High
mobility group box-1 (HMGB1) affects macrophage polarization and phagocytosis
in systemic lupus erythematosus patients.

F Schaper, G Horst, K de
Leeuw, H Bootsma, PC Limburg, P Heeringa, M Bijl, J Westra

Background/Purpose:
Decreased phagocytosis of apoptotic cells, leading to necrosis and exposure of
nuclear autoantigens, plays an important role in the pathogenesis of Systemic
Lupus Erythematosus (SLE). One of these nuclear autoantigens is High Mobility
Group Box
1 (HMGB1), which acts as an alarmin when secreted. Increased serum levels have
been found in SLE patients. We hypothesized that increased HMGB1 levels might
lead to skewing in the differentiation of monocytes into M1 macrophages, instead
of M2 macrophages.  As M1 macrophages are less efficient phagocytes this might
fuel SLE. We studied expression of M1 and M2 markers of peripheral blood
monocytes and in vitro differentiated macrophages of SLE patients were compared
to healthy controls (HC). Phagocytic capacity of macrophages and the effect of
HMGB1 on differentiation of macrophages and phagocytosis was also investigated.

Methods:
For this study SLE patients with quiescent or mild disease (SLEDAI 0-5) and age-
and sex-matched HC were included. Monocytes and differentiated M1 and M2
macrophages were phenotyped by flow cytometry for expression of CD86 (M1
marker) and CD163 (M2 marker). Monocytes of 19 patients and HC were isolated by
negative sorting and analyzed by RT-qPCR to determine mRNA levels of CD86, TLR2,
TLR4, TNF-α, IL-6 (M1) and CD163, Mannose Receptor, and IL-10 (M2). HMGB1
was added (4 or 24 hours) during M2 differentiation to investigate the effect
on macrophage phenotype and effect on phagocytosis of apoptotic Jurkat cells.

Results:
Expression of CD86 on monocytes was similar between patients and HC, but expression
of CD163 was significantly lower on monocytes from SLE patients (p=0.022). Moreover,
IL-6 mRNA levels were significantly increased in monocytes of patients
(P<0.0001). After cytokine induced differentiation no differences were
observed between M1 and M2 macrophages from SLE patients and HC regarding
surface receptor expression and phagocytic capacity. Addition of HMGB1 during
differentiation of M2 macrophages resulted in high IL-6 and TNF-α mRNA
expression in macrophages. Preincubation of HMGB1 with apoptotic Jurkat cells
resulted in reduced phagocytosis by M2 macrophages.

Conclusion:
Monocytes from SLE patients display an M1-like phenotype, shown by decreased
expression of CD163 and increased IL-6 mRNA expression. In vitro
differentiation abolishes these differences between SLE and HC. HMGB1 induces
differentiation into M1-like phenotype and reduces phagocytosis of apoptotic
cells. So, this study shows that the phenotype of monocytes or macrophages is
determined by their environment, such as presence of cytokines and HMGB1, and
thereby contributes to the pathogenesis of SLE.