Yan et al.

Cell Biosci (2020) 10:91

https://doi.org/10.1186/s13578-020-00448-6 Cell & Bioscience

RESEARCH Open Access

Chronic arsenic exposure induces

the time‑dependent modulation
of inflammation and immunosuppression
in spleen
Nan Yan1, Guowei Xu1, Chenchen Zhang1, Xuping Liu1, Xin Li1, Lin Sun1, Da Wang1, Xiaoxu Duan2* and Bing Li1*

Abstract
Background: Arsenic exposure has become a matter of worldwide concern, which is associated with immune-
related diseases. However, little is known about its effect on inflammatory immune-related homeostasis. The purpose
of our study was to understand the potential tuning of above responses exerted by chronic arsenic exposure.
Methods: Kunming mice were treated with 25 and 50 mg/L sodium arsenite for 1, 3 and 12 months via drinking
water. At different endpoints of arsenic exposure, all animals and the whole spleen of the mice were weighed. The
total arsenic levels of spleen were determined by the HPLC-HG-AFS method. Splenic NF-κB, MAPK and NRF2 protein
levels by treatment of 25 mg/L ­NaAsO2 for 1, 3 and 12 months and 25 mg/L and 50 mg/L ­NaAsO2 for 12 months were
assessed by western blot. Total RNA of spleen was isolated and relative mRNA levels of Foxp3, Il-10, Tnf-α, Il-6, Ifn-γ, Il-1β
and Il-12 were measured by real-time PCR.
Results: Our results shown that NF-κB were continuously activated with treatment of 25 mg/L arsenic from 1, 3 to
12 months and 50 mg/L arsenic for 12 months. The transcription factor Foxp3 increased at 1 month but decreased at
3 and 12 months no matter 25 or 50 mg/L arsenic exposure. However, cytokine Il-10 always showed increased trend
in mice treated with 25 or 50 mg/L arsenic for 1, 3 and 12 months. The transcriptional profiles of Tnf-α, Il-1β, Il-6, Ifn-γ
and Il-12 revealed transient elevation at 1 and 3 months but shown significant decrease at 12 months on the whole. In
addition, the sustained activation of inflammatory MAPK and anti-oxidative Nrf2 signaling pathways were observed in
mice exposed to arsenic for 1, 3 and 12 months.
Conclusion: In summary, our experiment in vivo suggested chronic arsenic exposure induces the time-dependent
modulation of the inflammation and immunosuppression in spleen, which may be related to the activation of Tregs
induced by MAPK/NF-κB as well as the increased transcription level of Foxp3 and Il-10.
Keywords: Arsenic, Inflammation, Immunosuppression, Spleen

Introduction
Arsenic contamination has become a global matter
*Correspondence: duanxiaoxu@symc.edu.cn; bli10@cmu.edu.cn
1
concerned. Studies have shown that arsenic exposure
Environment and Non‑Communicable Disease Research Center,
Key Laboratory of Arsenic‑Related Biological Effects and Prevention impedes cell growth and proliferation of immune func-
and Treatment in Liaoning Province, School of Public Health, China tion organs (spleen and thymus) [1–3]. A cohort study in
Medical University, No. 77 Puhe Road, Shenyang North New Area, Bangladesh has reported that exposure to arsenic dur-
Shenyang 110122, Liaoning, People’s Republic of China
2
Department of Toxicology, School of Public Health, Shenyang Medical ing pregnancy caused the thymus gland shrank and its
College, Shenyang 110034, Liaoning, China function impaired, leading to immunosuppression in

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Yan et al. Cell Biosci (2020) 10:91 Page 2 of 10

childhood [4]. In addition, it has been demonstrated that Materials and methods
arsenic could generate immunosuppressive responses Reagents and chemicals
by dose-dependent regulation of the NF-κB/Tregs/IL6/ Sodium arsenite ­(NaAsO2, ≥ 99.0%) was purchased from
STAT3 signaling axis of mouse thymus cells [5]. It was Sigma Chemical Co. (St. Louis, MO, USA). N ­ aAsO2 was
found that immunosuppression in arsenic-exposed peo- dissolved in distilled water and diluted to the desired con-
ple in India is related to T cell proliferation and cor- centrations. Primary antibodies of NF-κB (C-20: sc-372),
responding reduction of cytokines, such as TNF-α NRF2 (H-300: sc-13032), GSTO1/2 (FL-241: sc-98560),
(tumor necrosis factor), IL-2, IL-4, IL-5, IL-10, etc. [6]. GCLC (H-300: sc-28965), and β-actin (1-19: sc-1616)
It is known that the expression of these immune inflam- were bought from Santa Curz Biotechnology (Santa Cruz,
matory factors is inseparable from the involvement of CA, USA); P-ERK (#9101), ERK (#9102), P-JNK (#9251),
NF-κB. JNK (#9252), P-P38 (#9211), and P38 (#9212) were from
Duan et al. found that acute arsenic exposure activated Cell Signaling Technology (Cell Signaling, USA). The
MAPK/NF-κB signaling pathways in the thymus and corresponding secondary antibodies were all purchased
spleen of mice, which ultimately stimulated the inflam- from Santa Curz Biotechnology (Santa Cruz, CA, USA).
mation by the transcription and secretion of pro-inflam- Real-time polymerase chain reaction (real-time PCR) kits
matory cytokines such as TNF-α, IL-1β and IL-6 etc. [7]. were from Takara Co (Otsu, Japan). All other reagents
However, previous studies have failed to consider the were of the highest grade commercially available. Water
effect of long-term exposure to arsenic in drinking water used in all the preparations was distilled and deionized.
on inflammatory factors in vivo. Therefore, we are aimed
to figure out the changes of inflammatory factors in vivo Animals and experimental procedures
with the extension of arsenic exposure time. Ninety female Kunming mice (weighing 18–22 g,
A growing number of studies have shown that NF-κB 6–7 weeks old) were obtained from the Center for Exper-
maintains immune homeostasis through nonclassi- imental Animals at China Medical University (Shenyang,
cal pathways [5, 8]. It was found that NF-κB could exert China) with a National Animals Use License number of
immunosuppressive effect by Tregs (regulatory T cells) SCXK-LN2013-0007. All experiments and surgical pro-
[8]. The abundance of Treg cells was increased in vitro cedures were approved by Animals Care and Use Com-
culture of peripheral blood monocytes with arsenic mittee at China Medical University, which complies with
of 0.1–1.0 μM [9]. In addition, it has been demon- the National Institutes of Health Guide for the Care and
strated that arsenic could generate immunosuppressive Use of Laboratory Animals. All efforts were made to
responses by dose-dependent regulation of the NF-κB/ minimize the number of animals used and their suffering.
Tregs/IL6/STAT3 signaling axis of mouse thymus cells The mice were acclimatized to standard laboratory con-
[5]. In animal model of experimental automine encepha- ditions for 2 weeks before the experiments.
lopathy, arsenic (10 μg/L) resulted in an increase of Tregs Mice were group-housed in stainless steel cage (10
but a decrease with arsenic (> 100 μg/L) in the spleen and mice per cage) in an air-conditioning room with temper-
peripheral blood [9]. ature at 20 ± 2 °C, 12 h light: 12 h dark cycle, and year
The transcription factor Foxp3 is essential not only for round relative humidity of 50–60%. All animals were
the normal development of Tregs but also for their sup- allowed balanced food and drinking water ad libitum.
pressive effects [10]. Studies have shown that the loss of The desired concentrations of N ­ aAsO2 (25 mg/L
Foxp3 expression over time impair the suppressive activ- and 50 mg/L) were prepared freshly and provided to
ity of Tregs [11, 12]. One of the mechanisms by which the experimental mice to drink ad libitum for 1, 3 and
Tregs exert inhibitory activity is dependent on suppres- 12 months, respectively. Control mice were treated with
sive factors (IL-10, TGF-β, IL-35, etc.). So what is the deionized water parallelly. At the different endpoints of
effect of long-term exposure to arsenic on Foxp3 and arsenic exposure, all animals were weighed and sacrificed
IL-10, which is worth exploring. after deep anesthetization. The whole spleen of the mice
Nrf2 signaling pathway is a classic antioxidant was quickly removed and weighed, immediately frozen in
response element closely related to immunity [13]. liquid nitrogen and stored at − 80 °C for future use.
Fry et al. reported that activation of NF-κB was associ-
ated with oxidative stress [14]. Therefore, the purpose Determination of splenic arsenic levels
of our research is to observe the MAPK/NF-κB medi- The dissected spleen of mice was washed with nor-
ated immune inflammatory and immunosuppressive mal saline to remove blood, and then homogenized on
responses in the spleen of mice under long-term arsenic ice with deionized water. Arsenic species, including
exposure, and to provide a new treatment idea for long- arsenite ­(iAsIII), arsenate ­
(iAsV), monomethylarsonic
term arsenic exposure. (MMA) and dimethylarsinic (DMA) were measured by
Yan et al. Cell Biosci (2020) 10:91 Page 3 of 10

a high-performance liquid chromatography-hydride reactions were performed for each sample and the results
generation-atomic fluorescence spectrometer (HPLC- are presented as mean ± SD (n = 4).
HG-AFS, SA-10 Atomic Fluorescence Species Analyzer,
Titan, Beijing, China), consisting of a liquid chromato-
graphic column, a hydride generation equipment, and an Western blot analysis
atomic fluoresce detector, as published in previous exper- Spleen protein was extracted for western blot using
iments [15]. Total arsenic (T-As) levels of spleen were standard protocols. Briefly, total protein concentrations
calculated by summing up the levels of ­iAsIII, ­iAsV, MMA of spleen were determined by BCA reagent kit using
and DMA in each sample. All samples were analyzed in bovine serum albumin (Santa Cruz, CA, USA) as the pro-
triplicate, and the results were expressed as mean ± SD tein standard. An equal amount (30 μg) of protein was
(n = 3). separated by 10% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE), then transferred onto a
Total RNA isolation and real‑time PCR analysis PVDF membrane (Buckinghamshire, UK). The blotting
Total RNA of spleen was collected for RNA extraction membranes were saturated in blocking solutions (TBST,
using a Trizol Reagent (Invitrogen, USA). Real-time PCR the Tris-buffered saline containing 0.1% Tween 20 and 5%
was conducted using a two-step method and an ABI 7500 skim milk) for 2 h at room temperature, followed by incu-
real-time PCR system (ABI, USA). Then, equal amounts bating with the following primary antibodies of NF-kB
(500 ng) of RNA were used for reverse-transcription (RT) (1:2000), P-ERK (1:500), ERK (1:1000), P-JNK (1:500),
to cDNA synthesis by PrimeScript RT reagent kits with JNK (1:500), P-P38 (1:500), P38 (1:1000), NRF2 (1:1000),
gDNA eraser (Perfect Real Time, Takara, Japan), and GSTO1/2 (1:1000), GCLC (1:1000), β-actin (1:2000)
PCR amplification was performed by SYBR Premix Ex overnight at 4 °C, respectively. On the 2nd day, mem-
Taq II (Tli RNaseH Plus) kits (Perfect Real Time, Takara, branes were washed with TBST and then incubated with
Japan). The PCR step was performed with the thermal corresponding secondary antibodies (1:2000–1:5000) for
cycling conditions as follows: 1 cycle of initial denatura- 2 h at room temperature. After that, membranes were
tion (95 °C for 30 s), and 40 cycles of amplification (95 °C incubated with chemiluminescence reagents (PicoWest
for 5 s and 60 °C for 34 s). Primers for mouse genes were Super Signal, Pierce Biotechnology, USA). The signals
designed by PRIMER 3 software and synthesized by were visualized with electrophoresis gel imaging analy-
Sangon Biological Engineering Technology (Shanghai, sis system (MF-ChemiBIS 3.2, DNR Bio-Imaging System,
China) as provided in Table 1. Relative mRNA levels of Israel). The signals of β-actin (1:5000) were used to nor-
different target genes were normalized to Gapdh and malize protein loading. The results were representative of
finally expressed as folds of control groups. Triplicate three mice of each treatment group.

Table 1 Primer sequences used in real-time PCR analysis

cDNA Primer sequences Product
length
(bp)

Mus-Foxp3 (NM_054039) F:5′-CAG​C TC​TGC​TGG​CGA​AAG​TG-3′ 123

R:5′-TCG​TCT​GAA​GGC​AGA​GTC​AGGA-3′
Mus-Il-10 (NM-010548) F:5′-GGG​GCC​AGT​ACA​GCC​GGG​AA-3′ 92
R:5′-CTG​GCT​GAA​GGC​AGT​CCG​CA-3′
Mus-Ifn-γ (NM-008337) F:5′-AAG​CGT​CAT​TGA​ATC​ACA​CCTG-3′ 92
R:5′-TGA​CCT​CAA​ACT​TGG​CAA​TACTC-3′
Mus-Il-1β (NM_008361) F:5′-TGC​CAC​C TT​T TG​ACA​GTG​ATG-3′ 220
R:5′-AAG​GTC​CAC​GGG​AAA​GAC​AC-3′
Mus-Tnf-α (NM_013693) F:5′-CCT​GTA​GCC​CAC​GTC​GTA​G-3′ 148
R:5′-GGG​AGT​AGA​CAA​GGT​ACA​ACCC-3′
Mus-Il-12 (NM_001303244) F:5′-TGG​T TT​GCC​ATC​GTT​T TG​C TG-3′ 123
R:5′-ACA​GGT​GAG​GTT​CAC​TGT​T TCT-3′
Mus-Il-6 (NM_031168) F:5′-CTG​CAA​GAG​ACT​TCC​ATC​CAG-3′ 131
R:5′-AGT​GGT​ATA​GAC​AGG​TCT​GTTGG-3′
Mus-Gapdh (NM_008084) F:5′-TGT​GTC​CGT​CGT​GGA​TCT​GA-3′ 150
R:5′-TTG​C TG​T TG​AAG​TCG​CAG​GAG-3′
Yan et al. Cell Biosci (2020) 10:91 Page 4 of 10

Statistical analysis our observations of splenic weight or splenic index. So,

All the experiments were repeated three times to obtain the corresponding pathological results were not listed
the data and carried out at least in triplicate. All quanti- in this section. The splenic T-As levels of the study mice
tative data were expressed as mean ± SD. The statistical were determined and listed in Table 2.
significance among differences between experimental
groups was determined by one-way analysis of variation Chronic arsenic exposure leads to NF‑κB activation as well
(ANOVA) using least significant difference (LSD) method as regulatory T cells specification in spleen
(SPSS 11.0, SPSS Inc., Chicago, IL, USA). p < 0.05 was On the one hand, NF-κB is widely involved in the initia-
considered to be statistically significant. tion and development of inflammation [7, 16, 17]. Our
results found that the levels of NF-κB protein expres-
Results sion in spleens of mice treated with ­NaAsO2 (25 mg/L)
The general status, body weight gain, splenic weight for 1, 3 and 12 months were significantly increased than
and T‑As levels of the study mice those in the control group (Fig. 1a). Similarly, NF-κB was
Mice were treated with different concentrations of evidently activated in spleen of mice exposed to 25 and
­NaAsO2 for 1, 3 and 12 months through drinking water. 50 mg/L ­NaAsO2 for 12 months, respectively (Fig. 1b).
The amounts of water intake, food consumption, and Those suggested that the expression of NF-κB was not
the body weights were recorded every week. During the affected by the duration and dose of arsenic treatment.
whole study period, all the mice showed normal activ- On the other hand, a study in vitro found that arsenic-
ity, regular growth, and no other obvious abnormalities induced NF-κB/IL-6 axis triggered immune responses by
by visual inspection. We have not found any significant regulating the proliferation and differentiation of Tregs
differences in body weight gain, suggesting no obvious [5]. In our study, we observed an obvious increase in
growth inhibition happened among different experimen- the transcription level of Foxp3 (a transcription factor
tal groups. Organ index is a common used index in toxi- of Tregs) in the spleen of mice after 1 month of treat-
cological evaluation, since the changes of a certain organ ment with 25 and 50 mg/L N ­ aAsO2 (Fig. 1c), but with
index could reflect some toxic effects (such as edema or the increase of treatment time (such as 3 and 12 months),
atrophy) of a specific toxicant. In our experiments, the this kind of increasing trend disappeared (Fig. 1c). As the
splenic index decreased with the growth of the mice, main cytokine derived from Tregs, IL-10 also showed a
however, we found no obvious changes of the size and the rise in transcription level in groups treated with 25 mg/L
weight of the mice spleen, nor any other visible changes ­NaAsO2 for 1 and 12 months as well as 50 mg/L ­NaAsO2
at autopsy, among the control and the arsenic treatment for 1 and 3 months compared with the control group
groups within the same durations (1, 3 and 12 months). (Fig. 1d). However, IL-10 showed no signs of increased
Meanwhile, we also carried out splenic histomorphol- transcription level in mice exposed to 25 mg/L ­NaAsO2
ogy of each treatment group, whose results did not show for 3 months as well as 50 mg/L N ­ aAsO2 for 12 months
obvious pathological changes, which was consistent with (Fig. 1d). Taken together, these results suggested that

Table 2 The body weight gain, splenic weight, and the concentrations of total arsenic (T-As, ng As/g tissue) in spleen
of control and different experimental mice
Experimental Duration Dose (mg/L) Body weight gain (g) Splenic weight (g) T-As in spleen (ng/g)
groups (month)

Group 1 1 0 11.64 ± 1.83 0.11 ± 0.03 < LD

Group 2 1 25 11.98 ± 1.74 0.10 ± 0.03 31.46 ± 9.18
Group 3 1 50 11.76 ± 2.30 0.10 ± 0.02 39.95 ± 3.86
Group 4 3 0 19.61 ± 1.95 0.10 ± 0.03 < LD
Group 5 3 25 18.57 ± 3.86 0.10 ± 0.02 38.31 ± 5.87
Group 6 3 50 18.41 ± 1.27 0.10 ± 0.02 36.34 ± 19.82
Group 7 12 0 24.51 ± 3.59 0.10 ± 0.02 < LD
Group 8 12 25 23.21 ± 4.78 0.11 ± 0.02 47.80 ± 15.90
Group 9 12 50 24.80 ± 2.11 0.10 ± 0.03 52.42 ± 3.29
Mice were treated with 25 mg/L and 50 mg/L ­NaAsO2 for 1, 3 and 12 months through drinking water ad libitum, and the body weight of the mice was recorded every
week. At the treatment endpoints, the entire spleen was removed and weighed (mean ± SD, n = 10). The total arsenic (T-As) levels of spleen were determined by the
HPLC-HG-AFS method. Results were expressed as mean ± SD (n = 3). The limit of detection (LD) for T-As was 1 μg/L
* p < 0.05 compared with corresponding control mice
Yan et al. Cell Biosci (2020) 10:91 Page 5 of 10

Fig. 1 Chronic arsenic exposure leads to NF-κB activation as well as regulatory T cells specification in spleen. Mice were treated with N
­ aAsO2
(25 mg/L and 50 mg/L) for 1, 3 and 12 months through drinking water ad libitum. Splenic NF-kB protein levels by treatment of a 25 mg/L
­NaAsO2 for 1, 3 and 12 months and b 25 mg/L and 50 mg/L N ­ aAsO2 for 12 months were assessed by western blot. β-actin was blotted as the
internal control. The exposure duration of the control group was 12 months in a and b. The density quantification of each band was presented
as mean ± SD (n = 3). Total RNA of spleen was isolated and relative mRNA levels of Foxp3 (c) and Il-10 (d) were normalized to Gapdh and finally
expressed as folds of control by real-time PCR. Results were expressed as mean ± SD (n = 4), and two such independent experiments were carried
out. *p < 0.05 compared with corresponding control mice

long-term arsenic exposure induced the persistent acti- as well as 50 mg/L N ­ aAsO2 for 12 months in mice
vation of NF-κB as well as Tregs specification in a time- spleen. However, increased transcription level of Il-1β
dependent manner. occurred in mice exposed to 50 mg/L ­NaAsO2 for 1 and
3 months (Fig. 2b). Il-6, as a prominent pro-inflamma-
Chronic arsenic exposure affects cytokine profiles in spleen tory cytokine, showed dramatic increase in the mice
The disruption of arsenic on the immune inflammatory treated with 25 and 50 mg/L arsenic for 1 and 3 months
homeostasis was inseparable from the involvement of but decreased with 25 mg/L arsenic for 12 months
cytokines controlled by various immune cells [18–20]. (Fig. 2c). Ifn-γ, a hallmark cytokine of Th1 cells, is
Our research found that the classic inflammatory factor, widely involved in cellular immunity. We have detected
Tnf-α, did not change at 1 and 3 months but declined at a significant transcription increase in mice exposed
12 months under 25 mg/L N ­ aAsO2 treatment as well as to 50 mg/L arsenic for 1 and 3 months meanwhile no
decreased at 3 and 12 months in the case of 50 mg/L obvious change was observed in the other exposure
­NaAsO2 exposure in mice (Fig. 2a). Il-1β, another methods (Fig. 2d). Il-12 is also an important inflamma-
inflammatory cytokine, remained unchanged after tory factor. In our study, the transcription level of Il-12
exposure to 25 mg/L N ­ aAsO2 for 1, 3 and 12 months increased at 3 months but decreased after 12 months
of 50 mg/L arsenic exposure as well as decreased at
12 months of 25 mg/L arsenic (Fig. 2e).
Yan et al. Cell Biosci (2020) 10:91 Page 6 of 10

Fig. 2 Chronic arsenic exposure affects cytokine profiles in spleen. Mice were treated with N
­ aAsO2 (25 mg/L and 50 mg/L) for 1, 3 and 12 months
through drinking water ad libitum. Total RNA of spleen was isolated and relative mRNA levels of Tnf-α (a), Il-1β (b), Il-6 (c), Ifn-γ (d) and Il-12 (e) were
normalized to Gapdh and finally expressed as folds of control by real-time PCR. Results were expressed as mean ± SD (n = 4). *p < 0.05 compared
with corresponding control mice

Taken together, these results suggest that chronic Chronic arsenic exposure up‑regulates nuclear factor NRF2
arsenic exposure caused an immune-inflammatory and its downstream targets in spleen
imbalance in mice spleen. As a classical transcription factor for stress antioxidant
and adaptive cellular defense response, NRF2 path-
way has also been reported to play a regulatory role in
Chronic arsenic exposure activates MAPK pathway immune imbalance [22]. Our results showed that pro-
in spleen tein expression levels of NRF2 and its downstream pro-
As the upstream of NF-κB, MAPK was found to play an teins GSTO and GCLC increased after 25 and 50 mg/L
important regulatory part in immune imbalance [21]. ­NaAsO2 exposure for 12 months (Fig. 4b) as well as
We observed the phosphorylation levels of ERK and 25 mg/L ­NaAsO2 for 3 months (Fig. 4a). This demon-
P38 not JNK were obviously enhanced in mice treated strated arsenic largely up-regulated NRF2 and its down-
with 25 mg/L ­NaAsO2 for 1, 3 and 12 months (Fig. 3a) stream targets in mice spleen involved in maintaining
as well as 50 mg/L ­NaAsO2 for 12 months compared homeostasis.
with the control group (Fig. 3b). In summary, these
indicated that long-term arsenic exposure could acti- Discussion
vate the MAPK signaling pathway in a time and dose- The immune homeostasis is essential for maintaining
independent manner. body health. The organ index is usually calculated by
the ratio of organ weight to the whole-body weight, so
Yan et al. Cell Biosci (2020) 10:91 Page 7 of 10

Fig. 3 Chronic arsenic exposure activates MAPK pathway in spleen. Mice were treated with N ­ aAsO2 (25 mg/L and 50 mg/L) for 1, 3 and 12 months
through drinking water ad libitum. Splenic MAPK signaling pathway related protein levels by treatment of a 25 mg/L N­ aAsO2 for 1, 3 and 12 months
and b 25 mg/L and 50 mg/L N ­ aAsO2 for 12 months were assessed by western blot. β-actin was blotted as the internal control. The exposure
duration of the control group was 12 months in a and b. The density quantification of each band was presented as mean ± SD (n = 3)

Fig. 4 Chronic arsenic exposure up-regulates nuclear factor NRF2 and its downstream targets in spleen. Mice were treated with N ­ aAsO2 (25 mg/L
and 50 mg/L) for 1, 3 and 12 months through drinking water ad libitum. Splenic NRF, GSTO1/2 and GCLC protein levels by treatment of a 25 mg/L
­NaAsO2 for 1, 3 and 12 months and b 25 mg/L and 50 mg/L N ­ aAsO2 for 12 months were assessed by western blot. β-actin was blotted as the
internal control. The exposure duration of the control group was 12 months in a and b. The density quantification of each band was presented as
mean ± SD (n = 3)
Yan et al. Cell Biosci (2020) 10:91 Page 8 of 10

the weights and indexes of immune organs are gener- autoimmune diseases [34, 35]. Levels of Tregs in mice
ally used to evaluate the body immune functions [23]. deficient in NF-κB decreased significantly, and NF-κB
In our experiments, the splenic index decreased with was also shown to up-regulate Foxp3 expression [36].
the growth of the mice, however, no significant change Foxp3 cannot be used as a unique symbol of human
in splenic weight was observed in all treatment groups, Treg cells [37]. But for all Tregs, Foxp3 is nevertheless
which was consistent with our observations of histomor- an important gene to regulate development and function
phology. Totally, the current mice model did not initiate of them. Its constitutive expression is the decisive factor
serious toxicological abnormalities. Therefore, we were driving the immunosuppressive function of mouse and
aimed to explore the time-dependent modulation of the human Tregs [38]. In mice, for instance, the Foxp3 gene
inflammation and immunosuppression of spleen by long- mutation led to severe systemic autoimmune inflamma-
term arsenic exposure in vivo. tory diseases (Scurfy disease) [39]. Our results showed
At present, the effect of arsenic on it is currently con- that the transcription level of Foxp3 in the spleen of mice
troversial. Although most studies believe that arsenic was increased in the early stage (such as 1 month) of
has immunosuppressive effects [24–26], there are also arsenic exposure, and in the middle and late stages (such
studies that suggest arsenic could be used as an immune as 3 and 12 months) of exposure, this trend no longer
stimulant to induce allergic reactions and autoimmune appeared, which suggested that the early stage of chronic
diseases [5, 27–29]. Immune regulation is necessary to arsenic exposure may induce the production of immu-
control the occurrence and development of inflammation nosuppression in the spleen of mice. IL-10, as the main
and autoimmune diseases. In all mechanisms of arsenic- anti-inflammatory cytokine and immunosuppressive fac-
induced regulatory dysfunction of immunity, the activa- tor produced by regulatory T cells, also showed a signifi-
tion of NF-κB is inseparable. cant increase in the early stage of arsenic treatment. This
On the one hand, NF-κB is widely involved in the result further supported the induction of immunosup-
immune inflammatory response caused by arsenic. Duan pression in the spleen of mice at the beginning of chronic
et al. found that acute arsenic exposure induced NF-κB arsenic exposure.
activation and then stimulated the generation of inflam- Choudhury et al. found that arsenic triggered dose-
matory factors to induce the development of inflamma- dependent and differential immune responses in thy-
tion in the spleen and thymus of mice [7]. Arsenic could mocytes by regulating the NF-κB/IL-6 axis, that was,
induce inflammation, which also was occurred in humans high concentration of arsenic produced immunosup-
[14], rats [30], dendritic cells [18, 31] and avian brain tis- pression marked by high expression of IL-10 and Foxp3
sue [32], etc. As shown in our study, we found that the as well as the inflammatory response marked by increase
spleen continuous NF-κB of arsenic-exposed mice, that of TNF-α, IL-1β and IL-6 under low level arsenic [5].
was, it did not change with the extension of the exposure Taken together, it indicated that the immunosuppression
time (even for 12 months). This was not observed in pre- induced by arsenic was dose-dependent as well as the
vious studies. The transcription levels of various immune change of immune-inflammatory factors was involved
cytokines regulated by NF-κB also increased, such as in this process. We found that immunosuppression by
the classic inflammatory factor clusters (TNF-α, IL-1β arsenic may be time-dependent and it is important that
and IL-6) as well as non-classical immune inflammatory immune inflammatory factors played a vital role in this
factors (IFN-γ and IL-12). The increase in expression of process. It has been demonstrated that stress signals
NF-κB as well as controlled immune-inflammatory fac- elicited by pro-inflammatory cytokines lead to the deg-
tors of it with arsenic treatment suggested that chronic radation of Foxp3 through the action of the E3 ubiquitin
arsenic exposure induced the initiation and development [40]. For instance, IL-1β down-regulated TGF-β-induced
of inflammation as acute arsenic exposure [7]. However, Foxp3 expression [41] and TNF-α activated protein
unlike acute arsenic exposure, with the increase of arse- phosphatase1 dephosphorylation of the Ser418 site of
nic exposure time during the long-term arsenic exposure, Foxp3 [42], both possibly receding suppressive function
the increasing trend of these immune inflammatory fac- of Treg cells. In addition, IL-6 restrains Treg differentia-
tors gradually disappeared, and even a decline phenom- tion, which is an important mechanism involved in the
enon appeared at 12 months, which may be attributed to pathogenesis of SLE [41]. Therefore, in our study, we have
the alternative immune regulation mechanism of NF-κB. reasons to believe that the decrease in Foxp3 expres-
On the other hand, NF-κB (especially the subunits c-rel sion during mid-term arsenic exposure may be due to
and p65 in the classical pathway) is necessary for the increased transcription levels of inflammatory factors.
development and function of Tregs [33]. Recent studies In fact, IL-10 inhibits the release of pro-inflammatory
have found that the P65 subunit can maintain the abun- mediators from monocytes/macrophages and there-
dance of regulatory cells and prevent the occurrence of fore inhibits the LPS and IFN-γ-induced production
Yan et al. Cell Biosci (2020) 10:91 Page 9 of 10

of TNF-α, IL-1β and IL-6 [43]. IL-10 is a rather late Funding

This study was supported by grants from National Natural Science Foundation
cytokine being produced after the pro-inflammatory of China (Grant Number: 81673114) and Key Laboratory Basic Research Funds
mediators. Upon ­CD4+ T cells were activated in vitro, from Liaoning Education Department (Grant Number: LS201607).
the presence of IL-10 causes these cells to develop a reg-
Availability of data and materials
ulatory phenotype. In this manner, I type regulatory T All data are fully available without restriction.
cells (Tr1 cells) arise that secrete IL-10 but not Foxp3 and
suppress antigen-specific effector T cell responses via a Ethics approval and consent to participate
All mouse experiments and procedures were approved by the Animal Ethics
cytokine-dependent mechanism [44, 45]. Our results Committee of the China Medical University, Liaoning, China, and all experi-
suggested that the inconsistent rise in the levels of IL-10 ments were performed in accordance with the approved guidelines and
and Foxp3 in the middle and late stages of arsenic treat- regulations.
ment (3 and 12 months) may be due to the role of Tr1 Consent for publication
cells, which of course requires further researches to con- Written informed consent for publication was obtained from all participants.
firm this hypothesis. The continuous increase of IL-10 in
Competing interests
the later stage (such as 12 months) exerts its anti-inflam- The authors declare that they have no competing interests.
matory and immunosuppressive effects by inhibiting the
expression of various inflammatory cytokines, which also Received: 7 March 2020 Accepted: 19 June 2020
explains the phenomenon of the decline in the transcrip-
tion level of immune inflammatory factors.
As an upstream signal of NF-κB, MAPK is widely
involved in the arsenic-induced immune inflammatory
process. The continuous activation of MAPK in our References
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