Artesunate Switches Monocytes to an Inflammatory Phenotype with the Ability to Kill Leukemic Cells

Monocytes are components of the tumor microenvironment related to cancer progression and immune escape. Therapeutic strategies for reprogramming monocytes from a tumor-supporting phenotype towards a tumoricidal phenotype are of great interest. Artesunate (ART) may be an interesting option for cancer treatment; however, the role of ART in regulating the inflammatory tumor microenvironment has not yet been investigated. Our aim is to evaluate the immunomodulatory potential of ART in vitro in human primary monocytes. ART treatment induced an increase in inflammatory monocytes (CD14highCD16−) with HLA-DR high expression and MCP-1/IL-1β release. On the other hand, ART treatment reduced CD206 and CD163 expression, and abolished the monocyte population known as non-classical and intermediate. Leukemia cells in contact with monocytes programmed with ART presented enhanced in vitro apoptosis suggesting that monocytes acquired the ability to kill leukemic cells. ART induced changes in the monocyte phenotype were mediated by JAK2/STAT3 downregulation. The induction of immunosuppressive environment is an important step for cancer progression. ART showed an immunomodulatory activity, leading immune cells to an antitumor phenotype and could be a candidate for immunotherapy in cancer patients.


Introduction
Hematologic malignancies account for 10% of all annual deaths due to cancer [1]. Despite the poor prognosis of hematologic malignancies, the treatment of these disorders has remained largely unchanged over the past several decades. Currently, high dose chemotherapy with several adverse effects remains as the main therapy [2]. This has created an impetus to explore novel therapeutic approaches, such as immunotherapies [3].
Hematological tumor cells live in an immune cell-enriched microenvironment, unique and substantially different from that of other solid tumors [3,4]. Monocytes belong to this tumor microenvironment (TME) and after a tumor-induced "immunoediting," these monocytes, which are originally the first line of defense against tumor cells, undergo a phenotypic switch and become tumor-supportive and immunosuppressive [5,6]. Accordingly, repolarizing leukemia-associated monocytes with more M1-like inflammatory characteristics eliminate their pro-leukemic effects and reduce tumor progression [7]. Thus, monocyte plasticity highlights the reprogramming of monocytes as an attractive therapeutic strategy to inhibit tumor progression, enabling these cells to adapt their function to meet the needs of antitumor defense [3]. Therapies inducing systemic immune activation may also have the capability of reprograming monocytes before they arrive to the tumor site, probably exerting influence on their tumor infiltration activity.
Artemisinin is a semi-synthetic compound of the sesquiterpene lactone drug family, and is obtained from a Chinese plant, Artemisia annua L., used for centuries in traditional Chinese medicine and known for its antimalarial properties [8]. Artesunate (ART) is a more stable and soluble derivative of artemisinin that has been shown to exert several more stable and soluble derivative of artemisinin that has been shown to exert several pharmacological actions such as anti-inflammatory [9][10][11], anti-leishmanial [12,13], neuroprotective [14,15], immunomodulatory [16], and antitumoral activity [17]. However, the effects of ART in reprogramming monocytes from a tumor-supporting phenotype into an antitumor phenotype remain unexplored. Our objective was to investigate the potential of ART as a monocyte reprogramming agent.

ART Is Safe for Human Primary Monocytes
We first determined the ART cytotoxic profile by exposing monocytes cultures to ART for 24 h. ART did not induce monocytes cytotoxicity in a range between 25-5000 μM (IC50 = 2205 μM). These results indicated that the concentrations 100-500 μM were safe for monocytes ( Figure 1A). We next examined whether ART induced apoptosis of primary monocytes. Monocytes treated with 100, 200, and 500 μM of ART did not show a significant increase in apoptosis ( Figure 1B, C). Based on this, we chose non-cytotoxic concentrations (100, 200, and 500 μM) to perform the next experiments ( Figure 1D). In addition, monocytes showed no morphological changes after the treatment ( Figure S1). were considered 100%. *, *** and, **** indicate significant difference from the control group as determined by ANOVA followed by Tukey post hoc (p < 0.05, 0.001, and <0.0001, respectively). Monocytes were treated with 100, 200, and 500 μM of ART for 24 h. After treatment, the cells were stained with Annexin V/PI (B,C). Experimental design of the two protocols used in the study (D). Monocytes were obtained from buffy coats of healthy blood donors. In the prevention protocol, cells were treated with LPS for M1-like monocytes or IL-4 for M2-like monocytes and ART were added at the same time. In the reversal protocol, cells were treated with LPS and IL-4 for 6 h and then ART was added in fresh medium for an additional 18 h. Cells were then collected for the next experiments. were considered 100%. *, *** and, **** indicate significant difference from the control group as determined by ANOVA followed by Tukey post hoc (p < 0.05, 0.001, and <0.0001, respectively). Monocytes were treated with 100, 200, and 500 µM of ART for 24 h. After treatment, the cells were stained with Annexin V/PI (B,C). Experimental design of the two protocols used in the study (D). Monocytes were obtained from buffy coats of healthy blood donors. In the prevention protocol, cells were treated with LPS for M1-like monocytes or IL-4 for M2-like monocytes and ART were added at the same time. In the reversal protocol, cells were treated with LPS and IL-4 for 6 h and then ART was added in fresh medium for an additional 18 h. Cells were then collected for the next experiments.

ART Induces a Tumoricidal Phenotype
To study the apoptotic effects of ART, monocytes previously treated with ART were co-cultured with U937, HL60, and OCI-AML3 cell lines ( Figure 6A). U937 cell apoptosis increased after co-culture with monocytes ART-programmed (from 4.97 ± 0.3135 to 22.0 ± 2.373, p < 0.0001). In addition, an increase of U937 apoptotic cells was observed in IL-4 treated monocytes with ART (from 7.13 ± 0.5045 to 33.0 ± 1.0, p < 0.0001) ( Figure 6B,C). The same pattern was observed with HL-60 and OCI-AML3 cell lines ( Figure 6C). Taken together, these data indicate that ART induces monocytes to assume an attack phenotype, characterized by a tumoricidal activity and ability to kill leukemic cells in vitro.  with ART immunomodulatory activity. A reduction of 7.1, 4.1, and 3.5 times was observed in p-JAK2 when naive monocytes were treated with increased doses of ART (100, 200, and 500 μM, respectively). Moreover, IL-4 treated monocytes showed a reduction of 11.1, 7.1, and 3.0 times in p-JAK2 compared with M2-control ( Figure 5A). In addition, p-STAT3 increased four times in IL-4 monocytes compared with M0. Moreover, ART reduced in 4.0, 7.0, and 2.9 times p-STAT3 in IL-4 treated monocytes compared with M2-control (Figure 5B). No changes in NF-kB, IKKα/β, p-JNK, p-c-Jun, ERK, p-p38, p-eIF2, CHOP, or ATF4 were observed ( Figure 5C).

Discussion
The present study demonstrates that ART reduces in vitro leukemia growth by mo ulating monocytes activation state to an antitumor phenotype. First, we observed th ART was effective in inducing a strong inflammatory monocyte polarization, mainly cha acterized by high MCP-1 and IL-1β release, HLA-DR expression, NO production, and lo CD206/CD163 expression, which, in turn, promoted an antitumoral phenotype and i duced in vitro U937, HL-60 and OCI-AML3 apoptosis. This monocyte switch is in pa mediated by a decrease in the phosphorylation of JAK2/STAT3. We propose that ART an the consequent induction of an inflammatory monocyte phenotype may revert the tumo Figure 6. ART treatment reduces in vitro leukemia growth by modulating monocytes with the ability to kill leukemic cells. Experimental design of the co-culture protocol (A). Monocytes were treated with 500 µM of ART for 24 h in the presence or not of IL-4. Representative dot plot (B). U937, HL60, and OCI-AML3 were added in a fresh medium for more 24 h and the cells were stained with Annexin V (C). Data represent mean ± SEM of at least three independent experiments performed in triplicate. Data were analyzed by ANOVA followed by Tukey post hoc. *, **, and ****, significantly different from control RPMI cells or IL-4 cells.

Discussion
The present study demonstrates that ART reduces in vitro leukemia growth by modulating monocytes activation state to an antitumor phenotype. First, we observed that ART was effective in inducing a strong inflammatory monocyte polarization, mainly characterized by high MCP-1 and IL-1β release, HLA-DR expression, NO production, and low CD206/CD163 expression, which, in turn, promoted an antitumoral phenotype and induced in vitro U937, HL-60 and OCI-AML3 apoptosis. This monocyte switch is in part mediated by a decrease in the phosphorylation of JAK2/STAT3. We propose that ART and the consequent induction of an inflammatory monocyte phenotype may revert the tumor-induced blockade of immune surveillance, which permits antitumor responses and tumor regression.
Cancer is a public health problem. Conventional cancer therapy, such as chemotherapy, provides limited efficacy with side effects and drug resistance. Thus, there is an urgent need for novel drugs which target cancer cells specifically, without side effects. ART emerges as a good option for cancer treatment with IC values ranging from 0.092 to 1.5 µM in leukemic cell lines [17,18]. Here, we show that the IC 50 for ART in healthy primary immune cells is 2205 µM demonstrating that ART is extremely selective and non-cytotoxic for healthy cells.
Antitumor action of ARTs mainly involves induction of apoptotic cell death, ROS generation, and cell cycle arrest [17][18][19][20]. Nevertheless, our understanding of the ART/mechanisms is far from complete. Therefore, a few evidences indicate that ARTs antitumor affects may be related with immunoregulatory activities and cancer metabolism changes mainly by decreasing the secretion of immunosuppressive cytokines (TGF-β/IL-10) and the infiltration of inhibitory immune cells such as TRegs and MDScS, while increasing the infiltration of attack cells, T CD8+ and NK with increased antitumor potential [16,[21][22][23][24][25]. However, the immunomodulatory effect on monocytes/macrophages remains unclear.
Monocytes are a heterogeneous subset of cells with distinct subpopulations that can assume several phenotypes and secrete a variety of substances capable of impacting physiological processes or the development of diseases [6,26]. Recently, monocytes have emerged as important regulators of cancer progression and aggressiveness, with different phenotype subtypes appearing to have opposing impacts on tumor growth metastatic spread [6]. The most widely reported cancer-induced phenotypic alteration in circulating monocytes is the switch to an immunosuppressive and tumor friendly phenotype [27,28]. Thus, the identification of therapies with the ability of "re-educating" monocytes to a tumoricidal phenotype will most likely represent a useful therapy strategy for patients with cancer.
The present work demonstrates that ART modulates monocyte polarization, causing an increase in classical monocytes and completely abolishing intermediate and non-classical monocytes. Interestingly, classical monocytes are described as important cells during inflammatory and infectious processes, appearing to be critical effectors during the early phase of the antitumor response, in particular by killing tumor cells [6]. Our data show that monocytes treated for 24 h with ART assume an antitumor phenotype and induce apoptosis of leukemic cells. This suggests that ART is capable of leading monocytes to an antitumor phenotype and this phenotype remains even after the drug is withdrawn. In a clinical context, this may indicate that the effect of ART therapy may persist to eliminate the tumor via the reprogramming of immune cells to an inflammatory and tumor-killing phenotype. In addition, tumors often escape antitumor immune responses through critical immune checkpoint molecules. Thus, the immunomodulatory potential of ART herein described could be a new strategy to create synergy with immune checkpoint inhibitors.
Interestingly, ART also alters the cytokine release profile by increasing the release of MCP-1 and IL-1β. Both are important players in building a defensive immune response. The main activity of MCP-1 is to attract blood monocytes and activate monocytes for tumor cell killing phenotype [29]. In addition, IL-1β acts as an amplifier of immune reactions inducing MCP-1 expression in monocytes, regulating myeloid cell recruitment into tumor tissue, and leading to inflammation [30]. Therefore, both cytokines are important for the recruitment and activation of monocytes in the tumor site, allowing the assembly of an anti-tumor immune response, as well as the activation and maturation of other immune cells present in the TME.
The activation of JAK2/STAT3 is known as an important pathway related to IL-4 response in immune cells. Collectively, the present results suggest that inhibition of the JAK2/STAT3 pathway may be a potential mechanism, via which ART mediates monocyte switch to an inflammatory phenotype. Notably, STAT3 is an emergent target anti-cancer therapy [31], and data have indicated that STAT3 inhibition in immune cells may reprogram these cells to a tumoricidal phenotype, reduce tumor cell proliferation, angiogenesis, and metastasis [32,33]. The inhibition of JAK2/STAT3 activities in the monocytes programmed by ART may explain the switch to an antitumor phenotype in these monocytes. In addition, these data suggest a new mechanism of action for a previously established drug.
In conclusion, our data indicate that ART treatment induces monocytes to an inflammatory and antitumor phenotype (Figure 7), which may reduce tumor recurrence and progression. However, future studies are required to better understand the long-term effects of ART on the modulation of immune cell phenotypes.  [32,33]. The inhibition of JAK2/STAT3 activities in the monocytes programmed by ART may explain the switch to an antitumor phenotype in these monocytes. In addition, these data suggest a new mechanism of action for a previously established drug.
In conclusion, our data indicate that ART treatment induces monocytes to an inflammatory and antitumor phenotype (Figure 7), which may reduce tumor recurrence and progression. However, future studies are required to better understand the long-term effects of ART on the modulation of immune cell phenotypes. In an immunosuppressive environment, JAK2/STAT3 are activated after IL-4 release, which increases CD206 and CD163 expression, and decreases MCP-1/IL-1β release and NO production. In contrast, ART inhibits activation of JAK2/STAT3 pathway, increasing HLA-DR expression, NO production, and MCP-1/IL-1β release, and leads to an accumulation of monocytes with an inflammatory profile. In return, CD206, CD163, and CD16 expressions decrease. In co-culture with leukemic cells, monocytes-ART-programmed assumed a tumoricidal profile and induces apoptosis of leukemic cell. These data suggest that ART switches monocytes to an inflammatory and antitumoral profile that could be useful for control tumor progression and re-educates monocytes inside of a tumor microenvironment.

Monocytes
Peripheral blood mononuclear cells were isolated from buffy coats of healthy blood donors by Ficoll-Paque (17-1440-03, GE Healthcare Bioscience, Chicago, Illinois, USA) density gradient centrifugation. Monocytes were isolated by Percoll (17-5445-02, GE Healthcare Bioscience, Chicago, Illinois, USA) density gradient centrifugation followed by adherence to a plastic dish for 1 h as previously described [34]. All subjects donating blood specimens signed an informed consent approved by the Ethical Committee of University of Campinas Hospital number 1.242.880 approved on 09-24-2015.

Monocytes Treatment
ART was dissolved in DMSO and subsequently mixed with RPMI with 10% FBS to obtain solutions at final concentrations (DMSO final concentration 0.3%). To induce changes compatible with an immunosuppressive phenotype, in order to test the potential of ART in reprogramming monocytes, we used two different strategies, namely preven- In an immunosuppressive environment, JAK2/STAT3 are activated after IL-4 release, which increases CD206 and CD163 expression, and decreases MCP-1/IL-1β release and NO production. In contrast, ART inhibits activation of JAK2/STAT3 pathway, increasing HLA-DR expression, NO production, and MCP-1/IL-1β release, and leads to an accumulation of monocytes with an inflammatory profile. In return, CD206, CD163, and CD16 expressions decrease. In co-culture with leukemic cells, monocytes-ART-programmed assumed a tumoricidal profile and induces apoptosis of leukemic cell. These data suggest that ART switches monocytes to an inflammatory and antitumoral profile that could be useful for control tumor progression and re-educates monocytes inside of a tumor microenvironment.

Monocytes
Peripheral blood mononuclear cells were isolated from buffy coats of healthy blood donors by Ficoll-Paque (17-1440-03, GE Healthcare Bioscience, Chicago, IL, USA) density gradient centrifugation. Monocytes were isolated by Percoll (17-5445-02, GE Healthcare Bioscience, Chicago, IL, USA) density gradient centrifugation followed by adherence to a plastic dish for 1 h as previously described [34]. All subjects donating blood specimens signed an informed consent approved by the Ethical Committee of University of Campinas Hospital number 1.242.880 approved on 09-24-2015.

Monocytes Treatment
ART was dissolved in DMSO and subsequently mixed with RPMI with 10% FBS to obtain solutions at final concentrations (DMSO final concentration 0.3%). To induce changes compatible with an immunosuppressive phenotype, in order to test the potential of ART in reprogramming monocytes, we used two different strategies, namely prevention and reversal protocols ( Figure 1D,E). In the reversal protocol, the cells were first exposed to IL-4 (20 ng/mL, PeproTech Roch, city, state, country) for 6 h aiming to program monocytes into an immunosuppressive phenotype (M2-like) and were subsequently washed and treated in a fresh medium with ART (Sigma-Aldrich Corp., St. Louis, MO, USA) for an extra 18 h. In the prevention protocol, the potential of the ART was tested by blocking the immunosuppressive monocytes formation, the cells were exposed to different concentrations of ART and immediately treated with IL-4 (20 ng/mL) for 24 h. The control naive cells M0 were maintained in RPMI with 10% FBS and the M1-like positive cells were treated with LPS (10 ng/mL, Sigma-Aldrich Corp., St. Louis, MO, USA).

MTT Assay
Cell viability was determined using the (4,5-dimethyl)-2,5diphenyltetrazolium bromide assay (MTT) assay, which consists of measuring the number of cells with metabolically active mitochondria based on the reduction of the tetrazolium salt MTT to formazan. Briefly, monocytes (2 × 10 5 cells/well) were seeded in 96-well plates. After 1 h, cultures were replaced with fresh media in the absence or presence of increasing ART concentrations (25-5000 µM). Following 24 h of treatment, 0.5 mg/mL MTT were added and incubated for 3 h. Subsequently, 0.1 N HCl was added to anhydrous isopropanol to solubilize the formazan. The absorbance was determined in a microplate reader at 570 nm. Results were expressed as percentage of control. Cultures with the same volume of DMSO in RPMI 10% served as controls.

Apoptosis Assay
Monocytes (1 × 10 6 cells/well in a 24-well plate) were treated with ART for 24 h. Cells were washed with PBS and resuspended in a binding buffer containing 1 mg/mL propidium iodide (PI) and 1 mg/mL APC-labeled annexin V for 15 min at room temperature (RT) protected from light. 10.000 events were analyzed in the FACS Calibur (BD Bioscience, Franklin Lakes, NJ, USA).

Flow Cytometry Membrane Markers
Phenotypic analysis was performed using monoclonal antibodies against CD14, CD80, CD206, CD16, CD163, and human leukocyte antigen D related (HLA-DR) and the monocytes population was gated in CD14 positive cells. For this, after treatment as described in item 2.2, cells were suspended in the staining buffer with the Fluorochrome-conjugated antibodies listed in Supplementary Table S1 and incubated for 30 min at RT. After washing at least 2 times in the flow cytometry buffer, cells were immediately analyzed using an FACS Calibur (BD Bioscience, Franklin Lakes, NJ, USA).

Nitric Oxide Production
Intracellular NO generation was measured by flow cytometry following staining with 4-amino-5-methylamino-2 ,7 -difluorofluorescein diacetate (Sigma-Aldrich Corp., St. Louis, MO, USA). Acquisition of cells was performed on a FACS Calibur flow cytometer and analysis was carried out using the FlowJo software (v.10).

ELISA
TNF-α, IL-1β, IL-8, MCP-1, and RANTES levels were determined in the supernatant of monocytes cultures by ELISA, following the manufacturer's recommendations (R&D Systems, Minneapolis, MN, USA). Results were expressed as pg cytokine/mL based on a standard curve.

Western Blot
After 15 min of treatment, total cell protein was extracted with RIPA buffer. Protein concentrations were quantified by Bradford method [35]. Equal protein amounts (30-40 µg) were loaded on 8 to 10% SDS polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Membranes were then blocked by incubation with 5% fat-free dry milk, followed by an overnight incubation with a specific primary antibody (Supplementary Table S1). One hour of RT incubation was then carried out using horseradish peroxidase-conjugated secondary antibody. The immunoreactivities were visualized by ECL Western Blot Analysis System (Amersham Pharmacia Biotech, UK).