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).