in the SN (Fig. 2E). In contrast, clodronate liposome injected mice did not affect the proportion of CD45hiCD11c+ DCs in the SN compared with MPTP mice, although they were all elevated relative to the Control group (Fig. 2F). Among the three APCs detected in SN, microglia accounted for the highest proportion, followed by macrophages, while DCs had the significantly lowest (Fig. 2D, E and F). This suggests that the cells that mainly present antigens to T cells in PD model mice are less likely to be DCs.
We also detected the MHC II levels of these three APCs to assess their potential antigen-presenting capacity. There was no significant difference in the expression levels of MHC II among the above three cells in the SN of mice between each treatment group, suggesting that our previous finding that HMGB1 A Box inhibits Th17 levels in the SN of PD model mice may be irrelevant with affecting the antigen presentation ability of APCs in the SN (Fig. 2G, H and I). Comparing the differences between the three APCs, it can be seen that DCs have the highest expression levels of MHC II, microglia the lowest, and monocytes/macrophages have intermediate levels of MHC II expression (Fig. 2G, H and I). In the SN, Th2 and Th17 were elevated in the MPTP group, and both population-level inhibition was shown in both HMGB1 A Box and clodronate liposome injected mice, and Th17 was the main subset among the three Th cell subsets Th1, Th2, and Th17 (Fig. 2J, K and L).