Microglia and Microglia-Like Cell Differentiated from DC Inhibit CD4 T Cell Proliferation
Citation: Bai B, Song W, Ji Y, Liu X, Tian L, et al. (
Microglia and Microglia-Like Cell Differentiated from DC Inhibit CD4 T Cell Proliferation
Bo Bai 0
Wengang Song 0
Yewei Ji 0
Xi Liu 0
Lei Tian 0
Chao Wang 0
Dongwei Chen 0
Xiaoning Zhang 0
Minghui Zhang 0
Sudha Agarwal, Ohio State University, United States of America
0 1 Department of Neurobiology, Taishan Medical College , Taian, Shandong Province , People's Republic of China, 2 Institute of Immunology, School of Medicine, Tsinghua University , Beijing , People's Republic of China
The central nervous system (CNS) is generally regarded as a site of immune privilege, whether the antigen presenting cells (APCs) are involved in the immune homeostasis of the CNS is largely unknown. Microglia and DCs are major APCs in physiological and pathological conditions, respectively. In this work, primary microglia and microglia-like cells obtained by co-culturing mature dendritic cells with CNS endothelial cells in vitro were functional evaluated. We found that microglia not only cannot prime CD4 T cells but also inhibit mature DCs (maDCs) initiated CD4 T cells proliferation. More importantly, endothelia from the CNS can differentiate maDCs into microglia-like cells (MLCs), which possess similar phenotype and immune inhibitory function as microglia. Soluble factors including NO lie behind the suppression of CD4 T cell proliferation induced by both microglia and MLCs. All the data indicate that under physiological conditions, microglia play important roles in maintaining immune homeostasis of the CNS, whereas in a pathological situation, the infiltrated DCs can be educated by the local microenvironment and differentiate into MLCs with inhibitory function.
Funding: This work was supported by Grants from the National Natural Science Foundation of China (30770676, 30872322) http://www.nsfc.gov.cn/Portal0/
default106.htm, Tsinghua-Yu-Yuen Medical Sciences Fund (202400005-27) http://tuef.cic.tsinghua.edu.cn/. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Microglia are resident macrophages located in the central
nervous system (CNS) parenchyma, the number is about 515% of
the cells in the CNS[1,2]. Because of the low expression of MHC
class II, B7 and CD40, the essential molecules necessary for
antigen presentation, microglia seem to be potential APCs that can
mount the immune response in the CNS. But there are still no
evidences to demonstrate that microglia can present antigens to T
cells and initiate the immune response in the CNS. The
immunological role of microglia in physiological or pathological
immune response in the CNS is still a puzzle.
There are few mature DCs (maDC) and lymphocytes in
noninflammatory CNS due to the function of brain blood barrier (BBB),
so the CNS is generally regarded as a site of immune privilege[4,5].
But under pathological conditions, the BBB is broken down and large
numbers of immunocytes including monocytes and dendritic cells
(DCs) could be recruited to the CNS. In experimental autoimmune
encephalomyelitis (EAE), there is a considerable increase of DCs in
the CNS during the early stage[6,7]. But the function of DCs in the
CNS of EAE model is contradictory. Some researchers found that
DCs isolated from the CNS of EAE model can present peptides to T
cells, whereas others reported that DCs with the same origin can
not[9,10]. Our previous studies showed that immune
microenvironment has great effects on the functions of DCs, so we speculate that
the microenvironment of the CNS may lie behind the contradiction
of the DCs function in the CNS of EAE model.
Increasing data showed that some antigen presenting cell
subtypes, including regulatory dendritic cells and suppressive
macrophages with inhibitory function, are important in
terminating immune responses for maintaining immune homeostasis.
As the protagonist of APC in the CNS, whether microglia will be
involved in the local immune homeostasis should be illustrated and
the influence of the CNS microenvironment on the function of the
DCs recruited to the CNS should be addressed.
In this study, we isolated the microglia from the brain of
perfused mouse and tested the antigen presenting ability using
OVA-specific-TCR transgenic CD4 T cells as responser. In
addition, the CNS endothelia were isolated and cultured to mimic
the microenvironment of the CNS in vitro, the effects of the CNS
endothelia on the infiltrated DCs and the effects of the educated
DCs on T cell activation and proliferation were explored. Our
work suggests that microglia and the CNS-endothelia-educated
DCs can prevent the proliferation of CD4 T cells and indicates the
APCs in the CNS may play an important role in keeping immune
homeostasis of the CNS by inhibiting T cell proliferation.
Microglia Inhibit maDC-Initiated Proliferation of CD4 T
We used TCR transgenic CD4+ T cells specific for
OVA3232339 to test the APC function of microglia. As shown
in Figure 1a, OVA3232339-pulsed maDCs could stimulate T cells
to proliferate dramatically but OVA3232339-pulsed microglia did
not. Moreover, microglia could significantly inhibit
maDCsinitiated T cell proliferation when microglia were added into
maDC/T cells coculture system simultaneously. Similar result
was obtained using CFSE-labeled CD4 T cells (Figure 1b).
Furthermore, we found that microglia could inhibit the
proliferation of activated T cells which has been cocultured with
maDCs for 24 hours (Figure 1c). Putting the data together,
microglia not only failed to initiate the proliferation of nave T
cells but also could inhibit maDC-initiated proliferation of nave
or activated T cells.
The Characteristics of the Endothelial Cells from the CNS
To obtain stromal cells mimicking the microenvironment of the
CNS, newborn C57BL/6 mice brain was scissored into pieces and
attached to 24-well plate. After 2 weeks, the attached cells were
digested and CD11b2 CD31+ cells were isolated and used as the
CNS endothelial stroma cells. Pictures were taken to show the
morphology of the endothelial cells before and after enrichment.
The purity of CD11b2 CD31+ cells was tested by CD31 and
CD106 using FACS (Figure 2a). Moreover, the secretion of
cytokines, including IL-7, TGF-b, GM-CSF, M-CSF and VEGF
(Figure 2b), and chemokines including MCP-1, MCP-3, SDF-1a,
TECK and MDC (Figure 2c), were detected. The high
concentration of MCP-1 indicates that the endothelial cells might
chemoattract macrophages or DCs. The chemotactic assay
confirmed our hypothesis and the supernatant of LPS-treated
EC has a more potent ability to chemoattract monocytes or DCs
Endothelial Cells Induce maDCs to Differentiate into
To find out the effect of the CNS microenvironment on the
infiltrated DCs, bone marrow maDCs derived from EGFP
transgenic mouse were seeded on the endothelia stroma derived
from the CNS. The maDCs attached to the endothelia and along
with the continuous culture, proliferated on endothelia. After 14
days coculture, maDCs differentiated into cells with typical shape
of microglia (for the ensuing results, these differentiated cells were
nominated as microglia like cells, MLCs) (Figure 3a). The
phenotype of the differentiated EGFP+ cells (MLCs) was analyzed.
As shown in Figure 3b, these cells express relatively high level of
CD11b, low CD11c, MHC class II and costimulatory molecules,
such as CD80, CD86 and CD40 compared with mature DCs.
Notably, the phenotype of the differentiated cells (MLCs) was
quite similar to that of microglia and different from that of
maDCs. The high expression of CD11b, a crucial marker of
microglia, on the differentiated cells (MLCs) was confirmed under
the microscope (Figure.3a). Furthermore, the phagocytic
capability assay showed that the differentiated cells (MLCs) displayed
notable phagocytic capability, similar with microglia, while
maDCs showed poor phagocytic capability (Figure 3c). In cytokine
secretion, the results showed that the LPS-treated differentiated
cells (MLCs) could produce similar high levels of IL-12p40 as
microglia, different from the high secretion of IL-12p70 from
mature DC (Figure 3d). Since microglia has inhibitory function,
we detected the secretion of IL-10, TGF-b1, VEGF and NO
which have been reported to have potential inhibitory effect. The
results showed that LPS treatment could promote microglia and
the differentiated cells (MLCs) to secret higher level of IL-10 and
NO than maDC (Figure 3 d,e). But no difference was detected in
the level of TGF-b1, and VEGF (Figure 3d). Further analysis of
Figure 2. The characteristics of the endothelial cells from the CNS. (a) The CNS endothelial stroma cells before and after CD11b2 CD31+
selection were observed under the microscopy. The purity of CD11b2 CD31+ cells was tested by expression of CD31 and CD106. (b, c) The secretion
of cytokines including IL-7, TGF-b, GM-CSF, M-CSF and VEGF(b), and chemokines including MCP-1, MCP-3, SDF-1a, TECK and MDC(c) was detected.
(d)The chemotactic ability to monocytes or DCs of the supernatant of LPS-treated EC was assayed.
Figure 3. Endothelia induce maDCs to differentiate into MLCs. (a) GFP+ maDCs cultured on monolayers of CNS endothelia for 14 days and
were labeled with PE-conjugated anti-CD11b antibody. Fluorescence photos were taken with a Leica fluorescent microscope (objective 406). (b)
Comparison of phenotype among microglia, MLCs and maDCs. (c) Comparison of phagocytic ability among microglia, MLCs and maDCs. Numbers in
histograms indicate the geometric mean fluorescence. (d) The secretion of IL-12p70, IL-12p40, IL-10, TGF-b1 and VEGF by maDC, microglia and MLCs
stimulated by 10 ng/ml LPS for 24 h was detected. (e) The production of NO by maDC, microglia and MLCs stimulated by 10 ng/ml LPS for 24 h was
detected. (f) The expression of iNOS in maDC, microglia and MLCs was detected by RT-PCR.
iNOS confirmed the high expression of NO in MLCs and
microglia (Figure 3f).
Therefore, the CNS stromal cells can differentiate maDCs into a
kind of cell which has similar morphology, phenotype, phagocytic
capacity and cytokine secretion panel with microglia. So we
nominated the differentiated cells as microglia-like cells (MLCs).
MLCs Inhibit maDC-Initiated Proliferation of CD4 T Cells
Then we tested the immune function of MLCs. Using
CFSElabeled TCR transgenic CD4 T cells, we analyzed the antigen
presenting ability of MLCs. The results showed that
OVA3232339pulsed MLCs failed to drive the proliferation of nave TCR
transgenic CD4 T cells. Moreover, MLCs could inhibit
maDCsinitiated T cell proliferation when they were added into the
maDCs/T cells coculture system (Figure.4a). Further study showed
that even if the TCR transgenic CD4 T cells have been activated
24 hours before, MLC could exert similar inhibitory function
(Figure 4b). Since it has been demonstrated that TGF-b, M-CSF
and VEGF are key factors in regulatory DC modulation, to find
thier effects on the MLC differentiation, we used fixed endothelial
cells (EC) or endothelial supernatant to culture DC or added
neutralizing antibodies to the EC-DC coculture system. The results
showed that both fixed endothelial cells and endothelial supernatant
have effects to promote MLC differentiation, indicating that both
cell-to-cell contact and soluble factors played an important role in
promoting MLC differentiation. However, blocking any of TGF-b,
GM-CSF, M-CSF and VEGF did not abolish the inhibitory
function of MLC, indicating that these soluble factors are not the
key factors in MLC differentiation, but their roles in the MLC
differentiation remained to be investigated.
NO Is Involved in the Immune Inhibition by Microglia and
To elucidate the factors that lie behind the immune inhibiting
function of microglia and MLCs, we examined the inhibiting
capability of 4% paraformaldehyde-fixed microglia and MLCs,
and the supernatants of microglia and MLCs after culture for 5
days. The results show that 50% supernatant of either microglia or
MLCs in the culture system reproduced the immune inhibiting
function, while neither of the two kinds of fixed cells had inhibitory
We found both MLCs and microglia had a basic secretion of
NO, and there was a burst of NO production upon stimulation
with LPS at a dosage of 10 ng/ml or IFN-c(5 ng/ml). However
the NO production of maDCs was very low either in the presence
or absence of LPS (Figure 3e). Addition of NO donor NOC-18 at
a dosage of 40 mg/ml to the coculture system strongly inhibited the
proliferation of CD4 T cells. PBIT, the selective NO synthetase
inhibitor, could dramatically abolished the inhibiting effects of
microglia and MLCs (Figure 5b). As shown in figure 3d, compared
to DC, microglia and microglia-like cell could secret higher level of
IL-10, but similar level of TGF-band VEGF when they are
stimulated by LPS. The results indicated that TGF-band VEGF
might not involve in the inhibitory functions of microglia and
microglia-like cell. And using neutralizing antibody against IL-10
to block the secreted IL-10 in the microglia (or MLC)/mDC/CD4
coculture system could not abolish the inhibition(data not shown).
The data demonstrated that NO played a notable role in the
immunological suppressive function of microglia and MLCs.
Microglia originate from bone marrow hematopoietic cells and
populate the CNS during early fetal life and remain in the
parenchyma of the CNS as resident macrophages. There is much
controversy about the correlation of microglia with the CNS
immunity. Carson reported that microglia were of incomplete APC
phenotype and failed to present peptides to the responder T cells
with the defined TCR. We demonstrate further that microglia
not only are impotent in presenting peptides to nave CD4 T cells
with defined TCR but also can inhibit antigen activated CD4 T cell
expansion. With this immune inhibitory potential, microglia resided
in the CNS may contribute to the privilege status of the CNS.
Considering the overwhelming numbers of microglia against the
paucity of the immune competent DCs and nave T cells in normal
CNS[12,13], the immune response triggered in the CNS should be
strongly pressed by microglia and homeostasis in the CNS should be
Neurodegenerative diseases are often accompanied with
neuroinflammatory process in which large numbers of DCs and
CD4 T cells are recruited into the CNS and cause irreversible
neural impairment. For example, in EAE model, large
numbers of activated autoreactive CD4 T cells and DCs were
observed in the CNS. The entering of activated myelin specific T
cells to CNS is thought to be important for the initiation of
EAE[6,7]. Suter reported that DCs isolated from EAE mice CNS
were found to inhibit T cell proliferation stimulated by mature
bone marrow-derived maDCs. It is notable that these isolated
DCs exhibit a phenotype similar to imDCs characterized by
intermediate MHC class II and low CD80 expression, and we
presume these inhibitory DCs might be the redifferentiated cells
from maDCs under the influence of the CNS microenvironment.
We paid attention to this issue in the light of our previous
discovery that maDCs in the spleen can be induced by splenic
endothelia stroma to differentiate into regulatory DCs which are
capable of downregulating immune response by inhibiting T cell
proliferation. In this work, we found that maDCs could also be
induced by the CNS endothelia to differentiate into inhibitory
MLCs which share similar phenotype and phagocytic capability
with microglia. It has been reported that GFP positive microglia
were found in the brain of the C57BL/6 mice with induced
Parkinsons disease after the mice were irradiated and
intravenously injected with GFP mice derived bone marrow cells.
Taken together, we can conclude that microglia could be
replenished by maDCs which have infiltrated into the
inflammatory CNS and the CNS stroma might contribute to the
redifferentiation of infiltrated maDCs. Differentiation from
maDCs to microglia may be one of the fates of maDCs recruited
to the CNS in EAE model. This differentiation combined with
functional changes from stimulation to inhibition may terminate
the immune response in the CNS and result in a remitting course
In our study, we found that endothelia from the CNS can
differentiate maDCs into microglia-like cells (MLCs), which
possess similar phenotype and immune inhibitory function as
microglia. Similarly, it is reported that astrocytes secreted factors
could drive monocytes or macrophages to differentiate into a
microglial which has ramified morphology, overexpressed
substance P and the calcium binding protein Iba-1, dimly expressed
class II MHC and a potassium inward rectifier current.
Here we focused on the immunological function of microglia and
the differentiated cells under the influence of the endothelia
stromal cells. All the results indicate that different components of
the microenvironment in the CNS might play a key role in
different aspects of the infiltrated antigen-presenting cells.
Endothelia is one of the main component of the stromal cells of
different organ, which express many kinds of membrane associated
extracellular matrix (ECM) molecules, including fibronectin and
fibrinogen which are the bioligands of CD11b (integrin aM) and
CD11c (integrin aX). CD11b and CD11c are lineage markers
of monocyte-derived immunocytes, including macrophages,
microglia, kupffer cells and dendritic cells. In the organs,
monocytederived cells normally adhere to the endothelia of micrangium and
sinusoid through the binding between integrins and their ligands.
The adhesion is in favor of functional modulation of the
monocytederived cells. Endothelia of brain secret high level of TGF-b,
M-CSF and VEGF, which have been demonstrated that they are
key factors in regulatory DCs modulation[22,23]. It has been
demonstrated that stromal cells (endothelia and fibroblast) can drive
the differentiation of mDCs to regulatory DCs[15,24,25].
TGF-b, M-CSF and VEGF have been demonstrated to be
driven factors for the differentiation of regulatory DC in vitro
. In our manuscript our conclusion is TGF-b,M-CSF and
VEGF might not be involved in the inhibitory functions of
microglia and MLCs and blocking TGF-b, M-CSF and VEGF
can not abolish the differentiation from maDC to MLC. But their
effects in the differentiation of MLCs remain to be investigated.
Because the EC-DC coculture system is too complex, many
soluble factors might combine to promote the differentiation of
MLC. No great difference might be observed by blocking a single
one but we can not deny its role. Our previous experience showed
that no changes were observed by using neutralizing antibody
against some cytokine does not mean this cytokine does not play a
role. For example, in our previous study, neutralization of TGF-b
in the coculture system of splenic stormal cells and mature DC
does not alter the differentiation of maDC. But in others
study, it is reported that culture DC with TGF-b could induce
differentiation. So the detailed effects of TGF-b, M-CSF and
VEGF from endothelia in the CNS on the differentiation of MLC
remains to be investigated.
Furthermore, as to the effects of IL-10 in the immunological
suppressive function of microglia and MLCs, previous studies have
shown that IL-10 play a role in the suppression for immune
response. In our system, though MLC secret high level of
IL10, IL-10 is not the key factor of the inhibitory function by
blocking antibody. According to the same reasons as above, we
can demonstrate IL-10 is not a key factor, but can not deny its
effects in the inhibitory functions. As shown in Figure 6, the
detailed mechanisms of MLC differentiation and its inhibitory
function remain to be investigated.
We rise a hypothesis that in the process of CNS
immunopathology, antigen presenting cells (APC) entering the CNS will
undergo a functional transformation from priming APC to
suppressive APC under the influence of CNS microenvironment.
The transformation is dynamic and related with the status of
disease. The final transformation to suppressive microglia will
suppress the proliferation even induce the apoptosis of
autoreactive T cells and terminate the progress of the disease. The
microglia and microenvironment of CNS make up the immune
barrier of CNS (Figure 6).
Our unpublished data showed that peritoneal macrophages
with stimulatory function can be transformed to inhibitory cells by
the endothelia stroma of spleen, brain and liver. This indicates that
stromal cells have the propensity to induce stimulatory antigen
presenting cells to differentiate to inhibitory ones. Combined with
our previous study[15,24,25], this work might strengthen the
hypothesis that the endothelial cells are important in maintaining
local immune homeostasis by educating the infiltrated
Materials and Methods
C57BL/6(H-2Kb)mice and Balb/c(H-2Kd) mice were
purchased from Vitariver (Beijing, China). OVA323-339 peptide
specific TCR transgenic mice DO11.10(H-2Kd) and EGFP
transgenic mice C57BL/6-TgN(ACTbEGFP)1Osb(H-2Kb)were
obtained from the Jackson Laboratory(Bar Harbor, ME). All mice
were housed and cared according to the approved protocols of the
Tsinghua University Animal Care and Use Committee.
7-amino-actinomycin D (7-AAD) and CFSE were purchased
from Sigma (St Louis, MO). Magnetic beads-conjugated mAbs to
CD4, CD11b, CD11c, CD31, PE were purchased from Miltenyi
Biotec (Bergisch Gladbach, Germany). Fluorescein-conjugated
mAbs to CD4, CD11b, CD11c, CD40, CD80, CD86, CD31, Ia
and isotype control mAbs were purchased from BD Pharmingen
Newborn C57BL/6 mice brain was scissored into pieces and
attached to 24-well plate. After maintained for 2 weeks, the attached
cells were digested and incubated with magnetic beads-conjugated
mAbs to remove the CD11b positive cells and select the CD11b2
CD31+ cells which were used as the CNS endothelial stromal cells.
The CNS mononuclear cells were enriched from perfused brain
of adult C57BL/6 mice according to the method described[9,10].
Then the CD11b positive cells were sorted using FACSAria. The
purity of the sorted cells is about 95%.
Mature DCs were generated from bone marrow cells in the
presence of GM-CSF and IL-4 according to the established
MaDCs derived from C57BL/6 or
C57BL/6-TgN(ACTbEGFP)1Osb mice were respectively seeded on the CNS endothelia
monolayer (50% confluence) in 24-well plate. After 14 days of
coculture, CD11b+ cells were purified using magnetic microbeads
and used as MLCs.
RT-PCR Analysis of iNOS Expression
Total RNA was purified from the endothelial stromal cells using
an RNAfast200 purification kit (RNAfast200, Fastagen Biotech,
Shanghai, China), reverse transcribed and subjected to PCR
amplification using the following primers: forward: 59
ACCACCCTCCTCGTTC 39, reverse: 59
Cell migration was measured in 24-well culture plate with cell
culture inserts, First, 600 ml 50% endothelial stromal cell
supernatant or control RPMI-1640 medium supplemented with
10%FCS was added into wells in triplicate, then 3 mm pore size
insert was placed. The upper chamber of inserts was added with
200 ml monocytes or DC cell suspensions (56104/ml). After
incubation at 37uC in 5% CO2 for 6 hours, the insert was
removed, and the number of cells in the well was counted by flow
Phagocytic Capability and Phenotype Assay
MLCs, microglia and maDCs were incubated with
Alexa488conjuated OVA and the fluorescence intensity was detected with
For analysis of phenotype, MLCs, microglia and maDCs were
blocked with rat serum and 2.4G2 antibody before staining with
Fluorescein-conjugated mAbs. And data were acquired with
Cytokines, Chemokines and NO Measurement
Cytokines and chemokines concentration was assayed by
ELISA (ebioscience). NO production was tested by measuring
Assay for Antigen Presenting Function of Microglia and
According to the method described, CD4 T cells from
DO11.106C57BL/6 F1 hybrid mice were obtained by magnetic
cell sorting and then cocultured with either maDCs or microglia
(or MLCs) for 5 days at a ratio of 1:10 (DCs/T cells, microglia/T
cells, or MLCs/T cells) in 96-well plates (16105 T cells in 200 ml
per well) in the presence of OVA(3232339). Cells were then
double stained with anti-CD4-PE and 7-AAD, and the number of
CD4+ 7-AAD2 live cells was counted with FACSAria. For
inhibition test, microglia or MLCs were added to the DC/T
coculture system in a ratio of 1:1 (microglia:DC).
All experiments were performed at least 3 times. All data
analysis was performed using a 2-tailed Student t test. P value less
than 0.05 was considered as statistically significant.
We thank Ms Q Wan and F Ren for their excellent technical assistance.
We also thank Ms Z Guo and H He for helpful discussion.
Conceived and designed the experiments: XZ MZ. Performed the
experiments: BB WS YJ. Analyzed the data: MZ. Contributed reagents/
materials/analysis tools: BB YJ XL LT CW DC XZ. Wrote the paper: WS.
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