Discussion
Macrophages are prominent innate immune cells and divided into M1 and M2
subtypes, and new research also found that M3 switch phenotype also
exists[22,23].
Previous studies showed that increased macrophages were associated with
neuronal damage in MS: In the acute phase, M1 macrophage were activated,
along with the release of large amounts of pro-inflammatory cytokines,
neuro-inflammation, CNS demyelination, and neuronal
death[24-26]. However, as the disease
progressed, M2 macrophages increased gradually, and the maximum levels
of M2 and M1 macrophage reduced gradually from the peak of disease to
the remission of EAE. In the late stages of the disease, M2 macrophages
played a dominant role in the CNS and released a variety of
anti-inflammatory cytokines, which reduced the inflammatory response and
promoted tissue repair, while inhibiting the further progression of
EAE[25]. Our results is consistent
with the results of previous
studies[27].
Recent studies have shown that some treatment for MS like
glucocorticoids, fingolimod, glatirameracetate, teriflunomide and
interferon-beta can downregulate expression M1 macrophage or activate M2
macrophage[28-32]. Previous studies
have found that therapy with M2 macrophage or microglia can prevent the
occurrence of type 1 diabetes and the motor neurons damage caused by
spinal cord injured, and also alleviate EAE symptoms in
rats[33,13,14].
However, there are the limited macrophages source and needed to expand
more methods to obtain enough macrophages. In recent years, researchers
have applied cytokines and compounds that can stimulate the
differentiation of bone marrow stem cells into M2 macrophages, which
have proven to be effective in transforming stem cells into M2
macrophage. In our study, IL-4 and M-CSF could successfully induce bone
marrow stem cells to differentiate into M2 macrophages that have been
transferred into EAE mice at the early stage of the disease. In
comparison with the control group, the M2-treated group showed
simultaneous development of symptoms, but their peak symptoms were
alleviated, and symptom relief was faster. The degrees of spinal cord
inflammation and the degrees of inflammation and demyelination in spinal
codes and inflammatory factors secretion in blood were lower in
M2-treated group than those in control group, which is similar to other
results[34,12].
However, the difference from the previous studies is that our experiment
is using the M2 macrophages induced from bone marrow stem cells and
inject them by intravascular route, which will greatly facilitate future
clinical operations and make cell therapy more likely. Our experiment
also found that after treatment with M2 macrophage in EAE mice, the
macrophages in the spleen were decreased, which included both M1 and M2
macrophages, however, the M2 macrophage decreased more significantly.
Pro-inflammatory factors are decreased and anti-inflammatory factors are
increased in peripheral blood.
It has been evidenced that once the inflammatory reaction leads to the
immune system activation, and then the immune system tries to maintain
the internal environment in balance by various ways. In our study, after
a large number of M2 macrophages injected into EAE mice, the declined
macrophage activation in the spleen of EAE mice through reducing M2
macrophages mainly was for restoring internal environment homeostasis.
At the same time, due to the infusion of a large number of M2
macrophages, anti-inflammatory factors were significantly increased in
the blood, promoting repairing the damaged tissue and alleviating the
sings of EAE.
NF-κb is a key intracellular signal transduction molecule that can
regulate the expression of many molecules involved in the early stage of
immune and inflammatory responses. Induced NF-κb is mainly in the form
of p50/p65 isodimers which p65 played a major
role[15,16],
and that is involved in the occurrence and development of MS. Previous
research findings implied that changes in the activity or protein
expression of NF-κb are closely related to the severity of EAE and the
incidence of disease, clinical fraction, and inflammation of the CNS
were all shown to decrease in mice with NF-κb
defects[17,18].
Moreover, NF-κb also plays a central role in activating and
differentiating T cells and blocking NF-κb function is an effective
method to prevent autoimmune
encephalitis[35].
After we applied M2 macrophages, the symptoms of the mice were reduced,
and NF-kb expression in the spinal cord was also reduced too. Therefore,
we wondered whether NF-kb was involved in the polarization of
macrophages to M1 and M2 cells. Studies have shown that the NF-κB signal
transduction pathway participates in the inflammatory immune response
induced by macrophage[36]. NF-κB
activity is closely related to the number, state, degree, and product of
microglial activation, that is, the higher the NF-κB activity, the more
activated microglial cells and the more inflammatory
factors[37]. Therefore, we
hypothesized that blocking NF-kb pathway can reduce the polarization of
M1 macrophages, increase the number of M2 macrophages, and reduce the
symptoms of EAE.
We applied a NF-κb blocker BAY-11-7082, which can block the
phosphorylation of NF-κb65, and observed the effects on M1 and M2
macrophages in vitro and in vivo . The results showed that
the severity of EAE was alleviated after the application of the blocker,
and the therapeutic effect in the prevention group was better than that
in the treatment group, indicating that the effect of early blockade was
more obvious. The analysis of macrophages in the spleen of mice at the
peak of EAE displayed that the proportion of total macrophages was lower
in the NF-κb blockade group(prevented and treated group) than the
control group, in which the proportion of M1 macrophage declined
significantly, however, the proportion of M2 decreased a bit. Meanwhile,in vitro experiments revealed that the blocker blocked the
activity of both M1 and M2 macrophages simultaneously. However, the
blockade of activity of M1 was more obvious than activity of M2, which
is similar to in vivo study.
Our
results suggest that inhibition of NF-κb pathway can inhibit the
generation of macrophages, especially M1 macrophage and pro-inflammatory
factors. Therefore, early addition of this inhibitor can block the
pro-inflammatory effect of M1 more effectively and reduce the
inflammatory response in EAE mice.
The present study demonstrates that (a) macrophages are involved in the
occurrence and development of EAE disease: M1 macrophages have
pro-inflammatory effects, and M2 macrophages have anti-inflammatory
effects; (b)bone marrow stem cells could be successfully induced to
differentiate into M2 macrophages; (c) M2 macrophages blocked the NF-κb
pathway and had a promising therapeutic effect on EAE, which enable
rational therapeutic approaches for MS and other autoimmune diseases in
the future; (d)NF-κb blockade effectively prevented EAE development and
alleviated the severity of EAE via blockading NF-κb pathway. In the
future, we will further study how the NF-κb pathway involving in the
process of macrophage polarization.