IFN-g induced by IL-12 administration prevents diabetes by inhibiting pathogenic

IL-17 production in NOD mice

Jun Zhang, Zhan Huang, Rui Sun, Zhigang Tian**, Haiming Wei*

Institute of Immunology, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, China

article info

Article history:

Received 16 August 2011

Received in revised form

8 November 2011

Accepted 28 November 2011

Keywords:

Type 1 diabetes

Interleukin 12

IFN-g

Interleukin 17

Dendritic cells

abstract

Interleukin 12 （IL-12） is a pivotal Th1-associated cytokine and a potent immunoregulatory molecule.

However, the role of IL-12 in inducing immune tolerance that prevents insulitis and inhibits type 1

diabetes （T1D） remains unknown. The aim of this study was to investigate whether intermittent

administration of IL-12 could prevent the development of T1D in nonobese diabetic （NOD） mice. We

examined whether IL-12 treatment prevented diabetes by injecting different doses of IL-12 into NOD

mice and compared the incidence of diabetes and insulitis in NOD mice with the incidence in control

mice. Furthermore, we investigated the potential mechanisms of IL-12-mediated prevention of diabetes

and insulitis. The expression of pro-in?ammatory and immunoregulatory cytokines was measured before

and following therapeutic administration of IL-12 in NOD mice. Our data demonstrated that both the

absolute number and the function of DCs were impaired in NOD mice and that the levels of the Th17-

associated pro-in?ammatory cytokines, IL-1b, IL-6 and IL-23, were elevated in NOD mice compared

with age-matched BALB/c and C57BL/6 mice. However, treatment of NOD mice with IL-12 suppressed

insulitis and increased the number of healthy islets, and the levels of IL-17, IL-1b, IL-6 and IL-23 were

signi?cantly decreased. Moreover, IL-12 treatment of NOD mice induced the secretion of IFN-g, a potent

inhibitor of Th17 cells. These data indicated that intermittent administration of IL-12 prevented diabetes

by inducing IFN-g, suppressing the pathogenic IL-17-producing cells and reducing the expression of

Th17-associated pro-in?ammatory cytokines. Our results suggest a promising strategy for the treatment

of human T1D and other Th17 cell-mediated autoimmune diseases.

2011 Elsevier Ltd. All rights reserved.

1. Introduction

Type 1 diabetes （T1D） is an autoimmune disease thought to be

caused by autoantigen-reactive T lymphocytes that mediate the

destruction of insulin-producing b-cells located in pancreatic islets,

eventually resulting in b cell loss, insulin de?ciency, and hyper-

glycemia [1]. The nonobese diabetic （NOD） mouse spontaneously

develops insulin-dependent diabetes that strongly resembles

human T1D [2,3]. Long-term administration of insulin in appro-

priate doses is necessary to manage the blood glucose levels in T1D

patients. However, use of exogenous insulin cannot preciselymatch

endogenous insulin secretion, and this often leads to the risk of

hypoglycemia and other severe complications [4]. The events that

initiate T1D and the precise mechanisms of pancreatic b cell

destruction are incompley understood. Therefore, safe and

effective therapies for T1D are urgently needed.

DCs are professional antigen-presenting cells that initiate both

innate and adaptive immunity [5]. DCs have the ability to produce

large amounts of IL-12 and induce T cell maturation as well as Th1

responses, and these functions have been demonstrated to be

abnormal in both humans with T1D [6,7] and NOD mice [8]. Hence,

modulation of DC biology with the purpose of reshaping the

repertoire of T cells may be an attractive therapeutic option for the

treatment of T1D.

Increasing evidence from NOD mouse and human T1D studies

suggests that Th17 cells play a crucial role in the pathogenesis of

autoimmune diabetes. Several studies have shown an increase in

the number of IL-17-producing cells and the secretion of IL-17 in

NOD mice [9,10] as well as in the peripheral blood of patients with

T1D [11,12]. However, the mechanism behind this increase and its

relationship to the pathogenesis of T1D remain obscure. Substantial

evidence has indicated that IFN-g plays a protective role in the

* Corresponding author. School of Life Sciences, University of Science and Tech-

nology of China, 443 Huang-shan Road, Hefei 230027, China. .: þ86 551 360

7379; þ86 551 360 6783.

** Corresponding author.

addresses: jackey80@mail.ustc.edu.cn （J. Zhang）, zhhuang@mail.ustc.edu.

cn （Z. Huang）, sunr@ustc.edu.cn （R. Sun）, tzg@ustc.edu.cn （Z. Tian）, ustcwhm@ustc.

edu.cn （H. Wei）.

Contents lists available at SciVerse ScienceDirect

Journal of Autoimmunity

journal homepage: www.elsevier.com/locate/jautimm

0896-8411/$ e see front matter 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jaut.2011.11.017

Journal of Autoimmunity 38 （2012） 20e28experimental autoimmune encephalitismousemodel [13,14]. Here,

mice lacking IFN-g develop severe autoimmune disease compared

with wild-type mice, and this is attributed to the inhibitory activity

of IFN-g against Th17 cells [15e17]. A similar effect of IFN-g on the

inhibition of IL-17 production has been reported in autoimmune

diabetes [9]. However, the potent inducer of IFN-g, IL-12, has been

shown to be impaired in diabetes patients.

IL-12 is an immunoregulatory cytokine that promotes cell-

mediated immunity [18] and is produced mainly by activated

antigen-presenting cells [19]. It has been demonstrated that IL-12

plays a particularly important role in antitumor immunity

[20e22]. Results from mouse models of intracellular protozoan,

fungal and bacterial infections have indicated that IL-12 has a key

role in protection against pathogens [23e25]. The role of IL-12 in

autoimmunity is attracting increased attention. Previous studies

have shown that IL-12 administration induces Th1 cells and

accelerates autoimmune diabetes [26]. Consistent with these

studies, it has been shown that daily administration of IL-12 to NOD

mice induces a rapid onset of T1D in 100% of treated mice [27].In

addition, recent study revealed an IL-12 speci?c antibody protected

transplanted islets from in?ammatory damage [28]. However,

another study showed that intermittent administration of IL-12

markedly reduced the incidence of diabetes [29]. Moreover, IL-12

treatment can directly induce high levels of IFN-g in the circula-

tion. Taken together, the role of IL-12 is controversial, as it has been

shown to have both disease-promoting and disease-protective

roles in autoimmune diabetes. The reason for these opposing

roles of IL-12 is unclear, but administration of IL-12 likely affects

systemic immune regulation.

In the current study, we found that both the absolute number

and the function of DCs were impaired in NOD mice and that the

levels of the Th17-associated pro-in?ammatory cytokines, IL-1b, IL-

6 and IL-23, were elevated in NOD mice. We showed that the

intermittent administration of IL-12 to NOD mice suppressed

insulitis and increased the number of healthy islets. Finally, we

demonstrated that the IFN-g induced by IL-12 administration

prevented diabetes through a mechanism of inhibition of patho-

genic IL-17 production in NOD mice.

2. Materials and methods

2.1. Mice

Female NOD/Lt, BALB/c and C57BL/6 mice were obtained from

the Shanghai Experimental Animal Center （Shanghai, China）. All

mice weremaintained under speci?c pathogen-free conditions and

received care in compliance with the guidelines outlined in the

Guide for the Care and Use of Laboratory Animals.

2.2. Evaluation of diabetes

Diabetes was assessed bymonitoring blood glucose levels every

week using an Accu-Chek Active meter system （Roche）. Mice with

two consecutive blood glucose measurements 16.6 mmol/L were

considered diabetic. All mice were monitored for blood glucose

levels from 12 to 30 weeks of age.

2.3. Histological and immunohistological evaluation

Pancreata were harvested from NOD mice, ?xed in 10%

phosphate-buffered formalin （pH 7.2）, and embedded in paraf?n

for histological examination. Sections of 6 mm thickness were cut

100 mm apart to prevent double counting of the same islet. Three

sections per pancreaswere stainedwith hematoxylin and eosin and

analyzed by light microscopy. The pancreas from six animals was

counted in each experimental group. Insulitis scoring was per-

formed according to the following criteria: severe insulitis, 50% or

more of the islet area displayed in?ltration; mild insulitis, <50% of

the islet area displayed in?ltration; peri-insulitis, in?ltration was

restricted to the periphery of islets; and no insulitis, absence of cell

in?ltration. Sections were also stained for insulin to assess insulin

production （rabbit anti-insulin H-86; Santa Cruz Biotechnology）

following the manufacturer’s instructions. Positive reactions were

visualized with the peroxidase/DAB kit （Dako）, and the nuclei were

counterstained using hematoxylin.

2.4. In vivo treatments

NOD mice were givenweekly i.p. injections with different doses

of recombinantmurine IL-12 （Peprotech） or normal saline from6 to

12 weeks of age. These mice were monitored for blood glucose

levels beginning at week 12 until 30 weeks of age. For the IL-12

treatment group, 6w represents 6 weeks of intermittent treat-

ment, and other time points represent the time after a single

treatment. The mice were sacri?ced one week after the last treat-

ment in the 6w treatment group.

2.5. In vitro stimulation of dendritic cells

Splenic DCs were isolated from 8 week-old female mice by

FACSAria （BD Biosciences）, purity of FACS-sorted DCs was routinely

98e99%. After sorting, the DCs were cultured in 96-well ?at-

bottom plates （2 105

per well） in RPMI-1640 media supple-

mented with 10% FBS. In vitro stimulation of DCs was achieved by

exposure to LPS （1 mg/ml） for 6 h. Complementary DNA derived

from DCs after stimulation was assayed by real-time PCR to deter-

mine mRNA levels of cytokines.

2.6. Flow cytometry

Anti-CD4-PerCP-Cy5.5 （RM4-5）, anti-CD4-Allophycocyanin

（RM4-5）, anti-CD11c-Allophycocyanin （HL3）, anti-IL-17-PE （TC11-

18H10）, and anti-IFN-g-PE （XMG1.2） were purchased from BD

Pharmingen. For intracellular cytokine analysis of IL-17 and IFN-g,

the splenocytes （1 106

cell/ml） were stimulated with PMA （30 ng/

ml; SigmaeAldrich） and ionomycin （1 mg/ml; SigmaeAldrich）. One

hour later,monensin （5 mg/ml; SigmaeAldrich）was added for 4 h to

prevent the secretion of induced cytokines into the supernatant.

The antibodies used for intracellular analysis were anti-CD4-PerCP-

Cy5.5 （RM4-5）, anti-IL-17-PE （TC11-18H10）, and anti-IFN-g-PE

（XMG1.2）. Isotype-matched controls were included in all experi-

ments. Flow cytometry was performed on a FACS Calibur （BD）, and

data were analyzed using WinMDI2.9 software.

2.7. ELISA

The serum samples were kept at 80 C until cytokine

measurement. Levels of IL-1b, IFN-g, IL-6, IL-17, and IL-23 were

measured using commercially available ELISA kits （Cusabio） in

accordance with the manufacturer’s protocol.

2.8. Real-time quantitative RT-PCR

Total RNA was extracted using the TRIzol reagent （Invitrogen）.

One microgram of total RNA was reverse-transcribed with an oli-

go（dT）18 primer and quanti?ed on an ABI Prism 7000 Detection

System. Ampli?cationwas performed for 40 cycles in a total volume

of 30 mL, and productswere detected using SYBRGreen （Takara）. The

relative expression level of each target gene was determined by

normalizing itsmRNA level to the internal control gene, b-actin. The

J. Zhang et al. / Journal of Autoimmunity 38 （2012） 20e28 21primer sequences usedwere as follows: IFN-g,50

-TAG CCA AGA CTG

TGA TTG CGG-30

（forward） and 50

-AGA CAT CTC CTC CCATCA GCAG-

30

（reverse）; IL-1b,50

-GTT TTC CTC CTT GCC TCT GA-30

（forward） and

50

-GCT GCC TAA TGT CCC CTT G-30

（reverse）; IL-6, 50

-AGA CTT CCA

TCC AGT TGC CTT-30

（forward） and 50

-TCT CAT TTC CAC GAT TTC CC-

30

（reverse）; IL-12p40, 50

-GGA AGC ACG GCA GCA GAA TA-30

（forward） and 50

-AAC TTGAGG GAG AAG TAG GAA TGG-30

（reverse）;

IL-17, 50

-GCA AGAGAT CCTGGT CCT GA-30

（forward） and 50

-AGC ATC

TTC TCG ACC CTG AA-30

（reverse）; IL-23p19, 50

-CTT CTC CGT TCC

AAG ATC CTT CG-30

（forward） and 50

-GGC ACT AAG GGC TCA GTC

AGA-30

（reverse）; IL-12p35, 50

-GTG TCA ATC ACG CTA CCT CCT CT-30

（forward） and 50

-CCGTCT TCACCATGT CAT CTGT-30

（reverse）; IL-10,

50

- ATG CTG CCT GCT CTT ACT GAC TG-30

（forward） and 50

- CCC AAG

TAA CCC TTA AAG TCC TGC-30

（reverse）; TNF-a,50

-GGT GTT CAT CCA

TTC TCTACC C-30

（forward） and 50

-GTC ACT GTC CCAGCATCT TGT-30

（reverse）; b-actin, 50

-GCC GAT CCA CAC GGA GTA CTT-30

（forward）

and 50

-TTG CCG ACA GGA TGC AGA A-30

（reverse）.

2.9. Statistical analysis

The data are expressed as mean standard error of the mean.

Comparisons between two groups were performed using a two-

tailed unpaired t test. *, P < 0.05; **, P < 0.001.

3. Results

3.1. Intermittent administration of IL-12 prevents spontaneous T1D

in NOD mice

Six-week-old female NOD mice were treated with 50, 100 or

200 ng of IL-12 once perweek. Blood glucose levelsweremonitored

weekly between 6 and 30 weeks of age.Mice receiving 200 ng of IL-

12 once per week showed a striking delay in T1D incidence

（Fig. 1A）. Increased survival rates were also observed in mice

treated with 200 ng of IL-12 in NOD mice （Fig. 1B）. The prevention

of the development of diabetes in NOD mice following IL-12

（200 ng） treatment was associated with reduced insulitis and

blood glucose levels even though 2 of 16 mice showed insulitis and

high blood glucose levels （Fig. 1C）. In contrast, the weekly blood

glucose levels in control mice showed a consistent pattern of

hyperglycemia in 15 of 20mice （Fig.1D）. Overall, these data suggest

that intermittent treatment with 200 ng of IL-12 can prevent dia-

betes and enhance survival in NOD mice.

3.2. IL-12 treatment diminishes insulitis and increases the number

of healthy islets

To determine whether intermittent treatment with IL-12

diminished insulitis, histological examination of pancreata was

performed. As indicated in Fig. 2, most of the islets in control mice

exhibited intra-insulitis and low levels of insulin. In contrast, the

majorities of islets in treated mice were not in?amed or had only

mild peri-insulitis. The mice treated for one week had a higher

percentage of isletswith no insulitis （47 vs.15%） or peri-insulitis （35

vs. 28%） relative to the diabetic mice. The percentage of islets

exhibiting severe and mild intra-insulitis was reduced in the

treated versus diabetic mice （8 and 10% vs. 24 and 33%, respec-

tively）. Moreover, in the 6-week treatment group, although the

total number of islets was increased compared with that of the

diabetic group, the majority of islets exhibited no insulitis （67%）,

and only 20% and 11% of islets from the treated group showed peri-

insulitis or mild intra-insulitis, respectively （Fig. 2C）. In addition,

Fig. 1. Intermittent administration of IL-12 prevents spontaneous T1D in NOD mice. Six-week-old female NOD mice were treated for 6 weeks with 50, 100 or 200 ng of IL-12. Blood

glucose was monitored weekly, and mice with two consecutive blood glucose measurements 16.6 mmol/L were considered diabetic. All mice were monitored for blood glucose

from 12 to 30 weeks of age. A, The incidence of diabetes was measured following IL-12 treatment. B, Survival rate was measured following IL-12 treatment every day. C, D, Blood

glucose concentrations were detected in IL-12-treated or control mice.

J. Zhang et al. / Journal of Autoimmunity 38 （2012） 20e28 22enumeration of islets indicated that IL-12-treated mice had

a signi?cantly greater number of total islets than did control mice.

The number of insulin-positive islets also increased from 36 2in

the controlmice to 48 3 upon treatment with IL-12 in the 1-week

treatment group and from26 5 in the controlmice to 41 2 upon

treatment with IL-12 in the 6-week treatment group （Fig. 2D）.

These data indicate that the number of healthy islets signi?cantly

increases after IL-12 treatment.

3.3. The number and function of DCs is abnormal in NOD mice

Splenocytes from age-matched BALB/c and C57BL/6 mice were

analyzed for the expression of CD11c. Consistent with previous

reports, the frequency of DCs in the spleens of NODmice, especially

from diabetic （30w group） mice, was signi?cantly decreased

compared with BALB/c and C57BL/6mice （Fig. 3A, B）.Moreover, the

absolute number of DCs in NOD spleens was lower than that from

BALB/c or C57BL/6 mice （Fig. 3C）. Complementary DNA derived

from sorted DCs was assayed by real-time PCR to determine mRNA

levels of IL-12p35 and IL-12p40, two subunit of the Th1-associated

cytokine, IL-12. The result indicated that both IL-12p35 and IL-

12p40 were signi?cantly reduced in DCs isolated from NOD mice

compared with age-matched BALB/c and C57BL/6 mice （Fig. 3D, E）.

In addition, the level of IFN-g was signi?cantly lower in the serum

of diabetic mice than in controls （Fig. 3F）. Furthermore, in order to

demonstrate the cytokine production of NOD DCs, we performed

in vitro experiment to determine the functional abnormality of DCs

isolated from NOD mice. The result indicated the level of IL-6

transcript was signi?cantly elevated in DCs after LPS stimulation

from NOD mice compared with age-matched BALB/c and C57BL/6

mice. However, IL-12p35 and IL-12p40 were signi?cantly reduced

in DCs after LPS stimulation from NOD mice compared with the

control mice （Sup. 1）. Taken together, there were fewer DCs in NOD

mice, the ability of these DCs to produce IL-12was impaired and the

serum level of IFN-g, a major immunoregulatory cytokine, was also

decreased in NOD mice.

3.4. IL-12 treatment decreases pro-in?ammatory cytokines in NOD

mice

We next evaluated pro-in?ammatory cytokines,whichmay play

an important role in insulitis in NOD mice. The level of IL-1b

transcript was signi?cantly elevated in sorted DCs from diabetic

NODmice compared with the control mice （Sup. 2A）. Also in sorted

DCs, IL-6 mRNA levels showed a 4-fold increase in NOD mice

compared with age-matched BALB/c and C57BL/6 mice （Sup. 2B）.

However, the difference in IL-23 mRNA levels was not signi?cant

between these groups （Sup. 2C）. The mRNA levels of pro-

in?ammatory cytokines were also measured in the pancreata. IL-

1b and IL-23 were signi?cantly increased in the diabetic NOD mice

compared with the control mice; although the difference was not

statistically signi?cant, the level of IL-6 in diabetic NOD mice was

also higher than that of the controlmice （Sup. 2D, 2F）. Interestingly,

the level of IL-6was signi?cantly reduced in the NODmice （6weeks

old） compared with BALB/c and C57BL/6 mice （Sup. 2E）.

To establishwhether the IL-12 effect was due to a suppression of

the pro-in?ammatory cytokines in NOD mice, we measured pro-

in?ammatory cytokines at various time points following IL-12

Fig. 2. IL-12 treatment diminishes insulitis and increases the number of healthy islets. For the pancreatic histology, three sections per pancreas （6 mm thick, cut 100 mm apart） from

six untreated diabetic and IL-12-treated NOD mice were stained with hematoxylin and eosin （A） or anti-insulin antibody （B）; images are representative of three independent

experiments and analyzed at 400 magni?cation （scale bars indicate 50 mm）. For the untreated diabetic mice, sections were generated at the second consecutive positive blood

glucose reading. For the treated NOD mice, histology was performed 1-week or 6 weeks after the last treatment. C, Islets from untreated diabetic and IL-12-treated NOD mice were

scored as described in Materials and methods, and the percentages represent the number of islets with a given score divided by the total number of islets from （A）. D, Total islets per

pancreas as determined by hematoxylin and eosin staining from the two groups of treated mice or control mice described in A, and six mice were included in each experimental

group. Only structures with visible islet cells and incomplete in?ltration were counted. *P < 0.05, **P < 0.01.

J. Zhang et al. / Journal of Autoimmunity 38 （2012） 20e28 23treatment. The mean serum concentration of IL-23 following IL-12

treatment was lower than that of untreated control mice （Fig. 4C）,

but the levels of IL-1b and IL-6 were not signi?cantly decreased

（Fig. 4A, B）. The levels of these pro-in?ammatory cytokines were

also determined by real-time quantitative PCR in the pancreata.We

found that IL-1b, IL-6 and IL-23 transcripts were signi?cantly

decreased three days after IL-12 treatment in the pancreata.

Overall, these ?ndings suggested that pro-in?ammatory cytokines

were effectively suppressed following IL-12 treatment both

systemically and locally in the pancreas （Fig. 4C）.

3.5. IL-12 treatment interferes with IL-17 production

Th17 cells, distinct from Th1 and Th2 cells, represent a newly

de?ned subset of pathogenic T cells. IL-1b and IL-6 are the

differentiation factors necessary for Th17 cell development,

whereas IL-23 is dispensable for Th17 cell function, but necessary

for Th17 cell survival and expansion. In contrast, IFN-g, IL-25 and IL-

27 potently inhibit Th17 development [30]. The results above

indicated that IL-1b, IL-6 and IL-23 were signi?cantly increased in

NOD mice, especially in diabetic NOD mice, compared with age-

matched BALB/c and C57BL/6 mice. In addition, IL-12 treatment

modulated the expression of these pro-in?ammatory cytokines.

Recent data have indicated that IL-17-producing CD4þ T cells play

a pivotal role in the pathogenesis of T1D [9,31]. Similar results were

observed in our study when splenocytes were analyzed for intra-

cellular production of IL-17. The proportion of Th17 cells gradually

increased with age and disease progression in the spleen, and IL-12

treatment effectively interfered with IL-17 production （Fig. and

B）. In addition, the mean serum concentration of IL-17 clearly

Fig. 3. The number and function of DCs are abnormal in NOD mice. A, Flow cytometric analysis of CD11cþ DC populations in the splenocytes of NOD mice compare with BALB/c or

C57BL/6 mice. B, The frequency of DCs was analyzed in the spleens of NOD mice compared with control mice. C, DCs were counted in the spleens of NOD mice compared with

controls. Values are shown as means SE （n ¼ 6）. D and E, The cDNA derived from sorted DCs was assayed by real-time PCR for IL-12p35 and IL-12p40 mRNA levels. F, Concentration

of IFN-g was measured by ELISA in sera of NOD, BALB/c and C57BL/6 mice. Values are shown as means SE （n ¼ 6）.

J. Zhang et al. / Journal of Autoimmunity 38 （2012） 20e28 24increased when themice progressed to diabetes （Fig. 5C）; however,

the IL-17 levels were strongly suppressed after one week of IL-12

treatment. Similar results were observed in the pancreas by

quantitative real-time PCR （Fig. 5D）. These data, which are consis-

tent with previous reports [31], suggest that Th17 cells play

a crucial role in the pathogenesis of autoimmune diabetes.

3.6. IL-12 treatment induces protective IFN-g responses in NOD

mice

Previous studies have shown that IFN-g can potently inhibit

Th17 development and that the Th1-associated cytokine, IL-12, can

contribute to the production of IFN-g. Therefore, we examined the

production of IFN-g following IL-12 treatment. As expected, the

production of IFN-g from CD4þ T cells was markedly increased

following IL-12 treatment as determined by FACS analysis of sple-

nocytes （Fig. 6A and B）. The mean serum concentration of IFN-g

following IL-12 treatment was also higher than that found in

control mice （Fig. 6C）. In addition, IFN-g mRNA was signi?cantly

elevated in the pancreas （Fig. 6D）. In summary, IL-12 strongly

suppresses pathogenic Th17 development by promoting the

production of protective IFN-g.

4. Discussion

Previous studies have indicated that IL-12 promotes the acti-

vation of NK and CD8þ T cells and regulates memory CD8þ T cell

differentiation. In addition, IL-12 initiates tumor rejection and

regulates infectious diseases. However, the role of IL-12 in auto-

immune diabetes remains controversial. In the present study, we

demonstrated that intermittent administration of IL-12 resulted in

a protective effect in NOD mice, which is consistent with previous

reports [29]. Loss of IL-12 results in enhanced pro-in?ammatory

cytokine production and accelerated pathological damage of the

pancreas in NOD mice. This accelerated disease is also associated

with an increased number of IL-17-producing T cells. In our study,

we showed that T1D in NOD mice was a Th17-initiated process and

that known cytokines that strengthen Thl responses did not exac-

erbate disease. Furthermore, the Thl cytokine, IFN-g, displayed

inhibitory activity against Th17 cells. These results and those of

others [26,27] also indicate that injection of IL-12 can have very

different results depending on the dose and timing of administra-

tion. Weekly administration of IL-12 was more effective in pre-

venting the development of diabetes than when IL-12 was

administered more frequently. The half-life of IL-12 in vivo is

approximay 4e6h [27], but IL-12 levels and the cell-mediated

immunity induced by IL-12 are sustained for far longer periods.

In a previous report, Trembleau et al. administered IL-12 to IFN-g/

NOD mice, and this accelerated T1D development [26]. Based on

the present study, we conclude that IL-12 administration to IFN-g-

de?cient NODmice clearly could not induce the IFN-g that prevents

Th17 responses. Other cytokines have also been reported to have an

antagonistic effect on T1D development in NOD mice. For example,

systemic over-expression of the immunomodulatory cytokine, IL-

10, in NOD mice ameliorates diabetes through the induction of

regulatory T cells [32]. Also, local expression of transgenic tumor

necrosis factor-a （TNF-a） prevents diabetes onset in NODmice [33].

In addition, transgenic BALB/c mice expressing IFN-g in their

pancreatic b-cells are resistant to STZ-induced diabetes [34]. It has

also been reported that GM-CSF, IL-4 and TGF-b can delay or reduce

T1D development [35,36]. Here, we suggest that following inter-

mittent administration of IL-12, Th17-associated pro-in?ammatory

cytokines are effectively reduced and Th1-associated IFN-g is

elevated, which inhibits the pathogenic IL-17-producing T cells.

Ultimay, the balance of cytokines was restored in the IL-12-

treated NOD mice.

DCs are a primary source of IL-12. Patients with DC de?ciencies

can develop autoimmune diseases [37]. This phenomenon suggests

a role for DCs inmediating peripheral tolerance, T cell anergy or the

expansion of antigen-speci?c regulatory T cells [38]. Our results

demonstrated that DCs fromNODmicewere in a pro-in?ammatory

state and secreted high levels of IL-1b, IL-6 and IL-23. The latter pro-

Fig. 4. IL-12 treatment decreased pro-in?ammatory cytokines in NOD mice. A, B and C, The mean serum concentrations of the pro-in?ammatory cytokines were measured at

different time points following IL-12 treatment in NOD mice by ELISA. D, E and F, Relative levels of mRNA of the pro-in?ammatory cytokines were determined by real-time PCR from

the pancreas of NOD mice at different time points following IL-12 treatment. Values are shown as means SE （n ¼ 6）.

J. Zhang et al. / Journal of Autoimmunity 38 （2012） 20e28 25Fig. 6. IL-12 treatment induced protective IFN-g in NOD mice. A, Flow cytometric analysis of the production of IFN-g from the CD4þ T cells isolated from the spleens of NOD mice at

different time points following IL-12 treatment. B, The percentage of CD4þ IFN-gþ T cells is shown. Values are shown as means SE of six mice within each experimental group. C,

The mean serum concentration of IFN-g was measured at different time points following IL-12 treatment in NOD mice by ELISA. D, Relative levels of IFN-g mRNA in the pancreas of

NOD mice were determined by real-time PCR at different time points following IL-12 treatment. Values are shown as means SE （n ¼ 6）.

Fig. 5. IL-12 treatment interfered with IL-17 production. A, Flow cytometric analysis of IL-17-producing cells populations in the lymphocytes isolated from the spleen from different

ages of mice or from different time points under IL-12 treatment in NOD mice. B, The percentage of CD4þ IL-17þ T cells is shown. Values are shown as means SE of six mice within

each experimental group. C, The mean serum concentration of IL-17 was measured at different time points following IL-12 treatment in NOD mice by ELISA. D, Relative levels of

mRNA for IL-17 were determined from the pancreas of NOD mice at different time points following IL-12 treatment. Values are shown as means SE （n ¼ 6）.

J. Zhang et al. / Journal of Autoimmunity 38 （2012） 20e28 26in?ammatory cytokines were also elevated in pancreata. In

contrast, the level of IL-12 in DCs was signi?cantly decreased

compared with the levels observed in control mice. These results

suggest that IL-12 reduced the levels of pro-in?ammatory cyto-

kines and that higher levels of IL-12 may have a positive effect in

the clinical therapy of diabetes. Thus, in our study, IL-12 was

administered weekly to NOD mice from 6 weeks to 12 weeks and

was effective in suppressing the incidence of diabetes. The mech-

anism of this suppression was that IL-12 down-regulated the levels

of IL-1b, IL-6 and IL-23 and prevented the development of auto-

reactive Th17 cells in treated mice.

The balance of cytokines is a crucial determinant of resistance or

susceptibility in organ speci?c autoimmunity. Disease suscepti-

bility may correlate with the expression of pro-in?ammatory

cytokines, such as IL-17, IL-1b, IL-6, TNF-a and IFN-g, in experi-

mental autoimmune encephalomyelitis （EAE） [39]. Th17 cells,

distinct from Th1 and Th2 cells represent a newly de?ned subset of

pathogenic T cells and have recently been shown to play a key role

in the pathogenesis of type 1 diabetes in NOD mice. IL-1b and IL-6

are the factors necessary for Th17 cell differentiation,whereas IL-23

is dispensable for the function of Th17 cells but necessary for their

survival and expansion. In contrast, IFN-g, IL-25 and IL-27 potently

inhibit Th17 development.

To investigate whether IL-12 treatment in?uenced various cell

subsets, we analyzed the proportions of CD4þ Foxp3þ Tregs, CD8 T

cells, NK cells, NKT cells and gd T cells following the administration

of IL-12. We found that the changes in these cell types were not

signi?cant （data not shown）. These data suggest that IL-12 may

maintain homeostasis by regulating diverse in?ammatory cyto-

kines in NOD mice.

Our results showed that IFN-g produced downstream of IL-12

inhibited the development of Th17 cells. In addition, IL-12 indi-

rectly inhibited the Th17 cells by suppressing the Th17-associated

pro-in?ammatory cytokines, IL-1b, IL-6 and IL-23. Thus, IL-12

broadly regulated pathogenic Th17 cells and promoted the

balance of cytokines in a direct or indirect way. The present study

therefore provides the ?rst direct evidence that IL-12 plays

a protective role in the development of T1D in NOD mice and

suggests that IL-12, a possible therapeutic agent against infectious

diseases and tumors, may also be valuable in the clinical treatment

of diabetes.

Author contribution

Jun Zhang designed and performed the experiments, analyzed

and interpreted the data. Zhan Huang analyzed and interpreted the

data. Rui Sun established techniques of FACS and histochemistry.

Zhigang Tian provided strategic planning and conceived the

project. HaimingWei supervised the project, provided crucial ideas

and helped with data interpretation. Jun Zhang wrote the manu-

script with Haiming Wei and Zhan Huang.

Con?ict of interest

No potential con?icts of interest relevant to this article were

reported.

Acknowledgments

This work was supported by the Natural Science Foundation of

China （30730084, 31021061 and 91029303） andMinistry of Science

& Technology of China （973 Basic Science Project 2007CB815805,

2007CB512405 and 2009CB522403）.

The authors thank Weici Zhang （University of California, Davis）

for her expert technical assistance.

Appendix. Supplementary material

Supplementary material associated with this article can be

found, in the online version, at doi:10.1016/j.jaut.2011.11.017

References

[1] Tisch R, McDevitt H. Insulin-dependent diabetes mellitus. Cell 1996;85:

291e7.

[2] Anderson MS, Bluestone JA. The NOD mouse: a model of immune dysregu-

lation. Annu Rev Immunol 2005;23:447e85.

[3] Gallegos AM, Bevan MJ. Driven to autoimmunity: the nod mouse. Cell 2004;

117:149e51.

[4] Li L, Yi Z, Tisch R, Wang B. Immunotherapy of type 1 diabetes. Arch Immunol

Ther Exp （Warsz） 2008;56:227e36.

[5] Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immu-

nobiology of dendritic cells. Annu Rev Immunol 2000;18:767e811.

[6] Jansen A, van Hagen M, Drexhage HA. Defective maturation and function of

antigen-presenting cells in type 1 diabetes. Lancet 1995;345:491e2.

[7] Takahashi K, Honeyman MC, Harrison LC. Impaired yield, phenotype, and

function of monocyte-derived dendritic cells in humans at risk for insulin-

dependent diabetes. J Immunol 1998;161:2629e35.

[8] Serreze DV, Gaskins HR, Leiter EH. Defects in the differentiation and function

of antigen presenting cells in NOD/Lt mice. J Immunol 1993;150:2534e43.

[9] Jain R, Tartar DM, Gregg RK, Divekar RD, Bell JJ, Lee HH, et al. Innocuous

IFNgamma induced by adjuvant-free antigen restores normoglycemia in

NOD mice through inhibition of IL-17 production. J Exp Med 2008;205:

207e18.

[10] Mori Y, Kodaka T, Kato T, Kanagawa EM, Kanagawa O. Critical role of IFN-

gamma in CFA-mediated protection of NOD mice from diabetes develop-

ment. Int Immunol 2009;21:1291e9.

[11] Honkanen J, Nieminen JK, Gao R, Luopajarvi K, Salo HM, Ilonen J, et al. IL-17

immunity in human type 1 diabetes. J Immunol 2010;185:1959e67.

[12] Marwaha AK, Crome SQ, Panagiotopoulos C, Berg KB, Qin H, Ouyang Q, et al.

Cutting edge: increased IL-17-secreting T cells in children with new-onset

type 1 diabetes. J Immunol 2010;185:3814e8.

[13] Ferber IA, Brocke S, Taylor-Edwards C, Ridgway W, Dinisco C, Steinman L,

et al. Mice with a disrupted IFN-gamma gene are susceptible to the induction

of experimental autoimmune encephalomyelitis （EAE）. J Immunol 1996;156:

5e7.

[14] Krakowski M, Owens T. Interferon-gamma confers resistance to experimental

allergic encephalomyelitis. Eur J Immunol 1996;26:1641e6.

[15] Hofstetter HH, Ibrahim SM, Koczan D, Kruse N, Weishaupt A, Toyka KV, et al.

Therapeutic ef?cacy of IL-17 neutralization in murine experimental autoim-

mune encephalomyelitis. Cell Immunol 2005;237:123e30.

[16] Komiyama Y, Nakae S, Matsuki T, Nambu A, Ishigame H, Kakuta S, et al. IL-17

plays an important role in the development of experimental autoimmune

encephalomyelitis. J Immunol 2006;177:566e73.

[17] Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang YH, et al. A distinct lineage

of CD4 T cells regulates tissue in?ammation by producing interleukin 17. Nat

Immunol 2005;6:1133e41.

[18] Trinchieri G. Interleukin-12 and the regulation of innate resistance and

adaptive immunity. Nat Rev Immunol 2003;3:133e46.

[19] Trinchieri G, Sher A. Cooperation of toll-like receptor signals in innate

immune defence. Nat Rev Immunol 2007;7:179e90.

[20] Cui J, Shin T, Kawano T, Sato H, Kondo E, Toura I, et al. Requirement for

Valpha14 NKT cells in IL-12-mediated rejection of tumors. Science 1997;278:

1623e6.

[21] Tahara H, Zeh 3rd HJ, Storkus WJ, Pappo I, Watkins SC, Gubler U, et al.

Fibroblasts genetically engineered to secrete interleukin 12 can suppress

tumor growth and induce antitumor immunity to a murine melanoma in vivo.

Cancer Res 1994;54:182e9.

[22] Eisenring M, vom Berg J, Kristiansen G, Saller E, Becher B. IL-12 initiates tumor

rejection via lymphoid tissue-inducer cells bearing the natural cytotoxicity

receptor NKp46. Nat Immunol 2010;11:1030e8.

[23] Decken K, Kohler G, Palmer-Lehmann K, Wunderlin A, Mattner F, Magram J,

et al. Interleukin-12 is essential for a protective Th1 response in mice infected

with Cryptococcus neoformans. Infect Immun 1998;66:4994e5000.

[24] Park AY, Hondowicz BD, Scott P. IL-12 is required to maintain a Th1 response

during Leishmania major infection. J Immunol 2000;165:896e902.

[25] Cooper AM, Magram J, Ferrante J, Orme IM. Interleukin 12 （IL-12） is crucial to

the development of protective immunity in mice intravenously infected with

mycobacterium tuberculosis. J Exp Med 1997;186:39e45.

[26] Trembleau S, Penna G, Gregori S, Giarratana N, Adorini L. IL-12 administration

accelerates autoimmune diabetes in both wild-type and IFN-gamma-de?cient

nonobese diabetic mice, revealing pathogenic and protective effects of IL-12-

induced IFN-gamma. J Immunol 2003;170:5491e501.

[27] Trembleau S, Penna G, Bosi E, Mortara A, Gay MK, Adorini L. Interleukin 12

administration induces T helper type 1 cells and accelerates autoimmune

diabetes in NOD mice. J Exp Med 1995;181:817e21.

[28] Matsuoka N, Itoh T, Watarai H, Sekine-Kondo E, Nagata N, Okamoto K, et al.

High-mobility group box 1 is involved in the initial events of early loss of

transplanted islets in mice. J Clin Invest 2010;120:735e43.

J. Zhang et al. / Journal of Autoimmunity 38 （2012） 20e28 27[29] O’Hara Jr RM, Henderson SL, Nagelin A. Prevention of a Th1 disease by a Th1

cytokine: IL-12 and diabetes in NOD mice. Ann N Y Acad Sci 1996;795:241e9.

[30] Kleinschek MA, Owyang AM, Joyce-Shaikh B, Langrish CL, Chen Y,

Gorman DM, et al. IL-25 regulates Th17 function in autoimmune in?amma-

tion. J Exp Med 2007;204:161e70.

[31] Emamaullee JA, Davis J, Merani S, Toso C, Elliott JF, Thiesen A, et al. Inhibition

of Th17 cells regulates autoimmune diabetes in NOD mice. Diabetes 2009;58:

1302e11.

[32] Goudy KS, Burkhardt BR, Wasserfall C, Song S, Campbell-Thompson ML,

Brusko T, et al. Systemic overexpression of IL-10 induces CD4þCD25þ cell

populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice

in a dose-dependent fashion. J Immunol 2003;171:2270e8.

[33] Picarella DE, Kratz A, Li CB, Ruddle NH, Flavell RA. Transgenic tumor necrosis

factor （TNF）-alpha production in pancreatic islets leads to insulitis, not dia-

betes. Distinct patterns of in?ammation in TNF-alpha and TNF-beta transgenic

mice. J Immunol 1993;150:4136e50.

[34] Gu D, Arnush M, Sawyer SP, Sarvetnick N. Transgenic mice expressing IFN-

gamma in pancreatic beta-cells are resistant to streptozotocin-induced dia-

betes. Am J Physiol 1995;269:E1089e94.

[35] Falcone M, Sarvetnick N. Cytokines that regulate autoimmune responses. Curr

Opin Immunol 1999;11:670e6.

[36] Krakowski M, Abdelmalik R, Mocnik L, Krahl T, Sarvetnick N. Granulocyte

macrophage-colony stimulating factor （GM-CSF） recruits immune cells to the

pancreas and delays STZ-induced diabetes. J Pathol 2002;196:103e12.

[37] Ohnmacht C, Pullner A, King SB, Drexler I, Meier S, Brocker T, et al.

Constitutive ablation of dendritic cells breaks self-tolerance of CD4 T cells

and results in spontaneous fatal autoimmunity. J Exp Med 2009;206:

549e59.

[38] Ueno H, Klechevsky E, Morita R, Aspord C, Cao T, Matsui T, et al. Dendritic cell

subsets in health and disease. Immunol Rev 2007;219:118e42.

[39] O’Garra A, Steinman L, Gijbels K. CD4þ T-cell subsets in autoimmunity. Curr

Opin Immunol 1997;9:872e83.

J. Zhang et al. / Journal of Autoimmunity 38 （2012） 20e28 28

慧嘉生物您實驗身邊的好伙伴

為客戶提供“zui高質(zhì)量的產(chǎn)品”和“zui的服務”

歡迎廣大客戶咨詢，另有大量宣傳海報和小禮品贈送。

：www.biohj.com  

：

傳    真：

：382603320      1284882975

郵    箱：sale@biohj.com