Figure legends
Fig.1 HMGB1 A Box reduces macrophage infiltration in SN
parenchyma. (A and B) Brains of animals receiving saline, MPTP, MPTP +
HMGB1 A Box injection were separated and made into serial frozen
sections of 30μm thickness were prepared at the end of the 7-day
treatment, and sections of the SN site were subjected to CD45, F4/80,
CD11c, CD31 immunofluorescence staining.
Fig.2 HMGB1 A Box reduces macrophages and Th17 infiltration in
SN and protecting TH+ neurons. (A and B) Brains of
animals receiving saline, MPTP, MPTP + HMGB1 A Box, MPTP + clodronate
liposomes were separated after 7 days of treatment and serial frozen
sections were made and then the sections of SN site were subjected to TH
immunization group chemical staining, extract the protein from the SN
for western blot detection of TH, and finally, the gray value of the
results is counted (n=3; unpaired t-test; mean ± s.d.). (C) Digest mouse
SN cells to obtain single cell suspension, and perform flow cytometry
detection of CD45, CD11b, CD11c, MHC II, CD3, CD4, IFN-γ, IL-4 and
IL-17A. (D-F) Proportion of
CD45intCD11b+ microglia,
CD45hiCD11b+ macrophages and
CD45hiCD11c+ DCs in mouse SN (n=5;
unpaired t-test; mean ± s.d.). (G-H) MHC II mean fluorescence intensity
of CD45intCD11b+ microglia,
CD45hiCD11b+ macrophages and
CD45hiCD11c+ DCs in SN (n=5;
unpaired t-test; mean ± s.d.). (J) Numbers of
CD4+IFN-γ+ cells,
CD4+IL-4+ cells and
CD4+IL-17A+ cells in SN (n=3;
unpaired t-test; mean ± s.d.). *P < 0.05, **P <
0.01.
Fig.3 Peripheral monocytes/macrophages promote MPTP-induced Th17
differentiation in mouse SN and are inhibited by HMGB1 A Box. (A) Mouse
MPTP induction and midbrain cell isolation and culture timeline. (B)
Mouse primary cells from the midbrain and CD4+ naïve T
cells co-culture groups. (C) Co-cultured cells were collected for CD45,
CD11b, CD4 and IL-17A flow cytometry assays. (D and G) Ratio of
IL-17A+ cells to CD4+ cells in each
group of cells (n=4; unpaired t-test; mean ± s.d.). (E) Number of
CD45intCD11b+ microglial in each
group (n=4; unpaired t-test; mean ± s.d.). (F) Number of
CD45hiCD11b+ macrophages in each
group (n=4; unpaired t-test; mean ± s.d.). *P < 0.05, **P
< 0.01.
Fig.4 HMGB1 complexes with CXCL12 mediate macrophage migration.(A and B) Peritoneal macrophages were co-cultured with
MPP+-treated primary midbrain cells in transwell
system (n=4 random high-magnification fields of view; unpaired t-test;
mean ± s.d.). (C) Immunofluorescence staining of 6×His and CXCR4 after 6
hour-HMGB1 A Box treatment of peritoneal macrophages. (D) After the
culture supernatant of MPP+-treated midbrain cells was
concentrated, the binding of HMGB1/CXCL12 was detected by
immunoprecipitation assay. (E) Mouse peritoneal macrophages were
cultured with MPP+ -treated midbrain cell supernatant
for 6 hours, and the binding of CXCR4 to HMGB1 and CXCL12 was detected
by immunoprecipitation assay. (F) Peritoneal macrophages were treated
with HMGB1 A Box for 6 hours, and the binding of HMGB1 A Box-6×His and
CXCR4 were detected by immunoprecipitation assay. (G and H) Midbrain
microglia (mic), astrocytes (ast) and neurons (neu) HMGB1 were knocked
down separately and co-cultured with peritoneal macrophage in transwell
system in different combinations, and cells in the lower chamber were
treated with MPP+ (n=4 random high-magnification
fields of view; unpaired t-test; mean ± s.d.). ***P < 0.005,
****P < 0.001
Fig.5 CXCR4 of CD3+ T cell mediates their
infiltration in the SN. (A) Immunofluorescence staining of 6×His and
CXCR4 after HMGB1 A Box-6×His treatment of magnetic bead-sorted
CD3+ T cells for 6h. (B) CD3+ T
cells were treated with HMGB1 A Box for 6 hours, and the binding of
HMGB1 A Box-6×His and CXCR4 were detected by immunoprecipitation assay.
(C) FCM detection in peripheral blood of MPTP mice that were transfused
with CD3+ T cells from GFP mice (n=3; unpaired t-test;
mean ± s.d.). (D) GFP and CD3 tissue immunofluorescence staining of MPTP
mouse SN which were transfused with CD3+ T cells from
GFP mice. *P < 0.05, **P < 0.01,
Fig. 6 HMGB1/CXCL12-CXCR4 exists in the serum of PD patients.(A and B) Serum HMGB1 and CXCL12 levels of PD patients and healthy
people were detected by ELISA. (C) Correlation analysis between serum
HMGB1 levels and CXCL12 levels of PD patients. (D)
Co-immunoprecipitation experiments of HMGB1 and CXCL12 in serum of PD
patients. (E) Correlation analysis of serum HMGB1 level and age in PD
patients. (F) Correlation analysis between serum CXCL12 level and age in
PD patients. (G) Binding of HMGB1 and CXCL12 to CXCR4 in PD patient
CD14+ monocytes by co-immunoprecipitation. (H)
Immunofluorescence detection of CXCR4, HMGB1 and CXCL12 on
CD3+ T cells and CD14+ monocytes in
PD patients. (n=20; unpaired t-test; mean ± s.d.) *P<0.05,
**P<0.01.
Fig. 7 HMGB1 A Box suppresses immune cell responses in the SN of
PD mice and its mechanism. After neuronal damage in SN, frHMGB1
passively released from neurons binds to CXCL12 in the surrounding
environment to form frHMGB1/CXCL12 complex. This complex can bind to
CXCR4 on the surface of nearby microglia, macrophages in blood or
lymphatic capillaries and T cells to induce their migration and
infiltration into the SN parenchyma. The macrophages and microglia
infiltrated in the SN jointly promote T cells to differentiate into Th17
and continue to damage neurons. Intravenous injection of HMGB1 A Box
competitively inhibited this process by binding CXCR4 of these
peripheral immune cells.