Intravenous Immunoglobulin Attenuates Experimental Autoimmune Arthritis by Inducing Reciprocal Regulation of Th17 and Treg Cells in an Interleukin-10-Dependent Manner

Intravenous Immunoglobulin Attenuates Experimental Autoimmune Arthritis by

Inducting Reciprocal Regulation of Th17 and Treg in an IL-10-Dependent Manner

Seon-Yeong Lee1*, Young-Ok Jung

2*, Jun-Geol Ryu

1, Chang-Min Kang

1, Eun-Kyung Kim

1,

Hye-Jin Son1, Eun-Ji Yang

1, Ji-Hyeon Ju

1, 3, Young-Sun Kang

4, Sung-Hwan Park

1, 3, Ho-Youn

Kim1, 3

, Mi-La Cho1**,

1The Rheumatism Research Center, Catholic Research Institute of Medical Science, The

Catholic University of Korea, Banpo-dong, Seocho-gu, Seoul, 137-701, South Korea.

2Division of Rheumatology, Department of Internal Medicine, Kangnam Sacred Heart

Hospital, Hallym University, Seoul 137-701, Korea.3Center for Rheumatic Disease, Division

of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, The Catholic

University of Korea, Banpo-dong, Seocho-gu, Seoul, 137-701, South Korea. 4Department of

Biomedical Science and Technology, Institute of Biomedical Science and Technology,

Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea;

Authorship note: SYL and YOJ contributed equally to this work.

Funding:Funding:Funding:Funding: This study was supported by a grant of the Korean Health Technology R&D

Project, Ministry for Health & Welfare, Republic of Korea (HI09C1555) and also

supported by the Bio & Medical Technology Development Program of the National

Research Foundation (NRF) funded by the Korean government (MEST) (No.

2012M3A9C6049783)

Full Length Arthritis & RheumatismDOI 10.1002/art.38627

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article as an‘Accepted Article’, doi: 10.1002/art.38627© 2014 American College of RheumatologyReceived: May 30, 2013; Revised: Jan 07, 2014; Accepted: Mar 11, 2014

2

Conflict of Interests : None

**Address correspondence to: Mi-La Cho, Rheumatism Research Center, Catholic Institutes

of Medical Science, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul

137-040, Korea (South)

Phone: 82-2-2258-7467; Fax: 82-2-599-4287; E-mail: [email protected].

Keywords: Intravenous Immunoglobulin, Th17, Germinal Center, FcγIIB, Rheumatoid

Arthritis

Running Head: IVIG attenuate arthritis by Th17/Treg balance

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ABSTRACT

Objective. Intravenous immunoglobulin (IVIG) is used as a therapeutic agent in

various autoimmune diseases. The aims of this study were to investigate the therapeutic

effects of IVIG on collagen-induced arthritis (CIA) and the mechanism responsible for

any therapeutic effects.

Methods. IVIG was administered to CIA mice and the in vivo effects were

determined. T helper 17 (Th17) and regulatory T (Treg) cell frequencies were analyzed

by flow cytometry, and cytokine levels in the supernatant were measured by enzyme-

linked immunosorbent assays. Subpopulations of T cells and B cells of spleens were

assessed by confocal microscopy.

Results. The arthritis score and incidence of arthritis were lower in mice treated

with IVIG compared with untreated mice. Histopathological analysis showed less joint

damage in mice treated with IVIG. The expression of proinflammatory cytokines,

specific collagen type II antibody, and osteoclast markers was significantly reduced in

mice treated with IVIG. IVIG induced increased Foxp3 expression and inhibited Th17

cell development. The number of Foxp3+ Treg cells increased and the number of Th17

cells decreased in the spleens of mice treated with IVIG. The number of Foxp3+

follicular helper T cells increased and subsequent maturation of germinal center B cells

was inhibited by IVIG. IVIG also upregulated interleukin-10 (IL-10) and FcγγγγIIB

expression. IVIG lost its treatment effects on arthritis induced in IL-10 knockout mice.

Conclusion. Our results showed that IVIG has therapeutic effects by modulating

CD4+ T cell differentiation. The therapeutic effects of IVIG are dependent on IL-10.

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INTRODUCTION

Intravenous immunoglobulin (IVIG) is currently used in the treatment of a wide

variety of inflammatory and autoimmune diseases ((1, 2). Diseases for which IVIG has been

shown to be beneficial include Kawasaki disease, primary immunodeficiency disease, and

refractory polymyositis (1). IVIG is also used empirically to treat other autoimmune diseases

such as systemic lupus erythematosus and vasculitis (3-5).

The mechanism by which IVIG exerts beneficial effects in these diseases cannot be

explained. The classical action of IVIG includes the suppression or neutralization of

autoantibodies and cytokines, neutralization of the complement system, and prevention of the

autoantibody binding to the Fcγ receptor (FcγR) (1). IVIG upregulates the expression of

inhibitory FcγRIIB (6) and regulates cells of the innate immune system. IVIG therapy inhibits

the maturation of dendritic cells (7) and reduces the percentage of natural killer (NK) cells

and activity of NK cells in the peripheral blood (8). However, the beneficial effects of IVIG

extend beyond the half-lives of the infused immunoglobulins (Igs), and its treatment effects

occur in diseases whose pathology is attributed mainly to cellular immunity such as graft-

versus-host disease and cellular rejection (9, 10).

Regulatory T (Treg) cells play important roles in peripheral tolerance and in the

prevention of autoimmune diseases (11, 12). Patients with RA have defective

CD4+CD25

+Foxp3 Treg cells, and activation of Treg cells can improve clinical symptoms of

collagen-induced arthritis (CIA), regulate cytokine production, and inhibit osteoclastogenesis

in vitro and in vivo (13, 14).

The pathogenic roles of interleukin-17 (IL-17) and T helper 17 (Th17) cells, the

major producers of IL-17, are well known in inflammatory arthritis (15, 16). Th17 cells and

IL-17 increase synovial inflammation and joint destruction (15-19). Treg cells and Th17 cells

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are reciprocally regulated (20, 21) and skewing the balance toward Th17 cells makes the

body prone to inflammation. Therefore, regulating the Treg:Th7 cell ratio is of main interest

when treating autoimmune diseases.

Follicular helper T (TFH) cells, a special CD4+ T cell subset localized in the B cell

follicles, specialize in helping B cells in the germinal center (GC) reaction (22, 23).

Dysregulated TFH cells contribute to the development of autoreactive B cells, which produce

autoantibodies and induce autoimmune diseases (24). There has been no report on the effects

of IVIG on TFH cells and subsequent changes in GC B cells.

Several studies have suggested the possible regulatory effects of IVIG on T cell

subsets (25, 26). Several reports showed therapeutic effects of IVIG on inflammatory arthritis

(27) (28) . In this study, we examined the efficacy of IVIG in the treatment of CIA and the

underlying mechanisms by which IVIG modulated CIA. We investigated the changes in the

numbers of Treg and Th17, and the ratio elicited by IVIG and the effects of IVIG on TFH cells

within the GC B cell population. We also verified that the therapeutic effect of IVIG was lost

in IL-10 knockout (KO) mice.

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MATERIALS AND METHODS

Animals. Six-week-old male DBA1/J mice (SLC, Inc., Shizuoka, Japan) were

maintained in groups of five in polycarbonate cages in a specifically pathogen-free

environment and were fed standard mouse chow (Ralston Purina, Gray Summit, MO) and

water ad libitum. All experimental procedures were examined and approved by the Animal

Research Ethics Committee at the Catholic University of Korea.

Induction of arthritis and injection of IVIG. To examine the effect of IVIG on

CIA, IVIG was injected intravenously (i.v.) once on day 7 after CIA induction. To induce

CIA, 100 µg of bovine type II collagen (CII) and complete Freund’s adjuvant (Chondrex,

Inc.,Redmond, WA) were injected intradermally into the base of the tail. Starting the next day,

three independent observers examined the severity of arthritis three times a week. The

severity of arthritis was recorded using the mean arthritis index on scale of 0–4, as previously

reported (29).

Measurement of Ig concentrations. The serum concentrations of IgG, IgG1, and

IgG2a were measured using mouse IgG, IgG1, and IgG2a enzyme-linked immunosorbent

assay (ELISA) quantitation kits (Bethyl Laboratories, Montgomery, TX).

Immunohistochemistry. Mouse joint tissues were obtained 15 weeks after the immunization

and were fixed in 4% paraformaldehyde, decalcified in EDTA bone decalcifier, and

embedded in paraffin. Joint tissues were sectioned at 7-µm thickness, dewaxed using xylene,

dehydrated through a gradient of alcohol, and then stained with hematoxylin and eosin

(H&E), toluidine blue, Safranin O, and tartrate-resistant acid phosphatase (TRAP) to detect

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proteoglycans. The H&E stained sections were scored for inflammation and bone erosion.

Inflammation was scored according to the following criteria: 0 = no inflammation, 1 = slight

thickening of the lining layeror some infiltrating cells in the underlying layer, 2 = slight

thickening of the lining layer plus some infiltrating cells in the underlying layer, 3 =

thickening of the lining layer, an influx of cells in the underlying layer and the presence of

cells in the synovial space and 4 = synovium highly infiltrated with many inflammatory cells.

Cartilage damage was determined using safranin-O staining and Toluidine blue and the extent

of cartilage damage was scored according to the following criteria: 0 = no destruction, 1 =

minimal erosion limited to single spots, 2 = slight to moderate erosion in a limited area, 3 =

more extensive erosion and 4 = general destruction (30).

Immunohistochemistry was performed using the Vectastain ABC kit (Vector

Laboratories, Burlingame, CA). Joint tissues were incubated with the first primary

monoclonal antibodies (mAbs) at 4°C; the antibodies were goat anti-mouse tumor necrosis

factor-α (anti-TNF-α mAb), rabbit anti-mouse IL-1β mAb, rabbit anti-mouse IL-6 mAb,

rabbit anti-mouse IL-17, rabbit anti-mouse IL-10, and goat anti-mouse FcRIIB mAb. The

primary antibodies were detected with a biotinylated secondary linking Ab, followed by

incubation with streptavidin–peroxidase complex for 1 h. The final color product was

developed using 3,3′-diaminobenzidine chromogen (Dako, Carpinteria, CA). Positive cells

were counted and results are expressed as mean ± standard deviation (SD).

Real-time polymerase chain reaction (PCR). Relative expression of specific

mRNAs was quantified by real-time PCR using SYBR Green I (Roche Diagnostics). The

following sense and antisense primers were used: for TNF-α, 5′-AGC CCC CAG TCT GTA

TCC TT-3′ and 5′-CTC CCT TTG CAG AAC TCA GG-3′; for IL-6, 5′-ATT TGT GTG CTG

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AAG GAG GC-3′ and 5′-AAA GGA CAG GAT GTT GCA GG-3′; for IL-1β, 5′-CTT GGA

TGA GGA CAT GAG CAC CTT C-3′ and 5′-GGA AGA CAG GCT TGT GCT CTG C-3′;

for FcRIIB, 5′-CCC TGG GAA CTC TTC TAC CC-3′ and 5′-CA GCA GCC AGT CAG

AAA TCA-3′; and for β-actin, 5′-GAA ATC GTG CGT GAC ATC AAA G-3′ and 5′-TGT

AGT TTC ATG GAT GCC ACA G-3′.

Flow cytometric analyses of T and B cells. Cell pellets were prepared from the

spleens of CIA and IVIG-injected CIA mice. To examine the population of T helper cells, the

cells were stained with anti-CD4–peridin chlorophyll protein (PerCP) mAb (eBioscience) and

anti-CD25–allophycocyanin (APC) (eBioscience). Cells were permeabilized and fixed with

CytoFix/CytoPerm (BD Pharmingen) as instructed by the manufacturer, and stained further

with anti-Foxp3–phycoerythrin (PE) (eBioscience), anti- IFNγ-APC, anti-IL-4-PE, IL-17-

FITC or IL-10-APC (all purchase eBioscience). To examine the population of B cells, the

cells were stained with anti-CD4–PerCP, anti-B220-APC, GL7-FITC, CXCR5-APC and PD-

1-FITC.

Measurement of IL-17 concentration. The concentration of IL-17 in serum was

measured by sandwich ELISA. Anti-mouse IL-17 mAb (R&D Systems, Minneapolis, MN)

was added to a 96-well plate (Nunc, Roskilde, Denmark) and incubated overnight at 4°C. The

wells were treated with blocking solution (phosphate-buffered saline containing 1% bovine

serum albumin and 0.05% Tween 20), the samples and the standard recombinant IL-17 (R&D

Systems) were added to the 96-well plate, and the plate was incubated. Biotinylated IL-17

polyclonal Ab (R&D Systems) was added, and the reaction was allowed to proceed. The plate

was washed, 1:2,000 diluted ExtrAvidin–alkaline phosphatase (Sigma-Aldrich, St Louis, MO)

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was added, and the reaction was allowed to proceed. The plate was washed, and 50 µl of p-

nitrophenyl phosphate disodium salt (Pierce Chemical Company, Rockford, IL) diluted in

diethanolamine buffer was applied.

Staining for confocal microscopy. Spleen tissues were obtained 15 weeks after

primary immunization. The various cell populations were identified with specific antibodies

(all from eBioscience). To examine the populations of Th cells, the tissues were stained with

anti-CD4–PerCP, anti-interferon (IFN)-γ–fluorescein isothiocyanate (FITC), anti-IL-4–FITC,

anti-CD25–APC, anti-Foxp3–FITC, anti-IL-17–PE, and anti-IL-10–PE. To analyze the

populations of cells expressing signal transducer and activator of transcription (STAT), the

tissues were stained with (all from eBioscience) anti-CD4–PE, anti-p-STAT3 705–FITC,

anti-p-STAT3 727–FITC, and anti-p-STAT5–FITC. To analyze the populations of B cells,

the tissues were stained with anti-CD4–PerCP, anti-B220-APC, anti-CD138–PE, anti-ICOS–

PE, anti-GL7–FITC, anti-IgD–FITC, anti-IgM–biotin (BD Biosciences, San Jose, CA), and

streptavidin–PerCP. To analyze FcRIIB expression in cells, the tissues were stained with anti-

IL-10–PE, anti-FcRIIB–FITC, anti-CD4–APC, anti-CD19–APC, anti-F4/80–APC, and anti-

CD11c–APC. Stained sections were analyzed using a confocal microscopy system (LSM 510

Meta, Carl Zeiss). All images were analyzed with the MetaMorph software (Molecular

Devices, PA, USA).

Statistical analysis. All data were expressed as the mean ± SD. Statistical analysis

was performed using SPSS 10.0 for Windows (IBM Corp., Armonk, NY). Comparing

numerical data between groups was performed with nonparametric Mann-Whitney tests.

Statistical analysis was performed using SPSS 10.0 for Windows (SPSS, Chicago, IL, USA).

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P values <0.05 were considered significant.

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RESULTS

Prolonged IVIG-induced suppression of CIA and decreased expression of

inflammatory cytokines. We examined the effect of IVIG in the CIA animal model. IVIG

was given to CIA mice once on day 7 after the primary immunization. The arthritis score and

the incidence of arthritis were significantly lower in IVIG-treated mice compared with the

untreated group (Figure 1A). The single injection of IVIG after the first immunization

suppressed the arthritis score for >15 weeks.

Consistent with the arthritis score, the histological observations of tissues stained

with H&E, toluidine blue, and Safranin O showed less inflammatory infiltration and cartilage

damage in IVIG-treated CIA mice compared with the untreated group (Figure 1C). In

particular, the levels of CII antigen-specific IgG1 and IgG2a were decreased (Figure 1B). The

number of TRAP-positive cells was also lower in joint tissues from IVIG-treated mice

(Figure 1D). Next, we observed the effects of IVIG on the expression of inflammatory

cytokines in joint tissues of IVIG-treated CIA and CIA mice. The number of cells staining

positively for IL-1β, IL-6, TNF-α, and IL-17 in the immunohistochemically were lower in

joint tissues from IVIG-treated mice compared with CIA mice (Figure 1E). The mRNA levels

of IL-1β, IL-6, and TNF-α were also lower in IVIG-treated mice (Figure 1F). The mRNA

level of FcRIIB was higher in IVIG-treated mice (Figure 1G). These results suggest that

IVIG suppressed the induction and inflammation of CIA.

Counterregulatory effects of IVIG on Th17 and Treg cells in CIA mice. We

investigated the effects of IVIG on Th17 and Foxp3+ Treg cell populations. Confocal

microscopic examination and FACs analysis showed significantly more Th17 cells in spleens

of untreated CIA mice than in IVIG-treated CIA mice. By contrast, more Foxp3+ Treg cells

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were found in spleens of IVIG-treated mice than in untreated CIA mice (Figure 2A, B).

Confocal microscopic examination also showed more STAT3+ cells and fewer STAT5

+ cells

in spleens of untreated CIA mice than in IVIG-treated CIA mice (Figure 2C). All images were

analyzed with the MetaMorph software.

Serum IL-17 level also decreased by IVIG treatment (Figure 2D), but the mRNA

levels of IL-10 and suppressor of cytokine signaling 3 (SOCS3) increased in the serum from

IVIG-treated mice (Figure 2E). Confocal microscopic examination and FACs analysis of the

spleen showed more CD4+CD25

+Foxp3

+IL-10

+ Treg cells in IVIG-treated CIA mice than in

untreated CIA mice (Figure 2F, G). These results suggest that these effects seemed to be

related to counterregulatory control of Th17 and Treg cells through STAT3 and STAT5.

Regulatory effects of IVIG on immature and mature B cells. We investigated the

effects of IVIG on B cell populations. Confocal microscopic examination showed fewer

CD138+ plasma B cells and GL-7

+ GC B cells in spleens of IVIG-treated CIA mice. By

contrast, more IgM+B220

+ immature B cells were seen in the spleens of IVIG-treated mice

(Figure 3A). The B220+GL7

+ GC B cells significantly decreased in IVIG-treated CIA (Figure

3B). Additionally, there were fewer IL-17- and IFN-γ-expressing TFH cells and more Foxp3-

expressing TFH cells in the spleen from IVIG-treated mice (Figure 3C). In FACs analysis,

CD4+CXCR5

+PD-1

+ TFH cells were decreased in IVIG-treated. There were fewer IL-17-

expressing TFH cells and more Foxp3-expressing TFH cells increased (Figure 3D, E). These

results suggest that IVIG can control the production of pathogenic B cells in the GC of the

spleen by regulating TFH cells.

Upregulation of IL-10 and FcγγγγIIB by IVIG. IL-10 expression was significantly

increased in the spleen of IVIG-treated mice (Figure 4A). Double staining showed a

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significant increase in the number of CD4+IL-10

+ cells and F4/80

+IL-10

+ cells, suggesting

that CD4+ T cells and macrophages are the main source of IL-10 production induced by IVIG

(Figure 4B). FcγIIB expression was increased in CD11c+IL-10

+ cells and F4/80

+IL-10

+ cells

in the spleen from IVIG-treated CIA mice (Figure 4C). All images were analyzed with

MetaMorph software.

Requirement for IL-10 in the treatment effects of IVIG. We generated CIA in IL-

10 KO mice to examine whether IVIG maintains its treatment effects on arthritis induced in

IL-10 KO mice. IVIG was given to IL-10 KO CIA mice in the same manner as for the wild-

type CIA mice. There was no significant difference between the arthritis score and the

incidence of arthritis between IVIG-treated and untreated groups (Figure 5A). Consistent

with the arthritis score, histological findings and TRAP staining showed no difference

between the IVIG-treated CIA and the untreated groups (Figure 5B). The IgG concentration

did not differ significantly between groups (Figure 5C). Flow cytometric analysis showed

similar numbers of Th17 cells and Foxp3+ Treg cells in the spleens in the two groups (Figure

5D).

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DISCUSSION

In our experiments, we observed the effects of IVIG on CIA. IVIG decreased the

severity and incidence of arthritis for a prolonged period in this CIA animal model. IVIG also

decreased osteoclastogenesis, joint destruction, and the expression of inflammatory cytokines

TNF-α, IL-1β, IL-6, and IL-17 in joint tissues. IVIG increased the number of Treg cells with

a decrease in the number of Th17 cells causing a marked increase in the Treg:Th17 cell ratio.

Similarly, IVIG increased the expression of STAT5, the signal molecule essential for Treg cell

generation, but decreased the expression of STAT3, the signal molecule for Th17 generation.

The number of Foxp3+ TFH cells increased and subsequent induction of immature B cells in

the GC was observed in spleens of IVIG-treated mice. IVIG increased IL-10 expression, and

IL-10 KO mice showed no effects of IVIG, indicating that IL-10 is an essential factor in the

treatment effects of IVIG.

The therapeutic effects of IVIG in various autoimmune diseases are known, however,

the mechanism responsible for these therapeutic effects has not been elucidated. IVIG

interacts with the innate and adaptive immune systems in various ways (31). Recent reports

have shown that IVIG expands the CD4+CD25

+ Treg cell population (32) and decreases the

Th17 cell population (25).

We focused on the regulating effects of IVIG in T cells (26, 32) because T cells play

important roles in the pathogenesis of inflammatory arthritis. In an autoimmune

encephalomyelitis model, the number of CD4+CD25

+Foxp3

+ Treg cells increased in the

peripheral blood and the therapeutic effect of IVIG disappeared when Treg cells were

depleted (32) suggesting that Treg cells play a key role in the therapeutic effects of IVIG.

These experiments also reproduced the enhancing effect of IVIG on Treg cells. In the spleens

of IVIG-treated mice, the number of CD4+CD25

+Foxp3

+ Treg cells increased significantly. In

addition to the size of the Treg cell population, the Treg:Th17 cell ratio is also important in

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RA pathogenesis.

IVIG also inhibited Th17 cell generation in vitro (25). The Th17 cell population

decreased markedly in the spleens of IVIG-treated mice. The major cytokine produced by

Th17 cells, IL-17, upregulates synovial inflammation and bone injury by promoting

osteoclastogenesis (18, 19). We postulate that skewing the balance toward Treg cells and the

simultaneous decrease in Th17 cell number are responsible for the treatment effects of IVIG

on CIA. This is the first report to show a simultaneous regulatory effect of IVIG on Th17 and

Treg cells, and the ratio of these two cell types. An imbalance between Th17 and Treg cells

has been previously reported in patients with Kawasaki disease (33), an inflammatory disease

for which IVIG has a remarkable therapeutic effect. Therefore, it is possible that the

treatment effects of IVIG may be extended to patients with other inflammatory diseases with

a Treg:Th17 imbalance.

Autoimmune diseases are characterized as the loss of self-tolerance and generation of

autoreactive B cells that produce autoantibodies (34). TFH cells play an important role in the

selection of B cell clones in GCs. Dysregulated TFH cells have been found during the

development of systemic autoimmune disease including RA (24, 35). Surface molecules and

cytokines expressed on TFH cells determine the characteristics of these cells (24). We

observed a decrease in the number of IL-17- and IFN-γ-expressing TFH cells, but an increase

in the number of Foxp3-expressing TFH cells in the spleen of IVIG-treated mice. TFH cells

expressing Foxp3 have been reported to suppress the GC reaction (36).

Th cell differentiation from naïve CD4+ T cells is affected by microenvironmental

cytokines, which signal through STAT or other transcription factors, leading to the

differentiation to specific types of Th cells, which reciprocally inhibit the alternate

differentiation pathway. The proinflammatory cytokines IL-1β, IL-6, and IL-23 are known to

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be important to Th17 cell generation and Treg cell suppression, in which IL-6 plays a pivotal

role in the reciprocal relationship between Th17 and Treg cell generation (37). IL-6

concentration increased markedly in synovial membranes of RA patients favouring Th17

differentiation within the intra-articular environment (38). Both Th17 and TFH lineages

require IL-6, IL-21, and STAT3 (39) although Bcl6 acts as the specific transcriptional

regulator for TFH differentiation (39, 40). By contrast, STAT5 efficiently inhibits Th17 cells

(41) and TFH cells by inhibiting Blimp-1, a TFH cell suppressor (42). Mice with STAT5

deficiency show impairment of B cell tolerance. In our experiment, IVIG induced the

expression of STAT5 and suppressed the expression of STAT3, which explains the shifts to

Treg cells and Foxp3+ TFH cells.

FcγRs are important in many antibody-directed effector functions and blocking

FcγRs is effective in the treatment of inflammatory and autoimmune diseases. The anti-

inflammatory activity of IVIG is more closely linked to the upregulation of FcγIIB, the

inhibitory receptor (1). IVIG induces the expression of FcγRIIB on macrophages (6, 43). In

our experiments, the upregulation of FcγRIIB was seen in CD11C+ dendritic cells of IVIG-

treated mice and in F4/80+ macrophages.

Increased expression of FcRIIB is related to the decreased production of

autoantibodies by inhibition of the GC and GC B cell activation (44). We observed that the

GC of spleens of IVIG-treated mice was markedly inhibited and the expression of FcRIIB

and IL-10 in B19+ B cells increased (data not shown). Therefore, the anti-inflammatory

effects of FcRIIB and IL-10 seem to suppress GC B cells and the subsequent suppression of

GC B cells may explain the impaired production of CII-specific IgG in IVIG-treated mice.

We propose that IVIG controls adaptive immunity directly by regulating T and B cells in

addition to antigen-presenting cells. This finding suggests that induction of FcγRIIB in

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antigen-presenting cells is induced by IVIG and that this may be one explanation for the

treatment effects of IVIG.

Various cytokines are important in the pathogenesis of RA. IL-10 plays a role by

preventing inflammation and immune-mediated damage (45, 46). We have reported that IL-

10 plays an important role in the generation of Treg cells in RA (47). IL-10 has been shown

to effectively block the activation and effector function of T cells, monocytes, and

macrophages (48) to suppress Th17 cells, and to promote Treg cells within the CD4+ T cell

population in RA patients and a murine RA model (47, 49).

There have been few therapeutic trials of IL-10 in the treatment of RA even though

IL-10 is a potent anti-inflammatory cytokine. In our experiments, the development of CD4+ T

cells polarized toward Treg cells and subsequent therapeutic effects of IVIG were not

reproduced in IL-10 KO mice, indicating that IL-10 has critical effects on CD4+ T cell

development into Th17 or Treg cells.

The duration of the effect of IVIG in CIA mice lasted for >15 weeks, suggesting that

IVIG given in the early phase of disease has significant disease-controlling effects. The single

injection of IVIG showed continuous immunomodulatory effects by regulating macrophages,

B cells, and T cells.

In conclusion, our study showed that IVIG has treatment efficacy in CIA by

increasing the Treg:Th17 cell ratio and by reducing the population of pathogenic B cells by

modulating TFH cells. Our study suggests that IVIG may be a treatment option for patients

with inflammatory arthritis. The clinical applications of IVIG in treating RA should be

evaluated further.

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AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important

intellectual content.All of the authors have approved the final version to be published. M-L

Cho had full access to all of the data in the study and takes responsibility for the integrity of

the data and the accuracy of the data analysis.

Study conception and design. Seon-Yeong Lee, Young Ok Jung, Mi-La Cho

Acquisition of data. Seon-Yeong Lee, Jun-Geol Ryu, Chang-Min Kang, Eun-Kyung Kim,

Hye-Jin Son, Eun-Ji Yang, Young-Sun Kang

Analysis and Interpretation of data. Seon-Yeong Lee, Young-Ok Kim, Eun-Kyung Kim, Ji-

Hyeon Ju, Sung-Hwan Park, Ho-Youn Kim, Mi-La Cho

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FIGURE LEGENDS

Figure 1. IVIG suppressed induction of CIA and expression of inflammatory cytokines. IVIG

was injected i.v. once 7 days after CIA induction (number of mice per experiment=6). (A)

Arthritis severity was recorded as the mean arthritis index score and incidence score. (B)

Total IgG, IgG1, and IgG2a, and CII-specific IgG, IgG1, and IgG2a concentrations were

measured in serum. (C, D) Ankle joint tissues were obtained from CIA and IVIG-treated

mice in week 15 and stained with toluidine blue, Safranin O, H&E, and TRAP. The

inflammation and cartilage scores are shown in bar graphs (right). (E) Joint tissue from each

group was stained immunohistochemically with specific antibodies to TNF-α, IL-1β, IL-6,

and IL-17. (F, G) mRNA levels of TNF-α, IL-1β, IL-6, and FcγRIIB were analyzed by real-

time PCR. Data represent the mean ± SD of three independent experiments (*P < 0.05, **P <

0.005).

Figure 2. Counterregulatory effects of IVIG on Th17 and Treg cells in CIA. Spleen tissue was

obtained from each group in week 15. (A) The tissues were stained with specific antibody to

CD4 (red or green), CD25 (blue), IFNγ (green), IL-4 (green), Foxp3 (green), or IL-17 (red).

(B) Isolated spleen single cells were stained with CD4–PerCP, IFNγ-APC, IL-4-PE, IL-17-

FITC, CD25–APC, and Foxp3–PE for T helper cells analysis (C) For analysis of STAT-

positive T cells, the tissues were stained with specific antibodies for CD4 (red) and p-STAT3

727 (green), p-STAT3 705 (green), or p-STAT5 (green). Yellow-colored cells were considered

to be activated STAT-positive T cells. (D) In week 15, serum was collected from the mice,

and the protein level of IL-17 was measured by ELISA. (E) mRNA levels of IL-10 and

SOCS3 were measured by real-time PCR. (F) For IL-10-positive Treg cells, spleen tissues

were stained with specific antibodies to CD4 (red), CD25 (blue), Foxp3 (green), and IL-10

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(white). (G) Isolated spleen single cells were stained with CD4–PerCP, CD25–FITC, Foxp3–

PE and IL-10-APC. Positive cells of all confocal Images were analyzed with MetaMorph

software. Three images were analyzed and the percentage of showing positive cell in each

image was measured. Data are expressed as the mean ± SD of three independent experiments

(*P < 0.05, **P < 0.005).

Figure 3. Regulatory effects of IVIG on immature and mature B cells. To examine the effect

of IVIG on B cell activation and GC formation, spleen tissues were stained with B cell-

specific antibodies. (A) To analyze GC formation and B cell phenotype, the tissues were

stained with specific antibodies to CD4 (white), B220 (blue), CD138 (red), ICOS (red), GL-7

(green), IgM (green), and IgD (white). (B) For GC B cells, spleen single cells were stained

with specific antibodies to B220-APC, GL7-FITC. (C) To analyze TFH cells in the GC, spleen

tissues were stained with antibodies to CD4 (blue), B220 (white), GL-7 (green), IFNγ (red),

IL-17(red), and Foxp3(red). (D, E) Spleen single cells were stained with CD4-PerCP,

CXCR5-APC, PD-1-FITC, IL-17-PE or Foxp3-PE for subtypes of TFH cells. Data are

expressed as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.005).

Figure 4. Upregulation of IL-10 and FcγRIIB by IVIG. (A) Spleen tissues were stained

immunohistochemically with specific antibodies to IL-10. (B) To analyze IL-10-positive cells,

spleen tissues were stained with specific antibodies to IL-10, CD4, CD19, CD11c, and F4/80.

(C) Spleen tissue was stained with antibodies to CD11c, F4/80, IL-10, and FcγRIIB. Positive

cells of all confocal Images were analyzed with MetaMorph software. Three images were

analyzed and the percentage of showing positive cell in each image was measured. Data are


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Figure 5. Requirement of IL-10 for the treatment effects of IVIG. CIA was induced in IL-10

KO mice, and IVIG was injected i.v. once 7 days after CIA induction (number of mice per

experiment=6). (A) Arthritis severity was recorded as the mean arthritis index score and

incidence score. (B) Ankle joint tissues were obtained from CIA and IVIG-treated mice in

week 15 and stained with toluidine blue, Safranin O, H&E, and TRAP. The inflammation and

cartilage scores are shown in bar graphs (right). (C) Total IgG, IgG1, and IgG2a levels were

measured in serum from each group. (D) Isolated spleen single cells were stained with CD4–

PerCP, CD25–APC, Foxp3–FITC, and IL-17–PE to analyze the Treg and Th17 cells. Data are


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Figure 1. IVIG suppressed induction of CIA and expression of inflammatory cytokines. IVIG was injected i.v. once 7 days after CIA induction (number of mice per experiment=6). (A) Arthritis severity was recorded as the mean arthritis index score and incidence score. (B) Total IgG, IgG1, and IgG2a, and CII-specific IgG,

IgG1, and IgG2a concentrations were measured in serum. (C, D) Ankle joint tissues were obtained from CIA and IVIG-treated mice in week 15 and stained with toluidine blue, Safranin O, H&E, and TRAP. The

inflammation and cartilage scores are shown in bar graphs (right). (E) Joint tissue from each group was stained immunohistochemically with specific antibodies to TNF-α, IL-1β, IL-6, and IL-17. (F, G) mRNA levels of TNF-α, IL-1β, IL-6, and FcγRIIB were analyzed by real-time PCR. Data represent the mean ± SD of three

independent experiments (*P < 0.05, **P < 0.005). 287x411mm (300 x 300 DPI)

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Figure 2. Counterregulatory effects of IVIG on Th17 and Treg cells in CIA. Spleen tissue was obtained from each group in week 15. (A) The tissues were stained with specific antibody to CD4 (red or green), CD25 (blue), IFNγ (green), IL-4 (green), Foxp3 (green), or IL-17 (red). (B) Isolated spleen single cells were

stained with CD4–PerCP, IFNγ-APC, IL-4-PE, IL-17-FITC, CD25–APC, and Foxp3–PE for T helper cells analysis (C) For analysis of STAT-positive T cells, the tissues were stained with specific antibodies for CD4

(red) and p-STAT3 727 (green), p-STAT3 705 (green), or p-STAT5 (green). Yellow-colored cells were considered to be activated STAT-positive T cells. (D) In week 15, serum was collected from the mice, and

the protein level of IL-17 was measured by ELISA. (E) mRNA levels of IL-10 and SOCS3 were measured by real-time PCR. (F) For IL-10-positive Treg cells, spleen tissues were stained with specific antibodies to CD4

(red), CD25 (blue), Foxp3 (green), and IL-10 (white). (G) Isolated spleen single cells were stained with CD4–PerCP, CD25–FITC, Foxp3–PE and IL-10-APC. Positive cells of all confocal Images were analyzed with

MetaMorph software. Three images were analyzed and the percentage of showing positive cell in each image was measured. Data are expressed as the mean ± SD of three independent experiments (*P < 0.05, **P <

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0.005). 276x377mm (300 x 300 DPI)

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Figure 3. Regulatory effects of IVIG on immature and mature B cells. To examine the effect of IVIG on B cell activation and GC formation, spleen tissues were stained with B cell-specific antibodies. (A) To analyze GC

formation and B cell phenotype, the tissues were stained with specific antibodies to CD4 (white), B220

(blue), CD138 (red), ICOS (red), GL-7 (green), IgM (green), and IgD (white). (B) For GC B cells, spleen single cells were stained with specific antibodies to B220-APC, GL7-FITC. (C) To analyze TFH cells in the GC,

spleen tissues were stained with antibodies to CD4 (blue), B220 (white), GL-7 (green), IFNγ (red), IL-17(red), and Foxp3(red). (D, E) Spleen single cells were stained with CD4-PerCP, CXCR5-APC, PD-1-FITC,

IL-17-PE or Foxp3-PE for subtypes of TFH cells. Data are expressed as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.005).

249x305mm (300 x 300 DPI)

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Figure 4. Upregulation of IL-10 and FcγRIIB by IVIG. (A) Spleen tissues were stained immunohistochemically with specific antibodies to IL-10. (B) To analyze IL-10-positive cells, spleen tissues were stained with specific antibodies to IL-10, CD4, CD19, CD11c, and F4/80. (C) Spleen tissue was stained

with antibodies to CD11c, F4/80, IL-10, and FcγRIIB. Positive cells of all confocal Images were analyzed with MetaMorph software. Three images were analyzed and the percentage of showing positive cell in each image was measured. Data are expressed as the mean ± SD of three independent experiments (*P < 0.05, **P <

0.005). 210x226mm (300 x 300 DPI)

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Figure 5. Requirement of IL-10 for the treatment effects of IVIG. CIA was induced in IL-10 KO mice, and IVIG was injected i.v. once 7 days after CIA induction (number of mice per experiment=6). (A) Arthritis severity was recorded as the mean arthritis index score and incidence score. (B) Ankle joint tissues were

obtained from CIA and IVIG-treated mice in week 15 and stained with toluidine blue, Safranin O, H&E, and TRAP. The inflammation and cartilage scores are shown in bar graphs (right). (C) Total IgG, IgG1, and

IgG2a levels were measured in serum from each group. (D) Isolated spleen single cells were stained with CD4–PerCP, CD25–APC, Foxp3–FITC, and IL-17–PE to analyze the Treg and Th17 cells. Data are expressed

as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.005). 228x260mm (300 x 300 DPI)

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Documents

Intravenous Immunoglobulin Attenuates Experimental Autoimmune Arthritis by Inducing Reciprocal Regulation of Th17 and Treg Cells in an Interleukin-10-Dependent Manner