Acta Biochim Biophys Sin, 2021, 53(9), 1154–1165

https://doi.org/10.1093/abbs/gmab094
Advance Access Publication Date: 6 August 2021
Original Article

Original Article

Evaluation of the immunomodulatory effects of

C9-13-CPs in macrophages
Zimeng Xue, Jianbo Zhu, Xia Wang, Chunlei Yang, and Zhengwei Fu*

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College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China
*Correspondence address. Tel: +86-571-88320599; E-mail: azwfu@zjut.edu.cn
Received 16 December 2020; Editorial Decision 14 May 2021

Abstract
Short-chain chlorinated paraffins (SCCPs) have been listed as a new class of persistent organic pol-
lutants by the Stockholm Convention. SCCPs exhibit carcinogenic-, endocrine-, and metabolism-
disrupting effects. However, the knowledge of the immunomodulatory effects of SCCPs and their
underlying mechanisms, especially in specific immune cells, remains limited. In addition to SCCPs,
C9-13 -CPs have also been detected in humans. In this study, murine RAW264.7 macrophages were
exposed to C9-13 -CPs at environmentally relevant concentrations to investigate whether or how
C9-13 -CPs exhibit immunomodulatory effects. The results showed that the exposure of RAW264.7
cells to C9-13 -CPs increased cell viability, as assayed by MTT analysis at 490 nm, and also promoted
cell proliferation, as indicated by EdU uptake assay, which was measured at excitation and emis-
sion wavelengths of 488 and 512 nm, respectively. In addition, exposure to C9-13 -CPs not only led
to elevated ATP level and intracellular Ca2+ level but also caused AMPK signaling activation and
NF-κB signaling inhibition. Moreover, molecular docking showed that the β2 -AR receptor could
bind to C9-13 -CPs. Taken together, these results suggest that the immune dysfunction of RAW264.7
cells caused by C9-13 -CPs is closely related to the β2 -AR/AMPK/NF-κB signaling axis.
Key words: SCCPs, C9-13 -CPs, RAW264.7 cells, immunotoxicity, immunomodulatory cytokines

Introduction measured plasma concentrations of SCCPs collected from a general

Short-chain chlorinated paraffins (SCCPs) have been widely used as
plasticizers or flame retardants in plastics, rubber, sealants, paints,
textiles, and metal cutting fluids [1]. Due to their properties of
toxicity, persistence, long-range transport, and bioaccumulation,
SCCPs have been listed as a new class of persistent organic pollu-
tants (POPs) (UNEP, 2017) [2]. SCCPs have been widely detected
in the environment, including air, water, sediments, organisms, and
human diets. For example, the median level of SCCPs in indoor air
samples collected from Norway was 128 ng/m3 [3]. The total con-
centrations of SCCPs ranged from 15 to 1640 ng/l and from not
detected (ND) to 2020 ng/g in river water and sediment samples
collected in China in 2016, respectively [4]. The SCCP concentra-
tions in common barbell barbus collected from France in 2011 were
within the range of 0.3–10.6 ng/g wet weight (ww) [5]. The lev-
els of SCCPs in home-produced eggs collected in 2013 and 2016
from an electronic waste site in South China varied from 477 to
111,000 mg/g lipid weight (lw) [6]. Therefore, these observations
suggest that SCCP contamination is a worldwide problem. SCCPs
can pose exposure risk to humans via their breathing and diet. The
population in China in 2015 ranged from ND to 203 ng/g ww, with
an average value of 32 ng/g ww [7]. The concentration ranges of
SCCPs in paired maternal and cord serum collected in Beijing, China,
were 21.7–373 ng/g ww and 8.51–107 ng/g ww, respectively; in
addition, SCCPs were also detected in serum samples, with relative
abundances ranging from 4.35% to 28.4% [8]. The median SCCP
concentration in UK human milk samples collected between 2001
and 2002 was 180 ng/g lw, with a range of 49 to 820 ng/g lw [9].
Due to the abovementioned distribution, attention has been
paid to the biological toxicities of SCCPs, which are linked
to carcinogenic, endocrine-, and metabolism-disrupting effects.
To name only a few, liver and renal tumors were induced
in mice and rats by exposure to a commercial SCCP product,
Chlorowax 500 C, for 2 years; exposure to SCCPs significantly
decreased the circulating free thyroxine (T4) and triiodothyronine
(T3) levels in rats [10]; SCCPs caused metabolism disturbance
in human hepatoblastoma cell line HepG2, including aug-
mentation of β-oxidation, glutamate metabolism and the urea
cycle [11].

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Immunomodulatory effects of C9-13 -CPs in macrophages
The immune system is pivotal for human health and is a target
of many xenobiotics [12–14]. SCCPs can come into contact with the
immune system via the bloodstream and are potentially immuno-
toxic. However, limited studies have reported the immunomod-
ulatory effects of SCCPs. Our previous study demonstrated that
exposure to C9-13 -CPs elevated blood immune cell counts and spleen
weight in mice [15]. Nevertheless, the investigation of the immuno-
toxicity of SCCPs and its mechanism, especially in specific immune
cells, is still limited. Therefore, it is necessary to evaluate and study
the immunotoxicity of C9-13 -CPs and their underlying mechanisms
[2].
To investigate the immunomodulatory effects of C9-13 -CPs in
murine macrophages, RAW264.7 cells were exposed to C9-13 -CPs
at environmentally relevant concentrations. After exposure, cell
viability, cell proliferation, the mRNA expression of M1 and
M2 phenotype-related cytokines, antigen presentation-related genes,
and the activation of AMPK signaling were determined. The data
obtained in the present study provided a more comprehensive under-
standing of health risks caused by SCCPs.
Materials and Methods
Chemicals and cell culture
The mean detection level for SCCPs in Chinese blood samples was
32 ng/g bw and the maximum detection level was 203 ng/g bw [8].
Therefore, the in vitro exposure concentrations of C9-13 -CPs were
set at 1, 10 and 100 µg/l. To facilitate the development of sub-
sequent experiments, C9-13 -CPs (C9 :C10 :C11 :C12 :C13 = 1:1:1:1:1,
chlorine content = 52%) was purchased from Jinjinle Chemical
Co., Ltd (Shanghai, China) and dissolved in dimethylsulfoxide
(DMSO; Amresco, Englewood, USA) at 100 mg/l as a stock solu-
tion. Compound C (CC) was purchased from MedChemExpress
(Shanghai, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-
tetrazolium bromide (MTT) was purchased from Beyotime Biotech-
nology (Shanghai, China) and dissolved in phosphate-buffered
saline (PBS) at 5 mg/ml as a stock solution. EdU Apollo®488
In Vitro Imaging Kit was purchased from RiboBio Biotechnology
(Guangzhou, China). TRIzol reagent was purchased from Takara
(Dalian, China). Reverse Transcriptase Kit and SYBR Green PCR
Reagent Kit were purchased from Toyobo (Tokyo, Japan). Fetal
bovine serum (FBS) was purchased from Gibco (Gaithersburg, USA).
Mouse ATP enzyme-linked immunosorbent assay (ELISA) kit was
purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd
(Shanghai, China).
BCA protein qualitative kits, RIPA lysis buffer, mouse anti-
β-actin monoclonal antibody, and horseradish peroxidase (HRP)-
labeled anti-mouse/rabbit IgG secondary antibodies were purchased
from Multisciences (Hangzhou, China). Rabbit anti-phospho-
AMPK (Thr172) (p-AMPK) and AMPK monoclonal antibodies
were purchased from Cell Signaling Technology (Danvers, USA).
Enhanced chemiluminescence (ECL) detection kit was purchased
from 4a Biotech (Beijing, China).
RAW264.7 cells (American Type Culture Collection, Manassas,
USA) were cultured in RPMI 1640 medium (HyClone, Logan, USA)
supplemented with 10% FBS, 100 µg/ml penicillin and 100 U/ml
treptomycin at 37◦ C in humidified air and 5% CO2 .
MTT assay
RAW264.7 cells were seeded in 96-well plates at a density of 4000
cells/well and exposed to 1, 10, and 100 µg/l C9-13 -CPs for 12 or
1155
24 h. The control group (Con) received only 0.1% (v/v) DMSO vehi-
cle. After exposure, the medium was replaced by fresh complete
medium containing 5 mg/ml MTT and incubated for an additional
4 h. Afterwards, the medium was discarded, and the cells were
incubated with 100 µl of DMSO. The absorbance at 490 nm was
measured using a microplate reader (Bioteke, Winooski, USA). Rel-
ative cell viability was expressed as the ratio of absorbance of each
group to the control group. All experiments were conducted in
triplicate.
Cell proliferation assay
RAW264.7 cells were seeded into a 6-well plate at a density of 4000
cells/well and exposed to 1, 10, and 100 µg/l C9-13 -CPs for 24 h.
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The Con only received 0.1% (v/v) DMSO vehicle. In another exper-
iment, to study how CC, a specific AMPK inhibitor, reverses the
proliferation of macrophages promoted by C9-13 -CPs, 1 µM CC was
added to the medium 12 h before the end of C9-13 -CPs exposure. The
medium was replaced by fresh medium and incubated for 2 h in the
CC/C9-13 -CPs + CC group. After exposure, the EdU Apollo®488 In
Vitro Imaging Kit was used to determine cell proliferation. In brief,
the medium was replaced by fresh medium containing 50 µM EdU,
and the cells were incubated for 1 h. The cells were then collected,
fixed with 4% paraformaldehyde for 30 min, and neutralized with
2 mg/ml glycine for 5 min. After being washed with PBS, the cells
were incubated with 0.5% Triton X-100 for 10 min and washed once
with PBS. Then, the cells were resuspended in 500 µl of 488 staining
solution and incubated for 10 min in the dark. After the 488 stain-
ing solution was discarded, the cells were washed twice with 0.5%
Triton X-100. The cells were then resuspended in PBS and examined
with a fluorescence microscope (Becton, Dickinson and Company,
Franklin Lakes, USA) at excitation and emission wavelengths of 488
and 512 nm, respectively. All experiments were repeated three times.
Reverse transcription-quantitative real-time
polymerase chain reaction analysis
RAW264.7 cells were seeded in 6-well plates at a density of 4000
cells/well and exposed to 1, 10, and 100 µg/l C9-13 -CPs for 24 h.
The Con received only 0.1% (v/v) DMSO vehicle. Following expo-
sure, total RNA was extracted using TRIzol reagent, and 1 µg of
RNA was subject to cDNA synthesis using Reverse Transcriptase
Kits according to the manufacturer’s instructions (50◦ C for 15 min
and 85◦ C for 5 s). Quantitative reverse transcription PCR analy-
sis (RT-qPCR) was performed on an Eppendorf MasterCycler® ep
RealPlex4 (Eppendorf, Wesseling-Berzdorf, Germany) using SYBR
Green PCR Reagent Kit as previously described [16]. 18S rRNA was
used as the housekeeping gene to normalize the data. The primers
used in the study were shown in Table 1. All experiments were
conducted in triplicate.
Cytokine detection assay
RAW264.7 cells were seeded in a 6-well cell culture plate at a density
of 3× 105 cells per well and exposed to 1, 10, and 100 µg/l C9-13 -CPs
for 24 h. The Con received only 0.1% (v/v) DMSO vehicle. After
exposure, cells were centrifuged at 300 g for 10 min to collect the cell
supernatant. The levels of cytokines (IL-6, IL-1β, IL-4, and TNF-α)
in the cell supernatants were measured using commercial ELISA kits
according to the manufacturer’s instructions. All experiments were
conducted in triplicate.
1156
Table 1. Sequences of primer pairs used in the real-time quantita-
tive polymerase chain reaction reactions
Gene Primer sequence
H2-K1 Forward 5′ -GCGAGGGTGGCTCTCACACG-3′
Reverse 5′ -TCAGGGTGAGGGGCTCAGGC-3′
H2-Aa Forward 5′ -TGGGCACCATCTTCATCATTC-3′
Reverse 5′ -GGTCACCCAGCACACCACTT-3′
CD86 Forward 5′ -TGTTTCCGTGGAGACGCAAG-3′
Reverse 5′ -TTGAGCCTTTGTAAATGGGCA-3′
Il-1β Forward 5′ -GCAACTGTTCCTGAACTCAACT-3′
Reverse 5′ -ATCTTTTGGGGTCCGTCAACT-3′
Tnf-α Forward 5′ -GCCCAAGGCGCCACATCTCC-3′
Reverse 5′ -TTGGGGACCGATCACCCCGA-3′
Il-6 Forward 5′ -TAGTCCTTCCTACCCCAATTTCC-3′
Reverse 5′ -TTGGTCCTTAGCCACTCCTTC-3′
Sirt1 Forward 5′ -TGCCTCCTGAAGGCTGGGATTACAT-3′
Reverse 5′ -GGTGGTGCAAACACTGCTCCTC-3′
Il-4 Forward 5′ -TTACCTTGACGGTGTTCATACAG-3′
Reverse 5′ -TCTGCTCCTATTCGACCACTATC-3′
Il-13 Forward 5′ -ACCGAAATGTTGATAGCGACAG-3′
Reverse 5′ -ACAATGCTCTGACAAATGCGTA-3′
Arg1 Forward 5′ -CTCCAAGCCAAAGTCCTTAGAG-3′
Reverse 5′ -AGGAGCTGTCATTAGGGACATC-3′
18S Forward 5′ -CGAACGTCTGCCCTATCAACTT-3′
Reverse 5′ -CCGGAATCGAACCCTGATT-3′
Cd163 Forward 5′ -CTGGCTGTCCTGTCAAGGCT-3′
Reverse 5′ -GGGTCATTCAGAGGCACACTG-3′
iNos Forward 5′ -AATCTTGGAGCGAGTTGTGG-3′
Reverse 5′ -CAGGAAGTAGGTGAGGGCTTG-3′
Intracellular calcium ion detection
RAW264.7 cells were seeded in 6-well cell culture plates at 3 × 105
cells per well and exposed to 100 µg/l C9-13 -CPs for 4 h. For the
positive controls, cells were treated with 2 µg/ml ionomycin. Before
the end of the treatment, 0.5 µM Fluo-3/AM (MUTISCIENCES,
Hangzhou, China) was added to each well, incubated at 37◦ C
for 15–60 min to load the fluorescent probe, and then washed 1–
2 times with PBS. After washing, cells were incubated for another
20–30 minutes to ensure that Fluo-3/AM was completely trans-
formed into Fluo-3 in the cell. Finally, flow cytometric analysis was
conducted with a BD flow cytometer (Becton, Dickinson and Com-
pany) using excitation and emission wavelengths of 506 and 526 nm,
respectively.
Western blot analysis
RAW264.7 cells were seeded in 6-well cell culture plates at 3 × 105
cells per well and exposed to 1, 10, and 100 µg/l C9-13 -CPs for
24 h. The Con received only 0.1% (v/v) DMSO vehicle. After expo-
sure, the cells were washed with PBS and lysed using RIPA lysis
buffer. Protein concentrations were determined using BCA pro-
tein qualitative kits according to the manufacturer’s instructions.
Whole cell lysates containing 20 µg of protein were subject to 8%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) and transferred to polyvinylidene fluoride (PVDF) mem-
branes (Millipore, Billerica, USA). PVDF membranes were blocked
with TBST containing 5% non-fat milk for 1 h and then blot-
ted with antibodies against β-actin or p-AMPK overnight at 4◦ C.
After extensive wash with TBST, membranes were further incubated
with HRP-labeled secondary antibody for 1 h at room temperature.
Finally, membranes were visualized using an ECL detection kit and
Immunomodulatory effects of C9-13 -CPs in macrophages
exposed with a Luminescent Imaging Workstation (Tanon, Shang-
hai, China). The relative protein content was quantified by ImageJ
software. All experiments were conducted in triplicate.
Molecular docking
The crystal structure of the β2 -AR receptor (PDB code: 6
MXT) was obtained from the RCSB PDB (https://www.rcsb.org/
pdb). C9-13 -CP homologues were obtained from PubChem (http://
pubchem.ncbi.nlm.nih.gov/) as 3D sdf text, and they were converted
to pdbqt text by PyMOL (http://www.pymol.org, Schrödinger, Inc,
NY, USA) for use as input in AutoDock. AutoDock Vina (http://
vina.scripps.edu/) and MGLTools (The Scripps Research Institute)
(http://mgltools.scripps.edu/) were used to perform docking cal-
culations [17]. The output from AutoDock was rendered using
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PyMOL.
Statistical analysis
Data are shown as the mean ± SD. Statistical significance was cal-
culated by one-way analysis of variance (ANOVA) using Graph-
Pad 7.0. A statistical probability of P<0.05 was considered
significant.
Results
C9-13 -CPs increased the cell viability and proliferation
of macrophages
To investigate the toxicity of C9-13 -CPs on RAW264.7 cells, we
first analyzed the effect of C9-13 -CPs on the viability and prolifer-
ation ability of RAW264.7 cells. Exposure of RAW264.7 cells to
1, 10, and 100 µg/l C9-13 -CPs for 12 h did not affect cell viability
(Fig. 1A), whereas exposure for 24 h caused 8%± 3%, 16%± 3%,
and 18%± 3% increases in relative cell viability at 1, 10, and
100 µg/l, respectively, compared with the control (Fig. 1B). In addi-
tion, exposure to 100 µg/l C9-13 -CPs significantly promoted cell pro-
liferation in RAW264.7 cells, and compared with the Con, the pro-
portion of EdU-positive cells was increased significantly (Fig. 1C,D).
These data indicated that C9-13 -CPs increased cell viability and pro-
liferation of RAW264.7 cells in time- and concentration-dependent
manners.
C9-13 -CPs caused M2 polarization in macrophages
To understand the effect of C9-13 -CPs on inflammatory factor home-
ostasis, we explored the effect of C9-13 -CPs treatment on the expres-
sion of inflammatory factor-related genes in macrophages under dif-
ferent conditions. Exposure to C9-13 -CPs reduced the mRNA levels
of M1 phenotype-related cytokines, being 73%± 4%, 91%± 4%,
and 89%± 4% lower for Il-6 at 1, 10, and 100 µg/l, 26%± 6% and
27%± 6% lower for Tnf-α at 10 and 100 µg/l, and 56%± 14%
lower for iNos at 1 µg/l compared with the corresponding con-
trols, respectively (Fig. 2A,B,J), indicating that C9-13 -CPs reduced
M1 phenotype-related cytokine expression in RAW264.7 cells in a
concentration-dependent manner. In contrast, exposure to 100 µg/l
C9-13 -CPs improved the mRNA levels of M2 phenotype-related
cytokines, with 74%± 29% and 108%± 32% increase at the mRNA
levels of Il-4 and Il-13, respectively, compared with the corre-
sponding controls (Fig. 2D,E), indicating that high concentration of
C9-13 -CPs increased the M2 phenotype-related cytokine expression
in RAW264.7 cells. Exposure to C9-13 -CPs did not affect the mRNA
levels of Il-1β (an M1 phenotype-related cytokine), Arg1 and Cd163
(an M2 phenotype-related cytokine) (Fig. 2C,F,K).
Immunomodulatory effects of C9-13 -CPs in macrophages 1157

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Figure 1. Effect of C9-13 -CPs on the viability and proliferation of RAW264.7 cells After treatment, cell viability (A) and proliferation (B) were measured by MTT
assay and cell proliferation assay, respectively. The green fluorescence marker is the newly synthesized DNA of EdU-detected cells, i.e. cells in proliferation, and
the blue fluorescence marker is the nuclei of Hoechst 33,342-detected cells, i.e. the vast majority of cells in the assay. EdU-positive cell rates were determined
from five independent experiments (C-D). *P<0.05, **P<0.01, and ***P<0.001 vs control.

Meanwhile, exposure to C9-13 -CPs for 24 h significantly reduced
the secreted levels of M1 phenotype-related cytokines, being
14%± 4% lower at 100 µg/l for IL-6 (Fig. 2G), and 49%± 12% and
52%± 12% lower at 10 and 100 µg/l for TNF-α (Fig. 2H), respec-
tively, when compared with their corresponding controls. How-
ever, exposure to 100 µg/l C9-13 -CPs for 24 h significantly increased
the secreted level of IL-4, an M2 phenotype-related cytokine, by
19%± 8% (Fig. 2M), compared with the control. Exposure to
C9-13 -CPs for 24 h did not affect the secreted level of IL-1β (Fig. 2L).
C9-13 -CPs upregulated the expression of antigen
presentation-related genes in RAW264.7 cells
In order to investigate the effect of C9-13 -CPs on the antigen pre-
sentation function of RAW264.7 cells, we analyzed the effect of
C9-13 -CPs on the expressions of genes related to antigen presenta-
tion in RAW264.7 cells. After exposure to 10 and 100 µg/l C9-13 -
CPs for 24 h, the H2-K1 mRNA levels were significantly elevated
by 94%± 37% and 93%± 37%, respectively, compared with the
control (Fig. 3A). In addition, significant increases in the mRNA
levels of H2-Aa and Cd86 were only observed by exposure to
100 µg/l C9-13 -CPs for 24 h, being 102%± 36% and 81%± 25%
higher, respectively, compared with their corresponding controls
(Fig. 3B,C). These data indicated that C9-13 -CPs could interfere in
antigen presentation.
C9-13 -CPs caused NF-κB inhibition in macrophages
To further study the possible toxicity mechanism, the effects of
C9-13 -CPs exposure on the NF-κB pathway in RAW264.7 cells
were investigated. Exposure to 100 µg/l C9-13 -CPs for 24 h sig-
nificantly downregulated the expression of phosphorylated p-p65
in RAW264.7 cells (Fig. 4A,B) by 31%± 16% compared with
the control, while it significantly upregulated Sirt1 expression
in RAW264.7 cells by 62%± 23% compared with the controls
(Fig. 4C). These data indicated that exposure to C9-13 -CPs caused
a decrease in NF-κB activity and promoted the polarization of
macrophages to M2 type. At the same time, it is speculated that the
decline of NF-κB activity is caused by Sirt1 activation and its gene
silencing.
C9-13 -CPs led to AMPK activation in macrophages
AMPK also plays an important role in the regulation of inflam-
mation in immune cells (e.g. macrophages). To further investi-
gate the regulatory mechanism upstream of NF-κB, the effect of
C9-13 -CPs exposure on AMPK expression in RAW264.7 cells was
explored. Exposure to 100 µg/l C9-13 -CPs for 24 h caused a signif-
icant elevation in intracellular ATP level in RAW264.7 cells (Fig.
5A). Meanwhile, exposure to C9-13 -CPs caused AMPK activation
in RAW264.7 cells, being 83%± 26% higher in terms of rela-
tive change in the ratio of p-AMPK to β-actin at 100 µg/l, and
54%± 17% and 71%± 17% higher in terms of relative change in the
ratio of p-AMPK to AMPK at 10 and 100 µg/l, respectively, when
compared with their corresponding controls (Fig. 5B–E), indicat-
ing that C9-13 -CPs elevated the p-AMPK expression of RAW264.7
cells in a concentration-dependent manner. These data indicated
that exposure of C9-13 -CPs to RAW264.7 caused a decrease in the
ATP level, which may be a trigger for the increase in p-AMPK
expression.
Compound C reversed C9-13 -CPs-promoted
macrophage proliferation
To further explore the potential regulatory role of AMPK on
macrophages, we investigated the effect of 24 h of co-exposure with
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Figure 2. Effect of C9-13 -CPs on the expression of M1 and M2 phenotype-related cytokines in RAW264.7 cells After treatment, reverse transcription-quantitative
real-time polymerase chain reaction was conducted to determine the mRNA levels of M1 phenotype-related cytokine genes, including Il-6 (A), Tnf-α (B), iNos
(J), and Il-1β (C), and the M2 phenotype-related cytokine genes Il-4 (D), Il-13 (E), Cd163 (K), and Arg1 (F). ELISA was conducted to determine the levels of M1
phenotype-related cytokines, including IL-6 (G), IL-1β (L), and TNF-α (H), and the M2 phenotype-related cytokine IL-4 (M). Data are shown as the mean ± SD from
three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 vs control.

Figure 3. Effect of C9-13 -CPs on the expression of antigen presentation-related genes in RAW264.7 cells After treatment, reverse transcription-quantitative real-
time polymerase chain reaction was conducted to determine the mRNA levels of antigen presentation-related genes of RAW264.7 cells, including H2-K1 (A),
H2-Aa (B), and Cd86 (C). Data are shown as the mean ± SD from three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 vs control.

100 µg/l C9-13 -CPs and 1 µM compound C (CC, a specific AMPK
inhibitor) on the proliferation of RAW264.7 cells, with CC being
added 12 h before the end of exposure. As shown in Fig. 6A,B,
compared with that of the Con, the proliferative ability of the CC
group was decreased significantly. Similar to the results in the pre-
vious section, the proliferative ability of the C9-13 -CPs group was
increased significantly, while the co-exposure of C9-13 -CPs with CC
significantly reduced the proportion of EdU-positive cells, resulting
in no significant difference compared with that of the Con. These
data indicated that co-exposure with AMPK inhibitors reversed, to
some extent, the uncontrolled cell proliferation caused by exposure
to C9-13 -CPs.
Compound C reversed the effect of C9-13 -CPs on the
gene expression of AMPK and inflammatory factors in
macrophages
To further investigate the potential regulatory role of AMPK on
macrophages, we investigated the effect of 24 h of co-exposure with
100 µg/l C9-13 -CPs and CC on the inflammatory factor gene expres-
sion in RAW264.7 cells, with CC being added 12 h before the end
of exposure. Exposure to 100 µg/l C9-13 -CPs alone increased the
expression level of p-AMPK, while the combined exposure of C9-13 -
CPs with CC decreased the expression level of p-AMPK (Fig. 7A).
Similarly, exposure to 100 µg/l C9-13 -CPs resulted in a decrease in
the expression of Il-6 transcripts and an increase in the expression
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Figure 4. Effect of C9-13 -CPs on the protein expression of NF-κB in RAW264.7 cells After treatment, (A–B) the protein levels of p-p65 were determined by western
blot analysis, and (C) the mRNA levels of Sirt1 were determined by reverse transcription-quantitative real-time polymerase chain reaction. Data are shown as
the mean ± SD from three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 vs control.

Figure 5. Effect of C9-13 -CPs on the protein expression of AMPK in RAW264.7 cells After treatment (A) ATP level was determined by ELISA and (B) representative
western blot images for p-AMPK, AMPK, and the loading control β-actin. (C) The ratio of p-AMPK to β-actin and the relative expression level of p-AMPK were
determined by the gray-level semiquantitative method. (D) The ratio of AMPK to β-actin and the relative expression of AMPK were determined by the gray-level
semiquantitative method. (E) The ratio of p-AMPK to AMPK. Data are shown as the mean ± SD from three independent experiments. *P<0.05, **P<0.01, and
***P<0.001 vs control.

of Il-13, while the combined exposure of C9-13 -CPs with CC signif- level of Il-1β was significantly downregulated when CC alone was
icantly reversed these effects of C9-13 -CPs. However, CC altered the used for exposure, up to three times lower, because CC itself had a
mRNA level of Il-4 following exposure to C9-13 -CPs. The expression greater influence on the expression of Il-1β (Fig. 7B–E). These data
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Figure 6. Effects of C9-13 -CPs and CC co-exposure on the proliferation in RAW264.7 cells RAW264.7 cells were exposed to C9-13 -CPs (100 µg/l) for 24 h, and
C9-13 -CPs (100 µg/l) were added to CC 12 h before the end of exposure and divided into four groups, i.e., Con, CC, C9-13 -CPs, and C9-13 -CPs + CC groups. (A)
Typical fluorescence microscopy images of EdU assay used to detect cell proliferation. (B) Quantitative results of EdU assay expressed as the EdU-positive cell
percentage. Data are shown as the mean ± SD from three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 vs control.

Figure 7. Effects of C9-13 -CPs and CC co-exposure on the expression of AMPK and inflammatory factor genes in RAW264.7 cells Cells were exposed to C9-13 -CPs
(100 µg/l) for 24 h, and C9-13 -CPs (100 µg/l) were added to CC 12 h before the end of exposure and divided into four groups, i.e. Con, CC, C9-13 -CPs, and C9-13 -
CPs + CC groups. (A) Typical western blot images for p-AMPK and the loading control β-actin. Reverse transcription-quantitative real-time polymerase chain
reaction detection of the expression levels of the Il-6 gene (B), Il-1β gene (C), Il-4 gene (D), and Il-13 gene (E). Data are shown as the mean ± SD from three
independent experiments. *P<0.05, **P<0.01, and ***P<0.001 vs control.

indicated that co-exposure with AMPK inhibitors reversed, to some
extent, the inflammatory response disorder caused by exposure to
C9-13 -CPs.
C9-13 -CPs increased Ca2+ levels in macrophages
To further investigate the cause of AMPK activation, we explored
the effect of C9-13 -CP exposure on cellular Ca2+ level in RAW264.7
cells. Figure 8A,B are the results of the blank control and the posi-
tive control. Treatment with ionomycin was used to accurately find
the peak position of the Ca2+ positive signal combined with Fluo-
3/AM. As shown in Fig. 8C,D, 4 h of exposure to 100 µg/l C9-13 -CPs
increased the intracellular Ca2+ level in RAW264.7 cells. These
data indicated that exposure to C9-13 -CPs led to an increase in the
intracellular Ca2+ level, which can activate AMPK phosphorylation.
Immunomodulatory effects of C9-13 -CPs in macrophages
Figure 8. Effects of C9-13 -CP exposure on intracellular calcium levels in RAW264.7 cells RAW264.7 cells were exposed to C9-13 -CPs (100 µg/L) or positive control
ionomycin for 4 h, and the Ca2+ signal of Fluo-3/AM was detected by flow cytometry. (A) No C9-13 -CPs or ionomycin treatment. (B) Ionomycin treatment. (C)
No C9-13 -CPs treatment. (D) C9-13 -CPs treatment. Data are shown as the mean ± SD from three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 vs
control.
C9-13 -CPs were predicted to bind with the β2 -AR
receptor
Our previous in vivo studies showed that exposure to C9-13 -CPs can
cause transcriptome abnormalities in the G-protein-coupled recep-
tor signaling pathway [18]. Adrenergic receptors are a type of
G-protein-coupled receptor that are also expressed in immune cells
and are essential for the development of various inflammatory dis-
eases. Therefore, the receptor protein and small ligand molecules
(C9-13 -CP homologue and β2 -AR agonist) were docked through
AutoDock, and the binding affinity range was predicted to be
–7.32 to −4.87 kcal/mol (Supplementary Table S1). Among them,
the binding energies of the agonists, arformoterol, and levalbuterol,
were −6.49 kcal/mol and −7.32 kcal/mol, respectively.
As shown in Fig. 9, a considerable part of the binding energy
of CP molecules is distributed on both sides of the two agonists,
with a minimum of −7.06 kcal/mol. The binding energy highlights
the obvious dependence on chlorine content; the higher the degree
of chlorination, the stronger the binding capacity. In addition, the
binding capacity has a certain relationship with the carbon chain
length. The longer the carbon chain length, the stronger the binding
capacity (chain length does not exceed 13 carbons).
To further verify the experimental results, we used AutoDock
Vina to perform molecular docking analysis of C9-13 -CPs and β2 -
AR receptors and performed 10 independent docking runs for each
C9-13 -CP molecule. To verify the above rules, we used PYMOL to
visualize the binding mode (Supplementary Fig. S1). It was found
that the longer the carbon chain, the easier it is to fold in the binding
pocket to form the binding mode with the lowest binding energy. In
addition, the higher the degree of chlorination of the single chain
C9-13 -CPs molecules, the more likely they are to form hydrogen
bonds with surrounding amino acid residues in the binding pocket.
Discussion
Macrophages play an important role in both innate immunity and
adaptive immunity. Some environmental pollutants affect immune
function by interfering in the vitality of immune cells [19]. The effects
of different pollutants on cell viability, proliferation, and apoptosis
may be different. For example, a low concentration of bisphe-
nol A (BPA) (0.054–5.4 mg/l) induces the proliferation of zebrafish
lymphocytes and macrophages [20], but cells treated with a low con-
centration of 8:2 fluorotelomer alcohols (FTOH) showed decreased
1161
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viability and proliferation [13]. Currently, there are few in vitro stud-
ies on the cytotoxicity of SCCPs, and research on immune cells has
not yet been reported. Geng et al. [11] reported in 2015 that expo-
sure to 100 µg/l C9-13 -CPs (Cl% = 64.4%) for 24 h caused a 20%
decrease in HepG2 cell viability. Our previous study showed that in
vivo exposure to C9-13 -CPs in mice led to increases in white blood
cells and blood monocytes [18]. In agreement with this observation,
the present study demonstrated that C9-13 -CPs promoted the pro-
liferation of macrophages. Taking into account as far as possible
to cover the differences in the distribution of C9-13 -CP congener in
human serum, plasma, and placenta, as well as the availability of
chemicals, the C9-13 -CPs congener used in the present study were
C9 :C10 :C11 :C12 :C13 =1:1:1:1:1, chlorine content = 52% [2,7,8].
The cell viability and proliferative ability of RAW264.7 cells were
increased significantly with increasing concentrations of SCCPs in
the medium to high concentration group and the high concentra-
tion group, respectively. One of the main functions of macrophages
is antigen presentation, which links innate and adaptive immunity
[21,22]. Some surface molecules, including MHC-I, MHC-II, and
CD86, play a pivotal role in antigen presentation [23]. Xenobiotics
may exert immunomodulatory effects via interference in antigen pre-
sentation. For example, β-cypermethrin treatment of RAW264.7
cells reduces the phagocytic function of macrophages [24]. The expo-
sure of macrophages to PFOA causes their phagocytic ability to
decline [25]. In agreement with these observations, our study showed
that the exposure of macrophages to C9-13 -CPs led to the up regu-
lation of the expression of H2-K1, H2-Aa and Cd86 (Fig. 3A–C).
H2-K1, H2-Aa, and Cd86 gene expression levels were significantly
upregulated in either the medium to high or high concentration
groups compared with those in the Con, while none of the three
showed significant changes in the low concentration group, indicat-
ing that C9-13 -CPs can interfere in the antigen presentation function
of macrophages.
There are two types of macrophages, i.e. M1 and M2, which
secrete pro-inflammatory and anti-inflammatory factors, respec-
tively. The balance of the two types of macrophages plays a piv-
otal role in maintaining the body’s immune response [26]. Studies
have shown that xenobiotics can disturb the immune balance by
regulating macrophage polarization. PM2.5 upregulates the expres-
sion of miR-146a-3p and induces inflammatory M1 polarization
by inhibiting SIRT1 in RAW264.7 cells [27]. 2,4,6-Triiodophenol
(TIP) at 200 µM remarkably induced the M2-dominant polariza-
tion of macrophages, while 2,4,6-trichlorophenol (TCP) at 200 µM
1162 Immunomodulatory effects of C9-13 -CPs in macrophages

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Figure 9. Free energy of the binding (kcal/mol) between C9-13 -CPs and β2 adrenergic receptor predicted using the AutoDock program AutoDock predicts the
binding energy between SCCPs and β2 adrenergic receptors. The size of the dot indicates the length of the carbon chain, and the color change from green to
red indicates the degree of chlorination. The blue and red dashed lines indicate the binding energy of the two agonists of the β2 -AR receptor. The abscissa is
the binding energy value, and the ordinate is the different SCCP compounds.

induced an M1-dominant polarization of macrophages. 2,4,6-
Tribromophenol has a moderate ability to induce M1 and M2
polarization compared with TCP and TIP [14]. In the present study,
we demonstrated that exposure to C9-13 -CPs downregulated the
mRNA expression of M1 macrophage-related cytokines, including
Il-6 and Tnf-α, while it upregulated that of M2 macrophage-related
cytokines, including Il-4 and Il-3, indicating that C9-13 -CPs induced
M2 polarization of macrophages and disturbed the immune balance
in vivo.
NF-κB is a pivotal transcription factor mediating the immune
response, and this signaling pathway has been shown to play an
important role in macrophage polarization [28]. The uncontrolled
Immunomodulatory effects of C9-13 -CPs in macrophages
regulation of NF-κB is closely related to cancer occurrence, inflam-
matory response, autoimmune disease, and immune function devel-
opment disorders [29]. Many chemicals have been demonstrated to
dysregulate NF-κB activation. For example, the exposure of Jurkat
and HL-60 cells to caracanic acid promoted the activation of NF-
κB and cell apoptosis [30]. Carvacrol could protect RAW264.7
macrophages from lipopolysaccharide (LPS)-induced inflammation
by inhibiting NF-κB [31]. This study showed that C9-13 -CP exposure
inhibited the activation of NF-κB in RAW264.7 cells. Sirt1 can reg-
ulate the deacetylation of the RelA/p65 subunit of NF-κB on Lys310
to inhibit NF-κB-regulated gene expression. We found that C9-13 -
CPs increased the mRNA level of SIRT1, suggesting that exposure
to C9-13 -CPs reduced NF-κB activity and promoted the polarization
of macrophages towards the M2 type. It is also speculated that the
inhibition of NF-κB activity is due to SIRT1 activation and regu-
lation of its gene silencing. Therefore, we must consider that the
upstream signal of SIRT1 is involved in the regulation of NF-κB.
It has been reported that SCCPs can activate the AMPK signal-
ing pathways in HepG2 cells, zebrafish, and rats [32,33]. AMPK is
not only an enzyme that plays a role in cell energy homeostasis but
also in the regulation of inflammation in immune cells [34]. Studies
have shown that some chemicals can regulate AMPK activation and
the immune response. For example, quercetin induces the M2 polar-
ization of macrophages through TRPM2-dependent calcium influx
and AMPK/ATF3 activation, thereby conferring a protective effect
on murine sepsis [35]. Metformin inhibits the pro-inflammatory
expression of human monocytes/macrophages stimulated by LPS
through AMPK [36]. In the present study, we found that exposure
of RAW264.7 cells to C9-13 -CPs caused a decrease in the ATP level
and the activation of AMPK. In addition, we observed that expo-
sure to C9-13 -CPs caused an increase in intracellular Ca2+ level,
which may contribute to AMPK phosphorylation. Increasing intra-
cellular Ca2+ leads to the activation of AMPK. Ca2+ is essential
for the binding with its primary intracellular receptor calmodulin
(CaM) to trigger a variety of downstream processes and pathways,
and the pleiotropy of CaMKK2 downstream of Ca2+ /CaM is used to
coordinate energy homeostasis [37]. It has been reported that STO-
609 (a relative selectivity and cell permeability inhibitor of CaMKK)
causes a dose-dependent decrease in the activation of AMPK by
ionomycin, with an IC50 of 0.5 µg/ml. The concentration of STO-
609 was 10 µg/ml, which almost completely inhibited the activity
of CaMKK in HeLa cells, and STO-609 inhibited the activation
of AMPK by 95%. At this concentration, STO-609 directly inhib-
ited the in vitro activity of AMPK by ∼20% [38]. These results
indicate that Ca2+ /CaMKK/AMPK may mediate the disturbance of
macrophages by C9-13 -CPs.
It has been reported that pollutants usually bind to cell surface
receptors or biological macromolecules such as aromatic hydro-
carbon receptor, oestrogen receptor (ER), and androgen sterane
hydrocarbon receptor (CAR) [10,39,40]. For example, SCCPs bind
to ERα on the surface of Chinese hamster ovary cells (CHOs) and
interfere in normal endocrine function [41]. C9-13 -CPs can combine
with PPARα to interfere in lipid metabolism in the liver of rats [42].
It was also predicted that SCCPs can combine with CAR to affect
the homeostasis of thyroid hormones of the body [10]. In addi-
tion, our previous experiments revealed that the spleen of mice fed
with the 100 mg/kg/day C9-13 -CPs showed significantly affected gene
subsets as revealed by Gene Ontology (GO) enrichment, and the
most significant function in the biological process category was the
G-protein-coupled receptor signaling pathway [18]. β2 -Adrenergic
receptor (β2 -AR), a class of G-protein-coupled receptors, can be
1163
activated by the agonist salmeterol, activating β-arrestin 2 to block
the nuclear translocation of p65-NF-κB, weaken the activation of
NF-κB, and inhibit LPS induction of pro-inflammatory mediator
production in RAW264.7 and THP-1 cells [43]. In this experiment,
the main cause of RAW264.7 immune dysfunction induced by C9-13 -
CPs might be the regulation of NF-κB. Therefore, it can be speculated
that there are biological macromolecules or surface receptors that
can bind to CPs to inhibit NF-κB. According to various literature
studies, C9-13 -CPs may bind with β2 -AR. A large number of recent
studies have confirmed that SIRT1 inhibits NF-κB signaling and that
the activation of SIRT1 can relieve a variety of NF-κB-driven inflam-
matory processes. SIRT1 and AMPK are closely related in regulating
energy metabolism and inflammation because they can enhance each
other’s activity. SIRT1 activates AMPK through the deacetylation of
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LKB1, which then triggers AMPK activation. In addition, emerging
research has shown that AMPK is an effective inhibitor of NF-κB sig-
naling and inflammation [44]. In short, AMPK can inhibit RelA/p65
through the activation of SIRT1. The molecular docking results of
C9-13 -CPs with β2 -AR showed that C9-13 -CPs can bind to β2 -AR
and the binding capacity is positively correlated with the degree of
chlorination and chain length, suggesting that the immune function
of RAW264.7 is closely related to the β2 -AR/AMPK/NF-κB signal
axis.
In conclusion, C9-13 -CPs are immunotoxic to immune cells.
C9-13 -CPs lead to an imbalance in energy metabolism by disrupting
cellular homeostasis and activating AMPK, which regulates cellu-
lar homeostasis, thereby inhibiting NF-κB and ultimately promoting
macrophage polarization to the M2 type. In addition, molecu-
lar docking predicts another mechanism for NF-κB inhibition: the
β2 -AR/AMPK/NF-κB signaling axis.
Supplementary Data
Supplementary data is available at Acta Biochimica et Biophysica
Sinica online.
Funding
This work was supported by the grants from the Zhejiang Provincial
Natural Science Foundation of China (No. LQ19B070006) and the
Program for Changjiang Scholars and Innovative Research Team in
University (No. IRT17R97).
Conflict of Interest
The authors declare that they have no conflict of interest.
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