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Functional suppression of macrophages derived from THP-1 cells by environmentally-relevant concentrations of arsenite
Comparative Biochemistry and Physiology, Part C 214 (2018) 36–42
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Functional suppression of macrophages derived from THP-1 cells by environmentally-relevant concentrations of arsenite
T
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Huan Xua,b, , Xiaolei Wanga, Wei Wanga,b,c a
East China University of Science and Technology, School of Pharmacy, Department of Pharmaceutical Sciences, Shanghai 200237, China East China University of Science and Technology, State Key Laboratory of Bioreactor Engineering, Shanghai 200237, China c University of New Mexico, Department of Chemistry and Chemical Biology, Albuquerque, NM 87131, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Arsenite Macrophage THP-1 cells Environmentally-relevant concentrations Phagocytosis Cytokine production Nitric oxide
Environmental exposure to arsenic is known to induce immunotoxicity. Macrophages are the professional phagocytes that are important in the immune system. In this study, we utilized the macrophages derived from the THP-1 human monocyte cell line as the experimental model to study the functional suppression induced by arsenite (As+3), one of the most prevalent forms of inorganic arsenic, at environmentally-relevant concentrations. Apoptosis was observed in the THP-1 derived macrophages treated with 500 nM As+3 for 18 h. Suppression of phagocytosis was induced by 18 h As+3 treatment starting from 100 nM. Suppressive effects on the production of two pro-inflammatory cytokines, IL-1β and TNF-α, were also found with the treatment of low to moderate doses of As+3 in lipopolysaccharides-stimulated THP-1 derived macrophages. The nitric oxide production was also inhibited by As+3 treatments, which was negatively correlated with the production of superoxide. Collectively, the results from the study demonstrated that environmentally-relevant concentrations of As+3 induced cytotoxicity and suppressed the major cellular functions in THP-1 derived macrophages. The macrophages were showed to be relatively sensitive to As+3, and could be the essential target of the toxicity induced by environmental arsenic exposures.
1. Introduction Environmental arsenic exposure is a serious public health problem in many countries around world. Studies indicated that many diseases, such as skin lesions, diabetes, cardiovascular diseases, and cancers, were associated with the food, air and drinking water exposure to arsenic (Ahmed et al., 2014; Argos et al., 2010; Schuhmacher-Wolz et al., 2009; Vahter, 2008). Many of these diseases are known to be related to the dysfunction of immune system. The inorganic trivalent form of arsenic, arsenite (As+3), is the most prevalent form of arsenic in anaerobic environment such as the underground water (Vasques et al., 2018). Previous studies indicated that As+3 was immunotoxic and could induce genotoxicity, cell signaling inhibition, cell cycle arrest and functional suppression in different types of immune cells (Bolt et al., 2010; Ray et al., 2017; Soria et al., 2017; Taheri et al., 2016). As+3 is metabolized into the mono-/di-methylated organic forms in vivo to be excluded from the body. Our previous study in the 30 d As+3 drinking water-exposed mouse models revealed that total arsenic was accumulated to 50–100 nM in the primary immune organs of the 500 ppb As+3
exposed mice (Xu et al., 2016a). The accumulation of arsenic in the thymus, spleen and bone marrow can be detrimental to the development and function of T cells and B cells (Ezeh et al., 2014; Xu et al., 2016b). Monocytes are the largest cells in the leukocyte family, which can be differentiated into macrophages and dendritic cells. Macrophages are huge phagocytic cells that are involved in the removal of cellular debris and apoptotic cells generated in the body, we well as extrinsic pathogens (Mosser and Edwards, 2008). The phagocytotic function of macrophages is spontaneous and independent of other types of immune cells (Erwig and Henson, 2007). Besides its critical role as professional phagocytes, macrophages can also secrete pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β to promote inflammatory responses and induce insulin resistance to decrease nutrient storage (Jung et al., 2018). Production of nitric oxide (NO) is also an important function of macrophages, as NO is involved in the host defense mechanism to kill microbes (Wynn et al., 2013). Despite its vital functions in the innate immune system, macrophages are less studied than other types of immune cells due to the technical limitations of obtaining fresh cells. The
⁎ Corresponding author at: Department of Pharmaceutical Sciences, School of Pharmacy, ECUST, Rm 403, Lab Building 14 West, 130 Meilong Rd., Xuhui, Shanghai 200237, China. E-mail addresses: [email protected] (H. Xu), [email protected] (W. Wang).
https://doi.org/10.1016/j.cbpc.2018.08.010 Received 26 April 2018; Received in revised form 27 August 2018; Accepted 31 August 2018 Available online 03 September 2018 1532-0456/ © 2018 Elsevier Inc. All rights reserved.
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2.3. As+3 stock solution preparation and treatments
THP-1 monocytes can be differentiated into macrophages with the treatment of stimuli, and are often used as the in vitro model to study macrophage functions (Daigneault et al., 2010; Genin et al., 2015). The phenotype of THP-1 derived macrophages resembles the isolated macrophages from the human peripheral blood cells, and it can be stimulated to produce pro-inflammatory cytokines and NO (Daigneault et al., 2010). Arsenic exposure is known to impair the macrophage functions in vivo (Banerjee et al., 2009). Studies in sigmodon and chicken models showed that As+3 exposure decreased the phagocytosis efficiency and inhibited NO production of macrophages (Aggarwal et al., 2008; Savabieasfahani et al., 1998). Results from in vitro studies also indicated that As+3 disrupted cell adhesion, inhibited cytokine production and induced oxidative stress in human and THP-1 derived macrophages (Lemarie et al., 2006; Wang et al., 2011). However, the As+3 doses or concentrations used in these in vivo and in vitro studies were high above the environmentally-relevant levels. In the present study, THP-1 cells were differentiated into macrophages and stimulated with lipopolysaccharides (LPS) to induce cytokine secretion and NO production. The differentiated and stimulated macrophages were treated with 50 to 500 nM of As+3 for 18 h, and the effects of As+3 on cell viability, phagocytosis, cytokine secretion and NO production were evaluated. The results from the study are better reflection of the suppressive effects induced by environmental arsenic exposures than the previous studies using high concentrations of As+3.
To make the As+3 stock solution, sodium arsenite powder was weighed on a Shimadzu ATX224 balance and dissolved in sterile deionized water at the concentration of 0.1 M. The concentration of the stock solution was confirmed by ICP-MS and stored at 4 °C. To make the test solutions to treat the THP-1 derived macrophages, the 0.1 M As+3 stock solution was diluted to 50, 100, 200 and 500 μM in culture medium. 5 μl of the test solutions were added to 5 ml of cell cultures to achieve the final concentrations of 50, 100, 200 and 500 nM (1:1000 dilution). 5 μl of culture medium was also added to the 0 nM (control) samples. The cells were treated for 18 h in vitro at 37 °C for the evaluation of the effects of As+3 on THP-1 differentiated macrophages. 2.4. Cell apoptosis and phagocytosis assay by flow cytometry After the As+3 treatment, 1 × 105 THP-1 derived macrophages from each well were centrifuged and resuspended in 100 μl binding buffer supplied by the Annexin V-FITC/PI Apoptosis Detection Kit. 5 μl Annexin V and 10 μl PI were added to the cell suspension and stained at RT for 15 min. 400 μl binding buffer was added to dilute the samples to 500 μl. All the samples were analyzed within 1 h after the dilution on a BD AccuriC6 Plus flow cytometer. CCRF-CEM cells were also provided by Stem Cell Bank, Chinese Academy of Sciences. In order to induce apoptosis in the CCRF-CEM cells, cells were treated with 20 μM etoposide for 4 h, and > 80% rate of apoptosis was observed in the treated cells. The treated CCRF-CEM cells were stained with Cell Tracker Red at 37 °C for 15 min, washed twice, resuspended in cell culture medium at 1 × 106 cells/ml and combined with the same volume of 1 × 105 cells/ ml 18 h As+3-treated macrophages. After 2 h incubation at 37 °C, the combined cells were analyzed on a BD AccuriC6 Plus flow cytometer. Cytochalasin D was added at 5 μM to the negative control samples 20 min before the combination step to block the F-actin dependent phagocytosis.
2. Material and methods 2.1. Chemicals and reagents Sodium arsenite (As+3, CAS 7784-46-5, NaAsO2, Cat. No. S7400, 98.8% purity analyzed by HPLC-ICP-MS), RPMI 1640 medium and Dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). LPS (Cat. No. 60322ES10), Phorbol 12-myristate 13acetate (PMA, Cat. No. 50601ES03), Annexin V-FITC/PI Apoptosis Detection Kit (Cat. No. 40302ES60), Dihydroethidium (DHE, Cat. No. 50102ES02), 0.4% Trypan Blue (Cat. No. 40207ES60), 4-Amino-5Methylamino-2,7-Difluorofluorescein Diacetate (DAF-FM, Cat. No. 40769ES60) were purchased from Yeasen (Shanghai, China). Penicillin/Streptomycin (Pen/Strep, Cat. No. 15140122), Dulbecco's phosphate buffered saline (DPBS, Cat. No. 14190250), Fetal Bovine Serum (FBS, Cat. No. 10099141), Cell Tracker Red CMTPX Dye (Cat. No. C34552), Cytochalasin D (Cat. No. PHZ1063), IL-1β Human ELISA Kit (Cat. No. KHC0011) and TNF-α Human ELISA Kit (Cat. No. KHC3011) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Etoposide (CAS 33419-42-0, Cat. No. E0675) was purchased from Tokyo Chemical Industry (Shanghai, China). PerCP-Cy™5.5 Mouse Anti-Human CD14 Clone M5E2 (Cat. No. 550787) was purchased from BD Biosciences (San Jose, CA, USA).
2.5. IL-1β and TNF-α ELISA assay THP-1 derived macrophages were stimulated with 10 ng/ml LPS for 18 h with As+3 treatments. After the stimulation, supernatants were harvested and stored at −80 °C until assayed with the IL-1β and TNF-α ELISA kits. The experiments were performed according to the Novex® ELISA Kit Technical Guide and the kit manuals. Harvested cell supernatant samples were diluted at 1:10 with the culture medium for both IL-1β and TNF-α detection. Unstimulated samples were added for both assays as the controls for the LPS stimulation. The lower limits of detection (LLODs) were 0.84 pg/ml for IL-1β and 1.25 pg/ml for TNF-α. 2.6. Superoxide and NO detection by flow cytometry For superoxide detection, DHE was dissolved in DMSO and diluted to 25 μM in cell culture medium as the DHE working solution. For NO detection, DAF-FM diacetate was diluted to 25 μM in cell culture medium as the DAF-FM working solution. 100 μl from each working solution was added to 300 μl of the stimulated and treated macrophages to achieve the final DHE/DAF-FM concentration of 5 μM. After 30 min of staining at 37 °C in the incubator, cells were washed twice with ice cold DPBS and analyzed on a BD AccuriC6 Plus flow cytometer. The green fluorescence of DAF-FM was detected in FL1 and the red fluorescence of DHE was detected in FL3.
2.2. In vitro differentiation of THP-1 cells The THP-1 human monocyte cell line was kindly provided by Stem Cell Bank, Chinese Academy of Sciences. Cells were maintained in culture medium (500 ml RPMI 1640 supplied with 10% FBS and 100 U/ ml Pen/Strep) and subcultured every 3 d in T-75 flasks. PMA was diluted to 200 nM concentration for the differentiation of THP-1 cells, and CD14 was used as a differentiation cell surface marker (Daigneault et al., 2010). After 3 d treatment with PMA, THP-1 cells became adherent. The PMA-containing medium was removed and the differentiated cells were cultured in fresh cell culture medium for 5 d to increase their cytoplasmic volume. CD14 antibody was used to stain the differentiated cells at 1 μg/ml in DPBS in dark at RT. Stained cells were washed twice with DPBS and analyzed on a BD AccuriC6 Plus flow cytometer.
2.7. Statistics and correlation analysis Data were analyzed with Excel 2016 and GraphPad PRISM 5.01 softwares. Three independent experiments were performed and analyzed for each dose of As+3. One-way analysis of variance (ANOVA) and Dunnett's t-test were applied to determine the difference between 37
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Fig. 1. THP-1 derived macrophages by PMA treatment and resting. THP-1 cells were differentiated to macrophages by 200 nM PMA treatment for 3 d, followed by a 5 d resting step without PMA. A, representing pictures of the original THP-1 cells, PMA treated cells and the cells after the resting step, which was obtained with a Nikon Eclipse Ti-S microscope at 3 ms exposure time. B, mean channel fluorescence intensity (MFI) of CD14 on the cells at each stage. *Significantly different compared to the control (p < 0.05). Results are mean ± SD.
100 nM, indicating that low to moderate doses of As+3 can impair the phagocytosis function of macrophages (Fig. 3B).
the control and treatment groups. Pearson Correlation and polynomial linear regression were used in the analysis of the correlations between the MFI of DHE and DAF-FM. R-square (R2) was presented in the figure to show the correlation between the two factors.
3.3. Suppression of cytokine and NO production by As+3 in THP-1 derived macrophages
3. Results Besides its function of spontaneous phagocytosis, macrophages can also produce cytokines and NO to induce and mediate immune responses. IL-1β and TNF-α are two pro-inflammatory cytokines secreted by macrophages, which can be induced by LPS treatment in vitro (Daigneault et al., 2010). Therefore, we treated THP-1 derived macrophages with 10 ng/ml LPS and 0, 50, 100, 200, 500 nM As+3 for 18 h. The concentrations of IL-1β and TNF-α in the supernatant were analyzed by ELISA. Dose-dependent decreases of both IL-1β and TNF-α with the increase of As+3 concentrations (Fig. 4). TNF-α production was more sensitive to As+3-induced suppression, which was inhibited by As+3 at 100 nM (Fig. 4B). These results demonstrated that low to moderate concentrations of As+3 could inhibit the secretion of pro-inflammatory cytokines in macrophages. Similarly, the production of NO was also inhibited by As+3 at 200 nM in LPS stimulated THP-1 derived macrophages (Fig. 5A). In additional to NO production, we also examined the superoxide levels in As+3 treated macrophages. Significant increase of superoxide levels was observed with the 500 nM As+3 treatment, which may be due to the As+3-induced oxidative stress (Fig. 5B). Interestingly, regression analysis revealed that the decrease of NO production was correlated with increase of superoxide levels in these cells, indicating that the As+3-induced suppression of NO production in macrophages is likely to be associated with the oxidative stress induced by As+3 at environmentally-relevant concentrations (Fig. 5C).
3.1. Apoptosis induced by As+3 in THP-1 derived macrophages To set up the THP-1 cell derived macrophage model, we followed the protocol described in Daigneault et al., 2010. THP-1 cells were treated with 200 nM PMA for 3 d, followed by a 5 d resting step. Significant increase of cytoplasmic volume was observed in the PMAtreated cells, and the macrophage-like morphology was seen after the resting step (Fig. 1A). Decrease in monocyte-macrophage differentiation marker CD14 expression indicated that the PMA-stimulated and rested THP-1 monocytes were differentiated towards the macrophages (Fig. 1B). The differentiated cells were treated with 0 (Control), 50, 100, 200 and 500 nM of As+3 for 18 h, and Annexin V/PI staining was performed to evaluate the As+3-induced apoptosis (Fig. 2A). Significant decrease of cell viability and increase of apoptotic cells were only observed in 500 nM As+3 treated macrophages (Fig. 2B and C). In our previous study, 18 h 500 nM As+3 treatment did not induce apoptosis in the early developing T cells (Xu et al., 2016c). Therefore, these results indicated that THP-1 derived macrophages are relatively sensitive to As+3-induced cytotoxicity. 3.2. Inhibition of phagocytosis by As+3 in THP-1 derived macrophages Phagocytosis is the major function of macrophages. To examine if environmentally-relevant levels of As+3 can alter the phagocytosis of macrophages, apoptotic CCRF-CEM cells were labeled with Cell Tracker Red and combined with As+3 treated THP-1 derived macrophages, and the Cell Tracker Red intensity reflected the function of phagocytosis (Fig. 3A). A dose-dependent decrease of phagocytosis function was observed in As+3 treated THP-1 derived macrophages started at
4. Discussion Arsenic exposure is known to induce detrimental effects in different organs and cell types (Biswas et al., 2008; Cooper et al., 2013; SotoPeña et al., 2006). Results from our previous studies indicated both T 38
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Fig. 2. Apoptosis in THP-1 derived macrophages treated with As+3. THP-1 derived macrophages were treated with 0, 50, 100, 200 and 500 nM As+3 for 18 h. Cells were stained with Annexin V and PI, and the apoptosis of the cells were analyzed on a BD AccuriC6 Plus flow cytometer. A, representative plots of different treatments and the positive control. B, % of viable cells (Annexin V- PI-). C, % of viable cells (Annexin V+ PI- and Annexin V+ PI+). *Significantly different compared to the control (p < 0.05). Results are mean ± SD.
homeostasis. In this study, we induced apoptosis in the cells of a T cell line, CCRF-CEM, and combined THP-1 derived macrophages treated with low to moderate doses of As+3. The results clearly showed that the phagocytosis of apoptotic cells were inhibited by the As+3 treatment in the macrophages. While this is a good start as the initial insights into the functional inhibition induced by environmentally-relevant concentrations of As+3, more comprehensive studies with bacteria, cell debris, latex beads and other types of cancer cells are being conducted for a complete evaluation of the suppressive effects of As+3 on macrophage phagocytosis. Since the phagocytosis ability of macrophages is known to be actin-dependent and related to multiple signaling pathways, such as G-protein and MAPK signaling pathways, future studies should also be conducted to reveal the effects of As+3 on these signaling pathways in the macrophages to uncover the mechanisms of the suppression (Huang et al., 2014; Zhu et al., 2017). Macrophages display a high plasticity. They can be polarized to classically activated type 1 (M1) and the alternatively activated type 2 (M2) macrophages with the respective stimuli. M1 macrophages, which can be stimulated with LPS, secrete pro-inflammatory cytokines such as TNF-α and IL-1β, and have high capability of producing reactive oxygen and nitrogen species (Shiratori et al., 2017). Although the THP1 cell line has its limitations in evaluating the whole spectrum of
and B lymphocytes were sensitive to arsenic-induced toxicity in vitro and in vivo (Ezeh et al., 2014; Xu et al., 2016c). As the immune system is sensitive to arsenic-induced toxicity, the in vivo effects of environmental arsenic exposures are likely to be associated with the arsenic-induced immunosuppression. Therefore, more and more studies are focusing on the immunotoxicity induced by different arsenical species at environmentally-relevant levels. In this As+3-induced immunotoxicity study, we evaluated the alteration of macrophage functions with the treatment of low to moderate doses of As+3. In our previous studies, the intracellular concentrations of the arsenical species were quantified in 30 d As+3 drinking water exposed mice. Total arsenic amount was accumulated to the highest in the thymus of the 500 ppb exposed mice, which was about 100 nM (Xu et al., 2016a). Since the environmental arsenic exposures are usually below ppm levels, the in vitro studies should also be conducted within the nanomolar range for environmental relevance. Macrophages are the professional phagocytes that engulfs pathogens, cell debris, apoptotic and malignant cells. Macrophages are distributed in most tissues throughout the body, and its activation is closely related to inflammatory responses (Wynn et al., 2013). Phagocytosis is an important process not only for the host-defense mechanism, but also for the nutrient recycling in the metabolism and 39
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Fig. 3. Phagocytosis function of THP-1 derived macrophages treated with As+3. THP-1 derived macrophages were treated with 0, 50, 100, 200 and 500 nM As+3 for 18 h. Apoptotic CCRF-CEM cells induced by etoposide were labeled with Cell Tracker Red and combined with the treated macrophages. Phagocytosis was analyzed on a BD AccuriC6 Plus flow cytometer after 2 h of incubation. A, representative plots of different treatments and the negative control. B, phagocytosis function as the mean channel fluorescence intensity (MFI) of Cell Tracker Red. *Significantly different compared to the control (p < 0.05). Results are mean ± SD.
environmental exposure to As+3. Mechanistic future studies should be performed to analyze the molecular basis of the suppression. Also, the effects of As+3 and other arsenical species at low to moderate levels on the M2 macrophage functions should also be studied in the future with the primary macrophage models derived from isolated human and mouse monocytes with optimized techniques.
macrophage functions due to the low expression of M2 maker CD163 and defective migration ability after stimulation, the THP-1 derived macrophages are still the proper models to evaluate the M1 functions (Tedesco et al., 2018). Therefore, the As+3-induced suppression of TNFα and IL-1β productions indicated that the function of cytokine secretion of the M1 macrophages were likely to be inhibited by
Fig. 4. Cytokine secretion of THP-1 derived macrophages treated with As+3 and stimulated with LPS. THP-1 derived macrophages were treated with 0, 50, 100, 200 and 500 nM As+3 for 18 h with the stimulation of 10 ng/ml LPS. TNF-α and IL-1β levels in the supernatants were quantified by ELISA assay. A, IL1β concentrations in the supernatants. B, TNF-α concentrations in the supernatants. *Significantly different compared to the control (p < 0.05). Results are mean ± SD.
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Fig. 5. NO and superoxide production of THP-1 derived macrophages treated with As+3 and stimulated with LPS. THP-1 derived macrophages were treated with 0, 50, 100, 200 and 500 nM As+3 for 18 h with the stimulation of 10 ng/ml LPS. Cells were stained with DAF-FM (for NO detection) and DHE (for superoxide detection), incubated and analyzed on a BD AccuriC6 Plus flow cytometer. A, NO production as the mean channel fluorescence intensity (MFI) of DAF-FM. B, superoxide production as the mean channel fluorescence intensity (MFI) of DHE. C, correlation between the MFI of DAF-FM and DHE *Significantly different compared to the control (p < 0.05). Results are mean ± SD.
grant from East China University of Science and Technology (grant No. YC 0142125).
The suppressive effects of NO production induced by environmentally-relevant concentrations of As+3 should not be ignored. As we all known, arsenic exposure can induce oxidative stress and the production of reactive oxygen and nitric species (Zhou et al., 2011, 2016). We found that As+3 treatments at low levels decreased the secretion of NO in the THP-1 derived macrophage model. Interestingly, an epidemiological study reported that a significant decrease of serum NO levels was observed in the population exposed to arsenic via drinking water (Pi et al., 2000). The suggestion from that study was that As+3 suppressed NO production by inhibiting NO synthase in human umbilical vein endothelial cells. However, the suppression of another major NO producer. The macrophages, may also be involved in the decrease of the in vivo NO levels. Another interesting finding was that the decreased NO production was correlated with the increase of superoxide production, indicating that the oxidative stress induced by As+3 and the relative signaling pathways may also be involved in the As+3-induced inhibition on macrophage NO production. Therefore, the role of the NO-suppressive effect of As+3 in the regulation of the immune system and its association with the oxidative stress pathways in macrophages should be emphasized in future studies. In summary, we found that 500 nM As+3 induced apoptosis in THP1 derived macrophages. Low to moderate doses of As+3 suppressed the functions of phagocytosis, cytokine secretion and NO production of macrophages in vitro. The decrease of NO production induced by As+3 treatments were correlated with the increase of superoxide levels. The results from the study indicated that environmentally-relevant concentrations of As+3 could suppress the major functions of THP-derived macrophages, providing the initial evidence for the future mechanistic studies of the toxicity on macrophages induced by environmental arsenic exposures.
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Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc
Functional suppression of macrophages derived from THP-1 cells by environmentally-relevant concentrations of arsenite
T
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Huan Xua,b, , Xiaolei Wanga, Wei Wanga,b,c a
East China University of Science and Technology, School of Pharmacy, Department of Pharmaceutical Sciences, Shanghai 200237, China East China University of Science and Technology, State Key Laboratory of Bioreactor Engineering, Shanghai 200237, China c University of New Mexico, Department of Chemistry and Chemical Biology, Albuquerque, NM 87131, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Arsenite Macrophage THP-1 cells Environmentally-relevant concentrations Phagocytosis Cytokine production Nitric oxide
Environmental exposure to arsenic is known to induce immunotoxicity. Macrophages are the professional phagocytes that are important in the immune system. In this study, we utilized the macrophages derived from the THP-1 human monocyte cell line as the experimental model to study the functional suppression induced by arsenite (As+3), one of the most prevalent forms of inorganic arsenic, at environmentally-relevant concentrations. Apoptosis was observed in the THP-1 derived macrophages treated with 500 nM As+3 for 18 h. Suppression of phagocytosis was induced by 18 h As+3 treatment starting from 100 nM. Suppressive effects on the production of two pro-inflammatory cytokines, IL-1β and TNF-α, were also found with the treatment of low to moderate doses of As+3 in lipopolysaccharides-stimulated THP-1 derived macrophages. The nitric oxide production was also inhibited by As+3 treatments, which was negatively correlated with the production of superoxide. Collectively, the results from the study demonstrated that environmentally-relevant concentrations of As+3 induced cytotoxicity and suppressed the major cellular functions in THP-1 derived macrophages. The macrophages were showed to be relatively sensitive to As+3, and could be the essential target of the toxicity induced by environmental arsenic exposures.
1. Introduction Environmental arsenic exposure is a serious public health problem in many countries around world. Studies indicated that many diseases, such as skin lesions, diabetes, cardiovascular diseases, and cancers, were associated with the food, air and drinking water exposure to arsenic (Ahmed et al., 2014; Argos et al., 2010; Schuhmacher-Wolz et al., 2009; Vahter, 2008). Many of these diseases are known to be related to the dysfunction of immune system. The inorganic trivalent form of arsenic, arsenite (As+3), is the most prevalent form of arsenic in anaerobic environment such as the underground water (Vasques et al., 2018). Previous studies indicated that As+3 was immunotoxic and could induce genotoxicity, cell signaling inhibition, cell cycle arrest and functional suppression in different types of immune cells (Bolt et al., 2010; Ray et al., 2017; Soria et al., 2017; Taheri et al., 2016). As+3 is metabolized into the mono-/di-methylated organic forms in vivo to be excluded from the body. Our previous study in the 30 d As+3 drinking water-exposed mouse models revealed that total arsenic was accumulated to 50–100 nM in the primary immune organs of the 500 ppb As+3
exposed mice (Xu et al., 2016a). The accumulation of arsenic in the thymus, spleen and bone marrow can be detrimental to the development and function of T cells and B cells (Ezeh et al., 2014; Xu et al., 2016b). Monocytes are the largest cells in the leukocyte family, which can be differentiated into macrophages and dendritic cells. Macrophages are huge phagocytic cells that are involved in the removal of cellular debris and apoptotic cells generated in the body, we well as extrinsic pathogens (Mosser and Edwards, 2008). The phagocytotic function of macrophages is spontaneous and independent of other types of immune cells (Erwig and Henson, 2007). Besides its critical role as professional phagocytes, macrophages can also secrete pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β to promote inflammatory responses and induce insulin resistance to decrease nutrient storage (Jung et al., 2018). Production of nitric oxide (NO) is also an important function of macrophages, as NO is involved in the host defense mechanism to kill microbes (Wynn et al., 2013). Despite its vital functions in the innate immune system, macrophages are less studied than other types of immune cells due to the technical limitations of obtaining fresh cells. The
⁎ Corresponding author at: Department of Pharmaceutical Sciences, School of Pharmacy, ECUST, Rm 403, Lab Building 14 West, 130 Meilong Rd., Xuhui, Shanghai 200237, China. E-mail addresses: [email protected] (H. Xu), [email protected] (W. Wang).
https://doi.org/10.1016/j.cbpc.2018.08.010 Received 26 April 2018; Received in revised form 27 August 2018; Accepted 31 August 2018 Available online 03 September 2018 1532-0456/ © 2018 Elsevier Inc. All rights reserved.
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2.3. As+3 stock solution preparation and treatments
THP-1 monocytes can be differentiated into macrophages with the treatment of stimuli, and are often used as the in vitro model to study macrophage functions (Daigneault et al., 2010; Genin et al., 2015). The phenotype of THP-1 derived macrophages resembles the isolated macrophages from the human peripheral blood cells, and it can be stimulated to produce pro-inflammatory cytokines and NO (Daigneault et al., 2010). Arsenic exposure is known to impair the macrophage functions in vivo (Banerjee et al., 2009). Studies in sigmodon and chicken models showed that As+3 exposure decreased the phagocytosis efficiency and inhibited NO production of macrophages (Aggarwal et al., 2008; Savabieasfahani et al., 1998). Results from in vitro studies also indicated that As+3 disrupted cell adhesion, inhibited cytokine production and induced oxidative stress in human and THP-1 derived macrophages (Lemarie et al., 2006; Wang et al., 2011). However, the As+3 doses or concentrations used in these in vivo and in vitro studies were high above the environmentally-relevant levels. In the present study, THP-1 cells were differentiated into macrophages and stimulated with lipopolysaccharides (LPS) to induce cytokine secretion and NO production. The differentiated and stimulated macrophages were treated with 50 to 500 nM of As+3 for 18 h, and the effects of As+3 on cell viability, phagocytosis, cytokine secretion and NO production were evaluated. The results from the study are better reflection of the suppressive effects induced by environmental arsenic exposures than the previous studies using high concentrations of As+3.
To make the As+3 stock solution, sodium arsenite powder was weighed on a Shimadzu ATX224 balance and dissolved in sterile deionized water at the concentration of 0.1 M. The concentration of the stock solution was confirmed by ICP-MS and stored at 4 °C. To make the test solutions to treat the THP-1 derived macrophages, the 0.1 M As+3 stock solution was diluted to 50, 100, 200 and 500 μM in culture medium. 5 μl of the test solutions were added to 5 ml of cell cultures to achieve the final concentrations of 50, 100, 200 and 500 nM (1:1000 dilution). 5 μl of culture medium was also added to the 0 nM (control) samples. The cells were treated for 18 h in vitro at 37 °C for the evaluation of the effects of As+3 on THP-1 differentiated macrophages. 2.4. Cell apoptosis and phagocytosis assay by flow cytometry After the As+3 treatment, 1 × 105 THP-1 derived macrophages from each well were centrifuged and resuspended in 100 μl binding buffer supplied by the Annexin V-FITC/PI Apoptosis Detection Kit. 5 μl Annexin V and 10 μl PI were added to the cell suspension and stained at RT for 15 min. 400 μl binding buffer was added to dilute the samples to 500 μl. All the samples were analyzed within 1 h after the dilution on a BD AccuriC6 Plus flow cytometer. CCRF-CEM cells were also provided by Stem Cell Bank, Chinese Academy of Sciences. In order to induce apoptosis in the CCRF-CEM cells, cells were treated with 20 μM etoposide for 4 h, and > 80% rate of apoptosis was observed in the treated cells. The treated CCRF-CEM cells were stained with Cell Tracker Red at 37 °C for 15 min, washed twice, resuspended in cell culture medium at 1 × 106 cells/ml and combined with the same volume of 1 × 105 cells/ ml 18 h As+3-treated macrophages. After 2 h incubation at 37 °C, the combined cells were analyzed on a BD AccuriC6 Plus flow cytometer. Cytochalasin D was added at 5 μM to the negative control samples 20 min before the combination step to block the F-actin dependent phagocytosis.
2. Material and methods 2.1. Chemicals and reagents Sodium arsenite (As+3, CAS 7784-46-5, NaAsO2, Cat. No. S7400, 98.8% purity analyzed by HPLC-ICP-MS), RPMI 1640 medium and Dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). LPS (Cat. No. 60322ES10), Phorbol 12-myristate 13acetate (PMA, Cat. No. 50601ES03), Annexin V-FITC/PI Apoptosis Detection Kit (Cat. No. 40302ES60), Dihydroethidium (DHE, Cat. No. 50102ES02), 0.4% Trypan Blue (Cat. No. 40207ES60), 4-Amino-5Methylamino-2,7-Difluorofluorescein Diacetate (DAF-FM, Cat. No. 40769ES60) were purchased from Yeasen (Shanghai, China). Penicillin/Streptomycin (Pen/Strep, Cat. No. 15140122), Dulbecco's phosphate buffered saline (DPBS, Cat. No. 14190250), Fetal Bovine Serum (FBS, Cat. No. 10099141), Cell Tracker Red CMTPX Dye (Cat. No. C34552), Cytochalasin D (Cat. No. PHZ1063), IL-1β Human ELISA Kit (Cat. No. KHC0011) and TNF-α Human ELISA Kit (Cat. No. KHC3011) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Etoposide (CAS 33419-42-0, Cat. No. E0675) was purchased from Tokyo Chemical Industry (Shanghai, China). PerCP-Cy™5.5 Mouse Anti-Human CD14 Clone M5E2 (Cat. No. 550787) was purchased from BD Biosciences (San Jose, CA, USA).
2.5. IL-1β and TNF-α ELISA assay THP-1 derived macrophages were stimulated with 10 ng/ml LPS for 18 h with As+3 treatments. After the stimulation, supernatants were harvested and stored at −80 °C until assayed with the IL-1β and TNF-α ELISA kits. The experiments were performed according to the Novex® ELISA Kit Technical Guide and the kit manuals. Harvested cell supernatant samples were diluted at 1:10 with the culture medium for both IL-1β and TNF-α detection. Unstimulated samples were added for both assays as the controls for the LPS stimulation. The lower limits of detection (LLODs) were 0.84 pg/ml for IL-1β and 1.25 pg/ml for TNF-α. 2.6. Superoxide and NO detection by flow cytometry For superoxide detection, DHE was dissolved in DMSO and diluted to 25 μM in cell culture medium as the DHE working solution. For NO detection, DAF-FM diacetate was diluted to 25 μM in cell culture medium as the DAF-FM working solution. 100 μl from each working solution was added to 300 μl of the stimulated and treated macrophages to achieve the final DHE/DAF-FM concentration of 5 μM. After 30 min of staining at 37 °C in the incubator, cells were washed twice with ice cold DPBS and analyzed on a BD AccuriC6 Plus flow cytometer. The green fluorescence of DAF-FM was detected in FL1 and the red fluorescence of DHE was detected in FL3.
2.2. In vitro differentiation of THP-1 cells The THP-1 human monocyte cell line was kindly provided by Stem Cell Bank, Chinese Academy of Sciences. Cells were maintained in culture medium (500 ml RPMI 1640 supplied with 10% FBS and 100 U/ ml Pen/Strep) and subcultured every 3 d in T-75 flasks. PMA was diluted to 200 nM concentration for the differentiation of THP-1 cells, and CD14 was used as a differentiation cell surface marker (Daigneault et al., 2010). After 3 d treatment with PMA, THP-1 cells became adherent. The PMA-containing medium was removed and the differentiated cells were cultured in fresh cell culture medium for 5 d to increase their cytoplasmic volume. CD14 antibody was used to stain the differentiated cells at 1 μg/ml in DPBS in dark at RT. Stained cells were washed twice with DPBS and analyzed on a BD AccuriC6 Plus flow cytometer.
2.7. Statistics and correlation analysis Data were analyzed with Excel 2016 and GraphPad PRISM 5.01 softwares. Three independent experiments were performed and analyzed for each dose of As+3. One-way analysis of variance (ANOVA) and Dunnett's t-test were applied to determine the difference between 37
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Fig. 1. THP-1 derived macrophages by PMA treatment and resting. THP-1 cells were differentiated to macrophages by 200 nM PMA treatment for 3 d, followed by a 5 d resting step without PMA. A, representing pictures of the original THP-1 cells, PMA treated cells and the cells after the resting step, which was obtained with a Nikon Eclipse Ti-S microscope at 3 ms exposure time. B, mean channel fluorescence intensity (MFI) of CD14 on the cells at each stage. *Significantly different compared to the control (p < 0.05). Results are mean ± SD.
100 nM, indicating that low to moderate doses of As+3 can impair the phagocytosis function of macrophages (Fig. 3B).
the control and treatment groups. Pearson Correlation and polynomial linear regression were used in the analysis of the correlations between the MFI of DHE and DAF-FM. R-square (R2) was presented in the figure to show the correlation between the two factors.
3.3. Suppression of cytokine and NO production by As+3 in THP-1 derived macrophages
3. Results Besides its function of spontaneous phagocytosis, macrophages can also produce cytokines and NO to induce and mediate immune responses. IL-1β and TNF-α are two pro-inflammatory cytokines secreted by macrophages, which can be induced by LPS treatment in vitro (Daigneault et al., 2010). Therefore, we treated THP-1 derived macrophages with 10 ng/ml LPS and 0, 50, 100, 200, 500 nM As+3 for 18 h. The concentrations of IL-1β and TNF-α in the supernatant were analyzed by ELISA. Dose-dependent decreases of both IL-1β and TNF-α with the increase of As+3 concentrations (Fig. 4). TNF-α production was more sensitive to As+3-induced suppression, which was inhibited by As+3 at 100 nM (Fig. 4B). These results demonstrated that low to moderate concentrations of As+3 could inhibit the secretion of pro-inflammatory cytokines in macrophages. Similarly, the production of NO was also inhibited by As+3 at 200 nM in LPS stimulated THP-1 derived macrophages (Fig. 5A). In additional to NO production, we also examined the superoxide levels in As+3 treated macrophages. Significant increase of superoxide levels was observed with the 500 nM As+3 treatment, which may be due to the As+3-induced oxidative stress (Fig. 5B). Interestingly, regression analysis revealed that the decrease of NO production was correlated with increase of superoxide levels in these cells, indicating that the As+3-induced suppression of NO production in macrophages is likely to be associated with the oxidative stress induced by As+3 at environmentally-relevant concentrations (Fig. 5C).
3.1. Apoptosis induced by As+3 in THP-1 derived macrophages To set up the THP-1 cell derived macrophage model, we followed the protocol described in Daigneault et al., 2010. THP-1 cells were treated with 200 nM PMA for 3 d, followed by a 5 d resting step. Significant increase of cytoplasmic volume was observed in the PMAtreated cells, and the macrophage-like morphology was seen after the resting step (Fig. 1A). Decrease in monocyte-macrophage differentiation marker CD14 expression indicated that the PMA-stimulated and rested THP-1 monocytes were differentiated towards the macrophages (Fig. 1B). The differentiated cells were treated with 0 (Control), 50, 100, 200 and 500 nM of As+3 for 18 h, and Annexin V/PI staining was performed to evaluate the As+3-induced apoptosis (Fig. 2A). Significant decrease of cell viability and increase of apoptotic cells were only observed in 500 nM As+3 treated macrophages (Fig. 2B and C). In our previous study, 18 h 500 nM As+3 treatment did not induce apoptosis in the early developing T cells (Xu et al., 2016c). Therefore, these results indicated that THP-1 derived macrophages are relatively sensitive to As+3-induced cytotoxicity. 3.2. Inhibition of phagocytosis by As+3 in THP-1 derived macrophages Phagocytosis is the major function of macrophages. To examine if environmentally-relevant levels of As+3 can alter the phagocytosis of macrophages, apoptotic CCRF-CEM cells were labeled with Cell Tracker Red and combined with As+3 treated THP-1 derived macrophages, and the Cell Tracker Red intensity reflected the function of phagocytosis (Fig. 3A). A dose-dependent decrease of phagocytosis function was observed in As+3 treated THP-1 derived macrophages started at
4. Discussion Arsenic exposure is known to induce detrimental effects in different organs and cell types (Biswas et al., 2008; Cooper et al., 2013; SotoPeña et al., 2006). Results from our previous studies indicated both T 38
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Fig. 2. Apoptosis in THP-1 derived macrophages treated with As+3. THP-1 derived macrophages were treated with 0, 50, 100, 200 and 500 nM As+3 for 18 h. Cells were stained with Annexin V and PI, and the apoptosis of the cells were analyzed on a BD AccuriC6 Plus flow cytometer. A, representative plots of different treatments and the positive control. B, % of viable cells (Annexin V- PI-). C, % of viable cells (Annexin V+ PI- and Annexin V+ PI+). *Significantly different compared to the control (p < 0.05). Results are mean ± SD.
homeostasis. In this study, we induced apoptosis in the cells of a T cell line, CCRF-CEM, and combined THP-1 derived macrophages treated with low to moderate doses of As+3. The results clearly showed that the phagocytosis of apoptotic cells were inhibited by the As+3 treatment in the macrophages. While this is a good start as the initial insights into the functional inhibition induced by environmentally-relevant concentrations of As+3, more comprehensive studies with bacteria, cell debris, latex beads and other types of cancer cells are being conducted for a complete evaluation of the suppressive effects of As+3 on macrophage phagocytosis. Since the phagocytosis ability of macrophages is known to be actin-dependent and related to multiple signaling pathways, such as G-protein and MAPK signaling pathways, future studies should also be conducted to reveal the effects of As+3 on these signaling pathways in the macrophages to uncover the mechanisms of the suppression (Huang et al., 2014; Zhu et al., 2017). Macrophages display a high plasticity. They can be polarized to classically activated type 1 (M1) and the alternatively activated type 2 (M2) macrophages with the respective stimuli. M1 macrophages, which can be stimulated with LPS, secrete pro-inflammatory cytokines such as TNF-α and IL-1β, and have high capability of producing reactive oxygen and nitrogen species (Shiratori et al., 2017). Although the THP1 cell line has its limitations in evaluating the whole spectrum of
and B lymphocytes were sensitive to arsenic-induced toxicity in vitro and in vivo (Ezeh et al., 2014; Xu et al., 2016c). As the immune system is sensitive to arsenic-induced toxicity, the in vivo effects of environmental arsenic exposures are likely to be associated with the arsenic-induced immunosuppression. Therefore, more and more studies are focusing on the immunotoxicity induced by different arsenical species at environmentally-relevant levels. In this As+3-induced immunotoxicity study, we evaluated the alteration of macrophage functions with the treatment of low to moderate doses of As+3. In our previous studies, the intracellular concentrations of the arsenical species were quantified in 30 d As+3 drinking water exposed mice. Total arsenic amount was accumulated to the highest in the thymus of the 500 ppb exposed mice, which was about 100 nM (Xu et al., 2016a). Since the environmental arsenic exposures are usually below ppm levels, the in vitro studies should also be conducted within the nanomolar range for environmental relevance. Macrophages are the professional phagocytes that engulfs pathogens, cell debris, apoptotic and malignant cells. Macrophages are distributed in most tissues throughout the body, and its activation is closely related to inflammatory responses (Wynn et al., 2013). Phagocytosis is an important process not only for the host-defense mechanism, but also for the nutrient recycling in the metabolism and 39
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Fig. 3. Phagocytosis function of THP-1 derived macrophages treated with As+3. THP-1 derived macrophages were treated with 0, 50, 100, 200 and 500 nM As+3 for 18 h. Apoptotic CCRF-CEM cells induced by etoposide were labeled with Cell Tracker Red and combined with the treated macrophages. Phagocytosis was analyzed on a BD AccuriC6 Plus flow cytometer after 2 h of incubation. A, representative plots of different treatments and the negative control. B, phagocytosis function as the mean channel fluorescence intensity (MFI) of Cell Tracker Red. *Significantly different compared to the control (p < 0.05). Results are mean ± SD.
environmental exposure to As+3. Mechanistic future studies should be performed to analyze the molecular basis of the suppression. Also, the effects of As+3 and other arsenical species at low to moderate levels on the M2 macrophage functions should also be studied in the future with the primary macrophage models derived from isolated human and mouse monocytes with optimized techniques.
macrophage functions due to the low expression of M2 maker CD163 and defective migration ability after stimulation, the THP-1 derived macrophages are still the proper models to evaluate the M1 functions (Tedesco et al., 2018). Therefore, the As+3-induced suppression of TNFα and IL-1β productions indicated that the function of cytokine secretion of the M1 macrophages were likely to be inhibited by
Fig. 4. Cytokine secretion of THP-1 derived macrophages treated with As+3 and stimulated with LPS. THP-1 derived macrophages were treated with 0, 50, 100, 200 and 500 nM As+3 for 18 h with the stimulation of 10 ng/ml LPS. TNF-α and IL-1β levels in the supernatants were quantified by ELISA assay. A, IL1β concentrations in the supernatants. B, TNF-α concentrations in the supernatants. *Significantly different compared to the control (p < 0.05). Results are mean ± SD.
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Fig. 5. NO and superoxide production of THP-1 derived macrophages treated with As+3 and stimulated with LPS. THP-1 derived macrophages were treated with 0, 50, 100, 200 and 500 nM As+3 for 18 h with the stimulation of 10 ng/ml LPS. Cells were stained with DAF-FM (for NO detection) and DHE (for superoxide detection), incubated and analyzed on a BD AccuriC6 Plus flow cytometer. A, NO production as the mean channel fluorescence intensity (MFI) of DAF-FM. B, superoxide production as the mean channel fluorescence intensity (MFI) of DHE. C, correlation between the MFI of DAF-FM and DHE *Significantly different compared to the control (p < 0.05). Results are mean ± SD.
grant from East China University of Science and Technology (grant No. YC 0142125).
The suppressive effects of NO production induced by environmentally-relevant concentrations of As+3 should not be ignored. As we all known, arsenic exposure can induce oxidative stress and the production of reactive oxygen and nitric species (Zhou et al., 2011, 2016). We found that As+3 treatments at low levels decreased the secretion of NO in the THP-1 derived macrophage model. Interestingly, an epidemiological study reported that a significant decrease of serum NO levels was observed in the population exposed to arsenic via drinking water (Pi et al., 2000). The suggestion from that study was that As+3 suppressed NO production by inhibiting NO synthase in human umbilical vein endothelial cells. However, the suppression of another major NO producer. The macrophages, may also be involved in the decrease of the in vivo NO levels. Another interesting finding was that the decreased NO production was correlated with the increase of superoxide production, indicating that the oxidative stress induced by As+3 and the relative signaling pathways may also be involved in the As+3-induced inhibition on macrophage NO production. Therefore, the role of the NO-suppressive effect of As+3 in the regulation of the immune system and its association with the oxidative stress pathways in macrophages should be emphasized in future studies. In summary, we found that 500 nM As+3 induced apoptosis in THP1 derived macrophages. Low to moderate doses of As+3 suppressed the functions of phagocytosis, cytokine secretion and NO production of macrophages in vitro. The decrease of NO production induced by As+3 treatments were correlated with the increase of superoxide levels. The results from the study indicated that environmentally-relevant concentrations of As+3 could suppress the major functions of THP-derived macrophages, providing the initial evidence for the future mechanistic studies of the toxicity on macrophages induced by environmental arsenic exposures.
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