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
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