International Immunopharmacology 18 (2014) 98–105

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International Immunopharmacology

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Autophagy contributes to IL-17-induced plasma celldifferentiation in experimental autoimmune myocarditis

Jing Yuan 1, Miao Yu 1, Huan-Huan Li 1, Qi Long 1, Wei Liang, Shuang Wen, Min Wang, He-Ping Guo,Xiang Cheng ⁎, Yu-Hua Liao ⁎Laboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China

⁎ Corresponding authors at: Laboratory of CardiovasCardiology, Union Hospital, Jie-Fang Avenue 1277#, Wuh85726376; fax: +86 27 85727340.

E-mail addresses: nathancx@163.com (X. Cheng), liao1 Joint first authors: All of the four authors contributed

1567-5769/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.intimp.2013.11.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 August 2013Received in revised form 7 November 2013Accepted 7 November 2013Available online 21 November 2013

Keywords:AutophagyIL-17Plasma cellsApoptosisExperimental autoimmune myocarditis

Although IL-17 is considered to promote B cell differentiation into antibody-secreting plasma cells in some auto-immune diseases, its mechanism remains unclear. Recent studies revealed that autophagy, a lysosome-mediatedcatabolic process for providing nutrients under starvation, could regulate plasma cell homeostasis, so this studyaimed to explore whether and how autophagy participates in IL-17-mediated plasma cell differentiation byMyHC-α-induced experimental autoimmune myocarditis (EAM) mouse model. It showed that IL-17 could notonly induce B cell autophagy, but also facilitate the myocarditis severity, serum anti-MyHC-α autoantibody pro-duction and splenic CD38+ CD138+ B cell percentages, while the autophagy inhibitor 3-methyladenine attenu-ated these effects. Furthermore, serum anti-MyHC-α IgG autoantibody productions and CD38+ CD138+ B cellpercentages were positively correlated with B cell autophagy levels respectively. In vitro, we further revealedthat IL-17 could directly promote B cell autophagy, which boosted Blimp-1 expressions and CD38+ CD138+ Bcell percentages. Moreover, elevated autophagy mediated by IL-17 enhanced ubiquitin–proteasome system ac-tivity and B cell anti-apoptotic ability by Beclin-1 and p62 through Erk1/2 phosphorylation, and these changesbrought by IL-17 could be also inhibitedwith 3-methyladenine. Therefore, we concluded that autophagy contrib-uted to IL-17-mediated plasma cell differentiation by regulating Blimp-1 expression and Beclin-1/p62 associatedB cell apoptosis in EAM.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Antibodies specific for self-antigens produced by plasma cells arepathogenic in several autoimmune diseases [1]. We and others haveproved that Th17 cells and their characteristic cytokine IL-17 facilitateB cell function in humoral autoimmunity [2,3]. Doreau et al. furtherdemonstrated that IL-17 could promote B cell differentiation intoantibody-secreting plasma cells in systemic lupus erythematosus [4].However, the mechanism of IL-17-induced plasma cell differentiationremains unclear.

Autophagy, a lysosome-mediated catabolic process, is considered tobe a conserved system that plays critical roles in providing nutrientsunder starvation in eukaryotic cells [5]. Most recently, autophagyhas been shown to participate in plasma cell homeostasis [6], whichmight provide us the clues to explore the accurate relationship betweenIL-17 and plasma cell differentiation.

Thus, in the present study, we established an experimental autoim-mune myocarditis (EAM) mouse model with α-myosin-heavy chain

cular Immunology, Institute ofan 430022, China. Tel.: +86 27

yh27@126.com (Y.-H. Liao).to the work equally.

ghts reserved.

(MyHC-α) peptides, and investigated the relationship among IL-17, Bcell autophagy and plasma cell differentiation by using recombinantmouse-IL-17 (rIL-17) and autophagy inhibitor 3-Methyladenine (3-MA).Finally, we found that autophagy contributed to IL-17-mediated plasmacell differentiation by regulating Blimp-1 expression and B cell apoptosis.

2. Materials and methods

2.1. Mice

Male BALB/c mice aged 6 weeks were purchased from the experi-mental animal research centre (Hubei province, China). All of the ani-mals were kept in the pathogen-free mouse room in the experimentalanimal centre (Tongji Medical College of Huazhong University of Sci-ence and Technology), and the experiments were carried out accordingto the guidelines for the Care and Use of Laboratory Animals (Science &Technology Department of Huibei Province, PR China, 2005).

2.2. Interventions and groups

Mice were injected subcutaneously with 100 μg per mouse of theMyHC-α peptides [Ac-SLKLMATLFSTYASAD-OH] emulsified 1:1 withcomplete Freund's adjuvant (CFA, Sigma-Aldrich, Shanghai, China) ondays 0 and 7 to induce EAM [7]. BALB/c mice were randomly divided

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into five groups: EAM group (n = 10): mice were administrated withMyHC-α and PBS (200 μl per mouse); EAM + 3-MA group (n = 10):mice were administrated with MyHC-α and 3-MA (24 μg/g of bodyweight diluted in 50 μl PBS per mouse; Sigma); EAM + rIL-17 group(n = 10): mice were treated with MyHC-α and rIL-17 (1 μg dilutedin 200 μl PBS per mouse, PeproTech, Beijing, China); EAM + rIL-17 +3-MA group (n = 10): mice were injected with MyHC-α, rIL-17 (1 μgdiluted in 200 μl PBS per mouse) and 3-MA; normal group (n = 10):BALB/c mice were administrated the complete Freund's adjuvant only.The intraperitoneal injections of PBS, rIL-17 and 3-MA were on days 0,5, 10, 15 and 20 in the whole experiment. All animals of each groupwere killed separately on day 21, and their hearts and spleens were re-moved freshly and aseptically for measurements. Before killing, theblood of these mice was collected via retro-orbital bleeding and theserum was then prepared for detection.

2.3. ELISA and Elecsys

The determination of serum IL-17 was completed using a high-sensitivity mouse IL-17 ELISA kit (eBioscience, San Diego, CA, USA) ac-cording to the manufacturer's instructions. The sensitivity of this ELISAkit was 1.6 pg/ml, and there was no cross-reactivity detected. Anti-MyHC-α antibody was detected according to the ELISAmethod providedpreviously [8]. For the assay of anti-MyHC-α antibody isotype, thework was carried out with mouse Ig isotyping antibodies (SouthernBiotech, Birmingham, Alabama, USA) by ELISA. The blood concentra-tions of serum cardiac troponin (cTn) T weremeasured using the quan-titative rapid assay kit (Roche Diagnostics GmbH, Shanghai, China) byElecsys as previously described [9]. All of the samples were measuredin triplicate.

2.4. Histopathology

The ventricular tissues of the hearts were fixed in 10% phosphate-buffered formalin, trimmed and embedded in paraffin. Then, 5 μm sec-tions were cut longitudinally and stained with H&E (hematoxylin andeosin). The severity of impairment was assessed as the percentage ofthe heart section with inflammation compared with the overall size ofthe heart section, using a microscope eye piece grid with magnification×200 according to the following scoring system: grade 0, no; grade 1,b25% of the heart section is involved; grade 2, 25% to 50%; grade 3,50% to 75%; and grade 4, more than 75% [10]. Two independent re-searchers scored separately in a double-blind manner.

2.5. Magnetic activated cell sorting (MACS)

Spleen mononuclear cells from the mice in different groups wereused for negative selection of B cells by the mouse B cell isolation kit(Miltenyi Biotech, Shanghai, China) according to the manufacturer's in-structions. Briefly, cells were incubated with the cocktail of biotin-conjugated antibodies for 15 min and anti-biotin microbeads for 10 minat 4 °C. Afterwashing, the cellswere resuspended inMACSbuffer, appliedto a MACS LD separation column, and sorted in the magnetic field of theMACS separator, and the purified B cells were prepared for the furthertest.

2.6. Fluorescence assay

Freshly isolated B cells (2 × 106/ml) fromeachmousewere incubat-ed with 0.05 mmol/l monodansylcadaverine (MDC, Sigma) at 37 °C for1 h. After washingwith PBS, these cells were resuspended in 100 μl PBSin a 96-well plate separately. Then the MDC fluorescence intensity wasanalyzed by EnVisionMultilabel Plate Readers (PerkinElmer) with an ex-citation wavelength of 380 nm and an emission wavelength of 520 nm.

2.7. Transmission electron microscope

The formations of autophagic vacuoles were detected as previouslydescribed [11]. Briefly, the separated B cells were fixed in 3% glutaralde-hyde for 24 h and 1% osmium tetroxide for 2 h, dehydrated in gradedethanol and embedded in araldite. After cutting into ultrathin sections,these cells were stained with uranyl acetate and lead citrate. Then, thesections were observed with transmission electron microscope.

2.8. Flow cytometry

For plasma cell assay, the purified B cells were stained with anti-mouse-CD38-PE (Biolegend, San Diego, CA, USA) and anti-mouse-CD138APC (Biolegend) for 15 min at 4 °C. Then, cells were washedwith PBS and analyzed by flow cytometry with FACS Calibur. Forapoptosis detection by Annexin V-FITC Apoptosis Detection Kit(eBioscience), B cells at a density of 5 × 105 per 200 μl were resuspendedin the binding buffer. 5 μl Annexin V-FITC was added to 195 μl cellsuspension and incubated for 5 min at room temperature. Afterwashing, cells were resuspended in 190 μl binding buffer, incubatedwith 10 μl propidium iodide for 10 min and performed FACS analysis.

2.9. Western blot

The total proteins of isolated B cells were extracted with theTotal Protein Extraction Kit (Millipore, Billerica, MA, USA). Proteinconcentration was determined by the BCA Protein Assay Kit (Pierce,Rockford, IL, USA). 80 μg of total protein was separated on SDS-PAGEand electro transferred onto nitrocellulose membranes. Membraneswere sequentially blocked in TBST containing 5% skimmilk and then in-cubated with primary antibodies LC3B (1:500, Cell Signaling Technolo-gy, Boston, MA), Blimp-1 (1:800, Cell Signaling Technology), PAX-5(1:1000, Cell Signaling Technology), CD138 (0.1 μg/ml, R&D, Shanghai,China), Bcl-2 (1:1000, Cell Signaling Technology), ubiquitin (1:500, CellSignaling Technology), Beclin-1 (1:1000, Cell Signaling Technology), p62(1:1000, Cell Signaling Technology), Erk1/2 (1:1000, Cell Signaling Tech-nology), phospho-Erk1/2 (1:800, Cell Signaling Technology), AKT(1:1000, Cell Signaling Technology), phospho-AKT (1:500, Cell SignalingTechnology) or β-actin (1:1000, Cell Signaling Technology) at 4 °Covernight, followed by incubation with HRP-conjugated secondary anti-body (1:3000) for 2 h at room temperature. The target bands were per-formed with electro chemiluminesce and quantified by densitometryusing the BioRad image analysis program.

2.10. Luminescence assay

Purified B cells were sonicated in ice-cold buffer (pH 7.6, 50 mMTris/HCl, 1 mMDTT, 5 mMMgCl2, 0.5 mM EDTA, 2 mMATP and 50 mMNaCl and 0.05% Triton X-100) and the supernatants were prepared bycentrifugation for 15 min at 12,000 rpm. Then the protein concentrationsof extracts were adjusted to 1 mg/ml based on measurements with theBCA Protein Assay Kit (Pierce). The chymotrypsin-like proteasome activ-ity was assessed using 10 μl extracts by Proteasome-Glo Assay Systemsaccording to the manufacturer's instructions (Promega, Beijing, China)with luminometer. The blank and the positive control were used to de-termine the specificity of the experiment. The proteasome activitieswere normalized to the mean of the values in normal group.

2.11. In vitro studies on B cells

The isolated B cells (2 × 106 cells/ml) were cultured for 2 d at37 °C/5% CO2 in a 24-well culture plate in RPMI-1640 medium (Gibco,San Diego, CA, USA) containing 100 U/ml of penicillin, 100 μg/mlof streptomycin, 10% fetal calf serum (Gibco) with 10 μg/ml LPS,100 ng/ml rIL-17 or 100 μg/ml 3-MA. Then these B cells were collected

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for autophagy evaluation, plasma cell differentiation, proteasome activ-ity, and apoptosis detections.

2.12. Statistical analysis

Data were shown as the mean ± SEM. Statistical analysis was per-formed with one-way ANOVA, and the correlation between two vari-ables was tested by bivariate correlation analysis using SPSS11.0.p b 0.05 was considered statistically significant.

3. Results

3.1. The effects of IL-17 on B cell autophagy in vivo

The levels of serum IL-17 in EAM, EAM + 3-MA, EAM + rIL-17and EAM + rIL-17 + 3-MA groups were higher than those in normalBALB/c mice, and the serum concentrations of this cytokine were evenhigher in EAM + rIL-17 and EAM + rIL-17 + 3-MA groups thanthose in EAM and EAM + 3-MA groups (all p b 0.01). Therewas no ob-vious difference of serum IL-17 between EAM and EAM + 3-MAgroups, and neither did the difference between EAM + rIL-17 andEAM + rIL-17 + 3-MA groups (Fig. 1A).

To clarify the link of IL-17 and B cell autophagy, we evaluated theentire dynamic process of autophagy including the turnover of LC3,the formation of autophagosomes and most importantly the flux in ly-sosome [12] in splenic B cells from EAM mice, and found that both theratios of LC3B-II to LC3B-I and the numbers of autophagic vacuoles in cy-toplasm, as well as the fluorescence intensity of MDC in autophagic vac-uoles and lysosome were all increased along with the elevated IL-17levels in EAM, EAM + rIL-17 and EAM + rIL-17 + 3-MA groupscompared with those in normal group (all p b 0.05, Fig. 1B–F). Theseparameters for autophagy were not enhanced in EAM + 3-MA groupcompared with those in normal group, but these parameters weresignificantly reduced in EAM + 3-MA group compared with those

Fig. 1. IL-17 influenced B cell autophagy in EAMmice. (A) The levels of serum IL-17. (B)Westernratios between LC3B-II and LC3B-I. (D) The representative pictures for the autophagic vacuolescells. (F) Thefluorescence intensity ofMDC in autophagic vacuoles and lysosome inB cells. *p b

EAM group; $p b 0.05 vs EAM + rIL-17 group; $$p b 0.01 vs EAM + rIL-17 group. Each assay

in EAM group (all p b 0.05, Fig. 1B–F). The autophagy was boostedin EAM + rIL-17 group apparently compared with those in EAMgroup (all p b 0.01, Fig. 1B–F), but the differences between EAM +rIL-17 + 3-MA group and EAM group were not significant. More-over, the levels of B cell autophagy were declined evidently in EAM +rIL-17 + 3-MA groups compared with those in EAM + rIL-17 group(all p b 0.05, Fig. 1B–F).

3.2. The impact of autophagy in IL-17-mediated autoantibody production

It showed that the ratios of HW (heart weight)/BW (body weight),the pathological scores of heart sections, the levels of serum cTnT,and the productions of anti-MyHC-α IgM/IgG (especially IgG2aand IgG2b) autoantibodies in EAM, EAM + 3-MA, EAM + rIL-17 andEAM + rIL-17 + 3-MA were all elevated significantly comparedwith those in normal group (all p b 0.01, Fig. 2A–E). However,these four indexes evaluating for the severity of EAM were decreasedin EAM + 3-MA group compared with EAM group (all p b 0.05,Fig. 2A–E). At the same time, the severity of EAM were increasedin EAM + rIL-17 group compared with those in EAM group (allp b 0.05, Fig. 2A–E), and there was no significant difference betweenEAM + rIL-17 + 3-MA group and EAM group. But these four indexeswere decreased markedly in EAM + rIL-17 + 3-MA group comparedwith those in EAM + rIL-17 group (all p b 0.05, Fig. 2A–E). Furthermore,the levels of serum anti-MyHC-α IgG autoantibodies were positivelycorrelated with the fluorescence intensity of MDC in splenic B cells(R = 0.877, p b 0.01, Fig. 2F).

3.3. The influence of autophagy on IL-17-induced plasma cell differentiation

Upon plasma cell differentiation, B cell-specific surface proteinCD19 is faded away, while the glycoprotein CD138 is appeared andCD38 is still expressed highly [13]. Thus, we measured the CD38 andCD138 double positive B cells for accessing the levels of plasma cell

blotting for the alterations of LC3B-I and LC3B-II in B cells. (C) The statistical analysis of thein B cells. (E) The results of statistical analysis for the numbers of autophagic vacuoles in B0.05 vsNormal group; **p b 0.01 vsNormal group;#p b 0.05 vs EAMgroup; ##p b 0.01 vswas done in triplicate from ten animals per group. Values are means ± SEM.

Fig. 2.Autophagywas associatedwith IL-17-mediated autoantibody production. (A) The ratios ofHW/BW. (B) The representative pictures of histopathology (magnification×200). (C) Thepathological scores of heart sections. (D) The levels of serum cTnT. (E) The productions of anti-MyHC-α IgM/IgG/IgG1/IgG2a/IgG2b/IgG3 autoantibodies. (F) The correlation analysis be-tween the levels of anti-MyHC-α IgG autoantibody and the fluorescence intensity of MDC in B cells. *p b 0.05 vs Normal group; **p b 0.01 vs Normal group; #p b 0.05 vs EAM group;##p b 0.01 vs EAM group; $p b 0.05 vs EAM + rIL-17 group; $$p b 0.01 vs EAM + rIL-17 group. Results are representative of three independent experiments with ten mice pergroup. Values are means ± SEM.

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differentiation. The percentages of CD38+ CD138+ B cells wereenhanced in EAM, EAM + rIL-17 and EAM + rIL-17 + 3-MA groupscompared with those in normal group (all p b 0.01), but thesecells were declined obviously in EAM + 3-MA group compared withEAM group (p b 0.05, Fig. 3A and B). The levels of plasma cell differen-tiation were further boosted in EAM + rIL-17 group comparedwith those in EAM group (all p b 0.05, Fig. 3A and B), and the differ-ences between EAM + rIL-17 + 3-MA and EAM groups were not obvi-ous. However, the plasma cell differentiations were attenuated inEAM + rIL-17 + 3-MA groups compared with those in EAM + rIL-17group (all p b 0.05, Fig. 3A and B). Moreover, the percentages of CD38+

CD138+ cells were positively correlated with the MFI of MDC in Bcells (R = 0.745, p b 0.01, Fig. 3C).

The changes of Blimp-1, CD138, and the important molecules inautophagy and apoptosis including Becline-1, P62, p-AKT, and Bcl-2protein levels from isolated B cells were all consistent with the alter-ations of the percentages of CD38+ CD138+ B cells (all p b 0.05,Fig. 3D and E).

3.4. The effects of IL-17 on B cell autophagy in vitro

To explore the direct effect of IL-17 on autophagy, we cul-tured the isolated B cells from EAM mice with LPS, LPS + rIL-17, orLPS +rIL-17 + 3-MA for the research in vitro. The parameters for au-tophagy including the ratios of LC3B-II to LC3B-I, the numbers of autoph-agic vacuoles, and the fluorescence intensity of MDC were increased inLPS + rIL-17 group significantly compared with those in LPS group (allp b 0.01, Fig. 4A–E), but these autophagy parameters were notenhanced in LPS + rIL-17 + 3-MA group compared with those in LPSgroup. Moreover, the autophagy levels were decreased evidently in

LPS + rIL-17 + 3-MA groups compared with those in LPS + rIL-17group (all p b 0.01, Fig. 4A–E).

3.5. The role of autophagy in IL-17-induced plasma cell differentiationin vitro

Isolated B cells from EAMmice stimulatedwith LPS, LPS + rIL-17, orLPS + rIL-17 + 3-MAwere applied to investigate the direct effects andthe molecular mechanisms of autophagy in IL-17-mediated plasmacell differentiation. The percentages of CD38+ CD138+ B cells wereelevated in LPS + rIL-17 group (p b 0.01) and LPS + rIL-17 + 3-MAgroup (p b 0.05) markedly compared with those in LPS group (Fig. 5Aand B). But the levels of plasma differentiation were reduced evidentlyin LPS + rIL-17 + 3-MA groups compared with those in LPS + rIL-17group (p b 0.05, Fig. 5A and B).

The changes of Blimp-1 and CD138 protein levels were consistentwith the alterations of the percentages of CD38+ CD138+ B cells inthese three groups (all p b 0.05, Fig. 5C and D).

3.6. The influence of autophagy in B cell proteasome activity and apoptosis

To detect the effect of autophagy on B cell proteasome activity andapoptosis, we administered B cells from EAM mice with LPS, LPS +rIL-17, or LPS + rIL-17 + 3-MA. It revealed that increased proteasomecapacity, decreased poly-Ub protein accumulation and repressedapoptosis were found in LPS + rIL-17 group compared with those inLPS group, whereas there were no significant differences between LPSand LPS + rIL-17 + 3-MA groups. Moreover, these changes brought byIL-17 were attenuated in LPS + rIL-17 + 3-MA group compared withthose in LPS + rIL-17 group (all p b 0.05, Fig. 6A–D).

Fig. 3.Autophagy contributed to IL-17-induced plasma cell differentiation. (A) The purity of B cell sortingwas over 95% (left). The CD19+ B cells were gated (left), and the CD38+ CD138+

cellswere showed inflow cytometry charts (right). (B) The statistical analysis for thepercentages of CD38+CD138+B cells. (C) The correlation analysis between thepercentages of CD38+

CD138+ B cells and the fluorescence intensity of MDC in B cells. **p b 0.01 vs Normal group; #p b 0.05 vs EAM group; ##p b 0.01 vs EAM group; $$p b 0.01 vs EAM + rIL-17 group. Allmeasurements were performed with ten mice per group in at least three independent experiments. Values are means ± SEM.

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The levels of Beclin-1, Bcl-2 and p62 were raised along withenhanced Erk1/2 phosphorylation in LPS + rIL-17 and LPS + rIL-17 + 3-MA group compared with those in LPS group, and thesemolecule expressions were reduced in LPS + rIL-17 + 3-MAgroup compared with those in LPS + rIL-17 group (all p b 0.05,Fig. 6E and F).

4. Discussion

Autophagy could be regulated by a few characteristic cytokines fromCD4+ Th cells. Th1 cytokines IFN-γ and TNF-α induce autophagy in aMAPK-dependent manner, while the Th2 cytokines IL-4 and IL-13 in-hibit it through the Akt and STAT pathway [14–16]. As for IL-17,which is mainly secreted from Th17 cells and important for B cell differ-entiation in autoimmune diseases, however, its role in autophagy hasnot been clarified yet. In this study, it showed that B cell autophagywas increased along with the elevated IL-17 levels in EAM mice, sug-gesting a probable link of IL-17 and B cell autophagy. Accordingly, wetreated the EAMmice with rIL-17 and 3-MA, and found that IL-17 pro-moted B cell autophagywhile 3-MA attenuated IL-17-mediated autoph-agy obviously, which further indicated that IL-17 could induce B cellautophagy in EAM.

It has been reported that IL-17 exacerbated the disease by enhancingthe generation of autoantibodies against heart proteins such as anti-adenine nucleotide translocator (ANT) and MyHC-α in myocarditis

besides its proinflammatory function [2,17]. So in the course of investi-gating the effect of autophagy on IL-17-mediated anti-MyHC-α autoan-tibody production, we also observed the changes of the disease severityin EAM mice. It revealed that rIL-17 increased the secretions of anti-MyHC-α IgM/IgG (IgG2a/IgG2b) autoantibodies and the severityof EAM, but the autophagy inhibitor 3-MA inhibited these effects ofrIL-17, accompanied by decreased B cell autophagy. Furthermore, thelevels of serum anti-MyHC-α IgG autoantibodies were positively corre-lated with B cell autophagy levels, indicating that IL-17 might facilitateautoantibody production by regulating the autophagy in B cells.

Plasma cells are themain source of autoantibody. Sowe explored theroles of autophagy in the differentiations of B cells into plasma cells, andfound that IL-17 encouraged plasma cell differentiation while 3-MAalleviated it. Moreover, the levels of plasma cell differentiation werepositively correlated with the B cell autophagy, which suggested thatautophagy was involved in IL-17-induced plasma cell differentiation.

A variety of transcription factors control plasma cell transitionand commitment [18]. In particular, the transcriptional repressorBlimp-1 is the pivotal regulator of plasma cell differentiation by up-regulating plasma-cell-specific membrane protein CD138 synthesis[19,20]. Recently, although a few studies have discovered that IL-17could induce Blimp-1 expression in B cells, the mechanism of whichis still not clarified. Here we demonstrated that IL-17 boosted Blimp-1levels along with increased B cell autophagy and CD138 expression.After the inhibition of autophagy, the enhanced levels of Blimp-1

Fig. 4. IL-17 directlymodulatedB cell autophagy in vitro. (A)Western blotting for the alterations of LC3B-I and LC3B-II in B cells. (B) The statistical analysis of the ratios between LC3B-II andLC3B-I. (C) The representative pictures for the autophagic vacuoles in B cells. (D) The results of statistical analysis for the numbers of autophagic vacuoles in B cells. (E) The fluorescenceintensity ofMDC in autophagic vacuoles and lysosome in B cells. **p b 0.01 vs LPS group; #p b 0.05 vs LPS + rIL-17 group;##p b 0.01 vs LPS + rIL-17 group. Results are representative ofthree independent experiments. Values are means ± SEM.

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induced by IL-17 were diminished. At the same time, the reducedBlimp-1 protein effectively attenuated the alterations of CD138mediatedby IL-17. It then provided further evidence that autophagymight contrib-ute to IL-17-mediated plasma cell differentiation, which was performedby modulating Blimp-1 signaling.

It was different from B cells that plasma cells harbored the abilityof enhancing antibody synthesis and secretion [21]. The ubiquitin–proteasome system (UPS) and the autophagy-lysosomal system, thetwo major pathways in protein catabolism [22,23], might regulate the

Fig. 5. Autophagy was involved in IL-17-induced plasma cell differentiation by Blimp-1 signalinanalysis for the percentages of CD38+ CD138+ B cells. (C) Western blotting for the expressionsBlimp-1 and CD138 in B cells. *p b 0.01 vs LPS group; **p b 0.01 vs LPS group; #p b 0.05 vs LP

homeostasis during the process of plasma cell differentiation. Cenciet al. lately discovered that impaired UPS capacity could restrain termi-nal plasma cell differentiation by inducing cell apoptosis [24], but fornow the effects of autophagy-lysosomal system on B cell apoptosis havenot been studied. For these reasons, we investigated the relationshipamong autophagy, proteasome activity and apoptosis of splenic B cells,and found that IL-17 promoted proteasome capacity, inhibited accumula-tion of poly-Ub proteins, and repressed the apoptosis of B cells in EAM.The autophagy suppression alleviated the UPS capacity, causing the

g. (A) The CD38+ CD138+ cells were showed in flow cytometry charts. (B) The statisticalof Blimp-1 and CD138 in B cells. (E) The statistical analysis for the protein expressions ofS + rIL-17 group. Each assay was done in triplicate. Values are means ± SEM.

Fig. 6. Autophagy regulated proteasome activity and apoptosis of B cells in vitro. (A) The levels of the poly-Ub proteins accumulation in B cells. (B) The chymotrypsin-like proteasomeactivities in B cells. (C) The representative pictures of Annexin V positive B cells. (D) The statistical analysis for the percentages of Annexin V positive B cells. (E) The pictures of westernblotting for Beclin-1, p62, Bcl-2, Erk1/2 and phospho-Erk1/2 in B cells. (F) The statistical analysis for the protein expressions of Beclin-1, p62, Bcl-2 and phospho-Erk1/2 in B cells. *p b 0.05vs LPS group; **p b 0.01 vs LPS group; #p b 0.05 vs LPS + rIL-17 group; ##p b 0.01 vs LPS + rIL-17 group. Values are means ± SEM.

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decrease of B cell anti-apoptosis ability. Previously, Manz et al. found thatthe anti-apoptosis ability in B cells could prolong the lifespan of short-lived plasma cells and encourage the generation of long-lived plasmacells [25,26]. Therefore, we considered that the effect of autophagyon B cell apoptosis was also important in IL-17-mediated plasma celldifferentiation.

To further clarify the molecular mechanisms of autophagy in B cellapoptosis, we also detected the expression of the primary moleculesinvolving in autophagy. The autophagic protein Beclin-1 commonlyexists as the Beclin-1–Bcl-2 complex by interacting with the anti-apoptotic Bcl-2 familymembers through its Bcl-2-homology (BH)-3 do-mains, implying a crosstalk between autophagy and apoptosis [27]. Ad-ditionally, when autophagy occurs, this complex dissociates by theactivation of Erk1/2 signaling pathway, and the dissociative Beclin-1performs its function in autophagosome formation while the dissocia-tive Bcl-2 plays its role in apoptosis resistance [27]. In this study, wemeasured the changes of Beclin-1 expression, and indicated that IL-17facilitated Erk1/2 phosphorylation, Beclin-1 expression, and Bcl-2levels, but the repression of autophagy attenuated the alterations ofthesemolecules. It suggested that autophagy influenced apoptosis resis-tance through Beclin-1–Bcl-2 complex in IL-17-induced plasma celldifferentiation.

The adaptor protein p62 is another key molecular in autophagy,and it could deliver ubiquitylated proteins for autophagosome andproteasome, which links the autophagy and UPS in cell biology [28].In addition, the expressions of p62 could be also regulated by Erk1/2

signaling [29]. We represented that IL-17 boosted the levels of Erk1/2phosphorylation and p62. The increased p62 expression could delivermore ubiquitylated proteins for the enhanced autophagosome and pro-teasome, which promoted these protein degradation [29]. The ameliorat-ed protein accumulation resulted in attenuated endoplasmic reticulumstress and eventually inhibited endoplasmic reticulum stress induced ap-optosis through the increased Bcl-2 [30]. However, 3-MA inhibited theexpression of p62, and the impaired delivery of ubiquitylated proteinsled to protein accumulation, which would finally induced cell apoptosis[28,30]. So we revealed that autophagy also participated in apoptosis re-sistance in IL-17-mediated plasma cell differentiation via p62.

Herein, we firstly found that IL-17 could induce B cell autophagy inthe process of plasma cell differentiation, and the inhibition of autoph-agy reduced IL-17 mediated-B cell differentiation into plasma cells inEAM. Consistent with the down-regulated autophagy and plasma celldifferentiation were the decreased Blimp-1 expression and the en-hanced apoptosis in B cells. Consequently, we concluded that autophagycontributed to IL-17-mediated plasma cell differentiation by regulatingBlimp-1 signaling and Beclin-1/p62 associated B cell apoptosis. In fact,to clarify the association between endogenous IL-17 and autophagy inB cells, EAMmice treatedwith anti-IL-17 neutralizing antibodies shouldbe performed as the control besides the EAMmice injected with 3-MA.However, after anti-IL-17 neutralizing antibody injection in EAM micein our experiments, the levels of B cell autophagy, were not decreasedsignificantly compared with EAMmice treated with isotype control an-tibodies and EAM mice administrated with PBS, accompanied by the

105J. Yuan et al. / International Immunopharmacology 18 (2014) 98–105

enhanced IFN-γ levels (data not shown). And IFN-γ is a strong inducerof autophagy [14]. Thus, IFN-γ could interfere with the role of endoge-nous IL-17 in B cell autophagy, and we could not objectively evaluatethe effect of endogenous IL-17 on B cell autophagy in the EAM mice bytreating with anti-IL-17 neutralizing antibodies. Moreover, the morestudies were still underway to explore the accurate molecular mecha-nisms of IL-17 in inducing B cell autophagy. Additionally, in our previ-ous study, we indicated that IL-17 directly induced the apoptosis ofcardiac myocytes, and Chen et al. proved that the inhibition of autoph-agy could aggravate cardiomyocyte apoptosis [31,32]. Thus, autophagymight also influence IL-17-mediated cardiomyocyte apoptosis in themyocarditis progression, and these needed our further investigations.

Acknowledgments

This work was supported by the National Natural Science Foundationof China (81100158), National Basic Research Program of China (973Program) (2007CB512000, 2007CB512005), and National Science andTechnology Support Program (2011BAI11B00, 2011BAI11B23).

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