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\begin{document}

\title{Adoptive Transfer of Immunomodulatory M2 Macrophages Suppresses
Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice via Blockading
NF-\selectlanguage{greek}κ\selectlanguage{english}b Pathway\selectlanguage{english}}



\author[1]{Mingchao Shi}%
\author[1]{Fengna Chu}%
\author[1]{Yue Lang}%
\author[2]{Chao Zheng}%
\author[2]{Tao Jin}%
\author[1]{Li Cui}%
\author[1]{Jie Zhu}%
\affil[1]{Jilin University First Hospital}%
\affil[2]{First Hospital of Jilin University}%


\vspace{-1em}



  
  \date{May 19, 2024}


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\selectlanguage{english}
\begin{abstract}
Objectives: The aims of the study were to further elucidate the roles of
M1 and M2 macrophages in the pathogenesis of EAE and the effects of
treatment with M2 macrophages that target certain pro-inflammatory
cytokines and with immunomodulatory preparations that beneficially
influence the disease course should be in focus of future therapeutic
trials. Results: Enhanced the total number of macrophages at the onset
of clinical signs in the EAE group, consistent with an increased
proportion of M1 cells and low numbers of M2 cells. As the disease
progressed and the symptoms worsened, M1 cells were decreased and M2
cells were gradually increased till the peak. In the recovery stage, M2
cell numbers were gradually decreased. Treatment with M2 macrophages
inhibited the NF-\selectlanguage{greek}κ\selectlanguage{english}b pathway, alleviated the symptoms of EAE, reduced
inflammatory cell infiltration and demyelination in the central nervous
system, and decreased the numbers of macrophages in the spleens.
BAY-11-7082, an NF-\selectlanguage{greek}κ\selectlanguage{english}b blocking agent, could reduce the total number of
macrophages both in vivo and in vitro, effectively prevented the EAE
development, and significantly inhibited the symptoms EAE in mice.
Conclusions: Macrophages may play a crucial role in the pathogenesis of
EAE, while M2 macrophages have anti-inflammatory effects. Transfer of M2
macrophages to EAE mice can block the NF-\selectlanguage{greek}κ\selectlanguage{english}b pathway successfully and
relieve the EAE symptoms. Application of NF-\selectlanguage{greek}κ\selectlanguage{english}b blockers is useful in the
prevention and treatment of EAE.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Multiple sclerosis(MS) is an inflammatory demyelinating autoimmune
disease in the central nervous system(CNS) and it affects approximately
2.5 million people worldwide and poses a major burden to the healthcare
system{[}1,2{]}. Current therapeutic options for MS include
corticosteroids, beta interferon, immunosuppressive agents and
monoclonal antibodies like anti-CD20{[}3{]}, however, these treatments
are effective only in some patients, and have significant adverse
effects.

The pathogenesis of MS is unclear. Many studies have shown that both
patient characteristics{[}4{]}, and environmental factors such as
intestinal flora imbalance and also gene--environment(GxE) interactions
can affect the development of MS{[}5,6{]}.

Previous studies on model of MS, experimental autoimmune
encephalomyelitis(EAE), have shown that macrophages may play important
roles in the pathophysiology of MS{[}7{]}. At the onset of EAE and MS,
macrophages from peripheral tissues enter into the CNS, resulting in the
pathological changes and M1 macrophage increased{[}8,9{]}. M2
macrophages can antagonize and inhibit the inflammation caused by
inflammatory molecules related to M1 macrophages{[}10-12{]}. Previous
studies have reported that M2 microglia can promote recovery of motor
function after spinal cord injury in mice and delay the progression of
EAE{[}13,14{]}. Macrophages are homologous to microglia and are easier
to obtain and culture. Therefore, promotion of macrophage
differentiation into M2 or enhancement of M2 macrophage are expected to
be the novel strategies for the treatment of neuroinflammatory and
immunological diseases in the future, which has attracted attention as a
new method worldwide.

Nuclear factor kappa-light-chain-enhancer of activated B cells(NF-\selectlanguage{greek}κ\selectlanguage{english}b), a
protein complex mainly in the form of p50/p65 isodimers, 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{[}15,16{]}. NF-\selectlanguage{greek}κ\selectlanguage{english}b played an important role in the
immune response of the nervous system, which began to increase on the
9th day after EAE induction in Lewis rats, peaked at the 11th and 12th
days, and gradually decreased after the 14th day, and the dynamic
changes of NF-\selectlanguage{greek}κ\selectlanguage{english}b activity were consistent with the EAE
symptoms{[}17,18{]}. M2 macrophages can down-regulate the signal
transduction of NF-\selectlanguage{greek}κ\selectlanguage{english}b, which may have therapeutic effects on EAE via
inhibiting NF-\selectlanguage{greek}κ\selectlanguage{english}b activity{[}10{]}.

BAY-11-7082(Beyotime) is a kind of NF-\selectlanguage{greek}κ\selectlanguage{english}b pathway blocker that can
completely and specifically abolish NF-\selectlanguage{greek}κ\selectlanguage{english}b DNA binding, which also an
irreversible inhibitor of TNF-\selectlanguage{greek}α-\selectlanguage{english}induced inhibitor of NF-\selectlanguage{greek}κ\selectlanguage{english}b(I\selectlanguage{greek}κ\selectlanguage{english}B)-\selectlanguage{greek}α
\selectlanguage{english}phosphorylation that leading to NF-\selectlanguage{greek}κ\selectlanguage{english}b inactivation{[}19{]}. In the
present study, we applied BAY-11-7082 in EAE mice to observe whether
blocking NF-\selectlanguage{greek}κ\selectlanguage{english}b pathway can effectively alleviate the symptoms of EAE,
and to explore the possible mechanisms behind the therapeutic role of
BAY-11-7082 in EAE, in which investigation of its effect on macrophages
is focused on.

\textbf{Material and methods}

\textbf{Animals}

Female C57BL/6 mice aged 6-8 weeks and weighing 18-20g were purchased
from Vital River Laboratory Animal Technology Co., Ltd.(Beijing, China)
and bred in our animal facility at The First Hospital of Jilin
University. The mice were housed in pathogen-free conditions and had
free access to food and water. All experiments were performed in
accordance with the local ethics committee of The First Hospital of
Jilin University, Changchun of China(Ethical approval number: 2017-216).

\textbf{Induction of EAE and assessment of the clinical signs}

The EAE and control induction and assessment of the clinical signs was
the same with Xie's description{[}20{]}.

\textbf{Macrophage cultures and transfer to EAE mice}

Immunized C57BL/6 mice were sacrificed and bone marrow cells from
femoral and tibial bones were collected in RPMI-1640 complete
medium(Gibco, Waltham, MA) on days 2, 5, and 8 p.i. After red blood cell
lysis, cells from the bone marrow were cultured in RPMI-1640 complete
medium supplemented with penicillin and streptomycin(1\%; Hyclone,
Logan, UT), fetal bovine serum(10\%; Sigma Aldrich, St Louis,MO),
IL-4(10ng/ml; Pepro-Tech, Rocky Hill, NJ), and macrophage
colony-stimulating factor(M-CSF) (10ng/ml; Pepro-Tech, Rocky Hill, NJ).
On days 3, 5, and 7 post-culture, half of the previous medium was
replaced with fresh medium and the concentrations of IL-4 and M-CSF were
increased to 20ng/ml. M2 macrophages were harvested on day 8
post-culture using ethylene diamine tetra acetic acid(EDTA; Sigma) and
incubated with MOG35--55(20\selectlanguage{greek}µ\selectlanguage{english}g/ml) peptide for 4h at 37\selectlanguage{ngerman}°C. Then the cell
concentration was set to 10×10\textsuperscript{6}/ml, and the cells were
transferred to EAE mice(0.1ml, 1×10\textsuperscript{6} cells/mouse) on
days 10, 13 and 16 p.i. via the caudal vein. And the control group were
treated with PBS. Experiments were replicated three times(n = 10).

\textbf{Histological analysis}

The spinal cord was removed from the spinal canal and embedded in
paraffin. Multiple horizontal sections(5-6 \selectlanguage{greek}µ\selectlanguage{english}m thick) of the spinal cord
were stained with hematoxylin-eosin(HE) and replicate sections were
stained with luxol fast blue(LFB) violet for evaluation of the extent of
inflammatory cell infiltration and demyelination. The number of
infiltrated inflammatory cells and the degree of demyelination in each
mouse were averaged from 10 complete cross-sections of the spinal cord.

\textbf{Flow cytometric analysis}

After the spleen was taken out, rinsed in RPMI-1640 complete
medium,then lysed the red blood cell and adjusted the cell
concentration to 1\selectlanguage{ngerman}×10\textsuperscript{6}/ml with PBS and stained with
the following antibodies: F4/80-PE-CY7(Biolegend), CD11b-FITC(BD
Pharmingen\textsuperscript{TM}), CD206-BV650(Biolegend),
Arg-1-APC(R\&D), iNOS-PE(eBiosience), and CD40-BV421(BD Pharmingen TM).
For detection of extracellular molecules, the cells were incubated for
30 min at 4°C with antibodies(F4/80, CD11b, CD206, Arg-1, CD40). For
detection of intracellular molecules(iNOS), cells were permeabilized
with the Fixation/Permeabilization Solution Kit(BD
Pharmingen\textsuperscript{TM}) for 20 min, then incubated with the
antibodies for 30 min at 4°C. In FACS analysis, both F4/80 and CD11b
were positive to calculate the proportion of macrophages(Q2). In
macrophages, iNOS or CD40 positive was identified as M1
macrophages(Q1+Q2+Q3), and CD206 or Arg-1 positive was identified as M2
macrophages(Q5+Q6+Q7){[}7{]}.

\textbf{Cytokine cytometric bead array assay}

Serum was obtained after centrifugation at 2092\emph{g} for 10 min, and
serum levels of IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-4, IL-6, IL-10, IL-12p70, IL-17A, and tumor
necrosis factor(TNF) were measured using cytometric bead array(CBA)
kits(BD Bioscience) according to the manufacturer's instructions. The
CBA test was repeated twice(n=6) and results were expressed in pg/ml.

\textbf{Western blotting}

The spinal cords were cut into small fragments and added into the
lysates, and a mixture of protein phosphatase inhibitors was added into
the tissue cell lysates at a proportion of 1:100. The cracked samples
were centrifuged for 3-5 minutes at 10,000-14,000\selectlanguage{ngerman}×\emph{g} . The BCA
Protein Quantitation Kit(Bioteke Corporation) was used to draw the
standard curve and calculate the protein concentrations. For each 30\selectlanguage{greek}µ\selectlanguage{english}l
protein sample, 10 \selectlanguage{greek}µ\selectlanguage{english}l of buffer was added and blended, and proteins were
denatured by heating in a 100\selectlanguage{ngerman}°C water bath for 3-5 minutes. After
cooling to 25°C, the samples were centrifuged at 10,000-14,000rpm for
2-5 minutes. The supernatants were collected and directly used for
electrophoresis.

\selectlanguage{greek}\textbf{Blockade of the NF-κb pathway to treat and prevent EAE}\selectlanguage{english}

The EAE mice were divided into three groups: 1) EAE control
group(treated with PBS), 2) prevention group(treated with BAY-11-7082 on
day 2, 5, 8, 11, 14, and 17p.i., 20mg/kg/i.p), and 3) treatment
group(treated with BAY-11-7082 on days 10, 13, and 16 p.i, 20mg/kg/i.p).
The weight and clinical score were recorded every day to observe the
changes in the EAE symptoms. Experiments were replicated three
times(n=10).

\textbf{Culture of M1 and M2 macrophages}

The detail methods were as same as the process of cultured M2 macrophage
but M1 macrophage were added with LPS(10ng/ml, Solarbio) instead of
IL-4. BAY-11-7082(1 \selectlanguage{greek}µ\selectlanguage{english}M) were added on the day 8 of cultures and control
group were added with PBS, the wells were incubated for 48h, 20\selectlanguage{greek}µ\selectlanguage{english}l
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)
solution was added to each well and incubated for 4h. Then, 150\selectlanguage{greek}µ\selectlanguage{english}l
dimethylsulfoxide was added to each well and low-speed oscillation was
performed on the shaker for 10min to fully dissolve the crystals. The
absorbance value of each well was measured at 570nm by an enzyme-linked
immunodetection.

\textbf{Statistical analysis}

All experiments were repeated three times with each group had 10 mice
and data were expressed as mean \selectlanguage{ngerman}± standard error of mean(SEM). FLOWJO
7.6.1(Treestar) was used to analyze the flow cytometry data and GRAPHPAD
PRISM 5.0(GraphPad Software Inc., San Diego, CA) was used to draw the
graphs. Other statistical analyses were performed using SPSS 21.0(IBM
Deutschland, Ehningen, Germany). The student's t-test was used for
comparisons between two groups. All tests were two-tailed.
P\textless{}0.05 was considered statistically significant, represented
by *(P\textless{}0.05), **(P\textless{}0.01), ***(P\textless{}0.001),
and ****(P\textless{}0.0001).

\textbf{Results}

\textbf{Clinical course of EAE}

The mice developed clinical signs of EAE on day 10(onset) p.i. and on
day 22 p.i.(peak), the symptoms of EAE peaked, with an average score of
3.00±0.37. The signs began to ease gradually on day 25(recovery). The
weight of EAE group gradually decreased until the lowest point on day 22
p.i.(peak), and then increased gradually, which was consistent with the
changes of symptoms. Mice in the control group showed no clinical
sign(P\textless{}0.0001). (Figure 1a).

\textbf{Increased M1 macrophage at onset and M2 macrophage at peak of
EAE}

The percentage of macrophages in the spleen increased at the onset and
decreased at the peak phase(P\textless{}0.0001). At the recovery stage,
the percentage of macrophages decreased continuously(P\textless{}0.001).
The percentage of M1 macrophages increased at the EAE
onset(P\textless{}0.0001), then decreased at the peak and
recover(P\textless{}0.01). Conversely, the percentage of M2 macrophages
didn't increase at the onset(P\textgreater{}0.05) and increased at the
peak of EAE(P\textless{}0.0001) and decreased a little at recover
stage(P\textless{}0.05), but still higher than normal(Figure 1b).

\textbf{Pathological and inflammatory cytokine changes in EAE}

The main pathological changes in MS are inflammation and demyelination
in the white matter of the CNS, axonal injury and axonal loss, which can
coexist and are often associated with diffuse lesions of the
CNS{[}21{]}. As shown in Figure1c, a small number of monocyte and
lymphocyte infiltrations could be seen in the spinal cord of EAE mice,
with obvious perivascular inflammatory infiltrations by HE stainings.
LFB stainings showed demyelination in the spinal cord.

The pro-inflammatory cytokines, IL-17A, IL-6, TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-12p70 and IL-1\selectlanguage{ngerman}ß
increased at the disease onset(P\textless{}0.0001) and IL-17A, IL-6,
TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-12p70 decrease at the peak hour(P\textless{}0.0001). While
anti-inflammatory cytokines, IL-4 and IL-10, significantly increased
during the peak period(P\textless{}0.001)(Figure1d).

\par\null

\textbf{Clinical course, pathological and inflammatory cytokine changes
after M2 macrophage treatment}

The proportion of M2 macrophages cultured \emph{in vitro} was more than
97\% determined by flow cytology(Figure 2a). Those were transfused into
the mice when EAE mice showed clinical symptoms on day 10 p.i.(onset).
EAE treated with M2 macrophage(M2-treated) and PBS developed the disease
at the same time; however, the peak symptoms were lighter and recovered
earlier in the M2-treated group than controls. Similarly, weight records
of the two groups showed that the weight gradually decreased in the EAE
control group until day 22p.i. and dropped to the lowest at the peak
phase; while M2-treated group had lost weight until day 17p.i. and then
their weight increased gradually. On day 22 p.i., EAE symptoms reached a
peak with average scores of 3.08\selectlanguage{ngerman}±0.20. The M2-treated group showed the
highest score on day 17 p.i., with an average score of
2.36±0.26(P\textless{}0.0001)(Figure 2b).

During the peak hour, the spinal cord was removed for pathological
staining after perfusion. As shown in Figure 2c, the inflammatory cell
infiltrations in spinal cords were less in the M2-treated group than in
EAE control group measured by HE staining(P\textless{}0.0001). LFB
staining showed that spinal demyelination in the M2-treated group was
significantly less severe than that in the EAE control
group(P\textless{}0.0001).

Additionally, pro-inflammatory cytokines, IL-6, IL-17A, TNF, IL-1ß and
IL-12p70, are lower in the M2-treated group compared with the
controls(P\textless{}0.01). Anti-inflammatory cytokines, IL-4 and IL-10
are higher in M2 treated group(P\textless{}0.01)(Figure 2d).

\textbf{Decreased macrophages in spleens after M2 macrophage treatment}

The macrophage percentages in the spleens were higher in EAE groups than
that in M2-treated group at the peak of the EAE(P\textless{} 0.0001).
The percentage of M1 macrophages was lower in the M2-treated group than
in EAE group(P\textless{}0.05). The M2 macrophages were lower in the
M2-treated group than in EAE group(P\textless{}0.0001).(Figure 3a)

\selectlanguage{greek}\textbf{Low NF-κb activity in the spinal cord of M2-treated EAE mice}\selectlanguage{english}

At the peak hour, phosphorylated and non-phosphorylated NF-\selectlanguage{greek}κ\selectlanguage{english}b p65 levels
in the spinal cord were detected by western blotting. Phosphorylated p65
level was lower in the M2-treated group than in EAE group(Figure 3b).

\selectlanguage{greek}\textbf{NF-κb expression, clinical course and inflammatory response
after BAY-11-7082 prevented and treated}\selectlanguage{english}

NF-\selectlanguage{greek}κ\selectlanguage{english}b level of spinal cord was detected in NF-\selectlanguage{greek}κ\selectlanguage{english}b blocker prevention and
treatment group and EAE control group by western blot. As the Figure 4a
shows, phosphorylated NF-\selectlanguage{greek}κ\selectlanguage{english}b level were lower in the EAE prevented and
treated group than in the EAE control group(EAE vs. prevent:
P\textless{}0.05).

We conducted experiments according to the grouping and administration
methods introduced in the previous method. As shown in Figure 4b, the
prevention group had a later disease onset than the EAE control
group(P\textless{}0.001), with a lower symptom
incidence(P\textless{}0.0001) and a lower score at the peak
hour(P\textless{}0.0001). The treatment group had concurrent onset with
EAE group; however, the score at peak hour was lower than EAE
group(P\textless{}0.001).

After treated by the NF-\selectlanguage{greek}κ\selectlanguage{english}b blocker, the spinal inflammatory cell
infiltrations were more severe in the EAE control groups than in the
prevention and treatment groups(P\textless{}0.0001). LFB staining showed
that spinal demyelination in the prevention and treatment groups was
significantly less severe than that in the EAE control
group(P\textless{}0.0001)(Figure 4c).

The pro-inflammatory cytokines, TNF-a, IL-6, IL-12p70 andIL-17A, were
lower in the NF-\selectlanguage{greek}κ\selectlanguage{english}b blocker (prevent and treated) group than in the
control group(P\textless{}0.05). Anti-inflammatory cytokines, IL-4 and
IL-10 are higher in NF-\selectlanguage{greek}κ\selectlanguage{english}b prevent group(P\textless{}0.001), but no
differences in treatment group(Figure 5a).

\selectlanguage{greek}\textbf{Decreased macrophage in the spleens after blocking the NF-κb
pathway}\selectlanguage{english}

After using the NF-\selectlanguage{greek}κ\selectlanguage{english}b blocker, the total macrophage percentages were
lower in the NF-\selectlanguage{greek}κ\selectlanguage{english}b blocked (prevent and treated) groups than
controls(P\textless{}0.01)(Figure 5b). Spleen M1 macrophages in the EAE
control group were higher than that in the blocked
group(P\textless{}0.01), and spleen M2 macrophages were higher in the
EAE control group than in the blocked group(P\textless{}0.05)(Figure
5b).

\selectlanguage{greek}\textbf{Decreased macrophage via blocking the NF-κb pathway \emph{in
vitro}}\selectlanguage{english}

\emph{In vitro} proliferation and activity of M1(P\textless{}0.001) and
M2 cells(P\textless{}0. 01) were higher in the control group than those
in the NF-\selectlanguage{greek}κ\selectlanguage{english}b blocked group, which show that the blockade could block the
production of M1 and M2 at the same time, and inhibition of M1 was more
pronounced(Figure 5c).

\textbf{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-\selectlanguage{greek}κ\selectlanguage{english}b 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-\selectlanguage{greek}κ\selectlanguage{english}b 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-\selectlanguage{greek}κ\selectlanguage{english}b 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-\selectlanguage{greek}κ\selectlanguage{english}b defects{[}17,18{]}. Moreover, NF-\selectlanguage{greek}κ\selectlanguage{english}b also
plays a central role in activating and differentiating T cells and
blocking NF-\selectlanguage{greek}κ\selectlanguage{english}b 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-\selectlanguage{greek}κ\selectlanguage{english}B signal
transduction pathway participates in the inflammatory immune response
induced by macrophage{[}36{]}. NF-\selectlanguage{greek}κ\selectlanguage{english}B activity is closely related to the
number, state, degree, and product of microglial activation, that is,
the higher the NF-\selectlanguage{greek}κ\selectlanguage{english}B 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-\selectlanguage{greek}κ\selectlanguage{english}b blocker BAY-11-7082, which can block the
phosphorylation of NF-\selectlanguage{greek}κ\selectlanguage{english}b65, and observed the effects on M1 and M2
macrophages \emph{in vitro} and \emph{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-\selectlanguage{greek}κ\selectlanguage{english}b 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,\emph{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 \emph{in vivo} study. Our results suggest that inhibition
of NF-\selectlanguage{greek}κ\selectlanguage{english}b 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-\selectlanguage{greek}κ\selectlanguage{english}b
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-\selectlanguage{greek}κ\selectlanguage{english}b blockade effectively prevented EAE development and
alleviated the severity of EAE via blockading NF-\selectlanguage{greek}κ\selectlanguage{english}b pathway. In the
future, we will further study how the NF-\selectlanguage{greek}κ\selectlanguage{english}b pathway involving in the
process of macrophage polarization.

\textbf{Consent for Publication}

All authors agree to the publication of this manuscript.

\textbf{Acknowledgements}

This study was supported by grants from The First Hospital, Jilin
University, Changchun of China; International Science and Technology
Cooperation Program of Jilin Provincial Science and Technology
Development of China(No. 20150414011GH), Norman Bethune Cultivation Plan
of Jilin University(No. 2015320), the National Natural Science
Foundation(No.81471216, NO.81671186, No.81671177, No.31600820) as well
as from the Swedish Research Council(2015-03005).

\textbf{Disclosure of conflict of interest}

No competing interests to declare.

\textbf{Data availability statement}

The data used to support the findings of this study are available from
the corresponding author upon request.

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} The clinical scores, macrophage percentages in
spleen, pathology and cytokines changes in EAE. (a) EAE group developed
clinical signs on day 10 (onset) p.i. On day 22 p.i., the symptoms of
EAE peaked, then the signs began to ease gradually on day 25 (recovery).
The control group had no symptoms. At the same time, the weight of EAE
group gradually decreased until the lowest point on day 22 p.i., and
then increased, which was consistent with the changes of symptoms. (b)
Macrophage percentages in the spleens were measured at different stage
of EAE: Macrophages in the spleen increased at the onset, then will
gradually decreased at peakhour and recover stage; M1 macrophages
increased at the onset, and decreased at the peak hour and recover; M2
macrophages increased at the peak hour, then decrease at recover. (c)
Inflammatory cell infiltration around the blood vessels (HE stains) and
demyelination in the spinal cord (LFB stains are more severe in the EAE
mice than in the na\selectlanguage{ngerman}ïve mice (normal mice). (d) All the pro-inflammatory
cytokines increased at the disease onset and IL-17A, IL-6, TNF-\selectlanguage{greek}α,
\selectlanguage{english}IL-12p70 decrease at the peak hour. While anti-inflammatory cytokines,
IL-4 and IL-10, significantly increased during the peak period.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} Treated EAE with M2 macrophage that inducing in vitro
and observed pathological and cytokines changes in M2-treated and
control groups. (a) The proportions of macrophages and M2 macrophages
were 98.48\% \selectlanguage{ngerman}± 0.189\% and 97.60\% ± 1.407\%, respectively. IL-4 and
M-CSF can induce the M2 macrophages efficiently. (b) M2 macrophages were
harvested on day 8 post-culture and transferred to EAE mice on days 10,
13, and 16 p.i. via the caudal vein. After adoptive transfer of M2
macrophages, the weight of the animals in the M2-treated group gradually
increased after day 17 with the score decreased, while, the weight began
to increase from day 22 in the control group. (c) During the peak hour,
inflammatory cell infiltration and demyelination in the spinal cord are
more severe in the controls than in the M2-treated group. (d)
Pro-inflammatory cytokines, IL-6, IL-17A, TNF, IL-1ß and IL-12p70, are
lower in the M2-treated group compared with the controls.
Anti-inflammatory cytokines, IL-4 and IL-10 are higher in M2 treated
group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Macrophages and the activity of NF-\selectlanguage{greek}κ\selectlanguage{english}b changes in
M2-treated and EAE control mice. (a) Macrophage percentage is lower in
the M2-treated group than those in the EAE groups at peak hour; M1
macrophages are lower in the M2-treated group than in the control
groups; M2 macrophages are lower in the M2-treated group than in the EAE
groups. (b) Expression the ratio of phosphorylated in total NF-\selectlanguage{greek}κ\selectlanguage{english}b is
lower in the M2-treated group than in the EAE group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} NF-\selectlanguage{greek}κ\selectlanguage{english}b activity in the spinal cord, EAE score and
pathological differences between the EAE and NF-\selectlanguage{greek}κ\selectlanguage{english}b blocked groups. (a)
Phosphorylated NF-\selectlanguage{greek}κ\selectlanguage{english}b level is lower in the blocked group than in the EAE
group and the prevention group is statistically different from the EAE
group. (b) Intravenous BAY-11-7082 injection every 3 days from day 2
after the immunization until the peak hour showed that the prevention
group had a lower score at the peak phase; Intravenous BAY-11-7082
injection every 3 days from day 10 after the immunization until the peak
hour showed that the score was lower in the BAY-11-7082-injected
treatment group compared with the EAE group. (c) Inflammatory cell
infiltration and structural damage in the spinal cord are more severe in
controls than in the prevention and treatment groups.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5.} Cytokines and macrophage changes \emph{in vitro}
and\emph{in vivo} after NF-\selectlanguage{greek}κ\selectlanguage{english}b pathway blockade. (a)The cytokine levels
(IL-6, IL-17A, TNF and IL-12p70) are lower in the NF-\selectlanguage{greek}κ\selectlanguage{english}b blockade group
than in the EAE group. Anti-inflammatory cytokines, IL-4 and IL-10 are
higher in NF-\selectlanguage{greek}κ\selectlanguage{english}b blockade group. The results in the prevention group are
more significantly different. (b) Macrophages decreased in the spleen
after the NF-\selectlanguage{greek}κ\selectlanguage{english}b blockade; Spleen M1 macrophages were higher in the EAE
group than in the blockade group; Spleen M2 macrophages were higher in
the EAE group than in the NF-\selectlanguage{greek}κ\selectlanguage{english}b blockade group. (c) \emph{In
vitro}proliferation and activity of M1 and M2 cells are higher in the
control group than those in the NF-\selectlanguage{greek}κ\selectlanguage{english}b blockade group.

\selectlanguage{english}
\FloatBarrier
\end{document}

