3.3. Peripheral monocytes/macrophages promote Th17 differentiation in MPTP-induced mice.
Although depletion of monocyte-macrophages with clodronate liposomes reduced the number of Th17, and application of HMGB1 A Box also reduced the monocytes/macrophages in the SN and ultimately reduced the number of Th17, it cannot be confirmed in vivo whether microglia and monocytes/macrophages promote the proliferation, migration of Th17 or the differentiation and of naïve CD4+ T cells into Th17. Therefore, we took the midbrain of mice in the MPTP model group or MPTP+A Box group, stripped the meninges and blood vessels, and cultured them in vitro . Since adult mouse neurons are difficult to survive, each group was supplemented with midbrain neurons isolated from neonatal mice, and pre-stimulated mouse spleen naïve CD4+ T cells with anti-CD3 and anti-CD28 antibodies were added to this co-culture system (Fig.3A). Groups are: Control group (normal mouse midbrain cells + neonatal mouse midbrain neurons + naïve CD4+ T cells), MPTP group (MPTP model mouse midbrain cells + neonatal mouse midbrain neurons + naïve CD4+ T cells), MPTP-microglial group (MPTP model mice with microglia-depleted midbrain cells + neonatal mouse midbrain neurons + naïve CD4+ T cells), MPTP+A Box group (HMGB1 A Box intervened with MPTP mouse midbrain cells + neonatal mouse midbrain Neurons + naïve CD4+ T cells) and MPTP+A Box-microglia group (microglia-depleted midbrain cells from HMGB1 A Box treated MPTP mice + neonatal mouse midbrain neurons + naïve CD4+ T cells) (Fig.3B), since the addition of naïve CD4+ T cells, the entire co-culture system was maintained for 72h, and then all cells were collected and detected by FCM for CD45, CD11b, CD4, and IL-17A, as shown in the circle gate (Fig.3C). The results showed that the proportion of CD45hi CD11b+monocytes/macrophages in MPTP group was the highest, compared with that in MPTP+A Box group, CD45hiCD11b+monocytes/macrophages decreased significantly (Fig. 3F); Consistent with this, the proportion of Th17/CD4+ T cells in MPTP group was the highest, while that in MPTP + A Box group was significantly lower (Fig. 3D, G), indicating that monocytes/macrophages play an important role in promoting Th17 differentiation. In MPTP microglial group and MPTP+A Box microglial group, after removing microglia (Fig. 3E), the proportion of Th17/CD4+ T cells also decreased significantly (Fig. 3D,G), indicating that microglia can also promote Th17 differentiation in this system.