IL-33 changes CD25hi Tregs to Th17 cells through a dendritic cell-mediated pathway

Journal Pre-proof IL-33 changes CD25hi Tregs to Th17 cells through a dendritic cell-mediated pathway Su-Ho Park, Hak-Jun Jung, Tae Sung Kim

PII:

S0165-2478(19)30408-0

DOI:

https://doi.org/10.1016/j.imlet.2019.12.003

Reference:

IMLET 6403

To appear in:

Immunology Letters

Received Date:

5 August 2019

Revised Date:

30 November 2019

Accepted Date:

17 December 2019

Please cite this article as: Park S-Ho, Jung H-Jun, Kim TS, IL-33 changes CD25hi Tregs to Th17 cells through a dendritic cell-mediated pathway, Immunology Letters (2019), doi: https://doi.org/10.1016/j.imlet.2019.12.003

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IL-33 changes CD25hiTregs to Th17 cells through a dendritic cell-mediated pathway

Su-Ho Parka, Hak-Jun Junga, and Tae Sung Kima,*

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145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea.

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Division of Life Science, College of Life Science and Biotechnology, Korea University,

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Running title: The Change in the properties of Tregs to Th17 cells by IL-33-matDCs

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*Corresponding author: Division of Life Science, College of Life Science and Biotechnology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea. Tel.: +82-2-

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3290-3416; Fax: +82-2-3290-3921. E-mail address: [email protected] (T.S.Kim)

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Abbreviations: BMDCs, bone marrow-derived dendritic cells; IL-33-matDCs, IL-33-matured

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DCs; ImDCs, immature DCs; MFI, mean fluorescence intensity; Tregs, regulatory T cells; CD25hi Tregs, stable Tregs highly expressing CD25; CD25lo Tregs, unstable Tregs low expressing CD25; Th17 cells, T helper 17 cells

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Graphical abstract

Highlights

 IL33 interrupts Treg differentiation through DC maturation.

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 IL33-matDCs change stable Tregs highly expressing IL-6 receptor to Th17 cells.

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 The conversion of Tregs to Th17 cells is mediated by IL-33-matDCs-derived IL-6.

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 The interaction of IL-33-matDCs and Tregs is required for conversion to Th17 cells.

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ABSTRACT

Interleukin (IL)-33 is an alarmin factor that is highly secreted in a variety of autoimmune

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diseases, induces maturation of dendritic cells (DCs) and differentiation of T helper 17 (Th17) cells. As the balance between Th17 cells and regulatory T cells (Tregs) is important to

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maintain immune homeostasis, in this study, we investigated the effects of IL-33 on Treg cell response. We observed that direct treatment with IL-33 had no effect on Treg differentiation, whereas IL-33-matured DCs (IL33-matDCs) inhibited the differentiation of CD4+ T cells to Tregs by decreasing the expression of Foxp3. Furthermore, co-culture with IL-33-matDCs changed stable Tregs (CD25hiCD4+ Tregs) to IL-17-producing cells, whereas IL-33-matDCs 2

had little effects on unstable Tregs (CD25loCD4+ Tregs). The stable Tregs were demonstrated to express high levels of IL-6 receptors. Blocking of IL-6 secreted from IL-33-matDCs suppressed the conversion of Tregs to Th17 cells, indicating the greater propensity to convert stable Tregs to Th17 cells is due to IL-6 signaling. Taken together, these results demonstrate that IL-33 inhibits Treg differentiation and the conversion of stable Tregs to Th17 cells via

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DCs.

Keywords: IL-33; Matured DCs; Treg conversion; Th17 cells; Treg differentiation; IL-6

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signaling

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1. Introduction

IL-33, a pro-inflammatory cytokine, is associated with various autoimmune and inflammatory diseases [1]. IL-33 is expressed as an alarmin factor by endothelial cells, epithelial cells, and fibroblasts at the intestinal tract [2]. Inflammatory bowel disease (IBD) is exacerbated by increasing serum levels of IL-33, whereas inhibiting IL-33 signaling acts as a

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protective mechanism [3]. IL-33 is detected by utilizing ST2L (IL-33Rα), a component of the IL-33 receptor, which also contains another component, IL-1RAcP [4]. These receptors are

expressed on mast cells, macrophages, DCs, basophils, eosinophils, natural killer T cells, and Th2 cells, thereby enabling them to respond to IL-33 [1]. DCs are activated by IL-33

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signaling to orchestrate adaptive immune responses, inducing the differentiation of Th2 or

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Th17 cells via maturation of DCs [5-7]. In peripheral tissues, DCs are generally immature. Once they come in contact with antigens in the tissues, they undergo maturation and migrate

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to the secondary lymphoid organs for presenting antigens to T cells [8, 9]. Immature DCs (imDCs) promote tolerance by either the inhibiting function of antigen-specific T cells or by

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expanding Tregs, but mature DCs (matDCs) induce immunogenic responses [10, 11]. Tregs express the central transcription factor Foxp3 and play a pivotal role in the

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maintenance of tissue homeostasis [12], whereas Th17 cells are involved in the pathogenesis of autoimmune diseases [13]. CD25, the alpha subunit of the IL-2 receptor, is one of the

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major factors of Tregs, because IL-2 is needed for Treg development outside the thymus [14]. The level of CD25 in Tregs is an indicator of their stability. Tregs expressing high levels of CD25 (CD25hi Tregs) are stable, whereas Tregs expressing low CD25 levels (CD25lo Tregs) are unstable [15]. The developmental pathways of Tregs and Th17 cells are reciprocally regulated [16]. Tregs can lose the expression of Foxp3 and their suppressive ability under 4

inflammatory conditions [17]. These ex-Tregs reprogram to become Th17 cells, and thus contribute to the development of diseases such as psoriasis, IBD, rheumatoid arthritis (RA), and asthma by expressing IL-17 [18-22]. Conversion of Tregs to IL-17-producing cells occurs via IL-6 and/or IL-1β signaling [23-26]. In our previous report, DCs matured by IL-33 treatment were shown to promote the differentiation of Th17 cells by increasing IL-6 and IL1β levels [6]. However, whether IL-33 regulates Treg stability and changes Tregs into Th17

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cells is not well understood. As DCs are important for antigen presentation to T cells in autoimmune diseases, in this study, we investigated the effects of IL-33 on Treg stability and the DC-dependent conversion of Tregs to Th17 cells. Treg differentiation was interrupted by IL-33-matDCs, but not under conditions without DCs. Furthermore, we found that IL-33-

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matDCs convert Tregs to Th17 cells.

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2. Materials and Methods

2.1. Mice and IL-33 Female C57BL/6 mice at 7–11 weeks of age were purchased from Orient Bio (Kapyong, South Korea). OT-II Tg mice and Foxp3-eGFP mice on a C57BL/6 background were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). All mice were housed under

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specific pathogen-free conditions. All animal experiments were conducted according to the Korea University Guidelines for the Care and Use of Laboratory Animals (approval No. KUIACUC-2018-0045). Recombinant mouse mature IL-33 protein was purchased from

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YbdY biotechnology (Seoul, South Korea).

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2.2. Generation of bone marrow-derived dendritic cells (BMDCs)

The isolation of BM cells from C57BL/6 mice and generation of BMDCs were performed

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as previously described [6].

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2.3. CD4+ T cell isolation and sorting

CD4+ T cells were isolated from peripheral and mesenteric lymph nodes of mice by

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magnetic bead purification (MACS; Miltenyi Biotec), and the CD4+ T cell purity of each isolation was > 95%. Purified CD4+ T cells were activated with anti-CD3ε (clone 145-2C11;

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1 μg/ml) and anti-CD28 (clone 37-51; 1 μg/ml), which are Th0 cells. For the Treg polarization, TGF-β (5 ng/ml or 10 ng/ml) and IL-2 (5 ng/ml) were added to the cultures of CD4+ T cells together with anti-CD3ε/CD28. GFP+/GFP- cells and CD25hi/CD25lo cells were sorted by FACSAria (BD Biosciences) after Treg polarization.

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2.4. Co-culture with CD4+ T cells and BMDCs. Either naïve CD4+ T cells or Tregs were co-cultured with BMDCs for 4 d, as described with minor modification [6]. CD4+ T cells were isolated from OT-II mice and BMDCs from C57BL/6 mice were pulsed for 2 h with OVA323-339 (10 ng/ml) followed by incubation for 24 h with IL-33. Naïve CD4+ T cells were cultured with BMDCs at a 3:1 ratio in the presence of Treg-polarizing cytokines, TGF-β (5 ng/ml) and IL-2 (5 ng/ml), without anti-CD3ε and antiCD28 stimulation. After sorting CD25hi and CD25lo cell populations, either CD25hi or CD25lo

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Tregs were cultured with BMDCs at a 1:1 ratio in the presence of anti-CD3ε (1 μg/ml) and

TGF-β (1 ng/ml). To investigate cell-contact effect, DCs and CD25hi or CD25lo Tregs were

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separately cultured in a transwell (0.4 μm pore) with anti-CD3ε stimulation.

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2.5. Flow cytometric analysis

Cells were stimulated for 4 h with PMA (50 ng/ml), ionomycin (1 μg/ml), and brefeldin A

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(GolgiPlug) and were washed with PBS and resuspended in FACS washing buffer (0.5% FBS and filtered 0.05% NaN3 in PBS). The cells were then stained with FITC-conjugated anti-

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CD4 (RM4-5), PerCP-Cy5.5-conjugated anti-CD4 (RM4-5), Alexa Fluor 488-conjugated anti-CD25 (eBio7D4), PE-conjugated anti-Foxp3 (NRRF-30), APC-conjugated anti-Foxp3

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(FJK-16S), APC-conjugated anti-IL-17 (eBio17B7), PE-conjugated anti-CD126 (D7715A7), APC-conjugated anti-gp130 (KGP130), PE-conjugated anti-CD121a (JAMA-147). For

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blocking the Fc receptors of DCs, serum IgG was treated for 15 min. Surface molecules were stained by incubating mAbs for 20 min. For intracellular staining, cells were fixed for 30 min using a Foxp3/Transcription Factor Staining buffer set (eBioscience), and cells were performed intracellular staining for 1 h in perm/wash buffer (eBioscience). The stained cells were analyzed in a FACSAccuriC6 (BD Biosciences). 7

2.6. Treg suppression assay After Treg polarization, CD25hiCD4+ or CD25loCD4+ Tregs were isolated by FACS Aria. For assessing the suppressive capacity of CD25hiCD4+ or CD25loCD4+ Tregs, CFSE-labeled CD4+ Tconv cells (5 × 104 cells/well) were cultured with different Tregs in 96-well plate, as previously described [27]. For investigating the effect of IL-33-matDCs on regulatory activity, CD25hiCD4+ T cells were selected for co-cultured with OVA323-339-pulsed IL-33-matDCs (2.5

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× 105 cells/well) in 48-well plate for 4 d. CD4+ T cells were cultured with CFSE-labeled Tconv cells.

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2.7. RT-PCR

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Total mRNA of cells were extracted with RiboEX total RNA kit (GeneAll Biotechnology, Seoul, South Korea). RNA (1 μg) was converted to cDNA by RT-PCR using MyGenieTM 32

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Thermal Block (BioNeer, Daejeon, South Korea).

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2.8. Enzyme-linked immunosorbent assay (ELISA)

The quantities of IL-17 levels in culture supernatants were measured by a sandwich

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ELISA (ELISA Ready-SET-Go!®, eBioscience, San Diego, CA), as previously described [6].

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2.9. Statistical analyses

Groups were compared by using two-tailed Student’s t-test and analyzed by Microsoft®

Excel 2016. A P-value ≤ 0.05 was considered statistically significant. The data were presented as the mean ± SD of at least three independent experiments.

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3. Results

3.1. IL-33 inhibits DC-mediated Treg differentiation. IL-33 induces the maturation of DC to allow them to present antigens to T cells [5, 6]. Moreover, the differentiation of Tregs is reciprocally regulated with respect to the differentiation of Th17 cells [16], and IL-33 promotes Th17 cell differentiation in the

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presence of DCs [6]. Therefore, we hypothesized that IL-33 affects Treg differentiation in a DC-dependent manner. In Treg-polarizing conditions, IL-33-matDCs interrupted the

differentiation of Tregs in an IL-33 dose-dependent manner, compared with imDCs (Fig. 1A). However, Treg differentiation from naïve CD4+ T cells was not directly affected by IL-33 in

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the absence of DCs (Fig. 1B). Furthermore, to assess whether IL-33 reduces Treg stability,

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GFP-expressing Tregs polarized from naïve CD4+ T cells of Foxp3-eGFP mice were directly treated with IL-33. The treatment of Tregs with IL-33 did not affect the expression of Foxp3

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(Fig. 1C). These results indicated that, although IL-33 did not inhibit the differentiation or stability of Tregs in the absence of DCs, it did reduce Treg differentiation via DC maturation.

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Thus, we propose that IL-33 requires a DC-mediated pathway to affect CD4+ T cell immunity.

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3.2. IL-33-matDCs change stable Tregs into Th17 cells. Tregs that have lost Foxp3 expression (ex-Tregs) are able to convert to Th17 cells [28,

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29]. Therefore, we speculated if IL-33-matDCs induce the change of unstable Tregs or stable Tregs to Th17 cells. Thus, we determined the expression levels of Foxp3 in stable CD25hi and unstable CD25lo Tregs using flow cytometry. CD25hi Tregs expressed higher amounts of Foxp3 than CD25lo Tregs (Fig. 2A). Furthermore, CD25hi Tregs displayed a more suppressive capacity than CD25lo Tregs to inhibit the proliferation of conventional T (Tconv) cells 9

(Supplementary Fig. S1). To investigate the effect of IL-33-matDCs on the conversion of Tregs, either imDCs or IL-33-matDCs were co-cultured with CD25hi Tregs or CD25lo Tregs for 4 days. CD25hi Tregs stably express Foxp3, but CD25lo Tregs lost the expression of Foxp3 during incubation without DCs for 4 days (Fig. 2B). IL-33-matDCs down-regulated the expression of Foxp3 and up-regulated the expression of IL-17 in both Treg types (Fig. 2B, C). Interestingly, CD25hi Tregs that were changed to Th17 cells by IL-33-matDCs express

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significant levels of IL-17 (Fig. 2C). CD25hi Tregs decreased the suppressive ability by IL33-matDCs (Supplementary Fig. S2). To observe the process of the change of Tregs to IL-17producing cells, either CD25hi Tregs or CD25lo Tregs were co-cultured with IL-33-matDCs

for 2–4 days. The number of Foxp3-expressing cells decreased in a time-dependent manner,

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and that of IL-17-expressing cells markedly increased when CD25hi Tregs were co-cultured

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with IL-33-matDCs, but not when CD25lo Tregs (Fig. 2D). Moreover, IL-33 was able to induce IL-17 expression in CD25hi Tregs in the presence of OVA323-339-pulsed imDCs (Fig.

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3A, B). These results demonstrated that stable Tregs became Th17 cells under inflammatory

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conditions.

3.3. IL-6 is involved in the conversion of stable Tregs to Th17 cells by IL-33-matDCs.

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Tregs have been shown to convert into Th17 cells in the presence of IL-6 and/or IL-1β [23, 30]. We determined the expression levels of various receptors of IL-6, IL-1, and IL-33 in

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Th0 cells and Tregs. The expression levels of IL-6 (Il6Rα and Il6st) and IL-1 receptor (Il1r1) mRNA were elevated in Tregs, but not IL-33 receptor (St2) mRNA, when compared to those in Th0 cells (Fig. 4A). Next, to determine whether CD25hi Tregs expressed higher levels of receptors associated with the converting to Th17 cells than CD25lo Tregs, the levels of theses receptors in CD25hi and CD25lo Tregs were analyzed. The expression of CD126 (IL-6Rα) and 10

CD130 (gp130) were enriched in CD25hi Tregs, but not those of CD121a (IL-1R1) (Fig. 4B, C). We added IL-6 and/or IL-1β neutralizing Abs to a co-culture system with Tregs and IL33-matDCs to confirm the association of IL-6 and IL-1β with conversion of Tregs. The change of CD25hi Tregs to Th17 cells, which is mediated by IL-33-matDCs, was decreased by an anti-IL-6 neutralizing Ab and not by an anti-IL-1β neutralizing Ab (Fig. 4D). Moreover, the secretion level of IL-17 was inhibited when treated with neutralizing Ab against IL-6 (Fig.

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4E). These data showed that IL-6 inhibition successfully suppressed the conversion of CD25hi Tregs to Th17 cells. These results also provided evidence that a response to IL-6 secreted

from IL-33-matDCs by IL-6 receptors, which are highly expressed in stable Tregs, is required

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for their conversion to Th17 cells.

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3.4. The conversion of Tregs to Th17 cells by IL-33-matDCs is dependent on cell contact. In an inflammatory environment, antigen-specific Tregs lose their expression of Foxp3

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[31]. The presentation of antigens to Tregs and Tconv cells is mediated by interaction with DCs in lymphoid organs under inflammatory conditions [9]. In order to determine whether

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Treg conversion mediated by IL-33-matDCs requires a cell-to-cell contact, either CD25hi Tregs or CD25lo Tregs were maintained in a detached culture with IL-33-matDCs, in a

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transwell system. IL-33-matDCs convert CD25hi Tregs to IL-17-expressing cells without cellto-cell contact in a transwell (Fig. 5A). However, the amount of the converted IL-17-

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expressing cells from CD25hi Tregs were significantly lower, compared with those obtained from mixed co-cultures of IL-33-matDCs and CD25hi Tregs in a system of cell contact (Fig. 5A, B). These results indicated that the contact between IL-33-matDCs and Tregs was needed for efficient conversion of Tregs to Th17 cells, even though Treg conversion could occur by IL-6 without direct cell-to-cell contact. 11

4. Discussion

In several inflammatory diseases, Tregs can be converted to Th17 cells [32]. However, it has not been reported whether pro-inflammatory cytokines promote Treg conversion. Here, we showed that IL-33 induced DC-mediated conversion of Tregs to IL-17-producing cells. We have previously reported that IL-33 triggers the secretion of IL-1β and IL-6 from DCs, as well as the maturation of DCs, resulting in the induction of Th17 cell differentiation from

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naïve CD4+ T cells [6]. Migratory DCs and resident DCs are present in secondary lymphoid

organs [33]. Our results showed that IL-33-matDCs markedly inhibited Treg differentiation. As the inhibition of Treg differentiation is able to induce Th17 cells, we also found that IL-

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33-matDCs changed Tregs into Th17 cells. This may be because DCs uptake antigens, mature,

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and then migrate into lymphoid organs, where Tregs are located [8, 33]. We also determined that the change of Tregs to Th17 cells was induced by antigen-pulsed imDCs in the presence

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of IL-33, whereas IL-33 alone had no direct effect on the differentiation or conversion of Tregs. ST2L (IL-33 receptor) is not expressed in lymphatic and splenic Tregs, but it is

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expressed in Tregs of colon and lungs [34, 35]. IL-33 induces the dysregulation of lung Tregs into cells having Th2 cell characteristics [35]. In the colon, however, IL-33 amplifies local

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intestinal Treg responses to limit immune-mediated damage [34]. Hence, future studies are needed to further elucidate the effect of IL-33 and IL-33-matDCs on the conversion of tissue-

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resident Tregs.

Plastic Tregs contribute to pathogenesis of diseases [18-22]. The plasticity of Tregs is

related to the stability of Foxp3 expression [28]. The severity of arthritis is enhanced when Foxp3-lost Tregs (ex-Tregs) are adaptively transferred into mice, in which cells are converted to IL-17-producing cells by reducing the expression of phosphatase PTPN2 in Tregs [21]. In 12

addition, Komatsu et al. reported that adoptive transfer of autoreactive unstable Tregs increases the severity of arthritis by converting to Th17 cells [36]. Thus, we also studied the major origin of Th17 cells converted from Tregs by IL-33-matDCs. Interestingly, CD25hi Tregs expressed higher levels of IL-6 receptors than CD25lo Tregs, such that CD25hi Tregs were the main cell type turned into IL-17-producing cells by IL-33-matDCs-mediated IL-6. Additionally, more than 75 % of IL-6Rhi cells among CD25+ Tregs are TIGIT-negative cells,

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whereas IL-6RloCD25+ Tregs strongly express TIGIT [37]. The co-inhibitory molecule TIGIT suppresses the secretion of IL-17 in T cell-intrinsic effect [38]. These data are supporting the idea that conversion of Th17 cells from Tregs is developed by stable CD25hi Tregs. During inflammation, Foxp3+IL-17+CD4+ T cells are generated as an intermediate stage between

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Tregs and Th17 cells in the periphery [39, 40]. Although Foxp3+IL-17+CD4+ T cells were not

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observed in this study, it is clear that IL-17-producing cells were developed by Tregs, not naïve T cells, because CD25lo Tregs were slightly changed to Th17 cells in the presence of

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IL-33-matDCs. Furthermore, Foxp3 expression was down-regulated when co-culture with imDCs and CD25hi Tregs, compared to culture in a transwell system. These data suggest that

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conversion of CD25hiTregs to Th17 cells mainly occurs via IL-6 secreted from IL-33-matDCs, accompanying the decrease of Treg stability by DCs-Tregs contact.

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In conclusion, our results indicated that IL-33-matDCs may skew Th17 cell responses in lymphoid organs, thus interfering with Treg differentiation and inducing the conversion of

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Tregs to Th17 cells, as well as promoting Th17 cell differentiation. These data support the notion that IL-33, which is a highly expressed cytokine in inflammation environments, induces the severe pathogenic conditions by a DC-mediated pathway. Furthermore, these findings indicated that methods utilized to block IL-6 receptors in Tregs may be developed as a therapeutic strategy for autoimmune diseases. 13

Acknowledgment This research was supported by National Research Foundation of Korea (NRF) grant (Grant No.: NRF-2017R1A2B2009442), and also by a Korea University grant (to T.S. Kim). S.-H. Park was supported by Global PhD Fellowship Program through the National Research Foundation of Korea funded by the Ministry of Education (Project No.: 2015H1A2A1031021).

Korea University for careful maintenance of all animals.

Conflicts of interest

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We also would like to appreciate the Gyerim Experimental Animal Resource Center at

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The authors report no conflicts of interest regarding the publication of this paper.

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Figure Legends

Fig. 1. IL-33-matDCs inhibit the differentiation of CD4+ T cells to Tregs.

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(A) OVA323-339-pulsed ImDCs or IL-33-matDCs were co-cultured with naïve CD4+ T cells

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from OT-II mice, in the presence of the Treg-polarizing cytokines, TGF-β (5 ng/ml) and IL-2 (5 ng/ml), without anti-CD3ε/CD28 stimulation for 4 d. (B) Naïve CD4+ T cells were treated

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with IL-33 in the absence or presence of TGF-β and IL-2 with anti-CD3ε/CD28 for 3 d. (C) Either GFP-Tconv or GFP+ Tregs, sorted after Treg polarization for 2 d, were treated with IL-

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33 for 3 d in the presence of an anti-CD3ε (1 μg/ml) and TGF-β (1 ng/ml). The results are presented as the mean ± SD of three independent experiments. Statistical significance was

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assessed using a two-tailed Student’s t-test; ##P < 0.01 vs. untreated DCs, *P < 0.05 and **P

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< 0.01 vs. imDCs. N.S, not significant.

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Fig. 2. IL-33-matDCs change the characteristic of Tregs into that of Th17 cells.

(A) iTregs were polarized from naïve CD4+ T cells by TGF-β and IL-2 in the presence of

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anti-CD3ε and anti-CD28. The Foxp3 histogram was assessed by flow cytometry gated on

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either CD25hiCD4+ or CD25loCD4+ cells. The bar graph represents the MFI indices of the histogram. (B) For co-culture with Tregs and DCs, iTregs from OT-II mice were polarized by TGF-β (10 ng/ml) and IL-2 (5 ng/ml) for 2 d and then divided into CD25hi and CD25lo cells,

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and OVA323-339-pulsed DCs were activated by IL-33. Either CD25hi cells or CD25lo cells were co-cultured with DCs in the presence of anti-CD3ε (1 μg/ml) and TGF-β (1 ng/ml) for 4 d. (C)

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Bar graphs indicate the percentage of Foxp3+CD4+ T cells or IL-17+CD4+ T cells. (D) Tregs

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and DCs were co-cultured as in (B) for 2–4 d. Experiments were performed on three independent occasions and are presented as means ± SD. Statistical significance was assessed using a Student’s t-test; *P < 0.05 and **P < 0.01 in CD25hi Tregs; #P < 0.05 and ###P < 0.001 in CD25lo Tregs.

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Fig. 3. OVA323-339-pulsed imDCs induce the expression of IL-17 in CD25hi Tregs in the presence of IL-33.

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iTregs from OT-II mice were polarized by TGF-β (10 ng/ml) and IL-2 (5 ng/ml) for 2 d. CD25hiCD4+ Tregs and CD25loCD4+ Tregs were sorted by using FACSAria. (A) Eeach Tregs was co-cultured with OVA323-339-pulsed imDCs and IL-33 in the presence of an anti-CD3ε (1 μg/ml) and TGF-β (1 ng/ml) for 4 d. (B) Bar graphs display the percentage of Foxp3+CD4+ T

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cells or IL-17+CD4+ T cells. Results are presented as the mean ± SD after two independent

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experiments. *P < 0.05. N.S, not significant.

Fig. 4. DC-derived IL-6 is involved in the conversion of Tregs to Th17 cells by IL-33matDCs. 23

(A) Naïve CD4+ T cells were cultured for 2 d in the absence or presence of the Tregpolarizing cytokines, TGF-β (10 ng/ml) and IL-2 (5 ng/ml), after treating with both antiCD3ε and anti-CD28. The mRNA expressions were determined by RT-PCR. (B) The expression levels of CD126, CD130, and CD121a were accessed by flow cytometry after gating on either CD4+CD25hi cells or CD4+CD25lo cells. Bar graphs represent the MFI indices of histogram. (C) Bar graphs represent the MFI indices of histogram of CD126 and CD130. (D) CD25hi Tregs obtained from OT-II mice were co-cultured with OVA323-339-pulsed

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IL-33-matDCs in the presence of an anti-IL-6 (MP5-20F3, 1 μg/ml) and/or an anti-IL-1β

(B122, 1 μg/ml), or a rat IgG1 κ isotype Ab (eBRG1, 1 μg/ml) and an Arm Ham IgG isotype Ab (eBio299Arm, 1 μg/ml). Foxp3 and IL-17 levels were detected by flow cytometry, gated

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on CD4+CD25+ cells. (E) The concentration of IL-17 in the cell-free supernatant was

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measured by ELISA after co-culture with IL-33-matDCs and CD25hi Tregs. Results are

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presented as the mean ± SD after three independent experiments. *P < 0.05, **P < 0.01.

Fig. 5. Cell-to-cell contact is needed for efficient conversion of Tregs to Th17 cells by IL-33-

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matDCs.

(A) iTregs from OT-II mice were polarized by TGF-β (10 ng/ml) and IL-2 (5 ng/ml) for 2

d and then divided into CD25hiCD4+ Tregs and CD25loCD4+ Tregs cells by using FACSAria. Each Treg population was cultured with OVA323-339-pulsed DCs in the presence of an antiCD3ε (1 μg/ml) and TGF-β (1 ng/ml) for 4 d. Tregs were cultured in the bottom transwell 24

chamber and DCs were cultured in the upper chamber. (B) Bar graphs indicate the percentage of Foxp3+CD4+ T cells or IL-17+CD4+ T cells. Results are presented as mean ± SD of three independent experiments. Statistical significance was assessed using a two-tailed Student’s ttest; *P < 0.05 (transwell vs. no-transwell), #P < 0.05, ##P < 0.01, and ###P < 0.001 (culture with imDCs vs. IL-33-matDCs). N.S, not significant.

Supplementary Figure S1. CD25hi Tregs have more suppressive ability than CD25lo Tregs.

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Tregs were differenteated from naive CD4+ T cells by TGF-β (10 ng/ml) and IL-2 (5

ng/ml), and then isolated to CD25hi and CD25lo cells. Both CD25hi and CD25lo cells were cocultured with CFSE-stained Tconv cells at indicated ratio in 96-well plate for 3 d. Bar graph

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indicates the proliferation percentage of Tconv cells. Results are presented as mean ± SD of

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three independent experiments. Statistical significance was assessed using a Student’s t-test; *P < 0.05, **P < 0.01 (CD25hi Treg vs. CD25lo Treg), #P < 0.05, ##P < 0.01, and ###P <

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0.001 (vs. only Tconv)

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Supplementary Figure S2. IL-33-matDCs decrease the suppressive ability of CD25hi Tregs. CD25hi Tregs were polarized from naïve CD4+ T cells of OT-II mouse and co-cultured

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with imDCs or IL-33-matDCs at a 3:1 ratio in the presence of an anti-CD3ε (1 μg/ml) and TGF-β (1 ng/ml). After 4 d, each cell was cultured with CFSE-Tconv cells at a 1:1 ratio. Bar

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graph represents the proliferation percentage of Tconv cells. Data is representative of three independent experiments as mean ± SD. Statistical significance was assessed using a Student’s t-test; **P < 0.01 and ***P < 0.001 (vs. only CD25hiTregs), #P < 0.05 and ###P < 0.001 (vs. only Tconv). N.S, not significant.

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