Interleukin-1β produced in response to islet autoantigen presentation differentiates T-helper 17 cells at the expense of regulatory T-cells: Implications for the timing of tolerizing immunotherapy.

Abstract

The effectiveness of tolerizing immunotherapeutic strategies, such as anti-CD40L or dendritic cells (DCs), is greater when administered to young nonobese diabetic (NOD) mice than at peak insulitis. RelB(lo) DCs, generated in the presence of an nuclear factor-κB inhibitor, induce T-regulatory (Treg) cells and suppress inflammation in a model of rheumatoid arthritis. Interleukin (IL)-1β is overexpressed in humans and mice at risk of type 1 diabetes, dysregulates Treg cells, and accelerates diabetes in NOD mice. We investigated the relationship between IL-1β production and the response to RelB(lo) DCs in the prediabetic period.
We injected RelB(lo) DCs subcutaneously into 4- or 14-week-old NOD mice and tracked the incidence of diabetes and effect on Treg cell function. We measured the expression of proinflammatory cytokines by stimulated splenocytes and unstimulated islets from mice of different ages and strains and proliferative and cytokine responses of T effectors to Treg in vitro.
Tolerizing RelB(lo) DCs significantly inhibited diabetes progression when administered to 4-week-old but not 14-week-old mice. IL-1β production by NOD splenocytes and mRNA expression by islets increased from 6 to 16 weeks of age when major histocompatibility complex (MHC)-restricted islet antigen presentation to autoreactive T-cells occurred. IL-1 reduced the capacity of Treg cells to suppress effector cells and promoted their conversion to Th17 cells. RelB(lo) DCs exacerbated the IL-1-dependent decline in Treg function and promoted Th17 conversion.
IL-1β, generated by islet-autoreactive cells in MHC-susceptible mice, accelerates diabetes by differentiating Th17 at the expense of Treg. Tolerizing DC therapies can regulate islet autoantigen priming and prevent diabetes, but progression past the IL-1β/IL-17 checkpoint signals the need for other strategies.

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Interleukin-1? Produced in Response to Islet
Autoantigen Presentation Differentiates T-Helper 17
Cells at the Expense of Regulatory T-Cells
Implications for the Timing of Tolerizing Immunotherapy
Sebastien Bertin-Maghit,1Dimeng Pang,1Brendan O’Sullivan,1Shannon Best,1Emily Duggan,1
Sanjoy Paul,2Helen Thomas,3Thomas W.H. Kay,4Leonard C. Harrison,4Raymond Steptoe,1and
Ranjeny Thomas1
OBJECTIVE—The effectiveness of tolerizing immunotherapeu-
tic strategies, such as anti-CD40L or dendritic cells (DCs), is
greater when administered to young nonobese diabetic (NOD)
mice than at peak insulitis. RelBloDCs, generated in the presence
of an nuclear factor-?B inhibitor, induce T-regulatory (Treg) cells
and suppress inflammation in a model of rheumatoid arthritis.
Interleukin (IL)-1? is overexpressed in humans and mice at risk
of type 1 diabetes, dysregulates Treg cells, and accelerates
diabetes in NOD mice. We investigated the relationship between
IL-1? production and the response to RelBloDCs in the predia-
betic period.
RESEARCH DESIGN AND METHODS—We injected RelBlo
DCs subcutaneously into 4- or 14-week-old NOD mice and
tracked the incidence of diabetes and effect on Treg cell function.
We measured the expression of proinflammatory cytokines by
stimulated splenocytes and unstimulated islets from mice of
different ages and strains and proliferative and cytokine re-
sponses of T effectors to Treg in vitro.
RESULTS—Tolerizing RelBloDCs significantly inhibited diabetes
progression when administered to 4-week-old but not 14-week-old
mice. IL-1? production by NOD splenocytes and mRNA expression
by islets increased from 6 to 16 weeks of age when major histocom-
patibility complex (MHC)-restricted islet antigen presentation to
autoreactive T-cells occurred. IL-1 reduced the capacity of Treg
cells to suppress effector cells and promoted their conversion to
Th17 cells. RelBloDCs exacerbated the IL-1–dependent decline in
Treg function and promoted Th17 conversion.
CONCLUSIONS—IL-1?, generated by islet-autoreactive cells in
MHC-susceptible mice, accelerates diabetes by differentiating
Th17 at the expense of Treg. Tolerizing DC therapies can regulate
islet autoantigen priming and prevent diabetes, but progression
past the IL-1?/IL-17 checkpoint signals the need for other
strategies. Diabetes 60:248–257, 2011
N
with peri-insulitis and infiltration of macrophages and
dendritic cells (DCs) (1), followed by infiltration of auto-
reactive CD4?and CD8?T-cells (2). Expression of major
histocompatibility complex (MHC) class II I-Ag7is neces-
sary but not sufficient for diabetes susceptibility (3).
Presentation of proinsulin epitopes to islet autoreactive
CD4?T-cells triggers insulitis (4). After ?15 weeks, de-
structive insulitis is promoted by cytotoxic T-cells (5).
FoxP3?regulatory T (Treg) cells regulate autoreactivity,
interferon (IFN)-? production, and natural-killer cell lytic
activity (6). Treg infiltrate inflamed islets, but their func-
tion wanes prior to diabetes expression (7,8).
Mice and humans exhibit chronic inflammation at and
before diabetes onset. Nuclear factor (NF)-?B is constitu-
tively activated by DCs in NOD mice from at least 6 weeks
of age and in patients with type 1 diabetes, associated with
an interleukin (IL)-1–driven proinflammatory state (9–16).
We showed that IL-1? is overexpressed by effector T-cells
(Teff) in NOD mice and inhibits the capacity of Treg to
suppress Teff (17). IL-1? also exerts direct proapoptotic
effects on insulin-producing pancreatic ?-cells (17–20).
Progression to diabetes is slowed in IL-1 receptor-1 (IL-
1R1)-deficient NOD mice because of immunoregulatory
effects on hemopoietic cells (21). Newly diagnosed type 1
diabetic patients with high serum levels of the natural
antagonist, IL-1Ra, were more likely to preserve ?-cell
function (16). Further, sera from patients with recent-
onset type 1 diabetes or from at-risk relatives with islet
autoantibodies, but not healthy control subjects, induced a
gene expression signature characterized by upregulated
expression of members of the IL-1 family and associated
with IL-1 action when incubated with healthy peripheral
blood mononuclear cells (PBMCs) (15), and IL-1? also was
overexpressed in the gene expression signature of PBMCs
from children with newly diagnosed type 1 diabetes. IL-1
expression fell as hyperglycemia settled, suggesting that at
least some of the inflammatory drive is metabolic (22).
The effectiveness of tolerizing immunotherapies, such
as anti-CD40L, IL-10, rapamycin, or DCs treated with
NF-?B oligodinucleotides, is greater when administered to
young (4–6 weeks) NOD mice than mice with advanced
insulitis (10–14 weeks) (23–26). The RelB subunit of
onobese diabetic (NOD) mice spontaneously
develop autoimmune diabetes, driven by pro-
gressive inflammatory dysregulation. Autoim-
mune inflammation begins at ?4 weeks of age
From1The University of Queensland Diamantina Institute, Princess Alexandra
Hospital, Brisbane, Queensland, Australia; the2Queensland Clinical Trials
and Biostatistics Centre, School of Population Health, The University of
Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia;
the3Islet Biology Laboratory, St. Vincent’s Institute, Melbourne, Australia;
and the4Autoimmunity and Transplantation Division, Walter and Eliza Hall
Institute, Melbourne, Australia.
Corresponding author: Ranjeny Thomas, ranjeny.thomas@uq.edu.au.
Received 22 January 2010 and accepted 13 October 2010. Published ahead of
print at http://diabetes.diabetesjournals.org on 27 October 2010. DOI:
10.2337/db10-0104.
R.S. and R.T. contributed equally to this article.
© 2011 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
ORIGINAL ARTICLE
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NF-?B is a key determinant of DC antigen-presenting cell
(APC) function (27). Microbial, inflammatory, or T-cell–
derived signals induce the nuclear translocation and tran-
scriptional activity of RelB in DCs (28,29). Induction of
MHC class II and costimulatory molecules required for
effective TCR signaling and T-cell priming is impaired in
response to such activation of RelB?/?DCs (28,30,31).
T-cell stimulatory function of RelB?/?DCs is deficient in
vitro and in vivo, and transferred RelB?/?or RelBloDC,
generated in the presence of NF-?B inhibitors or RelB
siRNA, suppress primed T-cell effector responses through
induction of Tr1-type Treg in an IL-10–dependent manner
(31,32). RelBloDCs suppress disease in a mouse model of
rheumatoid arthritis (33). Here, we used RelBlotolerizing
DCs to prevent diabetes in NOD mice to define mecha-
nisms that determine the age-dependent effectiveness of
tolerizing therapies in type 1 diabetes.
RelBloDCs, which express reduced MHC class II and
CD40, suppress effector function both by reduced capacity
to signal Teff and through induction of Treg (31). The
induction of Treg by tolerizing immunotherapies depends
on the presence of FoxP3?natural Treg (34,35). Thus,
factors that dysregulate Treg function are candidates for
interference with effective suppression of autoimmune
disease by RelBloDCs. Because IL-1 plays an important
role in the pathogenesis of type 1 diabetes in mice and
humans, and its production in NOD mice dysregulates
Treg cell function, we hypothesized that IL-1 impairs the
response to RelBloDCs.
RESEARCH DESIGN AND METHODS
Mice. NOD.Lt, C57BL/6, NOD.I-Ak, NOD.CD45.2 (36), NOD.IL-1R1?/?(21),
and pINS.NOD (37) mice were obtained from the Animal Research Centre
(Perth, Australia), James Cook University, or bred at the Walter and Eliza Hall
Institute (WEHI) (Melbourne, Australia) or the University of Queensland. Mice
were housed at the Princess Alexandra Hospital (Brisbane, Australia) or
WEHI.
Cytokine assay. Cytokines were assayed in supernatants by enzyme-linked
immunosorbent assay (ELISA) for IL-1 (eBioscience, San Diego, CA) and IL-17
(Biolegend, San Diego, CA) and bead array (BD Bioscience, Franklin Lakes,
NJ). Supernatants in triplicate were pooled, and cytokine assays were made in
duplicate. Data represent means ? SD.
Intracellular staining. All surface antibodies were purchased from Bioleg-
end. Cells were stained fresh or after culture with anti-CD3ε mAb for 72 h or
phorbol myristate acetate, with addition of brefeldin A (Sigma, St. Louis, MO)
for 4 h. After surface mAb staining, cells were fixed and permeabilized then
incubated on ice with anti–IL-1 (eBioscience) or isotype control. When
biotinylated antibody was used, cells were washed and incubated for 15 min
with labeled streptavidin (Biolegend). Cells were suspended in 10% formalin,
read on an FACS Calibur (BD Bioscience), and analyzed using FlowJo
software (Tree Star).
Assessment of diabetes and insulitis. Mice were classified to be diabetic
and were killed following two consecutive weekly blood glucose readings ?12
mmol/l. For analysis of insulitis, pancreata were collected from four mice per
group at 12 weeks. Insulitis was graded in hematoxylin and eosin–stained
sections as described (38).
Islet purification and quantitative PCR. Islets of Langerhans were isolated
by collagenase P (dissolved in Hank’s Buffered Salt Solution containing 2
mmol/l Ca2?and 20 mmol/l HEPES) digestion and density gradient centrifu-
gation as described previously (39). RNA was extracted (RNeasy Mini Kit;
Qiagen) and cDNA synthesized. Quantitative PCR used Taqman primers on an
AB 7900 machine (Applied Biosystems).
Proliferation and suppression assay. CD11c?, CD4?CD25?, and CD4?CD25?
cells were isolated from spleen and lymph nodes using immunomagnetic
beads and an AutoMacs separator (Miltenyi Biotec, Bergisch Gladbach,
Germany). A total of 100,000 CD11c?, 105CD4?CD25?, and 7.5 ? 104
CD4?CD25?cells were incubated for 72 h in presence of anti-CD3ε antibody.
Supernatants were collected for cytokine assays and 1 ? Cu of tritiated
thymidine (Perkin Elmer, Waltham, MA) was added for 18 h before counting.
Data represent means ? SD of triplicates performed twice.
In vivo administration of RelBloDC and transfer of Treg. Bone marrow
was collected from long bones of NOD mice aged 8 ? 2 weeks. After
Ficoll-Histopaque gradient separation, cells were cultured in RPMI ? 10% FCS
in the presence of murine IL-4 and granulocyte macrophage colony–stimulat-
ing factor (GM-CSF) (Peprotech, Rocky Hill, NJ) and 2.5 ?mol/l BAY-11-7082
(Calbiochem, San Diego, CA) for 12 days, with media being refreshed every 3
days. RelBloDCs (5 ? 105per animal) were injected subcutaneously to the tail
base. For Treg transfer, CD4?CD25?were isolated from spleen and lymph
nodes of 4-week NOD.Lt or NOD.IL-1R1?/?mice using immunomagnetic
beads. Cells (2 ? 106) were transferred intravenously at the same time as DCs.
Statistical analysis. Student t tests or one-way ANOVA with Bonferroni
posttest compared means of two or multiple groups, respectively, and
Kaplan-Mayer based on log-rank analyses test for comparisons of survival
time. We made five comparisons, with “PBS treated” as the standard compar-
ator. The Bonferroni-corrected threshold was 0.01 based on family-wise
significance level at 0.05. Two-way ANOVA compared time-dependent changes
in Treg function.
RESULTS
RelBloDCs inhibit diabetes when administered to
young, but not insulitic, NOD mice. To investigate the
consequences of administration of RelBloDCs to young
or insulitic NOD mice, we generated RelBloDCs from
NOD mice by incubation of bone marrow cells in the
presence of GM-CSF, IL-4, and Bay11-7082 (an irrevers-
ible inhibitor of NF-?B and inflammasome, but not p38
mitogen-activated protein kinase [40,41]) as previously
described (33,35). CD40 and MHC class II expression
were reduced by addition of Bay11-7082 to NOD DC
cultures compared with control DCs without Bay11-7082, as
expected (supplementary Fig. 1 in the online appendix,
available at http://diabetes.diabetesjournals.org/cgi/content/
full/db10-0104/DC1). The concentration of inhibitor required
to inhibit expression of CD40 and MHC class II in NOD bone
marrow cell cultures was generally 50% of that required for
C57BL/6 RelBloDC cultures, consistent with the constitu-
tivelyhigherlevelsofNF-?BexpressioninNODcells(11).To
determine the potential of RelBloDCs to suppress disease
generation in an antigen-specific manner, we also generated
RelBloDCs from NOD mice where proinsulin is driven as a
transgene by the MHC class II promoter (pINS.NOD) (42).
Mean survival without diabetes was increased from 161 days
(95% CI 132–191) if mice were untreated to 232 days (215–
248) in mice administered 5 ? 105RelBloDCs subcutane-
ously, generated from NOD.Lt mice (P ? 0.002). The smaller
increases in survival time in mice administered pINS.NOD
DCs, or control DCs, at 28 days of age were not statistically
significant (Fig. 1). There was a trend for reduction in
insulitis at 85 days in mice treated with NOD.Lt or pINS.NOD
RelBloDCs compared with PBS, but because islets from only
four mice per group were examined, it was not meaningful to
analyze the data statistically (Fig. 1E). In contrast, diabetes
incidence was not reduced relative to saline-treated controls
bytransferofRelBloorcontrolNODorpINS.NODDCsat100
days of age and if anything, NOD RelBloDC reduced the
survival time without diabetes at this age (Fig. 1C and D).
Thus, as expected, administration of RelBloDCs prevented
development of diabetes when administered to 28- but not
100-day-old NOD mice. This was not dependent on trans-
genic expression of proinsulin autoantigen by the DCs.
IL-1? is overexpressed as islet inflammation devel-
ops, in response to self-antigen presentation. We
showed previously that IL-1? overproduction in NOD mice is
systemic and can be measured by stimulation of splenocytes
with anti-CD3; IL-1 production in humans is also systemic
and measurable in PBMCs (22). IL-1 dysregulates Treg cell
function, and RelBloDCs suppress inflammation through
Treg cells. We therefore determined the timing and mecha-
S. BERTIN-MAGHIT AND ASSOCIATES
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nism of systemic IL-1? overproduction prior to the onset of
diabetes relative to the timing of administration of RelBlo
DCs. We first analyzed supernatants from splenocytes stim-
ulated with anti-CD3 every 4 weeks from weaning till diabe-
tes. IL-1? secretion by NOD splenocytes was increased
relative to C57BL/6 controls, between weeks 6 and 16,
peaking at ?12 weeks of age (Fig. 2A), as insulitis develops
(43). IL-1? was not overexpressed by splenocytes from
NOD.I-Akcongenic mice, which do not express the MHC
class II proinsulin antigen restriction element, I-Ag7, or from
pINS.NOD transgenic mice. These mice express mouse pro-
insulin II under control of the I-E?
kMHC class II promoter,
are tolerant to proinsulin, and do not develop diabetes (but
remain susceptible to other autoimmune diseases) (44,45).
When anti-CD3–stimulated splenocytes were analyzed with
intracellular staining and flow cytometry, we found that
T-cells including both CD4?and CD8?subsets, and APCs
including CD11c?DC and F4/80?macrophages, produced
IL-1? (Fig. 2B). Together, the data indicate that IL-1? is
overexpressed by innate immune cells and T-cells, and,
unexpectedly, that IL-1? overproduction during the develop-
ment of islet inflammation is driven or accelerated by islet
autoantigen presentation in the context of the NOD genetic
background.
0 100
Age (days)
200300
0
25
50
75
100
NOD DC
NOD RelBloDC
PBS
*
A
Percent diabetic
0100
Age (days)
200300
0
25
50
75
100
pINS.NOD DC
pINS.NOD RelBloDC
PBS
B
Percent diabetic
0 100
Age (days)
200 300
0
25
50
75
100
NOD DC
NOD RelBloDC
PBS
C
Percent diabetic
0100
Age (days)
200 300
0
25
50
75
100
pINS.NOD DC
pINS.NOD RelBloDC
PBS
D
Percent diabetic
0
1
2
3
DC from pINS.NOD
DC from NOD
E
PBSDC
RelBlo DC
Insulitis score
FIG. 1. RelBlotolerizing DCs inhibit diabetes when adminis-
tered to young, but not insulitic, NOD mice. RelBloDCs were
generated from the bone marrow of NOD or pINS.NOD trans-
genic mice in the presence of GM-CSF, IL-4, and Bay11-7082.
Control DCs were generated from the same mice in the pres-
ence of cytokines and the absence of Bay11-7082. Twenty-eight-
day-old (A and B) or 100-day-old female NOD (C and D) mice
were injected subcutaneously with 5 ? 105DCs. Mice were
screened weekly for diabetes until 250 days of age. Diabetes
incidence curves are shown for groups each containing 12 mice.
*P < 0.05 (Kaplan-Meier survival analysis with Bonferroni
correction for multiple groups). Insulitis was assessed at 12
weeks (E) in four female NOD mice per group treated at 4
weeks of age with DCs as shown. Data represent means ? SD.
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Because sera from patients with or at risk of type 1
diabetes, when incubated with healthy PBMCs, promote a
microarray signature dominated by IL-1? (15), we as-
sessed whether a factor in mouse serum similarly pro-
motes IL-1? mRNA expression when incubated with
splenocytes from either C57BL/6 or 6-week-old NOD mice.
Although serum from diabetic NOD mice induced expres-
sion of IL-1? by healthy splenocytes, serum derived from
NOD mice during the insulitis phase did not (supplemen-
tary Fig. 2). Glucose at concentrations similar to those in
diabetic mice or humans promote IL-1 secretion by mono-
cytes in vitro (46). Thus, our data are consistent with the
conclusion that islet autoreactivity and not metabolic
factors, such as hyperglycemia, drive IL-1? overexpression
during the insulitis phase in NOD mice.
IL-1? blocks Treg function during the insulitis phase
in NOD mice. We next determined the effects of IL-1? in
NOD mice. We first tracked the capacity of NOD effector
T-cells (Teff) to be suppressed by regulatory (Treg) T-cells,
in parallel to IL-1? production, at different ages in NOD.Lt
mice. We incubated syngeneic splenic DCs, CD4?CD25?
Teff, CD4?CD25?Treg, and anti-CD3 and plotted the
percent suppression of Teff by Treg over time (Fig. 3).
Splenic Treg suppressed proliferation of Teff in vitro
before 6 weeks of age to 40% maximal. However, Treg
were unable to suppress Teff by 8 weeks of age, and this
function only returned by 18 weeks, associated with the
fall in splenocyte IL-1 secretion (Fig. 2A). The same
experiments were carried out using cells from NOD mice
lacking the IL-1R1 receptor (NOD.IL-1R1?/?). The capacity
of NOD.Lt Treg to suppress NOD.Lt Teff was significantly
different from the capacity of NOD.IL-1R1?/?Treg to
suppress NOD.IL-1R1?/?Teff over the same time period
(P ? 0.001 by two-way ANOVA). Thus, the reduced
capacity of NOD.Lt splenic CD4?CD25?T-cells to sup-
press Teff is IL-1? and time dependent. The results are
consistent with reduction in Treg function in NOD mice in
vivo after 4–6 weeks of age (7).
An inflammatory profile associated with IL-1?. Given
the marked change in proinflammatory IL-1? secretion
associated with altered Treg function during the insulitis
phase in NOD mice, we extended our analysis to deter-
mine whether other proinflammatory cytokines were pro-
duced simultaneously and whether these could be
suppressed by Treg cells. IL-6, tumor necrosis factor
(TNF), IL-17, IFN-?, and IL-10 were overexpressed at the
same time as IL-1? (8–12 weeks). Treg did not suppress
these cytokines when added to Teff (Fig. 4A). We did not
observe similar increases in IL-6, IL-17, IFN-?, or IL-10
when analyzing cells from NOD.IL-1R1?/?mice (Fig. 4B).
TNF was overexpressed in NOD.IL-1R1?/?mice between
048 12 1620 24
0
100
200
300
400
NOD
C57BL/6 NOD.I-Ak
pINS.NOD
mice age (week)
IL-1β (pg/mL)
A
B
CD4
CD8
F4/80
CD11c
IL-1β
102 103 104 105
102 103 104 105
102 103 104 105
102 103 104 105
20.2
9.4
19.5 4.6
4.0 1.211.12.9
FIG. 2. Secretion of IL-1? by NOD splenocytes in response to anti-CD3?
antibody during the phase of insulitis does not occur in the absence of
autoantigen presentation and responding T-cells. A: Splenocytes from
NOD.Lt, C57BL/6, NOD.I-Ak, or pINS.NOD mice of different ages were
incubated for 24 h in presence of anti-CD3? antibody, and IL-1? was
assayed in the supernatants of three mice per group by ELISA. B:
NOD.Lt splenocytes were incubated with anti-CD3? antibody for 24 h,
the last 4 h in presence of brefeldin A 5 ?g/ml, before staining for
surface markers as shown and intracellular IL-1?. Representative of
four experiments.
0
20
40
60
80
100
NOD.Lt
NOD.IL-1R1-/-
5 8 10 1218
Weeks
25diabetic
% suppression by Treg
***
FIG. 3. Transient inhibition of Treg cell function is IL-1? dependent,
corresponding to the peak of insulitis. CD11c?, CD4?CD25?, and
CD4?CD25?cells were purified from spleens and lymph nodes of NOD
mice of different ages (four mice pooled per group), using immunomag-
netic separation. A total of 100,000 CD11c?DCs, 105CD4?CD25?
T-cells, and 7.5 ? 104CD4?CD25?T-cells were incubated for 3 days in
presence of anti-CD3? antibody before determination of the T-cell
proliferative response by [3H]thymidine uptake. The regulatory capac-
ity is expressed as the percentage suppression of T-cell proliferation in
presence of CD4?CD25?T-cells relative to the maximum proliferation
observed in presence of CD4?CD25?T-cells and DCs alone. Data from
three separate experiments are expressed as means ? SE percent
suppression. ***P < 0.0001 analyzed by two-way ANOVA comparing
NOD and NOD.IL-1R1?/?over time.
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weeks 5 and 10 but was suppressed by the addition of
Treg. IL-23 was undetectable in these supernatants (data
not shown).
IL-17 production increased again in supernatants from
NOD mice at ?25 weeks and in IL-1R1?/?.NOD mice at 18
weeks, concomitant with reduction in Treg function (Fig.
FIG. 4. IL-1? drives IL-6, TNF, IL-10, and IL-17, which are not subject to Treg suppression, as insulitis develops in NOD mice. Cytokines were
assayed in supernatants from the T-cell proliferation assays (Fig. 3) from mice of the ages indicated using CBA kits and IL-17 ELISA. E, cytokine
production by CD4?CD25?T-cells in the absence of CD4?CD25?Treg; ?, cytokine production by CD4?CD25?T-cells in the presence of
CD4?CD25?Treg. A: Wild-type mice. B: NOD.IL-1R1?/?mice. Data represent mean ? SEM.
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4) and heralding the onset of diabetes. Levels of the IL-17
regulatory cytokine, IFN-?, dropped after 12 weeks,
whereas IL-17 levels increased. The data suggest that in
the presence of high levels of IL-1?, Treg are unable to
contain the expression of a set of proinflammatory cyto-
kines by effector T-cells or APCs.
Spontaneous pancreatic islet cytokine production is
time dependent and reflects production by stimu-
lated splenocytes. To determine the relevance of cyto-
kine production stimulated from splenocytes, we isolated
pancreatic islets from NOD mice between the ages of 4
and 15 weeks, extracted RNA from whole islet tissue and
quantitated IL-1?, TNF, IL-6, and IFN-? RNA by Taqman
real-time PCR. Islet IL-1? and IL-6 mRNA expression
increased between 4 and 15 weeks (P ? 0.05, P ? 0.0001,
respectively, one-way ANOVA with post hoc analysis for
linear trend), reflecting the time course of stimulated
splenocytes (Fig. 5). IL-17 signal was too low to amplify
from whole islets, likely because of a small proportion of
infiltrating Th17, as IL-17 was amplified from stimulated
NOD.Lt splenic T-cells (not shown). Thus, the Th17-
promoting cytokines, IL-1?, and IL-6 are also expressed by
inflamed islets.
IL-17 production is promoted by addition of Tregs to
Teff in an IL-1?–dependent manner at the time of
peak insulitis. We therefore tested the relationship be-
tween NOD mouse age, Treg, Teff, IL-1?, and IL-17 pro-
duction by adding Treg from NOD or NOD IL-1R1?/?mice
to syngeneic Teff stimulated by DCs and anti-CD3 (Fig. 6).
IL-17 increased approximately threefold with the addition
of Treg cells to Teff from 5-week NOD.Lt mice (Fig. 6A).
IL-17 secretion was dependent on IL-1? signaling in vitro
and in vivo, as anti–IL-1? mAb blocked the secretion
of IL-17 in vitro, and NOD.IL1R1?/?T-cells did not secrete
IL-17. By 10 weeks of age, both Teff and Treg secreted
IL-17 and this was no longer dependent on IL-1? in vitro
(Fig. 6B). However, because T-cells isolated from 10-week
NOD.IL1R1?/?mice did not secrete IL-17 ex vivo, the data
suggest that IL-1? (and concomitant IL-6) produced in
response to antigen presentation promotes the develop-
ment of Th17 from a young age in NOD.Lt mice through
FoxP3?Treg cell conversion to Th17 (47). To assess this
in vitro, Teff and Treg purified from 6-week CD45.1
NOD.Lt, CD45.2.NOD, and CD45.1 NOD.IL-1R1?/?mice
were stimulated with DCs and anti-CD3. Purified CD45.2
CD25?Teff included 4% CD4?FoxP3?cells (of which ?1%
were CD25?), and purified CD45.1 CD25?Treg comprised
86% FoxP3hiand 14% FoxP3locells. After 72 h, IL-17 was
measured in supernatants, and PMA-restimulated cells
were stained for CD4, CD45.2, FoxP3, and IL-17. Teff and
Treg secreted IL-17 in an IL-1–dependent manner. When
Teff were mixed with Treg, IL-17 was secreted if either Teff
or Treg but not both lacked IL-1R1 (Fig. 6C). The propor-
tion of CD45.1?FoxP3hicells fell to 30% in culture (Fig.
6D, i). Approximately 0.6% of the input CD45.2?Teff and
1.8% of input CD45.1?Treg expressed IL-17 (Fig. 6D, ii). Of
the IL-17?Treg, the majority were FoxP3lo. When all cells
were NOD.IL-1R1?/?, 0.3% of Teff and 0.1% of Treg cells
expressed IL-17 in culture (data not shown). However, if
NOD.IL-1R1?/?Teff were mixed with wild-type Treg, 4% of
input wild-type FoxP3loand FoxP3hiTreg expressed IL-17
(Fig. 6D, iii), and if NOD.IL-1R1?/?Treg were mixed with
wild-type Teff, 0.9% of Teff expressed IL-17 (Fig. 6D, iv).
Thus, NOD.IL-1R1?/?Treg were unable to prevent low
level of IL-17 production by wild-type Teff. Moreover,
FoxP3 expression is unstable after activation in the pres-
ence of effector cells, and conversion of FoxP3hiand
FoxP3loTh17 is IL-1 dependent.
In the IL-1–rich insulitic environment, RelBloDCs
reduce suppressive capacity of Treg cells at the
expense of Th17 cells. Given the IL-1–dependent dys-
regulation of Treg, coincident with the failure of RelBlo
DCs to prevent type 1 diabetes, we analyzed the relation-
ship between RelBloDC administration and Treg number
and function. As NOD mice age, Treg lose expression of
FoxP3 (48). The frequency of FoxP3?Treg in spleen and
intensity of FoxP3 expression 4 weeks later were not
affected by RelBloDC administration to 4- or 12-week mice
(Fig. 7A). Treg cells purified from untreated 8-week-old
NOD mice suppressed Teff purified from naïve 6-week-old
NOD mice, but this was significantly reduced by adminis-
tration of RelBloDC 4 weeks previously (Fig. 7B). Sup-
pressive capacity of Treg cells purified from 16-week-old
NOD mice was reduced relative to the activity of Treg cells
purified from 8-week-old mice and significantly reduced by
administration of RelBloDCs 4 weeks previously (Fig. 7B).
In contrast, suppressive activity of Treg cells isolated from
16-week-old NOD.IL-1R1?/?recipients of RelBloDCs ad-
ministered 4 weeks earlier and untreated NOD.IL-1R1?/?
mice was equivalent (Fig. 7B). The data demonstrate that
RelBloDCs exacerbate the age-dependent decline in Treg
cell function. Similarly, when 6-week wild-type or NOD.IL-
1R1?/?mice were untreated or injected with RelBloDCs,
IL-17 secretion by splenic Teff and Treg was promoted by
RelBloDCs in an IL-1–dependent manner 4 weeks later
(Fig. 7C). IL-1–dependent IL-17 secretion by anti-CD3–
stimulated pancreatic draining lymph node cells from
these mice was greater after treatment with RelBloDCs
(Fig. 7C). Consistent with this IL-1 dependence, promotion
FIG. 5. Age-dependent expression of proinflammatory cytokines by pancreatic islets. Pancreatic islets were purified from NOD.Lt mice (n ? 5 per
group) of the ages shown, and RNA was analyzed by Taqman PCR for relative expression of IL-1?, IL-6, TNF, and IFN-?. *P < 0.05; ***P < 0.0001
(one-way ANOVA with post hoc analysis for linear trend).
S. BERTIN-MAGHIT AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 60, JANUARY 2011 253

Page 7

of IL-17 production by RelBloDCs was not altered by
treatment of wild-type recipients with anti–IL-17 mAb (Fig.
7D). Consistent with the inability of IL-1R1?/?Treg to
suppress IL-17 production by wild-type cells in vitro (Fig.
6), adoptive transfer of wild-type or NOD.IL-1R1?/?Treg
purified from 4-week-old donors to 6-week-old recipients
did not impact IL-17 secretion in the presence of RelBlo
DCs (Fig. 7D). By staining, IL-17 was almost all produced
by host FoxP3loTreg (data not shown). The data demon-
strate that RelBloDCs exacerbate the age-dependent de-
cline in Treg cell function at the expense of conversion to
Th17 in NOD mice including in pancreatic draining lymph
node, in an IL-1–dependent manner.
IL-17 impairs Treg function systemically. Although
IL-1 impairs the suppressive function of Treg (Fig. 4) (17),
a recent publication (49) showed that inhibition of IL-17
for 10 days in 10-week NOD mice was sufficient to impair
progression to diabetes, associated with increased Treg
infiltration of islets. When we treated 6-week NOD with
anti–IL-17, Teff were suppressed by splenic Treg signifi-
cantly better than those of untreated mice (Fig. 8). The
data support the conclusion that in addition to the broad
impact of IL-1, IL-17 itself impacts suppression by FoxP3?
Treg.
DISCUSSION
Many newly diagnosed type 1 diabetic patients exhibit
high levels of IL-1? or a microarray signature, including
IL-1? (22,50). Although metabolic disturbance is impli-
cated in the IL-1? expression by such patients, there are
suggestions of IL-1 overexpression earlier in the disease
course. For example, sera from at-risk first-degree rela-
tives induced an IL-1–related gene expression signature,
when incubated with healthy PBMCs (15). We show here
that as NOD mice age, IL-1 is produced systemically,
including in the pancreatic islet, and is implicated in the
immune dysregulation that occurs as islet autoantigen is
presented in at-risk mice, by virtue of their predisposing
MHC class II alleles. Tolerant pINS.NOD mice also fail to
upregulate IL-1. These data suggest that IL-1 production by
self-reactive peripheral blood T-cells could be a useful
early prognostic indicator in children with high-risk MHC
alleles, even before development of islet autoantibodies.
Not all type 1 diabetic patients show elevated IL-1? levels
or gene signature. Delayed diabetes progression in
NOD.IL-1R1?/?mice suggests that IL-1? is not necessary
for this form of diabetes; however, high IL-1 might predict
a more aggressive disease course associated with high-risk
MHC alleles.
0
1000
2000
3000
4000
5000
NOD.Lt 5wks
NOD.Lt 5wks + anti-IL1β
NOD.Lt 5wks + anti-IL17 Ab
025 5075 100Treg only
NOD.IL1R1-/- 5wks
IL-17 (pg/mL)
0
1000
2000
3000
4000
5000
NOD.Lt 10-12wks
NOD.Lt 10wks + anti-IL1β
NOD.Lt 12wks + anti-IL17
NOD.IL1R1-/- 10-14wks
0 255075 100 Treg only
Number of added CD4+CD25+ T cells (x10-3)
D
(i)
AB
C
0
1000
2000
3000
wt
IL1R-/-
wt Teff + IL1R-/- Treg
IL1R-/- Teff + wt Treg
TeffTreg Teff + Treg
******
***
***
***
IL-17 (pg/mL)
FoxP3
FoxP3
IL-17
CD45.2
hi
lo
Input Teff
Input Treg
30.7%
wt
Input IL-1R-/- Teff
Input IL-1R-/- Treg
38%
Input wt Treg
44.1%
Input wt Teff
5%
IL-17
FoxP3
(ii)
(iv)
(iii)
0.3%
1.5%
0.5%
0.1%
2%
1.9%
0.7%
0.2%
10%
3%
65%
22%
67.5%
95.4%
4%
61.1%
0.9%
94%
0%
52%
0.4%
96.2%
0.1%
3.3%
FIG. 6. IL-17 is produced by Teff and reprogrammed Tregs in an IL-1?–dependent manner during insulitis. IL-17 was assayed at 5 (A) or 10–14
weeks (B) in supernatants from the T-cell proliferation assay by ELISA from NOD.Lt cells with or without anti–IL-1 or anti–IL-17 mAb (10 ?g/ml)
or from NOD.IL-1R1?/?cells. C: CD4?CD25?Teff and CD4?CD25?Treg purified from 6-week CD45.1 NOD.Lt, CD45.2.NOD, and CD45.1
NOD.IL-1R1?/?mice were stimulated with DCs and anti-CD3. After 72 h, IL-17 was measured in supernatants. ***P < 0.0001 (one-way ANOVA).
D: Cells from the same experiment were restimulated with PMA in the presence of brefeldin A and stained for CD4, CD45.2, FoxP3, and IL-17.
Cells are gated on CD4 and the relevant congenic marker to analyze Teff and Treg FoxP3 and IL-17 expression individually.
TIMING OF TOLERIZING IMMUNOTHERAPY
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Page 8

In NOD mice, early IL-1 overproduction has functional
consequences: systemic (including islet) induction of IL-6
and IFN-?, reduction in Treg suppressor function, FoxP3
instability, reprogramming of Treg to Th17, and dysregula-
tion of the mechanisms by which tolerizing DCs suppress
autoimmunity. Similar to NOD mice, IL-6 downregulated
FoxP3 expression and (with IL-1 and transforming growth
factor-?) reprogrammed Treg to Th17 (47,51–53). Repro-
gramming is of particular concern in the context of autoim-
munity, because FoxP3?Treg are selected in the thymus for
their reactivity toward self-antigens, and FoxP3?Th17 could
contribute to pathogenic self-reactivity (54). In experimental
allergic encephalomyelitis, ?10% of splenic FoxP3?cells
produced IL-17 (47). Th17 differentiation is initiated but not
maintained by IL-1 in NOD mice and can be initiated by other
factorsinNOD.IL-1R1?/?mice.Thus,IL-1?acceleratesdiabetes
by reprogramming Treg to Th17 at the expense of suppressor
function, between 6 and 18 weeks in NOD mice. Moreover, IL-1
enhances T-cell IFN-?, which drives insulitis, and must be
contained by FoxP3?Treg (6,17).
Many immunotherapeutic strategies, which induce Treg,
prevent diabetes when administered to young NOD mice
but not during insulitis (25,55–58). This is likely reflected
in human type 1 diabetes, where the outcome of preven-
tive trials of antigen-specific tolerizing immunotherapy has
varied (59,60). Nasal insulin, which was ineffective, was
administered to very high-risk children in the late preclin-
ical phase (60) when IL-1 was likely already expressed.
Similar to previous studies, RelBloDC immunotherapy in
young NOD mice did not depend on presentation of
proinsulin antigen (25). This could be because nontrans-
genic RelBloDCs present a range of self-antigens that
tolerize relevant autoreactive T-cells, or because of non-
specific Th2 immune deviation by DCs cultured in FCS,
presenting FCS-derived epitopes (61,62). The latter is less
likely because DCs cultured without NF-?B inhibitor were
0
2000
4000
6000
8000
10000
Teff TregTeff + Treg
Untreated
RelBloDC treated
wt
IL-1R1-/-
wt
IL-1R1-/-
wt
IL-1R1-/-
Recipient:
*
***
***
**
**
***
***
IL-17 (pg/ml)
0
2000
4000
6000
8000
10000
TeffTregTeff + Treg
wt IL-1R1-/-
wt
IL-1R1-/-
wt IL-1R1-/-
Treg transferred
to wt:
***
***
***
IL-17 (pg/ml)
0
2000
4000
6000
8000
10000
TeffTregTeff + Treg
Anti-IL-17 treatment of wt
***
***
IL-17 (pg/ml)
25 5075100
0
untreated RelBlo DC treated
T cells alone
NOD treated at
4 weeks
[3H] thymidine incorporation (cpm)
***
100
75
50
25
CD4+/CD25+cells added (x10-3)
255075100
0
NOD.IL-1R1-/- treated
at 12 weeks
100
75
50
25
255075100
0
NOD treated at
12 weeks
***
100
75
50
25
A
FoxP3+ of CD4+ splenocytes (%)
B
C
0
50
100
150
200
400
800
1200
1600
wt
IL-1R1-/-
***
***
***
PLN IL-17 (pg/ml)
0
5
10
15
20
25
UntreatedRelBloDC treated
4wks
12wks
IL-1R1-/-
Age:
wt
12wks
0
20
40
60
80
100
4wks
12wks
IL-1R1-/-
Age:
wt
12wks
FoxP3+ splenocytes MFI
D
FIG. 7. In the IL-1–rich insulitic environment, tolerizing DCs reduce suppressor function at the expense of IL-17. Four-week-old, 12-week-old
NOD mice, and 12-week-old NOD.IL1R?/?mice were injected subcutaneously with RelBloDCs or saline. A: The percentage of CD4?cells
expressing FoxP3 in spleen and the mean fluorescence intensity (MFI) of FoxP3 expressed by CD4?T-cells were enumerated. B: Four weeks
later, CD4?CD25?T-cells were isolated from spleen and various numbers were added to 1 ? 105CD11c?splenic DCs and 1 ? 105CD4?CD25?
T-cells purified from naïve 6-week NOD mice and stimulated with 0.5 ?g/ml anti-CD3. Proliferation was assessed by incorporation of [3H]
thymidine. Data represent the mean of triplicate wells ? SE. A total of 6–9 mice were pooled in each of two separate experiments. ***P < 0.0001
(two-way ANOVA). C and D: Six-week wild-type or NOD.IL-1R1?/?mice were untreated or injected with RelBloDCs. As DCs were administered,
some groups of wild-type mice were administered anti–IL-17 mAb intraperitoneally on alternate days for 10 days or CD4?CD25?Treg purified
from either wild-type or NOD.IL-1R1?/?mice intravenously once. After 4 weeks, Teff and Treg were purified from each group and incubated with
DCs in the presence of anti-CD3. IL-17 levels were measured in supernatants by ELISA (C) and cells restimulated with PMA in the presence of
brefeldin A were stained for CD4, CD45.2, FoxP3, and IL-17 (D). *P < 0.05; **P < 0.001; ***P < 0.0001 (one-way ANOVA) (C and D).
S. BERTIN-MAGHIT AND ASSOCIATES
diabetes.diabetesjournals.orgDIABETES, VOL. 60, JANUARY 2011255

Page 9

ineffective, and neither Teff nor Treg secreted more IL-4
after administration of RelBloDCs (not shown) (61,62).
In a recent study (49), anti–IL-17 mAb administered to
10-week but not 4- to 5-week NOD mice prevented diabe-
tes. The current studies demonstrate that 10 weeks is ideal
and 4 weeks is too early to block IL-17. Furthermore, IL-17
may impact the capacity of Teff to be suppressed by Treg.
This may be indirect, as anti–IL-17 reduces DC differenti-
ation and promotes IL-10 production (63,64).
Tolerizing DCs delivered to young mice likely regulate
autoantigen presentation, preventing expression of proin-
flammatory triggers for Th17. RelBloDCs, which suppress
inflammatory arthritis, also prevent diabetes in NOD mice
when administered at 4 weeks but not 14 weeks. At 10–14
weeks, RelBloDCs exacerbated the IL-1–dependent decline
in Treg cell function and conversion to Th17. When IL-1? and
IL-6 are overexpressed by NOD mice, reprogramming Th17,
the ground is no longer fertile for therapies which induce
Treg. In contrast, GAD-specific Ig-GAD immunotherapy,
which promotes IFN-? and blocks IL-17, was only effective
when delivered after 8 weeks of age (65). Our data in NOD
mice have important implications for appropriate timing of
immunotherapy in humans at risk of type 1 diabetes. IL-1 and
IL-17 would be interesting early biomarkers in children at
risk of type 1 diabetes to identify similar pathogenetic stages
and to stratify treatment to tolerizing immunotherapy or
cytokine blockade (66).
ACKNOWLEDGMENTS
This work was supported by National Health and Medical
Research Council (NHMRC) grants 351439 and 569938 and
Juvenile Diabetes Research Foundation grants 1-2006-149
and 5-2003-269. R.T. was supported by Arthritis Queensland
and an Australian Research Council Future Fellowship. R.S.
was supported by an NHMRC Career Development Award.
B.O. was supported by a Queensland Smart State Fellowship.
No potential conflicts of interest relevant to this article
were reported.
S.B.-M., D.P., S.B., E.D., S.P., and R.S. researched data.
S.B.-M., D.P., S.P., R.S., and R.T. wrote the manuscript.
B.O., S.P., L.C.H., and T.W.H.K. contributed to the discus-
sion and reviewed/edited the manuscript.
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