3.4. HMGB1 A Box binds CXCR4 to inhibit HMGB1/CXCL12
complex-mediated infiltration of monocytes/macrophages and T cells into
the SN.
Mario Tirone et al . previously reported that the fully reduced
form of HMGB1 (frHMGB1) binds CXCR4 of myofibroblasts to induce their
migration and promote tissue repair(Tirone et al. , 2018).In vitro, cell experiments by Milena Schiraldi et al .
confirmed that the complex formed by HMGB1 and CXCL12 significantly
induced the migration of human monocytes(Schiraldi et al. , 2012).
Given that monocytes/macrophages infiltration in SN was significantly
inhibited by HMGB1 A Box in our study, it is possible that HMGB1 A Box
inhibited monocytes/macrophages migration by targeting this pathway.
Therefore, intact midbrain cells from newborn C57BL/6J mice were
isolated and co-cultured with peritoneal macrophages in a transwell
system, and the results showed that MPP+-treated mixed
cultures of midbrain cells caused the greatest degree of macrophage
migration, whereas HMGB1 A Box, HMGB1 neutralizing antibody, CXCR4
neutralizing antibody, or AMD3100-treated cultures all showed
significantly less migration of macrophages. This suggests an important
role for HMGB1, CXCL12 and CXCR4 in the
MPTP/MPP+-based PD model (Fig.4 A, B).
Compared with CXCL12 alone, the chemotaxis of HMGB1 complexed with
CXCL12 on CXCR4-transfected cells was enhanced more than a
hundred-fold(Schiraldi et al. , 2012). HMGB1/CXCL12-CXCR4 may also
exist in the MPP+-induced PD cell model. The
co-immunoprecipitation experiments of HMGB1 and CXCL12 in the
supernatants of MPP+-treated midbrain cells showed
that HMGB1/CXCL12 complexes existed in this in vitro co-culture
system (Fig. 4D). And after primary macrophages were cultured in the
supernatant of MPP+-treated midbrain cells, CXCR4 on
macrophages was detected to bind HMGB1 and CXCL12 (Fig. 4E). As a domain
of HMGB1, HMGB1 A Box has receptor binding potential similar to HMGB1,
so we used co-immunoprecipitation and immunofluorescence to evaluate the
possibility of HMGB1 A Box competitively binding to CXCR4 on
macrophages. The result showed that HMGB1 A Box partially co-localized
with CXCR4 (Fig. 4C), and HMGB1 A Box-6×His was pulled down using a
CXCR4 monoclonal antibody. However, to exclude influence by the 6×His
tag, HMGB1 B Box-6×His, as a control, was not detected, which indicated
that HMGB1 A Box indeed binds to CXCR4 on macrophage (Fig. 4F).
Neurons, microglia, and astrocytes in the SN during PD are the source of
elevated HMGB1 (Sasaki et al. , 2016). To determine the main
source of HMGB1 which causes cell migration in PD, midbrain neurons,
microglia and astrocytes from neonatal mice were isolated and cultured,
and after knockdown of intracellular HMGB1 using lentivirus, different
combined ways of these cells were co-cultured with peritoneal
macrophages in the lower chambers of transwell system, and
MPP+ treatment was added to the lower chamber to
induce neuronal damage at the same time. It was found that the mixed
culture of all the three cells without knockdown of HMGB1 had the
strongest effect on the migration of macrophages. and the application of
AMD3100, an antagonist that specifically inhibits CXCR4-CXCL12 binding,
significantly inhibited the migration of macrophages, and in the
mic+ast+neusi group which knockdown neuronal HMGB1,
the number of macrophage migration was also significantly reduced (Fig.
4G, H). This indicates that HMGB1 released by midbrain neurons during PD
plays an essential role in the migration of peripheral monocytes to the
SN.