Lipid mediator lipoxin A4 inhibits tumor growth by targeting IL-10-producing regulatory B (Breg) cells

Cancer Letters 364 (2015) 118–124

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

Lipid mediator lipoxin A4 inhibits tumor growth by targeting IL-10-producing regulatory B (Breg) cells Zheng Wang a,1, Qiong Cheng b,1, Ke Tang c, Yanling Sun a, Keke Zhang d, Yi Zhang c, Shunqun Luo a, Huafeng Zhang c, Duyun Ye b, Bo Huang a,c,* a

Department of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China Department of Pathophysiology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China c State Key Laboratory of Medical Molecular Biology & Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China d Department of Gynecology and Obstetrics, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China b

A R T I C L E

I N F O

Article history: Received 23 March 2015 Received in revised form 15 April 2015 Accepted 15 April 2015 Keywords: Lipoxin A4 Breg cells Tumor Inhibition

A B S T R A C T

Lipoxin A4 (LXA4), an arachidonic acid-derived anti-inflammatory lipid mediator, shows anti-tumor potential by regulating tumor immune microenvironments. However, the underlying molecular and cellular basis of this function remains unclear. IL-10-producing B (Breg) cells display tumor-promoting effects by negatively regulating anti-tumor immunity. Here we show that LXA4 inhibits tumor growth by suppressing the generation of Breg cells in tumor-bearing mice. The administration of LXA4 inhibited the induction of Breg cells. Breg cell deficiency, in turn, resulted in LXA4 losing its anti-tumor properties. Intriguingly, regulatory T (Treg) cells also had a role in this process. Targeting Breg cells by LXA4 decreased the number of Treg cells in draining lymph nodes and tumor tissues as well as enhanced cytotoxic T cell activities. In addition, we further demonstrated that LXA4 inhibited Breg cells through its dephosphorylating STAT3 and ERK. These findings unveil a new anti-tumor mechanism underlying LXA4 targeting Breg cells with potential clinical applications. © 2015 Elsevier Ireland Ltd. All rights reserved.

Introduction B lymphocytes are well known to function as antibodysynthesizing machines to protect the host against invasive pathogens. However, mounting evidence indicates that B cells also play an essential role in various immune pathologies including malignant diseases [1,2]. Previous studies showed that B cells inhibited the induction of T cell-dependent tumor immunity and B cells can induce CD8 T cells to become anergic [3]. On the other hand, depletion of B cells enhances anti-tumor cytotoxic T cell response, leading to effective clearance of tumor cells [4]. In clinic, there are reports that the presence of B cells is correlated with worse outcome [5,6], regardless of other contrary findings. It has now become clear that a subset of IL-10-producing regulatory B (Breg) cells play a pivotal role in mediating immunosuppression. It has been reported that Breg cells can suppress Th1 and Th17 responses [7]. In murine models, adoptive transfer of Breg cells can ameliorate the symptoms of experimental autoimmune encephalomyelitis (EAE) [8]. In human systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA),

* Corresponding author. Tel.: +86 010-65229258; fax: +86 010-65229258. E-mail address: [email protected] (B. Huang). 1 Z. Wang and Q. Cheng contributed equally to this work. http://dx.doi.org/10.1016/j.canlet.2015.04.030 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.

disease activities are closely related to the number and function of Breg cells [9,10]. Initially, IL-10 production is considered as a hallmark of Breg cells; however, other cytokines like lymphotoxin and IL-35 produced by B cells also display suppressive properties [11,12]. In addition to producing immunosuppressive cytokines, Breg cells also act directly on natural killer (NK) and dendritic cells to restrain their activities [13,14]. Furthermore, Breg cells indirectly induce and maintain Treg cells as immune suppressors [15]. Currently, though the mechanisms by which Breg cells regulate immune responses have been widely investigated, how Breg cell numbers and their function are controlled remains poorly understood. Lipoxin A4, an endogenous eicosanoid, is highlighted in the regulation of inflammation [16]. Lipoxin A4 is synthesized locally from arachidonic acid at inflammation sites via a transcellular biosynthesis mechanism [17]. By specifically binding to two distinct receptors: formyl peptide receptor 2 (FPR2), a surface membrane G-protein-coupled receptor, and the aryl hydrocarbon receptor (AhR), a ligand-activated nuclear transcription factor, LXA4 strongly inhibits the trafficking of leukocytes to the inflammatory site and stimulates the phagocytosis of apoptotic cells by tissue macrophages [18,19]. Roles of LXA4 in tumorigenesis have been explored using different tumor models [20–22]. Although those results have shown anti-tumor effects of LXA4, the underlying mechanism remains unclear. In this study, we provide evidence that LXA4 exerts

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the anti-tumor effect through inhibiting the generation of IL-10 producing Breg cells. Our findings suggest that LXA4 functions as an endogenous immune modulator against tumors. Materials and methods Cell lines and mice Murine hepatocarcinoma H22 (BALB/c), melanoma B16 (C57BL/6) and colorectal carcinoma CT26 (BALB/c) cell lines were purchased from the China Center for Type Culture Collection (Wuhan, China) and cultured according to the guidelines given. Female BALB/c and C57BL/6 mice, 8- to 10-week-old, were purchased from the Center of Medical Experimental Animals of Hubei Province (Wuhan, China). Female BALB/c nude and CB-17 SCID mice, 6- to 8-week-old, were purchased from Vital River Laboratory Animal Technology Company (Beijing, China). All animals were purchased for studies approved by the Animal Care and Use Committee of Tongji Medical College. Animal experiments 2 × 105 H22, B16 or CT26 tumor cells were subcutaneously injected to the syngenic mice. One day later, mice were intraperitoneally (i.p.) injected with BML-111 (1 mg/kg) or LXA4 (10 μg/kg) every two days. LXA4 and BML-111 were purchased from Cayman Chemical. Proliferation assay Purified T or B cells were labeled with 5 μM CFSE for 10 min at 37 °C in FBSfree 1640 culture medium. CFSE was quenched by adding an equal volume of cold FBS and suspension was incubated for 5 minutes on ice before washing three times with PBS containing 0.1% FBS. Then cells were cultured with 50 ng/ml phorbol-12myristate-13-acetate (PMA) and 750 ng/ml ionomycin for 48 hours before being analyzed by flow cytometry. Flow cytometry Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, PE-Cy5-, or allophycocyanin (APC)-labeled monoclonal antibodies to CD19, IL-10, CD8a, IFN-γ, CD4, Foxp3, CD11b and Ly6C were purchased from eBioscience. Appropriate isotype controls were used. For intracellular cytokine staining, lymphocytes prepared from draining lymph nodes and tumor tissues were restimulated with PMA (50 ng/ml) and ionomycin (750 ng/ml) in the presence of monensin (2 μM) for 5 hours and then stained with anti-CD8 antibody. After surface staining, the cells were treated with Fix/Perm solution and restained with anti-IFN-γ antibody.

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Histology Cell purity was always observed to be ≥95%. Primers of the LXA4 Receptor were as follows: 5′-AGACCTCAGCTGGTTGTGCAG-3′ (forward); 5′-TGATGGAGACAACCACC ATTGA-3′ (reverse). The tissues were fixed in 4% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4) for 48 h and then embedded into paraffin sections (4 μm). For histologic assessment, the sections were stained with hematoxylin and eosin (H&E) for conventional morphologic evaluation under light microscope (Nikon eclipse 90i). For immunohistochemical assay, CD3 expression was detected by anti-CD3 primary antibody (1:100, Abcam). The secondary antibody was conjugated to horseradish peroxidase. Quantitative real-time PCR RNA was isolated from sorted cells with Trizol and reverse transcribed into cDNA with the Reverse Transcription System. Quantitative real-time PCR was performed on an ABI 7900 System with SYBR green. Primers of the LXA4 Receptor were as follows: 5′-AGACCTCAGCTGGTTGTGCAG-3′ (forward); 5′-TGATGGAGACAACCACC ATTGA-3′ (reverse). Transcripts were quantified with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as an internal standard. Statistical analysis Results were expressed as mean values ± SEM and interpreted by unpaired twotailed Student’s t test. Differences were considered to be statistically significant when the P-value was < 0.05.

Results Anti-tumor effects of LXA4 in different murine tumor models To investigate the anti-tumor effect of LXA4, a murine H22 hepatocarcinoma tumor model was first tested. After subcutaneous injection of H22 tumor cells (2 × 105), the tumor-bearing mice were intraperitoneally administered with BML-111, an agonist of LXA4 receptor, once per two days. The result showed that the administration of BML-111 significantly inhibited H22 tumor growth (Fig. 1A). Besides the H22 tumor model, murine B16 melanoma and CT26 colon cancer tumor models were also conducted. Consistently, tumor growth was also substantially inhibited by BML-111 in both settings (Fig. 1B and C). We then directly injected LXA4 to H22 tumor-bearing mice to further confirm its anti-tumor effect.

Induction of Breg cells IL-10-producing B cells were induced in vitro according to a previously described method [23]. Briefly, 2 × 106 isolated splenocytes or purified B cells were cultured in complete 1640 medium and stimulated with LPS (10 μg/ml, Sigma), PMA (50 ng/ml, Sigma), ionomycin (500 ng/ml, Sigma) and monensin (2 μM, eBioscience) for 5 hours in 48-well plates. Cell isolation and adoptive transfer B cells, CD3+ T cells or CD4+ T cells were isolated from naive mice using MACS (Miltenyi Biotech). Cell purity was always observed to be ≥95%. For SCID mice adoptive transfer, 5 × 106 sorted CD3+ T cells were injected via the lateral tail vein of mice 1 day before and 7 days after tumor inoculation. Isolation of tumor-infiltrating lymphocytes Large tumors were digested with collagenase and hyaluronidase for 1.5 h at 37 °C and homogenized with semifrosted slides. After lysis of red blood cells (RBCs), the dissociated cells were underlaid with 5 ml lymphocyte separation solution and centrifuged at 2200 rpm for 20 minutes. Tumor-infiltrating lymphocytes were then harvested from the interface for flow cytometric analysis. Western blot B cells or splenocytes were isolated from naive or tumor-bearing mice for protein extraction and then SDS-PAGE gels were loaded with 40 μg of protein and transferred to PVDF membranes (Millipore). Membranes were probed with specific antiERK (1:1000), anti-STAT3 (1:2000), anti-p-ERK (1:2000), and anti-p-STAT3 (1:2000) overnight at 4 °C. The membranes were washed three times and signals were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies. The immunoreactivity was visualized by enhanced chemiluminescence according to the manufacturer’s protocol (ECL kit, Thermo Scientific). All the antibodies were purchased from Cell Signaling Technology.

Fig. 1. LXA4 suppresses tumor growth in mice. (A–C) Administration of LXA4 receptor agonist BML-111 inhibited tumor growth in mice. 2 × 105 tumor cells (H22, B16 or CT26) were inoculated subcutaneously to mice (n = 6). From day 1, the mice were i.p. injected with BML-111 (1 mg/kg) once per two days. The tumor growth was measured. The results were combined from three reproducible experiments. (D) LXA4 treatment suppressed tumor growth. 2 × 105 H22 tumor cells were inoculated to mice. From day 1, the mice were i.p. injected with LXA4 (10 μg/kg) once per two days. The tumor volume was measured on day 16. * P < 0.05; ** P < 0.01.

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Once again, LXA4 treatment markedly inhibited tumor growth (Fig. 1D). These data suggest that LXA4 possesses an intrinsic ability to suppress tumor growth. Anti-tumor effect of LXA4 is mediated by inhibiting Breg cells Next, we wondered how LXA4 mediated tumor suppression. When we added LXA4 to the tumor cell culture medium, we did not find that tumor cell growth was affected by LXA4 (Fig. S1A). This result combined with the known effect of LXA4 on inflammation promoted us to test the possible role of LXA4 in a tumor immune microenvironment. Recent studies highlight the important role myeloid-derived suppressor cells (MDSCs) play in various inflammatory conditions, including tumor microenvironments where they promote tumor development [24,25]. Given the primary role of LXA4 in inflammation regulation, we speculated that LXA4 might negatively regulate the accumulation of MDSCs, leading to disarming the tumor-promoting effect of MDSCs. However, in LXA4-treated tumorbearing mice, we did not observe the alteration of MDSCs (Fig. S1B). Whether adaptive immune cells such as T and B cells were involved in LXA4-mediated anti-tumor process was then examined. We used T and B cell-deficient SCID mice to address this question. In H22

tumor-bearing SCID mice, the injection of LXA4 did not suppress tumor growth and even favored it (Fig. 2A), indicating that T cells and/or B cells are involved in LXA4-mediated antitumor effects. We then adoptively transferred CD3 T cells (5 × 106) to tumor-bearing SCID mice. Here tumor growth was also not influenced by LXA4 treatment (Fig. 2A), implicating that B cells are required in LXA4mediated anti-tumor effects. The potential impact of LXA4 on B cells was studies as follows. We firstly examine the germinal center formation of lymph nodes by H&E staining, which appeared not to be affected by LXA4 (Fig. 2B). In parallel, when we used PMA and ionomycin to stimulate CFSElabeled B cells in the presence or absence of LXA4, we found that LXA4 did not affect the proliferation of B cells (Fig. S1C). In addition, the number of CD138 positive plasma cells was not changed (Fig. S1D). However, when we used PMA and ionomycin to stimulate splenic B cells of naïve mice, the induction of IL-10-producing Breg cells was significantly inhibited by LXA4 ( Fig. 2C). Moreover, in LXA4-treated tumor-bearing mice (H22, B16 and CT26), the induction of IL-10-producing Breg cells was also inhibited (Fig. 2D), suggesting that LXA4 targets Breg cells. IL-21 is a critical factor for the development of Breg cells. In our study, IL-21 effectively induced IL-10-producing Breg cells. However, this effect was also inhibited

Fig. 2. Anti-tumor effect of LXA4 is mediated by inhibiting the generation of Breg cells. (A) 2 × 105 H22 tumor cells were inoculated s.c. into SCID mice. From day 1, the mice were i.p. injected with LXA4 once per two days. 5 × 106 purified CD3+ T cells were adoptively transferred to the mice at day 0 and day 7, respectively. The tumor growth was measured. The results were combined from three reproducible experiments. (B) Hematoxylin and eosin (H&E) staining of germinal center structure of spleen. 2 × 105 H22 tumor cells were inoculated s.c. into mice. From day 1, the mice were i.p. injected with LXA4 once per two days. Mice were sacrificed and the spleen was used for H&E staining at day 15. Scale bars: 200 μm. (C) LXA4 suppressed IL-10 production by Breg cells in vitro. Splenocytes of naive mice were stimulated with 50 ng/ml PMA and 750 ng/ml ionomycin for 48 hours. The number of IL-10-producing Breg cells was determined by flow cytometry. Data are mean ± SEM from five reproducible experiments. (D) LXA4 suppressed IL-10-producing Breg cells in vivo. 2 × 105 H22, B16 or CT26 tumor cells were inoculated s.c. into mice (n = 5). From day 1, the mice were i.p. injected with LXA4 once per two days. On day 7, splenocytes were isolated for the induction of Breg cells. (E) IL-21 could not relieve the inhibitory effect of LXA4. Splenic B cells isolated by MACS were stimulated with PMA and ionomycin in the presence or absence of IL-21 (100 ng/ml) and LXA4. 24 hours later, IL-10-producing Breg cells were analyzed by flow cytometry. (F) LXA4 treatment generated a durable effect on Breg cells. 2 × 105 H22 tumor cells were inoculated s.c. to mice (n = 10). From day 1 to day 5, the mice received LXA4 treatment once per day. Mice (n = 5) were sacrificed and splenocytes were isolated for IL-10-producing Breg cell analysis by flow cytometry on day 5 and day 15, respectively.

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by LXA4 (Fig. 2E). Notably, when we injected LXA4 to tumorbearing mice once per day for 5 days, the inhibitory effect of LXA4 on Breg cells was still maintained 10 days later (Fig. 2F), suggesting that LXA4 might exert a durable effect on Breg cells. LXA4 decreases Treg cells in tumor-bearing mice through inhibiting Breg cells Next, we wondered how LXA4-mediated inhibition of Breg cells resulted in tumor suppression. We found that the induction of IL10-producing Breg cells in tumor-bearing nude mice was not impaired, compared to that in wild type mice (Fig. 3A). However, when we treated these nude mice with LXA4, LXA4 did not generate anti-tumor effects (Fig. S1E), suggesting that T cells are also required for LXA4 antitumor action, since T cells are absent in nude mice. However, when we stimulated either wild-type mousederived CD4 or CD8 T cells with PMA and ionomycin, the addition of LXA4 did not affect T cell activation, evaluated by T cell proliferation and IFN-γ production (Fig. S2A and B). In line with these results, LXA4 receptor was very lowly expressed in T cells but very highly expressed in B cells (Fig. S2C), implying thereby that LXA4 probably indirectly influences T cells through a pathway directly affecting Breg cells. Here, we concentrated on Treg cells, considering the induction of Treg cells by Breg cells [15,26]. When the spleen, draining lymph nodes and tumor tissues from tumor-bearing wild-type mice were analyzed, it was found that the number of CD4+CD25+FOXP3+ splenic Treg cells and the production of IFN-γ by

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splenic CD8 T cells were not affected by LXA4 treatment (Fig. S2D and E). However, Treg cell numbers in draining lymph nodes and tumor tissues were significantly decreased after LXA4 treatment (Fig. 3B and C). Meanwhile, the activities of the corresponding CD8 T cells were enhanced (Fig. 3D and E). In line with the decrease of Treg cells in tumor, more CD3+ cells were recruited to tumor sites, as seen from immunohistochemical staining of the cells (Fig. 3F). In addition, the decrease of Breg cells was confirmed and less B cell infiltration inside the tumor (Fig. S2F and G). Moreover, although it did not directly affect the induction of Treg cells, the addition of LXA4 blocked the induction of Treg cells by either resting or activated B cells in vitro (Fig. 3G and S2H). Together, these data suggest that LXA4 down-regulates Treg cells by inhibiting Breg cells, leading to improve anti-tumor immune responses. The inhibition of Breg cells by LXA4 is mediated through ERK and STAT3 inactivation pathway Finally, we studied the molecular basis through which LXA4 inhibited the generation of IL-10-producing Breg cells. Previous studies showed that the expression of IL-10 was upregulated by the activation of ERK and STAT3 molecules. LXA4 has also been shown to inhibit the activation of ERK and STAT3 [27]. We thus checked these two signal molecules. The tumor-bearing mice were continually treated with LXA4 for 7 days and sacrificed. The bulk splenic cells did not show the alteration of phosphorylation of ERK and STAT3 by western blot. However, the phosphorylation of ERK and STAT3

Fig. 3. LXA4 decreases Treg cells in tumor-bearing mice through inhibiting Breg cells. (A) LXA4 suppressed IL-10-producing Breg cells in nude mice. 2 × 105 H22 tumor cells were inoculated s.c. into nude mice. From day 1, the mice were i.p. injected with LXA4 once per two days. On day 15, mice were sacrificed and splenocytes were cultured in the presence of PMA and ionomycin. The induction of Breg cells was analyzed by flow cytometry. (B–E) The influence of LXA4 treatment on Treg cells and CD8 T cells. The above LXA4 treatment decreased Treg cell numbers in draining lymph nodes (B) and tumor tissue (C). LXA4 treatment increased IFN-γ+CD8+ T cells in draining LNs (D) and tumor tissue (E). These results were representative of three repeated experiments. (F) The above LXA4 treatment promoted CD3+ T cell infiltration into tumor tissues by immunohistochemical staining. Scale bars: 200 μm. (G) LXA4 suppressed Treg cell differentiation in the presence of B cells. Purified CD4+ T cells co-culture with purified B cells at a 1:1 ratio and were stimulated with anti-CD3 (0.5 μg/ml) and LPS (1 μg/ml). Three days later, cells were harvested for Treg cell analysis by flow cytometry.

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Fig. 4. LXA4 suppressed IL-10 production of B cells by decreasing the phosphorylation level of ERK and STAT3. (A and B) LXA4 treatment inhibited the phosphorylation of ERK and STAT3 in splenic B cells. 2 × 105 H22 tumor cells were inoculated s.c. into mice. From day 1, the mice were i.p. injected with LXA4 every two days. Mice were sacrificed and splenocytes or B cells were isolated for protein extraction at day 7. The phosphorylation levels of STAT3 (A) and ERK (B) were determined by Western blot and data were analyzed. (C) The ERK and STAT3 inhibitors decreased IL-10 production in splenocytes. Splenocyes were isolated from naive mice. Then Breg cells were induced with U0126 (10 μM ERK inhibitor, Abcam) or cryptotanshione (20 μM STAT3 inhibitor, Beyotime Biotechnology) treatment for 5 hours. IL-10 production was analyzed by flow cytometry.

was overtly decreased in splenic B cells (Fig. 4A and B). Moreover, we used PMA and ionomycin to stimulate splenocytes in the presence or absence of U0126 (ERK inhibitor) or cryptotanshione (STAT3 inhibitor); we found the induction of IL-10-producing Breg cells was significantly inhibited by ERK or STAT3 inhibitors (Fig. 4C). Together, these data suggested that LXA4 inhibits the generation of IL10-producing Breg cells through inhibiting phosphorylation of ERK and STAT3. Discussion Lipid metabolites are critical in the immunoregulation of both innate and adaptive immune responses. Many lipid mediators show immunosuppressive action, and potentially favor tumor immune evasion. However little is known about the process of restraining tumor immune evasion by lipid mediators. In this study, we demonstrate that arachidonic acid-derived lipid mediator LXA4 can

upregulate anti-tumor immunity by targeting an important immunosuppressive B cell subset, Breg cells. The role of B cells in tumors remains incompletely understood. Some clinical studies indicate that B cell infiltration in the tumor microenvironment is associated with increased patient survival [28,29]. B cells are also shown to be required for optimal T cell tumor immunity in a murine tumor model [30]. On the other hand, many studies show the tumor-promoting effect of B cells by inhibiting T cell immune responses [31–37]. These conflicting results might be reconciled by the activation status as well as the subset of B cells in different contexts. For example, under inflammatory conditions, IL-1β, IL-6 and IL-33 seem to be capable of inducing IL-10producing Breg cells [38,39]. Although selectively targeting Breg cells in the tumor microenvironment is an ideal strategy in tumor immunotherapy, how to target this subset of B cells remains unresolved. In this study, we provide evidence that lipid mediator LXA4 can target Breg cells, based on several lines of results: (1) administration of

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LXA4 resulted in the suppression of tumor growth in tumor models; (2) such anti-tumor effect vanished in SCID mice; and (3) the adoptive transfer of T cells could not rescue the anti-tumor effect. These data support the involvement of B cells; however, in mature T cell-deficient nude mice, LXA4 still could not exert its antitumor effect. We further provide evidence to reconcile this discrepancy. We found that Treg cells may be induced by Breg cells, consistent with previous reports [2,40]. On the basis of these results, we propose the immune mechanism through which LXA4 exerts the anti-tumor effect: (1) LXA4 targets Breg cells first; (2) Treg cell number and function in tumor microenvironment were impaired due to the decreased Breg cells; and (3) the anti-tumor T cell immune responses are consequently enhanced. Treg cells play critical roles in tumor immune evasion. In addition to TGF-β, IL-10 is another pivotal cytokine for Treg cell development in vivo. Although several types of immune cell subsets such as M2 type macrophages, tolerant dendritic cells, myeloid-derived suppressor cells and certain help T cells are capable of producing IL-10, IL-10-producing regulatory B cells seem to be a major cell type for IL-10 production in vivo. IL-10-producing Bregs can convert naive CD4+ T cells into Foxp3+ Treg cells by releasing IL-10 [26]. In contrast, Breg deficiency exacerbated EAE development due to the reduced frequency of Tregs, which resulted from the reduction of IL-10 [7,41]. Consistently, in B cell-deficient μMT mice, the development of Foxp3+ Treg cells was impaired and the antitumor response was subsequently enhanced [40,42]. Notably, Breg cells may also use IL-10-independent means to induce Treg cell development. CD5 and CD72 molecules on the surface of IL-10-producing Breg cells have been reported to be involved in the induction of Treg cells [43]. In this study, it is interesting to find that although LXA4 can target Breg cells, it seems not to influence conventional B cells, including their proliferation, differentiation and germinal center formation. Although this Breg cell selection by LXA4 is not elucidated in this study, we speculate that some signal molecules such as ERK and STAT3 play important roles. We found that LXA4 inhibited the phosphorylation of ERK and STAT3 and the latter were required for the development of Breg cells. Nevertheless, further elucidating how LXA4 selectively targets Breg cells is worthy of investigation. LXA4 is a locally and endogenously synthesized eicosanoid from arachidonic acid by 5-lipoxygenase (5-LO) and 12-LO or 15-LO. Meanwhile, the metabolism of arachidonic acid also generates prostaglandins and leukotrienes, two popular types of lipid mediators that usually show tumor-promoting effects. In contrast to prostaglandins and leukotrienes, LXA4 seems to be an inherent antitumor agent. In this study, the general anti-tumor effect of LXA4 was confirmed in three different tumor models, including hepatocarcinoma, melanoma and colon cancer. Cancer has been thought as wounds that never heal and chronic inflammation is commonly braided with the initiation, promotion, and progression of tumorigenesis. The endogenous anti-inflammatory nature of LXA4 confers its unique merits in tumor immunotherapy. All in all, in this study, we identify a new anti-tumor pathway of LXA4 through inhibiting Breg cells and emphasizing their important role in tumor immune evasion. Acknowledgments This work was supported by National Basic Research Program of China (2014CB542100), National Science Fund for Distinguished Young Scholars of China (81225021), National Natural Science Foundation of China (81472653), and Special Fund of Health Public Welfare Profession of China (201302018). Conflict of interest The authors declare no conflict of interest.

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Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.04.030.

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