médecine/sciences 2018 ; 34 (focus issue, F1) : 116-20

médecine/sciences Complement protein

C5a enhances the
b-amyloid-induced
> Objective: The dysregulation of neuro-
neuro-inflammatory
inflammation is one of the attributes of the response in microglia
pathogenesis of Alzheimer’s disease (AD). in Alzheimer’s disease
Over-expression of complement proteins co- Xiao-qun An1*, Wei Xi1, Chen-yun Gu1, Xiao Huang2*
localizes with neurofibrillary tangles, thereby
indicating that a complement system may
be involved in neuro-inflammation. Here, we 1
MD, Department of Psychiatry,
report the influence of complement activation Yangpu District Mental Health
Center of Shanghai, 585 Jungong
on the neuro-inflammation using a microglial Road, Shanghai (200090), China.
cell line. 2
MD, Department of
Methods: first, we performed a cytotoxic Psychological Medicine,
assay using the microglial cells BV-2. Second, Zhongshan Hospital, Fudan
University, 180 Fenglin Road,
after treatment of BV-2 cells with Aβ42 and/ Shanghai (200032), China.
or C5a, the anaphylatoxin derived from C5, *The authors contributed
we determined the expression levels of the equally to this work. They are
co-corresponding authors
pro-inflammatory factors TNF-α, IL-1β, and anxion@163.com
IL-6. Finally, we explored whether this neuro- huangxiao320@126.com
inflammatory response was mediated by JAK/ Introduction
STAT3 signaling.
Alzheimer’s disease (AD) is one of the neurodegenerative diseases that
Results: C5a had an enhanced effect on the frequently occur in elderly populations; it is characterized by cognition
neural cell viability of BV-2 cells treated impairment and progressive memory loss [1]. An estimated 60 million
with Aβ42. In addition, C5a also increased people suffer from AD, and approximately 10 million new cases are
the Aβ-induced neuro-inflammatory response, diagnosed each year worldwide [2]. Currently, accumulation of fibril-
and these effects were blocked by the C5aR lary amyloid β (fAβ) surrounding the microglial cells and inducing the
antagonist, PMX205. Finally, we demonstrated inflammatory response is believed to be a pathogenesis of AD [3];
that the neuro-inflammatory responses microglial cells surrounding the senile plaques are further recruited,
and neurotoxic factors (such as IL-1, TNF-α, and IL-6) are released.
induced by Aβ and C5a were mediated through
It aggravates the inflammatory responses, although microglial cells
JAK/STAT3 signaling. By blocking this pathway normally function as macrophages to help eliminate Aβ [4]. Treat-
with an antagonist, AG490, the expression of ment with Aβ oligomer in primary microglial cells can increase the
TNF-α, IL-1β, and IL-6 was alleviated. expressions of IL-1β and TNF-α, which are significantly increased in
Conclusion: The complement protein C5a serum and cerebrospinal fluid of AD patients [5,6]. Afterward, these
could exaggerate the Aβ-induced neuro- inflammatory factors would feed back to the neurons and microglial
inflammatory response in microglia, and C5aR cells, thereby promoting the production of other inflammatory factors.
Hence, a vicious cycle is developed between the microglial cells that
may be a potential therapeutic tool for AD are activated by accumulation of Aβ and the inflammatory mediators
treatment. < released by microglial cells, thereby resulting in the denaturation and
Key words: Alzheimer’s disease; β-amyloid; necrosis of neurons [7,8].
complement system; neuro-inflammatory res- Complement system plays an important role in the identification and
ponse; STAT3. removal of invasive pathogenic microorganisms as one part of self-
defense immune system [9,10]. Misfolded and aggregated proteins
or reactive microglia found in neurodegenerative diseases can acti-
vate the complement pathways [11,12]. C5a and its receptor (C5aR)

­116 m/s, vol. 34 (focus issue, F1), october 2018

https://doi.org/10.1051/medsci/201834f120
 RNA and cDNA synthesis kit were from Takara, Japan;
120 ELISA kits of TNF-α and IL-6 were from eBioscience,
100 USA; AG490 and JNK/STAT3 specific inhibitor were from
80 Sigma, USA; BCA protein assay kit was from Thermo,
60 USA; rabbit anti-p-STAT3 and STAT3 polyclonal anti-
% control

40 bodies were from Cell Signaling Technology, USA; PVDF

REVUES
membrane and ECL kit were from Millipore, Germany.
20
0 Cell culturing
Aβ – + – + + Microglial BV-2 cells were from the Shanghai Cell
C5a – – + + +
Bank of Chinese Academy of Sciences. BV-2 cells were
cultured in a medium containing DMEM supplemented
PMX205 – – – – + with 10% FBS in the 37 °C, 5% CO2 humid atmosphere
incubator.
Figure 1. Cell viability was measured by a CCK8 assay after treatment with Ab42,

SYNTHÈSE
C5a, and PMX205 in BV-2 for 24 h.
CCK-8 proliferation assay
Microglial cells were inoculated onto a 96-well plate
are prominently up-regulated in microglia co-localized with amyloid (105/well). After 12 h, Aβ1-42 oligomers at different
plaques in AD mouse models [13]. Brain tissues of AD patients revea- concentrations were added, and BV cells were cultured
led an increased expression of two C5 receptors, CD88, and C5L2, which for another 24 h. The supernatant was replaced with a
was associated with abundant neurofibrillary tangles when compared fresh culture medium, after which 10 μL CCK-8 reagent
with age-matched counterparts [14]. Continuously activated comple- were added to each well and incubated for 2 h. Sub-
ment system results in excessive production of C5a and subsequently sequently, OD450 values were determined using a micro-
exaggerates the neuro-inflammatory response [15]. In addition, C5a plate reader.
enhances the injury of fibrillary amyloid β to the primary neurons [16).
Hence, blocking the C5a/C5aR signaling activation axis alleviates the Western blot
neuro-inflammatory alterations to AD pathologies. Both C5a-tar- After cell lysis and centrifugation of the cell extracts
geting vaccines and C5a receptor antagonist, PMX205, have shown at 4 °C for 15 min at 12,000 rpm, the supernatant that
improved contextual memory and reduced cerebral amyloid plaque contains the cellular proteins was collected and used in
[17, 18]. However, the underlying mechanisms by which C5a/C5aR are Western-blot experiments. 20 mg of total proteins were
involved in AD pathogenesis have not been yet elucidated. loaded and separated through sodium dodecyl sulfo-
Janus kinase-signal transducer and activator of transcription (JAK- nate polyacrylamide gel electrophoresis (SDS-PAGE)
STAT) has become one of the important regulatory pathways associa- and then transferred onto a PVDF membrane. After
ted with AD pathogenesis [19]. STAT3, a critical nuclear transcription incubation in blocking solution for 2 h, the PVDF mem-
factor in this pathway, is involved in the neuro-inflammatory response brane was probed with primary antibodies to p-STAT3
caused by the activation of microglial cells at the onset of AD, thereby (1:1,000), STAT3 (1:500), and β-actin (1:5,000) at 4 °C
producing multiple inflammatory factors [20]. C5a was previously overnight. After washing with TBST and probing with the
reported to induce STAT3 activation [21]. However, the influence of relevant secondary antibodies, an ECL kit was used to
this effect in the context of AD has not been studied. Therefore, we reveal specific protein expression.
used an Aβ oligomer to induce the in vitro inflammatory response of
microglial cells and investigated the role of C5a/C5aR pathway in this qRT-PCR
process [22]. We also tried to explore the effects of C5a-induced STAT3 Total RNA was extracted using Trizol according to the
activation on Aβ oligomer-induced neuro-inflammatory response. manufacturer’s instructions. Then, 500 ng total RNA
were reverse transcribed into cDNA. PCR conditions:
Materials and methods 37 °C for 15 min, 85 °C for 5 s, and termination at 4 °C
in a 10 μL system. The product of cDNA was added into
Reagents the RT-PCR reaction system as template for the fol-
Cell culture medium high-glucose Dulbecco’s modified eagle medium lowing procedures: initial denaturation for 30 s at 95 °C;
(DMEM) and fetal bovine serum (FBS) were from Gibco, USA; 0.25% PCR reaction for 5 s at 95 °C and 30 s at 60 °C followed
trypsin and penicillin-streptomycin were from Hyclone, USA; CCK-8 by 40 cycles; and termination at 95 °C for 15 s, 60 °C
kit was from Dojindo, Japan; Aβ1-42 peptides, hexafluoroisopropanol for 1 min, and 95 °C for 15 s in 20 μL system. The pairs
(HFIP) and DMSO were from Sigma, USA; Trizol for extraction of total of primer sequences were: STAT-3: (Sense) 5’-TCGTGG

m/s, vol. 34 (focus issue, F1), october 2018 ­117

 A
7
p-STAT3
Relative mRNA expression
6
5
4
(Folds)

TNF-α STAT3
3
2 IL-1β
1 IL-6 Aβ – + – + +
0
Aβ – + – + + C5a – – + + +
C5a – – + + + PMX205 – – – – +
PMX205 – – – – + Figure 3. Phosphorylation of STAT3 was detected in both control
and treatment groups.

B
2500
Results
Cytokines production (pg/mL)

2000
1500 C5a enhances the cytotoxic effect of Ab42 on BV-2 cells
Aβ42 and C5a inhibited the cell viability of microglia.
1000 TNF-α
Thus, we tried to examine the effects of co-treatment
IL-6
500 with Aβ42 and C5a on microglia. After a 24 h treatment,
0 the cell viability of BV-2 was tested with a CCK8 assay.
Aβ – + – + + As shown in Figure 1, the cell number was reduced by
about 50% in the Aβ42 and C5a co-treatment group
C5a – – + + + compared with Aβ42 alone group. Cell growth was res-
PMX205 – – – – + tored after adding PMX205.

Figure 2. Effects of C5a on the inflammatory response stimulated by Ab42. A. C5a raises the neuro-inflammatory response to Ab42
mRNA expression levels of TNF-α, IL-1β, and IL-6 were detected by qRT-PCR To explore the role of C5a on the inflammatory response
after cells were treated with Aβ42, C5a, and PMX205. B. Levels of TNF-α and IL-6 in BV-2 cells after exposure to Aβ42, BV-2 cells were
in cell culture medium were measured by ELISA. incubated with Aβ42, C5a, Aβ42 plus C5a, or PMX205 for
24 h. The production of inflammatory factors, TNF-α,
IL-1β, and IL-6, were analyzed. As shown in Figure 2A,
AGCTGTTCAGTTCAGAAAC-3’, (Antisense) 5’-GGAAATTTGACCAGCAA- the expression of these pro-inflammatory molecules
CCT-3’; IL-1β: (Sense) 5’-GGGCCTCAAAGGAAAGAATC-3’, (Antisense) increased in both Aβ42 and C5a groups and was further
5’-TACCAGTTGG GGAACTCTGC-3’; TNF-α: (Sense) 5’-TATGGCTCAGGGTC up-regulated in Aβ42 plus C5a group, whereas their
CAACTC-3’, (Antisense) 5’-TCCCTTTGCAGAACTCAGG-3’; β-actin: (Sense) expression was substantially reversed in Aβ 42 plus
5’-GTGCTATGTTGCTCTAGA CTTCG-3’, (Antisense) 5’-ATGCCACAGGATTC- PMX205 group. We further measured the secreted TNF-α
CATACC-3’. All primers were synthesized by Sangon Co., Ltd Biotech and IL-6 with ELISA kits. Similarly, more TNF-α and IL-6
(Shanghai, PR China). The transcription level of target gene was eva- were produced in the Aβ42 plus C5a group than in the
luated using 2-△△ct. Aβ42 or C5a alone group and were decreased in the Aβ42
plus PMX205 group (Figure 2B).
Enzyme-linked immunosorbent assay (ELISA)
After treatment, cell supernatants were collected to quantify the C5a enhances the Ab42-induced activation of STAT3
protein expression levels of IL-6 and TNF-α using ELISA detection kits in BV-2 cells
according to the manufacturer’s instructions. Next, we examined whether STAT3 was involved in the
stimulation process of Aβ42 and C5a in BV-2 cells.
Statistical analysis Figure 3 shows the increase in the phosphorylation
Statistical analysis was performed using the SPSS 19.0 software. In the of STAT3 after treatment with either Aβ42 or C5a. This
present study, data are presented as mean ± SEM. Single-factor ANOVA increase was further reinforced in the Aβ42 and C5a
was applied to compare multiple groups. t test was adopted for paired combination group. As expected, blocking C5a with
comparison. P<0.05 was set as the statistical significance. PMX205 reduced the activation of STAT3.

­118 m/s, vol. 34 (focus issue, F1), october 2018

 C5aR1, one of two receptors of C5a, is expressed on
A 7 the surface of primary microglia isolated from wild-
Relative mRNA expression
6 type mice when compared with C5aR1 knockout mice.
(Folds)
5 A previous experiment demonstrated that C5aR1+ cells
4 TNF-α surrounded Aβ plaques in AD mouse models [13],
IL-1β thereby suggesting that C5a/C5aR plays a role in the
3

REVUES
IL-6 pathogenesis of AD. Other studies have reported that
2
the levels of complement components increase in an
1 age-dependent manner, thereby subsequently being
0 able to activate more strongly complement pathways
Aβ – + – + +
in neurons and microglia [22]. This dysregulation of
C5a – – + + + complement cascade enhances the neuro-inflammatory
PMX205 – – – – + response to fibrillary Aβ plaques in AD [23]. Hence,
approaches to block C5a/C5aR activation are hypothe-
B sized to help preventing neural damage and cognitive

SYNTHÈSE
decline. In our study, we confirmed that utilization of
C5aR1 antagonist, PMX205, restored cell viability to
3000 some extent. Moreover, blocking the C5a/C5aR interac-
Cytokines production (pg/mL)

2500 tion had no influence on other complement proteins,

2000 such as C1q, C3, and C4 [13, 24], thereby suggesting
1500 that the benefit of part of the complement system is
TNF-α preserved.
1000
IL-6 Chronic inflammatory response induced by persistently
500
activated microglia is considered one of the major
0 pathogenesis of AD. C5a, a pro-inflammatory factor, is
Aβ – + – + + actively produced after complement activation induced
C5a – – + + + by Aβ deposits, which is one of the major mechanisms
PMX205 – – – – + for microglia activation [14, 25]. The activation of
microglia and the activation of the complement system
further induces neuro-inflammation [23, 26]. Constant
Figure 4. Effects of the pharmacological modulation of STAT3 on inflammatory
production of pro-inflammatory and complement com-
response stimulated by Ab42 and C5a. A. mRNA expression levels of TNF-α,
IL-1β, and IL-6 were detected by qRT-PCR after cells were exposed to Aβ42, C5a,
ponents enhances the release of amyloid peptides [27,
and AG490. B. Levels of TNF-α and IL-6 in cell culture medium were measured
28]. This self-sustaining neuro-inflammation loop
by ELISA.
between the activated microglia, complement sys-
tem, and Aβ plaques ultimately results in the loss of
synapses and the decline of cognitive function in AD
The neuro-inflammatory response to Ab42 and C5a is mediated patients [29, 30]. In the present study, we demonstra-
through STAT3 activation ted that targeting C5a and prohibiting its receptor C5aR
AG490, a STAT3 inhibitor, was used to investigate whether STAT3 acti- obviously suppressed the production of pro-inflamma-
vation mediates the neuro-inflammatory response to Aβ42 and C5a. tory factors induced by Aβ oligomers; this finding was
The production of TNF-α, IL-1β, and IL-6 was reduced after treatment consistent with a previous suggestion that C5aR could
with AG490 in cells exposed to Aβ42 and C5a (Figure 4A). Moreover, less be a promising therapeutic target for AD.
inflammatory factors were produced when the activation of STAT3 was We further demonstrated that the down-regulation
blocked (Figure 4B). of pro-inflammatory factors via treatment with C5aR
antagonist was mediated through JAK/STAT3 signaling.
Discussion STAT3, an important nuclear transcription factor of STAT
family, mediates the signal transduction of multiple
In the present study, C5a aggravated the cytotoxic effect induced cytokines into the nucleus, thereby subsequently affec-
by Aβ42 in BV-2 cells, consistent with previous findings, in which C5a ting the transcription of target genes and regulating
resulted in less Aβ42-induced damage to primary neurons isolated from cell function. Increasing evidence supports that the
C5a receptor knockout (C5aR1KO) mice [16]. Moreover, C5a enhanced dysregulation of STAT3 is associated with the chronic
the neuro-inflammatory response stimulated by Aβ42 in BV-2 cells. inflammatory injuries in AD [31]. STAT3 is highly acti-

m/s, vol. 34 (focus issue, F1), october 2018 ­119

 17. Landlinger C, Oberleitner L, Gruber P, Noiges B, Yatsyk K, Santic R, et al.
vated in brain tissue in AD mouse model [32]. In addition, STAT3 is Active immunization against complement factor C5a: a new therapeutic
involved in regulating the transcription of multiple inflammatory fac- approach for Alzheimer‘s disease. J Neuroinflammation 2015; 12: 150.
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effective alternative for AD treatment. Moreover, these results may Stat3 activation in cell-cell interaction between B-cell lymphoma and
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Dr. Huang was financially supported by an educational grant from Zhongshan Hospital, increasing vulnerability to cognitive decline and neurodegeneration: a
Fudan University and Dr. An was financially supported by an grant from Yangpu District microarray study. J Neuroinflammation 2012; 9: 179.
Mental Health Center of Shanghai. The authors have no potential conflict of interests. 24. Wyss-Coray T and Rogers J. Inflammation in Alzheimer disease-a brief review
of the basic science and clinical literature. Cold Spring Harb Perspect Med
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