Interleukin 35: Inhibitory regulator in monocyte-derived dendritic cell maturation and activation

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Interleukin 35: Inhibitory regulator in monocyte-derived dendritic cell maturation and activation

T

Xi Chena, Shengnan Haoa, Zhonghua Zhaob, Jia Liua, Qianqian Shaoc, Fang Wanga, Dong Sunc, ⁎ Ying Hec, Wenjuan Gaoc, Haiting Maoa, a

Department of Clinical Laboratory, The Second Hospital of Shandong University, Jinan, Shandong Province 250033, PR China Department of Oncology, The Affiliated Hospital of Binzhou Medical College, Binzhou, Shandong Province 256603, PR China c Institute of Basic Medical Sciences, Qi Lu Hospital, Shandong University, Jinan, Shandong Province 250012, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Interleukin 35 Monocyte-derived dendritic cell CD4+T cell CD8+T cell Immunosuppression

IL-35, a novel IL-12 family member, is a potent inhibitory cytokine predominantly produced by regulatory T and B lymphocytes that exerts optimal suppression in immune response. However, it remains unclear whether IL-35 plays an inhibitory role on human dendritic cells. In the present study, we focused on the possible immunosuppressive effect of IL-35 on the differentiation, maturation and function of monocyte-derived DCs (MoDCs). Addition of exogenous IL-35 was able to partially suppress MoDCs differentiation in vitro. Subsequently, LPS was used for the maturation of MoDCs and IL-35 was found to mainly restrain the maturation of MoDCs, characterized by the remarkable down-regulation of costimulatory molecules, CD83 and HLA-DR as well as a reduced production of pro-inflammatory cytokines (IL-12p70, IFN-γ, and TNF-α). Furthermore, IL-35treated MoDCs exhibited strong inhibition in the proliferation of allogeneic CD4+/CD8+ T lymphocytes. Meanwhile, IL-35-treated MoDCs also suppressed the polarization of naïve CD4+ T lymphocytes towards Th1 phenotype and impaired CD8+ T cells allogeneic responses. And the foregoing suppression of MoDCs maturation and function by IL-35 might be due to the aberrant activation of STAT1/STAT3 and inhibition of p38 MAPK/NFκB signaling pathway. Our results demonstrated for the first time that IL-35 played a critical role in modulating not only adaptive immune response, but also innate immune response. The inhibitory effect of IL-35 on MoDCs maturation and function may facilitate the development of promising therapeutic interventions in tumors and other diseases.

1. Introduction Dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a critical role in immune surveillance protecting against malignancy and infection [1]. Their functions directly control the results of immune response mediated by T helper (Th) cells and cytotoxic T lymphocytes (CTLs) [2–4]. The molecular signatures of human DC subsets located in tissues strongly suggests that in addition to classical DCs derived from dedicated precursors (the pre-DCs), monocyte-derived DCs (MoDCs) also exist in human tissues [5]. In their immature state, DCs are characterized by the strong ability to capture antigens coincident with the low expression of co-stimulatory molecules and cytokines [6]. On stimulation with microbial antigens or other damage-associated molecular signals, immature DCs (imDCs) undergo the process of maturation. This process includes the up-regulated expression of the co-stimulatory molecules (CD40, CD80 and CD86), the MHC class II molecules and inflammatory cytokines such as ⁎

IL-12 and TNF-α, and a strong T-cell stimulatory ability [6–8]. However, a number of studies have shown that DCs can be arrested at the semimature status following coculture with regulatory T cells (Tregs), expressing low levels of co-stimulatory molecules, making them incapable of initiating T-cell proliferation [9]. The possible mechanisms of suppression by Tregs are being explored in mice and humans, which mainly through the secretion of inhibitory cytokines (IL-10 and TGF-β) and cell-to-cell contact [10,11]. Collison et al have discovered that Tregs can also secret IL-35 which contributes to their suppressive functions [12]. IL-35 is a novel dimeric protein composed of Epstein-Barr virusinduced gene 3 (EBI3) and IL-12p35 subunits, signaling through a unique heterodimer of receptor chains gp130 and IL-12Rβ2 or the homodimers of each chain in target cells [13,14]. IL-35 belongs to the enigmatic IL-12 cytokine family including other three cytokines IL-12, IL-23, and IL-27, while the source of IL-35 is different from others [15]. Early reports have demonstrated that IL-35 is specifically expressed by

Corresponding author at: Department of Clinical Laboratory, The Second Hospital of Shandong University, 247# Beiyuan Street, Jinan 250033, Shandong Province, PR China. E-mail address: [email protected] (H. Mao).

https://doi.org/10.1016/j.cyto.2018.03.008 Received 12 October 2017; Received in revised form 13 February 2018; Accepted 9 March 2018 1043-4666/ © 2018 Elsevier Ltd. All rights reserved.

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Fig. 1. IL-35 expression on monocytes and MoDCs. A. MoDCs were stimulated with LPS in the presence of IL-35 (25 ng/ml) or not for 48 h. The expression of IL-35 (IL-12p35 and EBI3) was examined by flow cytometry. iTr35 was chosen for the positive control. B. Flow cytometry analysis of IL-35 receptor (gp130 and IL-12Rβ2). CD14+ monocytes and MoDCs were stained on their surfaces with designated antibodies. Isotype antibodies were used as negative controls. The results were representative of three independent experiments.

resting and activated CD4+Foxp3+ Tregs and is considered to be a crucial anti-inflammatory cytokine, which could suppress Th1, Th2 and Th17 cell-responses in a context-dependent manner [12,16–18]. Interestingly, the newest studies have shown that IL-35, rather than TGF-β or IL-10, is required in Tregs-mediated maximal immune suppression [12,19]. It is well known that TGF-β secretion and CTLA-4 expression on Tregs are necessary to inhibit immune responses by affecting the function of DCs to activate T cells [10,20]. However, to our knowledge, the role of IL-35 on the modulation of human DCs phenotypic and functional properties remains unknown. Our study aimed to extend limited knowledge on IL-35-induced impairment of DC maturation and function and related signal pathways and sought to gain further insight

into dysfunction of IL-35-treated DCs on target T cells. To address these research questions, we firstly investigated the expression of IL-35 and its receptor on monocytes and MoDCs. Then we explored the effect of IL-35 on the differentiation and Lipopolysaccharide (LPS)-induced maturation of MoDCs. The functional impact of IL-35-treated MoDCs was investigated by examining their ability to promote T lymphocyte proliferation, skew naïve CD4+ T cell polarization and elicit CD8+ T cell alloreactive response. Further analysis of the underlying mechanisms indicated that the aberrant activation of STAT1/STAT3 and inhibition of p38 MAPK/NF-κB signaling pathway was involved in the suppression of LPS-induced maturation and function of MoDC by IL-35.

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Fig. 2. Exogenous IL-35 partially inhibits the differentiation of MoDCs. Human CD14+ monocytes (1 × 106/ml) were induced to differentiate into imDCs with the stimulation of IL-4 and GM-CSF in the presence of IL-35 (25 or 50 ng/ml) or not for 5 days, that is imDC group, imDC + IL-35(25 ng/mL) group, and imDC + IL-35(50 ng/mL) group. Above cell-free supernatants were harvested, and the production of IL-1β, IL-2, IL-10, IL-12p70, IL-17a, TNF-α, and IFN-γ was detected by the Bio-Plex protein array system. And the cells were collected for flow cytometric analysis. A, The expression of CD14, CD1a, CD11b, CD11c, CD209, CD80 and CD86 was analyzed by flow cytometry. The numbers in the dot plots indicated the percentage of the positive cells. The figure represented one of three similar experiments. B. The cytokine profile (IL-1β, IL-2, IL-10, IL-6, IL-12p70, IL-17a, TNF-α, and IFN-γ) in the culture supernatants of imDCs with different treatment. The data were represented as mean ± SD of duplicate samples. *P < 0.05.

CD14+ monocytes were cultured in the complete RPMI 1640 with GMCSF (1000 U/ml) plus IL-4 (500 U/ml) in the presence of IL-35 (25 or 50 ng/ml) or not for 5 days to generate the immature MoDCs (imDCs). For mature MoDCs (mDCs), 1 μg/ml of LPS was added in imDCs for another 2 days. To investigate the effect of IL-35 on MoDC maturation, 25 or 50 ng/ml IL-35 was added during the process of maturation (mDC + IL-35). The use of PBMCs from healthy donors was approved by the Human Investigation Committee of Qilu Hospital, Shandong University, and informed consent was obtained from each subject.

2. Materials and methods 2.1. Antibodies and reagents FITC-labeled anti-CD4, CD8a, CD14, CD40, CD83, HLA-DR monoclonal antibodies (mAbs); PE-labeled anti-CD1a, CD80, CD86, CD209 mAbs; PE-Cy5.5-labeled anti-CD11b, CD11c mAb; APC-labeled anti-IL12p35 mAb and related isotype control antibodies were obtained from BD Biosciences. PE-labeled anti-IFN-γ, IL-4, IL-17a, indoleamine 2, 3dioxygenase (IDO), PD-L2, EBI3 and gp130 mAbs; APC-labeled anti-PDL1 mAb and related isotype control antibodies were purchased from eBioscience. APC-labeled anti-IL-12Rβ2 mAb was purchased from Miltenyi Biote. Lipopolysaccharide (LPS) was from Escherichia coli (Sigma-Aldrich). Recombinant human IL-2, IL-4, GM-CSF were obtained from R&D systems. Recombinant human IL-35 was purchased from Peprotech.

2.3. Allogeneic mixed lymphocyte reaction (MLR) CD4+ and CD8+ T cells were selected from PBMCs using antihuman CD4 or CD8 MACS beads (Miltenyi Biotec). Purity of CD4+ and CD8+ T cells was > 97% as assessed by FACS analysis. Monocyte-derived imDCs were stimulated with LPS in the presence of IL-35 or not for another 2 days. Before coculturing with CD4+ and CD8+ T cells, different conditioned MoDCs (imDCs, mDCs, mDCs + IL-35) were harvested and washed twice in order to remove the residual IL-35. Then 2 × 104 conditioned MoDCs were incubated with allogeneic CD4+ or CD8+ T cells (1 × 105) at a ratio of 1:5 in RPMI 1640 containing 50 U/ ml IL-2 and placed into 96-well microplate. CD4+ and CD8+ T cells cultured in the absence of MoDCs were used as the control. The proliferation rate was detected on day 0, 3 and 5 of coculture using cell counting Kit-8 (CCK8) (Beyotime) method as previously described [21].

2.2. Generation of human monocyte-derived DCs (MoDCs) Human peripheral blood mononuclear cells (PBMCs) were isolated from leukocyte-enriched buffy coats of healthy volunteers according to the standard protocol using Ficoll-Paque Plus (Sigma-Aldrich). CD14+ monocytes were purified by positive selection using anti-CD14-conjugated magnetic microbeads (Miltenyi Biotec). The purity reached 98% which was determined by flow cytometry (BD Biosciences). 45

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Fig. 3. Exogenous IL-35 suppresses the maturation of MoDCs. Human CD14+ monocytes were differentiated into imDCs in the presence of GM-CSF and IL-4 for 5 days. Then imDCs were stimulated with LPS in the presence of IL-35 (25 or 50 ng/ml) or not for another 2 days. The cell-free supernatants were harvested, and the cytokine production of IL-1β, IL-2, IL-6, IL-10, IL-12p70, IL-17a, TNF-α, and IFN-γ was detected by the Bio-Plex protein array system. A. The expression of HLA-DR, CD40, CD80, CD86 and CD83 on DCs was analyzed by flow cytometry. Histograms illustrated staining with isotype-matched control (solid profiles) and the positive cells (black line). In these histograms, the expression of CD83 was presented as gated cells (%), while the others were presented as geometric mean fluorescence intensities (GM). The results are representative of at least three independent experiments. B. The cytokine profile (IL-1β, IL-2, IL-6, IL-10, IL-12p70, IL-17a, TNF-α, and IFN-γ) in different conditioned MoDCs culture supernatants. The data were represented as mean ± SD of duplicate samples. * P < 0.05, **P < 0.01, ***P < 0.001. C. Expression of IDO was analyzed by flow cytometry. The numbers in the dot plots indicated the percentage of the positive cells. The figure represented one of three similar experiments.

Fig. 4. IL-35 impairs the ability of MoDCs to induce the proliferation of allogeneic T lymphocytes. Different conditioned MoDCs (imDCs, mDCs, mDCs + IL-35) collected, washed twice and then cocultured with allogeneic CD4+ (A) or CD8+ (B) T lymphocytes for 5 days. The proliferation folds were analyzed by CCK8 method on days 0, 3, 5 and the pure T lymphocytes proliferation was monitored as control. The bar graphs exhibited the ratio of CD4+ and CD8+ T lymphocytes proliferation as mean ± SD of triplicate samples. **P < 0.01, *** P < 0.001.

IL-35 and other stimulations and then were used to stimulate allogeneic CD8+ T cells at a DC/T cell ratio of 1:5. The mixed cells were continuously cultured for 5 days with 50 U/ml IL-2 added on day 3. Then the supernatants were collected and assessed for cytokine production by the Bio-Plex Protein Array system (Bio-Rad) and the cells were examined for their intracellular expression of Granzyme B.

Each experiment was repeated three times. 2.4. Naïve T cell polarization Human CD4+ naïve T cells were isolated from PBMCs by immunomagnetic selection using a naïve CD4+ T cells isolation kit (Miltenyi Biotec). The purity was > 97% assessed by flow cytometry. The method of iTr35 induction was consulted the Collison’s study [22]. Different conditioned MoDCs (imDCs, mDCs, mDCs + IL-35) were harvested and extensively washed twice in order to remove the residual IL-35 and other stimulations before incubation with allogeneic naïve CD4+ T cells. T cells (1 × 106 cells) were stimulated with MoDCs (2 × 105 cells) under different conditions in RPMI 1640 medium. After allogeneic T-DC coculture for 5 days under 37 °C, the supernatants were collected and assessed for cytokine production by the Bio-Plex Protein Array system (Bio-Rad). The collected T cells were restimulated with 1 × cell stimulation cocktail (eBioscience) for 15–16 h. Cells were then fixed, permeabilized, and analyzed by flow cytometry using PE-conjugated anti-IL-4, IFN-γ and IL-17 mAbs.

At day 5 (imDCs) or day 7 (mDCs) of DC culture, cells were harvested and phenotype of MoDC was detected by FITC-, PE-, PE-Cy5.5labeled mAbs or appropriate isotypic controls. Intracellular staining was performed according to manufacturer-recommended protocols after intracellular fixation & permeabilization (eBioscience). For T cells, cytokine secretion was induced by 1 × cell stimulation cocktail (eBioscience) for 15–16 h. The stained cells were detected for single or double color immunofluorescence with a FACSCalibur flow cytometer, and the mean fluorescence intensities were analyzed using FCS express V3 (De Novo).

2.5. Antigen-specific CD8+ T cell activation

2.7. Cytokine measurement

The conditioned MoDCs (imDCs, mDCs and mDCs + IL-35) were harvested and extensively washed twice in order to remove the residual

The concentration of cytokines (IL-1β, IL-2, IL-4, IL-6, IL-10, IL12p70, IL-17a, TNF-α and IFN-γ) in the cultured supernatant was

2.6. Flow cytometry

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Fig. 5. IL-35-treated MoDCs restrain naïve CD4+ T lymphocytes polarization. A. Frequencies of IFN-γ/IL-4/IL-17a-secreting CD4+ T cells were determined after cocultivation of different conditioned MoDCs and naïve CD4+ T lymphocytes. MoDCs were excluded from analysis by gating on CD4+ T lymphocytes. The figure represents one of three similar experiments. B. Cytokine secretion profile (IL-1β, IL-4, IL-6, IL-10, IL-12p70, IL-17a, TNF-α, and IFN-γ) of CD4+ T lymphocytes that stimulated by imDCs, mDCs and IL-35-treated MoDCs. The data were represented as mean ± SD of duplicate samples. *P < 0.05, **P < 0.01, ***P < 0.001.

simultaneously determined by the commercially available Human High Sensitivity Panel (eBioscience) according to the manufacture’s protocols.

3. Results

2.8. Western blot analysis

IL-35 is described to be produced predominantly by Tregs as well as regulatory B cells (Bregs) and contributes to their suppressive activities. Furthermore, IL-35 induces a distinct subset of IL-35-producing regulatory T cells (iTr35) and IL-35+Bregs, respectively [23]. In light of the close relationship between these immune cell populations, we speculated that IL-35 might also play a role on human MoDCs. To validate this, we first measured EBI3 and IL-12p35 expression by intracellular flow cytometry staining on both MoDCs and IL-35-treated MoDCs. iTr35 was chosen for the positive control. As shown in Fig. 1A, comparing with the expression of IL-35 in iTr35, neither MoDCs nor IL35-treated MoDCs had the expression of IL-35. Subsequently, we investigated the expression of IL-35 receptor (gp130 and IL-12Rβ2) on CD14+ monocytes and MoDCs by flow cytometry. IL-35 receptor was detectable on both CD14+ monocytes and MoDCs, and the expression level was similar between the MoDCs group and CD14+ monocytes group (Fig. 1B). These data indicated that although MoDC itself did not secrete IL-35, it did express IL-35 receptor, which might be immunoregulated by IL-35 through binding to the receptor.

3.1. Not IL-35 but its receptor was detectable on MoDCs

To examine the activation of phosphorylated p38, extracellular regulated kinase (ERK), nuclear factor κB (NF-κB) and signal transducer, activator of transcription 1 (STAT1), STAT3 and STAT4, freshly isolated imDCs were stimulated with LPS in the presence of IL-35 (25 ng/mL) or not for 30 and 60 min. Cells were lysed with 100 μl lysis buffer (Beyotime). The lysates were then resolved by SDS-PAGE and transferred to PVDF membranes (Merck Millipore). The membranes were blocked with 5% non-fat dry milk for 1 h before being incubated with indicated primary antibodies (Cell signaling technology) overnight at 4 °C. The membranes were then treated with horseradish peroxidase conjugated secondary antibodies and the signals were detected by enhanced chemiluminescence (Life Technology). 2.9. Statistical analysis All experiments were performed in triplicate and data were shown as the mean ± SD. Differences between groups were examined by Student’s t-test. The statistical analysis was done by using Graphpad Prism Version 5 for Windows (GraphPad Software). P values < 0.05 were considered significant. 48

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Fig. 6. IL-35-treated MoDCs impair CD8+ T lymphocytes function. A. Different conditioned MoDCs (imDCs, mDCs and IL-35-treated DCs) were cocultured with allogeneic CD8+ T lymphocytes to induce T cell response. MoDCs were excluded from analysis by gating on CD8+ T lymphocytes. The percentage of Granzyme B+ effectors was assessed by the intracellular staining on day 5. The figure represents one of three similar experiments. B. Cytokine profile (IL-1β, IL-4, IL-6, IL-10, IL-12p70, IL-17a, TNF-α, and IFN-γ) of CD8+ T lymphocytes. The data were represented as mean ± SD of duplicate samples. *P < 0.05, **P < 0.01.

3.2. IL-35 partially inhibited differentiation of CD14+ monocytes to imDCs

to the above results, we selected 25 ng/ml as the proper concentration of IL-35 for the further study. Whereafter, we detected that IL-35 (25 ng/mL) treated-MoDCs secreted lower levels of cytokine IL-1β, IL-2, IL-12p70, IFN-γ, and TNF-α than control mDCs, but the production of IL-6, IL-10 and IL-17a was comparable between two groups (Fig. 3B). Furthermore, the expression of immunosuppressive enzyme IDO in MoDCs was not significantly altered by IL-35 treatment (Fig. 3C). The same results were also explored in the inhibitory receptor PD-L1 and PD-L2 (supplementary data 1). In order to detect whether IL-35-treated MoDCs displayed a delay in apoptosis, we examined the apoptosis related proteins by western blot (Bcl2, Bax and caspase 3) and assayed for apoptosis using annexin V/PI staining by flowcytometry. the results showed that there was no significant differences between mDCs and IL35 treated MoDCs in the expression of apoptosis related protein, including Bcl2, Bax and caspase 3 (supplementary data 2A). Similar results were found in the apoptosis detection kit by flowcytometry (supplementary data 2B). As a consequence, IL-35 suppressed the LPSinduced maturation of human MoDCs without a delay in apoptosis and the mechanism might need further exploration.

Human PBMC-purified CD14+ monocytes were cultured with GMCSF and IL-4 in the presence or absence of IL-35 for 5 days. As shown in Fig. 2A, IL-35 (25 ng/ml) partially suppressed the expression of CD1a and CD209 and remarkably decreased the expression of costimulatory molecules CD80 and CD86 on imDCs. But we did not observe any significant changes in the expression of CD14, CD11b or CD11c on IL-35treated MoDCs. Cytokine secretion profiles demonstrated the reduced production of IFN-γ in IL-35 (25 ng/ml)-treated MoDCs (Fig. 2B). Thus, exposure to IL-35 only partially impaired the differentiation of CD14+ monocytes to imDCs. 3.3. IL-35 suppressed maturation of MoDCs After 5 days of culture, LPS was added to promote the maturation of MoDCs. As expected, LPS induced the maturation of MoDCs by increased expression of HLA-DR, CD83 and co-stimulatory molecules (CD40, CD80 and CD86) (Fig. 3A) and elevated secretion of cytokine (IL-1β, IL-2, IL-10, IL-12p70, IL-17a, IFN-γ and TNF-α) (Fig. 3B). To examine the effect of IL-35 on the phenotypic maturation and cytokine secretion in human MoDCs, exogenous IL-35 was applied in the maturation process of MoDCs. It was found that IL-35-treated MoDCs exhibited obviously down-regulation of HLA-DR, CD83 and costimulatory molecules (CD40, CD80 and CD86) (Fig. 3A). Notably, Stimulation with IL-35 (25 ng/ml) was more functional than IL-35 (50 ng/ml). According

3.4. IL-35-treated MoDCs restrained allogeneic T lymphocytes proliferation Mature MoDCs were able to induce allogeneic T lymphocyte proliferation. To fully ascertain the function of IL-35-treated MoDCs on T lymphocyte-proliferation, imDCs, mDCs and IL-35-treated MoDCs were cocultured with allogeneic CD4+ or CD8+ T lymphocytes, and the 49

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Fig. 7. Signaling pathways responsible for impairing MoDC maturation. Immature MoDCs were stimulated with LPS in the present or absent of IL35 (25 ng/ml) for 30 min or 60 min. Treated cells were then collected and the whole cell extracts were prepared. The protein levels of phosphorylated (p) and total STAT1/STAT3/STAT4 and ERK/P38 MAPK/NF-κB were determined by western blot. The figure represents one of three similar experiments. Right insets: the quantitative analysis of protein ratio, as measured by ImageJ analysis of band intensity. Values were presented in arbitrary units.

CD4+ T lymphocytes polarization towards Th1 type.

extents of T lymphocyte proliferation were examined. The results verified that mDCs were potent inducers of CD4+/CD8+ T lymphocytes proliferation (Fig. 4A and B). Conversely, IL-35-treated MoDCs were significantly hampered in their ability to stimulate either allogeneic CD4+ or CD8+ T lymphocytes proliferation regardless of the usage of maturation stimulus (Fig. 4A and B).

3.6. IL-35-treated MoDCs were incapable to induce CD8+ T lymphocytes activation To add the current findings on the effect of IL-35-treated MoDCs on CD8+ T lymphocytes proliferation, we examined the capacity of CTLs response. IL-35-treated MoDCs exhibited a significantly reduced ability to stimulate CD8+ T lymphocytes to secrete Grazyme B compared with mDCs (Fig. 6A). To further confirm the single-cell level results of the cytokine production, we measured the cytokine production by the whole-cell population. The levels of cytokines IFN-γ, IL-1β, IL-10, IL12p70, IL-17a and TNF-α secreted by the CD8+ T lymphocytes were much lower in the IL-35-treated MoDCs group than in mDCs group (Fig. 6B). While the secretion of cytokine IL-4 and IL-6 was comparable between two groups (Fig. 6B). Therefore, IL-35-treated MoDCs were inefficient at activation of CD8+ T lymphocytes and these data lended further support to our hypothesis that IL-35 triggered the functional impairment of DCs.

3.5. IL-35-treated MoDCs prevented naïve CD4+ T lymphocytes polarization towards Th1 effectors DCs are the most potent APCs capable of priming the polarization of naïve CD4+ T lymphocytes. But, to date, there is no available evidence in the literature with regard to whether IL-35-treated MoDCs would influence naïve CD4+ T lymphocytes polarization. We monitored that, compared with mDC stimulation, naïve CD4+ T lymphocytes stimulated by IL-35-treated MoDCs showed a significantly reduced ability to produce the Th1 cytokine IFN-γ at intracellular levels (Fig. 5A). No significant differences were observed in Th2 and Th17 lineage development indicated by comparable IL-4 and IL-17a at intracellular levels, irrespective of stimulation with mDCs or IL-35-treated MoDCs (Fig. 5A). We further analyzed the cytokine secretion profiles in the coculture system, including IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17a, TNF-α and IFN-γ. Consistent with the intracellular cytokine staining, naïve CD4+ T lymphocytes stimulated by IL-35-treated MoDCs exhibited reduced secretion of Th1 type cytokines (IFN-γ, IL-1β, IL-12p70 and TNFα) compared with mDCs group (Fig. 5B). However, Th2 type cytokines (IL-4, IL-6 and IL-10) secretion remained unchanged in the presence of IL-35 (Fig. 5B). Interestingly, the production of IL-17a also decreased in IL-35-treated MoDCs groups while the intracellular staining was not hampered. It might need further exploration. Together this certified that IL-35 indeed inhibited the capacity of MoDCs to stimulate naïve

3.7. Cell signaling pathways induced by IL-35 The observations in the phenotypic and cytokine secretion changes of IL-35-treated MoDCs spurred our interest to search for the relative signaling pathways that could lead to further understanding of IL-35mediated modulation of MoDC function. Since IL-35 receptor utilizes IL-12Rβ2 and gp130, we hypothesized that signaling would be mediated through the STAT family. It has been reported that IL-35-treated wild-type conventional T cells results in the phosphorylation of STAT1 and STAT4 [14]. In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor [24]. In addition, MAPK and NF-κB are probably the 50

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expression between IL-35-treated MoDCs and mDCs. Therefore, the inhibitory effect of IL-35 was not mediated via the expression of intracellular IDO, PD-L1 and PD-L2. In recent study, it was reported that IL-35 inhibited proliferation and promoted apoptosis of fibroblast-like synoviocytes isolated form mice with collage-induced arthritis [33]. So we further explored whether IL-35 could induce apoptosis in MoDCs and we found that IL-35 induced little apoptosis in MODCs. These phenomena clearly indicated that IL-35 played a suppressive role mainly through restraining the LPS-induced maturation of MoDCs, rather than the differentiation of MoDCs. Another novel finding of this study was that CD4+/CD8+ T lymphocytes allostimulatory function of mDCs was dramatically decreased after treatment with IL-35. A study from asthmatic mice induced by ovalbumin suggested that IL-35 markedly inhibited the ovalbumin-induced conversion of recruited monocytes into MoDCs and then caused less T lymphocytes proliferation in lymph node in vivo [30]. In the present study, IL-35-treated MoDCs not only restrained CD4+/CD8+ T lymphocytes proliferation, but also suppressed naïve CD4+ T lymphocytes polarization towards Th1 phenotype and impaired CD8+ T lymphocytes alloreactive responses. It was further confirmed by the cytokine secretion profiles: T cells stimulated by IL-35-treated MoDCs secreted lower levels of IFN-γ, TNF-α, and comparable levels of IL-4 than control groups. The secretion of IL-17a was also repressed strongly in the supernatant of IL-35-treated MoDCs group, similar results were obtained in the previous studies that Treg expressing IL-35 inhibited the differentiation of CD4+ T cells into Th17 effector cells [34–36]. Here, we provided evidence that IL-35-treated MoDCs were poor stimulators in mixed lymphocyte reactions and skewed the proliferation and differentiation of T lymphocytes. The signaling pathways by which IL-35 initiates in MoDCs is also explored in this study. It has been shown that signaling through IL-35 receptor requires the transcription factors STAT1 and STAT4 in T cells, which forms a unique heterodimer that binds to distinct sites within the IL-12p35 and EBI3 promoters [14]. In B cells, IL-35 signals via heterodimeric receptors comprising of IL-12Rβ2 and IL-27Rα and activates STAT1 and STAT3 [24]. Moreover, STAT3 activation in DCs has been linked to defects in differentiation, maturation and function [37]. Both the intensity and duration of STAT3 signaling determine whether DC dysfunction develops [37]. Given that the IL-35 receptor utilizes IL12Rβ2 and gp130, it seemed logical that signaling would be mediated via the STAT family of transcription factors in DCs. Our study confirmed for the first time that IL-35 treatment of MoDCs resulted in phosphorylation of STAT1 and STAT3, but no activation of STAT4. The transcription factor NF-κB plays an important role in the complex multistep process of MoDC maturation, including regulation of CD83, CD80 and CD86 expression as well as IL-12 induction [38]. Inhibition of NF-κB activation using pharmacological agents results in a failure to upregulate the expression of CD80, CD86 and MHC II [38]. In addition, MAPK p38 activation also up-regulates CD80, CD40 and ICAM-I in DCs [39]. In line with previous reports, we discovered that the activation of NF-κB and p38 was restrained in IL-35-treated MoDCs. Collectively, IL35 blocks MoDC maturation, antigen presentation capacity and proinflammatory cytokine secretion of MoDCs, through the inhibition of NF-κB, p38 MAPK and the activation of STAT1/STAT3. And these IL-35treated MoDCs further suppress the expansion of effective CD4+/CD8+ T lymphocytes and downstream immune responses. In addition to iTr35 and IL-35+Bregs, IL-35 is expressed on some non-immune cells, including human placental trophoblasts and various tumor cells [26,40–42]. We previously demonstrated that IL-35 was highly expressed on tumor-infiltrating lymphocytes (TILs) and breast cancer tissues along with poor prognosis [43]. In the tumor microenvironment, whether tumor-derived or immunosuppressive cell-derived IL-35 can play an inhibitory effect on DC function and indirectly lead to tumor progression needs to be further explored. In summary, our study potentially informs a novel role of IL-35 as an immunosuppressive regulator through inhibiting MoDCs function, in

main signaling pathways in the regulation of DC functions. Both p38 MAPK and NF-κB activation could lead to DC maturation and secretion of pro-inflammatory cytokines, such as IL-12, TNF-α, IL-1β [25]. In contrast, ERK negatively regulates DCs maturation by decreasing surface expression of co-stimulatory molecules and enhancing secretion of immunosuppressive cytokines such as IL-10 and TGF-β [25]. In the present study, we used western blotting to examine the signaling pathways associated with IL-35 and MoDC maturation. As shown in Fig. 7, decreased pp38 and pNF-κB levels and increased pSTAT1 and pSTAT3 expression were observed in IL-35-treated DCs compared with control DCs, and this phenomenon was time-dependent. However, the expression of pERK was not significantly changed and the expression of pSTAT4 was undetectable between IL-35-treated MoDCs and control MoDCs. These findings suggested that IL-35 inhibits the maturation and function of MoDCs through activation of STAT1/STAT3 pathway and suppression of P38 MAPK and NF-κB signal pathways. 4. Discussion Dendritic cells play a critical role in the induction of antigen-specific adaptive immune response by presenting the antigens to T lymphocytes and activating appropriate subtypes of T lymphocytes [2,6]. IL-35, a new anti-inflammatory and immunosuppressive cytokine, is responsible for the suppressive function of Tregs and Bregs [26]. Despite several studies have proven that IL-35 appears to play an immunomodulatory role in a wide variety of disease conditions, the effects of IL-35 on DCs is not yet elucidated at present. Here we demonstrated for the first time that IL-35 restrained the maturation of human MoDCs in vitro and further inhibited the proliferation and activation of allogeneic T lymphocytes. In this study, we first verified that MoDCs did not express IL-35. Furthermore, the induction of MoDCs in the presence of IL-35 also did not lead to the development of a DC population which producing IL-35. However, IL-35 receptor could be expressed on the cell surface of MoDCs, in other words, exogenous IL-35 might exert biological effect through specifically binding to its receptor and modulate MoDCs function. We then found that MoDCs exhibited obvious down-regulation of CD80, CD86 and a moderate reduction of CD1a and CD209 expression in the presence of IL-35. CD1a and CD209 are often regarded as a differentiation markers for immature DCs [27,28]. The down-regulation of CD1a and CD209 in IL-35-treated imDCs definitely suggested that IL-35 partially suppressed MoDC differentiation. In paralleled with the results of imDC phenotypic analysis, we observed that IL-35 did not influence the cytokine secretion in the process of MoDC differentiation. Dendritic cell maturation is a prerequisite for the induction of adaptive immune response. Many previous studies have demonstrated that Tregderived TGF-β and IL-10 have an inhibitory effect on DC maturation and the T cell stimulatory ability of DCs [29]. Here we chose LPS as the inducer for MoDCs maturation and observed that IL-35 induced a remarkable decrease in the expression of CD40, CD80, CD83, CD86 and HLA-DR on maturation of MoDCs. Moreover, the down-regulation of costimulatory molecules expression on IL-35-treated MoDCs was accompanied by a decrease in cytokine production of MoDCs, such as IL-1β, IL-2, IL-12p70, IFN-γ and TNF-α. Therefore, like other immunosuppressive cytokines TGF-β and IL-10, IL-35 also appears to be capable of inhibiting LPS-induced MoDC maturation. Notably, a recent study from mice also confirmed that IL-35 treatment could display decreased expression of CD86 on DCs in lymph nodes [30]. IDO is a tryptophan-catabolizing enzyme expressed by professional antigen presenting cells such as DCs. IDO plays an important role in DC-regulatory T cells crosstalk and has been implicated as an immunosuppressive effector mechanism of Tregs. Tregs could condition DCs to express IDO, which then indirectly suppress T-cell response [31]. The inhibitory checkpoint PD-L1 and PD-L2 could limit T-cell immunity, causing T-cell exhaustion [32]. However, in this study, we did not observe any significant difference of IDO, PD-L1 and PD-L2 51

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addition to direct suppressor effect on CD4+/CD8+ T cells, which may help to provide new therapeutic approach for disease treatment. [17]

Author contributions

[18]

Conception and design: Haiting Mao and Xi Chen. Development of methodology: Xi Chen, Shengnan Hao, Zhonghua Zhao, Jia Liu, Qianqian Shao, Fang Wang and Dong Sun. Acquisition of data: Xi Chen, Shengnan Hao, Zhonghua Zhao, Jia Liu and Qianqian Shao. Analysis and interpretation of data: Haiting Mao, Xi Chen, Ying He and Wenjuan Gao. Writing, review and revision of the manuscript: Haiting Mao, Xi Chen. Administrative, technical or material support: Ying He and Wenjuan Gao. Study supervision: Haiting Mao.

[19]

[20]

[21]

Acknowledgements

[22]

This work was supported by the Natural Science Foundation of China under Grant (No: 31270970 and 31570919); Taishan Scholar Foundation; the Science and Technology Project of Jinan of China under Grant (No: 201202197); the Science and Technology Project of Shandong, China under Grant (No: 2008GG10002035 and 2012G0021821).

[23]

[24]

[25]

[26]

Conflict of interest

[27]

The authors report no conflict of interest.

[28]

Appendix A. Supplementary material [29]

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.cyto.2018.03.008.

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References

[31]

[1] A.K. Palucka, B. Laupeze, C. Aspord, H. Saito, G. Jego, J. Fay, et al., Immunotherapy via dendritic cells, Adv. Exp. Med. Biol. 560 (2005) 105–114. [2] M.L. Kapsenberg, Dendritic-cell control of pathogen-driven T-cell polarization, Nat. Rev. Immunol. 3 (2003) 984–993. [3] J. Zhu, W.E. Paul, CD4 T cells: fates, functions, and faults, Blood 112 (2008) 1557–1569. [4] H. Shin, Y. Kumamoto, S. Gopinath, A. Iwasaki, CD301b+ dendritic cells stimulate tissue-resident memory CD8+ T cells to protect against genital HSV-2, Nat. Commun. 7 (2016) 13346. [5] F. Faure, M. Jouve, I. Lebhar-Peguillet, C. Sadaka, F. Sepulveda, O. Lantz, et al., Blood monocytes sample MelanA/MART1 antigen for long-lasting cross-presentation to CD8(+) T cells after differentiation into dendritic cells, Int. J. Cancer 142 (2018) 133–144. [6] S.K. Kim, C.H. Yun, S.H. Han, Induction of dendritic cell maturation and activation by a potential adjuvant, 2-hydroxypropyl-beta-cyclodextrin, Front. Immunol. 7 (2016) 435. [7] A.M. Dudek, S. Martin, A.D. Garg, P. Agostinis, Immature, semi-mature, and fully mature dendritic cells: toward a DC-cancer cells interface that augments anticancer immunity, Front. Immunol. 4 (2013) 438. [8] F. Osorio, C. Fuentes, M.N. Lopez, F. Salazar-Onfray, F.E. Gonzalez, Role of dendritic cells in the induction of lymphocyte tolerance, Front. Immunol. 6 (2015) 535. [9] E. Mavin, L. Nicholson, S. Rafez Ahmed, F. Gao, A. Dickinson, X.N. Wang, Human regulatory T cells mediate transcriptional modulation of dendritic cell function, J. Immunol. (2016). [10] N. Misra, J. Bayry, S. Lacroix-Desmazes, M.D. Kazatchkine, S.V. Kaveri, Cutting edge: human CD4+CD25+ T cells restrain the maturation and antigen-presenting function of dendritic cells, J. Immunol. 172 (2004) 4676–4680. [11] K. Mahnke, T.S. Johnson, S. Ring, A.H. Enk, Tolerogenic dendritic cells and regulatory T cells: a two-way relationship, J. Dermatol. Sci. 46 (2007) 159–167. [12] L.W. Collison, C.J. Workman, T.T. Kuo, K. Boyd, Y. Wang, K.M. Vignali, et al., The inhibitory cytokine IL-35 contributes to regulatory T-cell function, Nature 450 (2007) 566–569. [13] O. Devergne, M. Birkenbach, E. Kieff, Epstein-Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin, PNAS 94 (1997) 12041–12046. [14] L.W. Collison, G.M. Delgoffe, C.S. Guy, K.M. Vignali, V. Chaturvedi, D. Fairweather, et al., The composition and signaling of the IL-35 receptor are unconventional, Nat. Immunol. 13 (2012) 290–299. [15] C.E. Egwuagu, C.R. Yu, L. Sun, R. Wang, Interleukin 35: critical regulator of immunity and lymphocyte-mediated diseases, Cytokine Growth Factor Rev. 26 (2015) 587–593. [16] W. Niedbala, X.Q. Wei, B. Cai, A.J. Hueber, B.P. Leung, I.B. McInnes, et al., IL-35 is

[32]

[33]

[34]

[35]

[36]

[37] [38]

[39]

[40]

[41]

[42]

[43]

52

a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells, Eur. J. Immunol. 37 (2007) 3021–3029. L.W. Collison, D.A. Vignali, Interleukin-35: odd one out or part of the family? Immunol. Rev. 226 (2008) 248–262. C.H. Huang, E.X. Loo, I.C. Kuo, G.H. Soh, D.L. Goh, B.W. Lee, et al., Airway inflammation and IgE production induced by dust mite allergen-specific memory/ effector Th2 cell line can be effectively attenuated by IL-35, J. Immunol. 187 (2011) 462–471. V. Chaturvedi, L.W. Collison, C.S. Guy, C.J. Workman, D.A. Vignali, Cutting edge: human regulatory T cells require IL-35 to mediate suppression and infectious tolerance, J. Immunol. 186 (2011) 6661–6666. C. Oderup, L. Cederbom, A. Makowska, C.M. Cilio, F. Ivars, Cytotoxic T lymphocyte antigen-4-dependent down-modulation of costimulatory molecules on dendritic cells in CD4+ CD25+ regulatory T-cell-mediated suppression, Immunology 118 (2006) 240–249. X. Chen, Q. Shao, S. Hao, Z. Zhao, Y. Wang, X. Guo, et al., CTLA-4 positive breast cancer cells suppress dendritic cells maturation and function, Oncotarget 8 (2017) 13703–13715. L.W. Collison, V. Chaturvedi, A.L. Henderson, P.R. Giacomin, C. Guy, J. Bankoti, et al., IL-35-mediated induction of a potent regulatory T cell population, Nat. Immunol. 11 (2010) 1093–1101. S. Haller, A. Duval, R. Migliorini, M. Stevanin, V. Mack, H. Acha-Orbea, Interleukin35-producing CD8alpha+ dendritic cells acquire a tolerogenic state and regulate T cell function, Front. Immunol. 8 (2017) 98. R.X. Wang, C.R. Yu, I.M. Dambuza, R.M. Mahdi, M.B. Dolinska, Y.V. Sergeev, et al., Interleukin-35 induces regulatory B cells that suppress autoimmune disease, Nat. Med. 20 (2014) 633–641. F. Arce, G. Kochan, K. Breckpot, H. Stephenson, D. Escors, Selective activation of intracellular signalling pathways in dendritic cells for cancer immunotherapy, Anticancer Agents Med. Chem. 12 (2012) 29–39. B. Kong, G.B. Liu, J.A. Zhang, X.X. Fu, W.Y. Xiang, Y.C. Gao, et al., Elevated serum IL-35 and increased expression of IL-35-p35 or -EBI3 in CD4(+)CD25(+) T cells in patients with active tuberculosis, Am. J. Transl. Res. 8 (2016) 623–633. B. Coventry, S. Heinzel, CD1a in human cancers: a new role for an old molecule, Trends Immunol. 25 (2004) 242–248. T.B. Geijtenbeek, R. Torensma, S.J. van Vliet, G.C. van Duijnhoven, G.J. Adema, Y. van Kooyk, et al., Identification of DC-SIGN, a novel dendritic cell-specific ICAM3 receptor that supports primary immune responses, Cell 100 (2000) 575–585. J. Zong, A.A. Keskinov, G.V. Shurin, M.R. Shurin, Tumor-derived factors modulating dendritic cell function, Cancer Immunol. Immunotherapy: CII 65 (2016) 821–833. J. Dong, C.K. Wong, Z. Cai, D. Jiao, M. Chu, C.W. Lam, Amelioration of allergic airway inflammation in mice by regulatory IL-35 through dampening inflammatory dendritic cells, Allergy 70 (2015) 921–932. Y. Han, Z. Chen, Y. Yang, Z. Jiang, Y. Gu, Y. Liu, et al., Human CD14+ CTLA-4+ regulatory dendritic cells suppress T-cell response by cytotoxic T-lymphocyte antigen-4-dependent IL-10 and indoleamine-2,3-dioxygenase production in hepatocellular carcinoma, Hepatology 59 (2014) 567–579. Y. Tanaka, A.M. Maeshima, J. Nomoto, S. Makita, S. Fukuhara, W. Munakata, et al., Expression pattern of PD-L1 and PD-L2 in classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, and gray zone lymphoma, Eur. J. Haematol. (2018). Y. Li, S. Wu, Y. Li, S. Jiang, T. Lin, L. Xia, et al., Interleukin-35 (IL-35) inhibits proliferation and promotes apoptosis of fibroblast-like synoviocytes isolated from mice with collagen-induced arthritis, Mol. Biol. Rep. 43 (2016) 947–956. J.Q. Liu, Z. Liu, X. Zhang, Y. Shi, F. Talebian, J.W. Carl Jret al., Increased Th17 and regulatory T cell responses in EBV-induced gene 3-deficient mice lead to marginally enhanced development of autoimmune encephalomyelitis, J. Immunol. 188 (2012) 3099–3106. G.S. Whitehead, R.H. Wilson, K. Nakano, L.H. Burch, H. Nakano, D.N. Cook, IL-35 production by inducible costimulator (ICOS)-positive regulatory T cells reverses established IL-17-dependent allergic airways disease, J. Allergy Clin. Immunol. 129 (207–215) (2012) e1–e5. B.M. Olson, J.A. Sullivan, W.J. Burlingham, Interleukin 35: a key mediator of suppression and the propagation of infectious tolerance, Front. Immunol. 4 (2013) 315. M. Tang, J. Diao, M.S. Cattral, Molecular mechanisms involved in dendritic cell dysfunction in cancer, Cell. Mol. Life Sci.: CMLS (2016). E. Mavin, L. Nicholson, S. Rafez Ahmed, F. Gao, A. Dickinson, X.N. Wang, Human regulatory T cells mediate transcriptional modulation of dendritic cell function, J. Immunol. 198 (2017) 138–146. D. Escors, L. Lopes, R. Lin, J. Hiscott, S. Akira, R.J. Davis, et al., Targeting dendritic cell signaling to regulate the response to immunization, Blood 111 (2008) 3050–3061. J. Long, X. Zhang, M. Wen, Q. Kong, Z. Lv, Y. An, et al., IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells, Biochem. Biophys. Res. Commun. 430 (2013) 364–369. Z. Wang, J.Q. Liu, Z. Liu, R. Shen, G. Zhang, J. Xu, et al., Tumor-derived IL-35 promotes tumor growth by enhancing myeloid cell accumulation and angiogenesis, J. Immunol. 190 (2013) 2415–2423. H. Mao, W. Gao, C. Ma, J. Sun, J. Liu, Q. Shao, et al., Human placental trophoblasts express the immunosuppressive cytokine IL-35, Human Immunol. 74 (2013) 872–877. Z. Zhao, X. Chen, S. Hao, R. Jia, N. Wang, S. Chen, et al., Increased interleukin-35 expression in tumor-infiltrating lymphocytes correlates with poor prognosis in patients with breast cancer, Cytokine 89 (2017) 76–81.