A Crucial Role of RORγt in the Development
of Spontaneous Sialadenitis-like Sjögren's
Syndrome
This information is current as
of June 17, 2017.
Mana Iizuka, Hiroto Tsuboi, Naomi Matsuo, Hiromitsu
Asashima, Tomoya Hirota, Yuya Kondo, Yoichiro Iwakura,
Satoru Takahashi, Isao Matsumoto and Takayuki Sumida
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Material
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J Immunol 2015; 194:56-67; Prepublished online 19
November 2014;
doi: 10.4049/jimmunol.1401118
http://www.jimmunol.org/content/194/1/56
The Journal of Immunology
A Crucial Role of RORgt in the Development of Spontaneous
Sialadenitis-like Sjögren’s Syndrome
Mana Iizuka,* Hiroto Tsuboi,* Naomi Matsuo,* Hiromitsu Asashima,* Tomoya Hirota,*
Yuya Kondo,* Yoichiro Iwakura,† Satoru Takahashi,‡ Isao Matsumoto,* and
Takayuki Sumida*
S
jögren’s syndrome (SS) is a chronic autoimmune disease
characterized by infiltration of lymphocytes into lacrimal
and salivary glands, and clinically by dry eyes and dry
mouth (1). Immunohistochemical studies have shown that most
infiltrating lymphocytes in the labial salivary and lacrimal glands
are CD4+ ab T cells (2). Autoantigens recognized by T cells infiltrating the labial salivary glands of patients with SS have been
analyzed and several candidate autoantigens such as Ro/SSA 52
kDa (3), a-amylase (4), heat shock protein and TCR BV6 (5), and
M3 muscarinic acetylcholine receptor (M3R) (6, 7) have been
identified. However, the pathological role of the T cell subsets in
SS remains to be elucidated.
Infiltrating Th1-type cells that mainly produce IFN-g have been
found in salivary glands of patients with SS (8, 9). These cells
enhance the activity of T cells, B cells, and macrophages, resulting
in the destruction and dysfunction of these glands (8, 9). In experimental models, transgenic (Tg) expression of IL-12 in the
*Department of Internal Medicine, Faculty of Medicine, University of Tsukuba,
Tsukuba, Ibaraki 305-0006, Japan; †Research Institute for Biomedical Sciences,
Tokyo University of Science, Noda, Chiba 278-0022, Japan; and ‡Department of
Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Tsukuba,
Ibaraki 305-8575, Japan
thyroid gland also leads to mononuclear infiltration of the salivary
glands (10). Thus, the Th1 response is considered to play a role in
the pathogenesis of SS.
However, a high level of IL-17 and Th17-related cytokines
(TGF-b, IL-6, IL-23) have recently been found in the salivary
glands and plasma of patients with SS and the mice model of SS
(11–13). We also have reported the involvement of IL-17 production by M3R-reactive T cells in the pathogenesis of sialadenitis
based on studies in a mouse model of M3R-induced sialadenitis,
which is also considered as a mouse model of SS (14). Moreover
Th17-associated cytokines, IL-17, IL-1b, and IL-23 are reported
to form ectopic germinal centers in patients with SS (15).
The nuclear receptor retinoic acid–related orphan receptor
(ROR)gt is a transcription factor induced by TGF-b and IL-6, and
it directs the differentiation of inflammatory Th17 cells as well as
cytokine production (16, 17). RORgt cooperates with RORa and
IkBz to enhance Il17a expression by binding directly to the regulatory region of the Il17a gene (18). Although several studies
have indicated that Th17-related cytokines may be involved in
development of SS, there is little information on the pathological
role of RORgt in SS. To address this issue, we used RORgt Tg
mice, which have been found to spontaneously develop severe
sialadenitis-like SS.
Received for publication May 1, 2014. Accepted for publication October 22, 2014.
This work was supported by Research Program for Intractable Diseases Health and
Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare,
Japan.
Materials and Methods
Address correspondence and reprint requests to Prof. Takayuki Sumida, Department
of Internal Medicine, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba City, Ibaraki 305-0006, Japan. E-mail address: [email protected]
RORgt Tg mice, which were generated from C57BL/6 mice, were provided by Prof. S. Takahashi (University of Tsukuba, Ibaraki, Japan). We
used female mice for analysis in this study. IL-17A knockout RORgt Tg
(IL-172/2/Tg) mice were generated by crossing RORgt Tg mice with IL17A knockout mice provided by Prof. Y. Iwakura (Tokyo University of
Science). For the isolation of regulatory T (Treg) cells, Tg and C57BL/6
mice were crossed with knock-in mice with Foxp3-IRES-GFP (RORgtFoxp3GFP, C57BL/6-Foxp3GFP). C57BL/6-Foxp3GFP mice were provided
by Prof. B. Malissen (Université de la Méditerranée, Marseille, France).
All animals were maintained in specific pathogen-free conditions in the
The online version of this article contains supplemental material.
Abbreviations used in this article: ANA, anti-nuclear Ab; cLN, cervical lymph
node; CNS, conserved noncoding sequence; LTi, lymphoid tissue inducer; M3R,
M3 muscarinic acetylcholine receptor; ROR, retinoic acid–related orphan receptor;
SS, Sjögren’s syndrome; Teff, effector T; Tfh, T follicular helper; Tg, transgenic.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401118
Mice
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The nuclear receptor retinoic acid–related orphan receptor (ROR)gt is required for the generation of Th17 cells, which are involved
in various autoimmune diseases, including Sjögren’s syndrome (SS). However, the pathological role of RORgt in SS remains to be
elucidated. The present study was designed to clarify the role of RORgt in the pathogenesis of sialadenitis-like SS. Histological
analysis of RORgt transgenic (Tg) mice was determined, and then Tg mice developed severe spontaneous sialadenitis-like SS. The
analysis of infiltrating cells showed that most infiltrating cells were CD4+ T cells. RORgt-overexpressing CD4+ T cells induced
sialadenitis as a result of transferred CD4+ T cells from Tg mice into Rag22/2 mice. The examination of IL-17–deficient Tg mice
indicated that IL-17 was not essential for the development of sialadenitis. The number of CD4+CD25+Foxp3+ regulatory T (Treg)
cells was significantly decreased in Tg mice, and CD25 expression and IL-2 stimulated STAT5 activation were inhibited in Treg cells.
The inhibitory function of Treg cells of Tg mice was equal to that of wild-type mice in vitro. The abundant Treg cells of Tg mice could
suppress the development of sialadenitis, but the reduced Treg cells of Tg mice could not inhibit the induction of sialadenitis in
Rag22/2 mice transferred with effector cells from Tg mice. These results suggest that both RORgt-overexpressed CD4+ T cells and
reduced Treg cells might contribute to the development of SS-like sialadenitis. The Journal of Immunology, 2015, 194: 56–67.
The Journal of Immunology
Laboratory Animal Resource Center. All experiments were performed
according to the Guide for the Care and Use of Laboratory Animals at
Tsukuba University.
57
In vitro suppression assay and Treg cell culture
Histological score
Detection of autoantibodies by ELISA
The inflammatory lesions were graded histologically (histological focus
sore) using the method proposed by Greenspan et al. (19) as follows: focus
score 1, a single focus composed of .50 mononuclear cells per 4 mm2
tissue. The salivary glands were evaluated histologically by researchers
blinded to the experiment, and at least one tissue section from each salivary gland was examined.
The anti-M3R Abs were detected by ELISA as previously described (14). The
peptide solution containing all types of M3R extracellular (N terminus, first,
second, and third extracellular loop) peptides (each 5 mg/ml) was mixed
with 0.1 M Na2CO3 buffer (pH 9.6) absorbed onto a Nunc Immunoplate
(Nalge Nunc International, Rochester, NY) at 4˚C overnight. Serum was
incubated for 2 h at room temperature, and HRP-conjugated anti-mouse IgG
Ab diluted 1:1000 was added for 1 h at room temperature. After washing,
100 ml tetramethylbenzidine solution (Kirkegaard & Perry Laboratories,
Gatithersburg, MD) was added as a substrate and then the OD was measured
at 450 nm by plate spectrophotometry (PerkinElmer Cetus, Norwalk, CT).
Anti-nuclear Abs (ANAs), autoantibodies against SSA/Ro and SSB/La,
were measured by a commercially available ELISA assay according to
the protocols of the manufacture (Alpha Diagnostic International, San
Antonio, TX).
Measurement of saliva volume
TUNEL staining
Apoptotic cells were detected by in situ apoptosis detection kit (Takara Bio,
Otsu, Japan). Briefly, frozen sections were fixed with 4% paraformaldehyde
for 30 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxidase/methanol. After washing in PBS, the sections were
treated for 5 min on ice with the permeabilization buffer and then incubated
with TdT enzyme reaction for 90 min at 37˚C. Nuclei were counterstained
with DAPI (Sigma-Aldrich, St. Louis, MO).
Isolation of lymphocytes from salivary glands
For flow cytometric analysis of infiltrating lymphocytes in salivary glands,
tissue was cut into small pieces, digested by 2 mg/ml collagenase
(Worthington Biochemical, Lakewood, NJ), and homogenized, and the
lymphocytes were purified by centrifugation with Ficoll-Paque Plus (GE
Healthcare Biosciences, Uppsala, Sweden).
Quantitative PCR analysis
Total RNA was prepared using Isogen (Nippon Gene, Toyama, Japan). Firststrand cDNA was synthesized at 37˚C for 15 min using the PrimeScript
reverse transcriptase master mix (TaKaRa Bio) and 1 ml of this 10 ml
reaction mixture was used for the PCR. A TaqMan Assay-on-Demand gene
expression product was used for real-time PCR (Applied Biosystems,
Foster City, CA). The expression levels of Ifng, Il17a, Il17f, Ccr6, Ccl20,
Il6, Il12p35, Tnf, Il22, Il21, Il4, Il23r, Tbx21, Rorc, Bcl6, Irf4, and Ahr
were expressed relative to the expression of hprt1. Analyses were performed with an ABI Prism 7500 apparatus (Applied Biosystems).
Flow cytometry
Staining for CD3, CD4, CD8, CD19, CD25, CD44, and CD62L (all from
BioLegend, San Diego, CA) expression was performed for 20 min using
a mixture of Abs. Intracellular staining for Foxp3, CTLA-4, GITR, and
CD103 (all from BioLegend) was performed after fixation and permeabilization according to the protocol supplied by the manufacturer
(eBioscience, San Diego, CA). Samples were analyzed with a FACSCalibur
flow cytometer (Becton Dickinson, Mountain View, CA), and data were
analyzed with FlowJo software (Tree Star, Ashland, OR). For phosphoSTAT5 (Cell Signaling Technologies, Danvers, MA) staining, Phosflow
Lyse/Perm buffer and Perm Buffer III (BD Biosciences, San Jose, CA) were
used according to the instructions provided by the manufacturer.
Immunohistochemical analysis
Sections of the salivary glands were thawed, dried, and then fixed with 1%
formalin for 10 min. Immunofluorescence staining was performed as follows: the sections were incubated with Alexa Fluor 488–conjugated CD4
and Alexa Fluor 647–conjugated B220 Abs (all from Invitrogen, Carlsbad,
CA) for 30 min at room temperature. The nuclear DNA was stained with
DAPI.
Adoptive transfer and cell sorting
CD4+ T cells were sorted from splenocytes of mice by using the CD4+
MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were
resuspended in PBS and inoculated intravenously in Rag22/2 mice. For the
isolation of Treg cells, CD4+ T cells were selected with CD4+ MACS bead,
and CD4+CD25+GFP+ or CD4+CD252GFP2 cells were further purified
from C57BL/6-Foxp3GFP or RORgt-Foxp3GFP mice using a MoFlo cell
sorter (DakoCytomation, Glostrup, Denmark).
Results
Spontaneous development of autoimmune sialadenitis in
RORgt Tg mice
Various body organs from RORgt Tg mice were analyzed histologically to determine the effects of RORgt overexpression in
T cells under hCD2 promoter. Infiltrating mononuclear cells were
detected in salivary glands and lacrimal glands of RORgt Tg mice
(Fig. 1A). We also detected mild cell infiltration in lungs, which is
a frequent complication of SS (20), but not in other organs of
RORgt Tg mice and all organs of littermates control (C57BL/6)
(Supplemental Fig. 1). The histological scores of inflammatory
lesions in the salivary glands of RORgt Tg mice at 8, 12, 16, and
20 wk were significantly higher than those of C57BL/6 mice (Fig.
1B). We also detected the infiltrating mononuclear cells in salivary
glands and lacrimal glands, as well as mild cell infiltrations in
lungs from male RORgt Tg mice (Supplemental Fig. 2A, 2B), and
the focus score of inflammatory lesions in salivary glands of those
mice was significantly higher than that of male C57BL/6 mice
(Supplemental Fig. 2C).
To verify the secretory function of the salivary glands, saliva was
collected from C57BL/6 and RORgt Tg mice. The amount of
saliva collected from RORgt Tg mice was significantly lower than
that from C57BL/6 mice at 8 and 12 wk (Fig. 1C). However, there
was no difference in saliva secretion between C57BL/6 and
RORgt Tg mice at 3 wk, which is the predisease time point. To
investigate tissue-specific autoantibodies, we examined sera for
Abs against M3R, which are highly expressed in the exocrine
glands and are candidate autoantigens in patients with SS and
mice with experimentally induced autoimmune sialadenitis. The
levels of anti-M3R Abs in sera of RORgt Tg mice were significantly higher than those in C57BL/6 mice (Fig. 1D). There was
also a significant difference in the titer of anti-SSA Abs between
C57BL/6 and RORgt Tg mice, and anti-SSB and ANAs were
prone to increase in sera of RORgt Tg mice (Fig. 1D). These
results indicated that overexpression of RORgt in T cells results in
spontaneous autoimmune sialadenitis-like SS.
Characterization of age-dependent infiltration of inflammatory
cells in salivary glands
The infiltrating cells were isolated from the inflammatory lesions of
the salivary glands and then analyzed by flow cytometry. Most
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Mice were first anesthetized with i.p. injection of pentobarbital (25 mg/kg),
and then injected s.c. with pilocarpine (1.0 mg/kg). This was followed
15 min later by collection of saliva from the oral cavity using a 200-ml
micropipette. The volume of the sample was measured and expressed
relative to body weight.
Responder cells (CD4+CD252GFP2) were labeled with 5 mM CFSE
(Invitrogen) and then cultured with or without unlabeled Treg cells at the
indicated ratio for 96 h in the presence of Dynabeads mouse T-activator
CD3/CD28 (Invitrogen) in round-bottom 96-well dishes at a density of one
bead per cell. The percentage inhibition rate on the proliferation of responder was calculated as [1 – (CFSE percentage of Treg cell plus responder cell coculture/responder cells alone)] 3 100%. After collection of
supernatant from Treg cultures, the levels of IFN-g, IL-17, and IL-10 were
measured by ELISA (R&D Systems, Minneapolis, MN).
58
A PATHOLOGICAL ROLE OF RORgt IN SJÖGREN’S SYNDROME
infiltrating cells were CD3+ T cells although a few B cells were
observed at the early phase of sialadenitis. B cells gradually increased in number with age and most infiltrating cells at later
stages were B cells in RORgt Tg mice. The infiltrated CD3+ cells
were mainly CD4+ T cells, whereas CD8+ T cells formed a minor
population. The CD4/CD8 T cell ratio did not change throughout
the different stages of sialadenitis (Fig. 2A). Immunofluorescence
staining showed similar results in salivary glands of RORgt Tg
mice (Fig. 2B).
Using the TUNEL method, we were able to identify a few
apoptotic cells in the inflammatory lesions of salivary glands
isolated from RORgt Tg mice but not from C57BL/6 mice
(Fig. 2C). This finding might account for the destruction and
dysfunction of the salivary glands.
Critical role of RORgt overexpression on CD4+ T cells in the
development of sialadenitis
Because the main population of infiltrated cells in the early stage
of sialadenitis were CD4+ T cells, we then characterized these cells
in RORgt Tg mice. CD4+ T cells harvested from the spleen,
cervical lymph nodes (cLN), and salivary glands were mainly
effecter memory cells (CD44hiCD62L lo). Alternatively, only
a few naive cells (CD44 lo CD62L hi) and central memory cells
(CD44hiCD62Lhi) were found in the spleen and cLN of RORgt Tg
mice relative to C57BL/6 mice (Fig. 3A, 3B).
We also analyzed the expression of Rorc, Ahr, Irf4, and Ccr6,
which are related to Th17 induction, and found higher expression
among splenic CD4+ T cells from RORgt Tg mice compared with
C57BL/6 mice. However, the expression of other T cell subset–
related transcription factors Tbx21, Gata3, and Bcl6 had similar
levels among RORgt Tg and C57BL/6 mice (Fig. 3C). A similar
result was found in CD4+ T cells of cLN (Supplemental Fig. 3).
To understand the pathological role of RORgt overexpressed
CD4+ T cells in the development of sialadenitis, we injected purified splenic CD4+ T cells into Rag22/2 mice. Histological
analysis showed marked cell infiltration in the salivary glands of
Rag22/2 mice inoculated with CD4+ T cells of RORgt Tg mice,
but not in those inoculated with CD42 cells of RORgt Tg mice or
any other cell types from C57BL/6 mice (Fig. 3D). The histological score of inflammatory lesions in the salivary glands of
RORgt Tg CD4+→Rag22/2 mice was significantly higher than in
RORgt Tg CD42→Rag22/2 mice (Fig. 3E). These results emphasize the pathological role of overexpression of RORgt on
CD4+ T cells in the development of sialadenitis-like SS.
Various T cell subsets in inflammatory lesions of salivary
glands
We checked the involvement of effector subsets of Th1, Th2, Th17,
and T follicular helper (Tfh) cells in the pathogenesis of sialadenitis
in RORgt Tg mice, because expression of various T cell subsets was
present in the salivary glands of patients with SS. First, examination
of the expression of Th17-related molecules (Rorc, Il17a, Il23r, and
Ccl20 [the ligand of CCR6]) showed significantly high mRNA
expression levels of Rorc, Il17a, Il23r, and Ccl20 in the salivary
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FIGURE 1. Development of spontaneous sialadenitis-like SS in RORgt
Tg mice. (A) H&E-stained sections
of the salivary and lacrimal glands
from C57BL/6 and RORgt Tg mice.
Scale bars, 30 mm (left panel), 70 mm
(right panel). Data are representative
of five tissue samples with similar
results. (B) Histological score of inflammatory lesions in salivary glands
of C57BL/6 and RORgt Tg mice at
8, 12, 16, and 20 wk of age. Six mice
were analyzed per group. (C) Saliva
volume from C57BL/6 and RORgt
Tg mice at 3, 8, and 12 wk of age.
Data are representative of two independent experiments with similar
results. (D) Anti-M3R, anti-SSA, antiSSB, and ANAs Abs in sera of C57BL/
6 and RORgt Tg mice detected by
ELISA. Data are representative of
two independent experiments with
similar results. *p , 0.05 (Mann–
Whitney U test).
The Journal of Immunology
59
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FIGURE 2. Infiltrating cells in salivary glands isolated from RORgt Tg mice. (A) Flow cytometric analysis of lymphocyte-gated cells (15–20%) stained
with CD19, CD3, CD4, and CD8 on CD3+ subsets in infiltrated cells isolated from the salivary glands of RORgt Tg mice. Four mice per group were
analyzed and representative data are shown. (B) Immunofluorescence analysis of salivary glands from RORgt Tg mice at 4, 6, and 12 wk of age. Data are
representative of two tissue samples with similar results. (C) Apoptotic cells in salivary glands of C57BL/6 and RORgt Tg mice detected by the TUNEL
method. Data are representative of three tissue samples with similar results. Original magnification 3100 (B) and 3600 (C).
glands of RORgt Tg mice compared with C57BL/6 mice (Fig. 4A).
Second, overexpression of Th1 (Ifng, Il2, Il12, Tnf, Tbet)-, Th2 (Il4,
Gata3)-, and Tfh (Il21, Bcl6)-related factors was also noted in the
inflammatory lesions of salivary glands of RORgt Tg mice compared
with C57BL/6 mice (Fig. 4B). Comparison of the expression of
transcriptional factors related to Th cells in CD4+ T cells of salivary
glands of RORgt Tg mice showed significantly higher expression of
Rorc, Tbet, Gata3, and Bcl6 in CD4+ T cells than those of the spleen
(Fig. 4C). Moreover, infiltrating CD4+ T cells isolated from salivary
glands of RORgt Tg mice produced IL-17, INF-g, IL-4, and IL-21
60
A PATHOLOGICAL ROLE OF RORgt IN SJÖGREN’S SYNDROME
(Fig. 4D). These data suggest the involvement of not only Th17 but
also Th1, Th2, and Tfh cells in the pathogenesis of sialadenitis.
IL-17 is not essential for the induction of sialadenitis in RORgt
Tg mice
To determine the significance of IL-17 in the development
of sialadenitis, we generated IL-17–deficient RORgt Tg mice
(IL-172/2/Tg). Sialadenitis occurred spontaneously in IL-172/2/
Tg mice and was similar to that in RORgt Tg mice (Fig. 5A).
Furthermore, the focus score of inflammatory lesions in the
salivary glands (Fig. 5B) and the saliva flow (Fig. 5C) of these
mice was comparable to that of RORgt Tg mice. Immunoflu-
orescence (Fig. 5D) and flow cytometric analyses (Fig. 5E) of
infiltrated cells in salivary glands showed the presence of CD4+
T and B cells in the salivary glands of IL-172/2 /Tg mice. The
percentages of infiltrating CD3, CD19, CD4, and CD8 T cells
in IL-172/2 /Tg mice were not different from those counted in
RORgt Tg mice (Fig. 5E). Quantitative PCR showed that
mRNA expression of Il17a was absent in salivary glands of IL172/2 /Tg mice. The expression of other molecules (Ccr6, Tnf,
Il6, Il22, Il17f, Rorc, Tbx21, and Gata3) was not suppressed in
those mice (Fig. 5F). These results indicate that the production
of IL-17 is not essential for the development of sialadenitis in
RORgt Tg mice.
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FIGURE 3. RORgt-overexpressed CD4+ T cells in RORgt Tg mice. (A) Flow cytometric analysis of naive and effector CD4+ T cells in spleen, cLN, and
salivary glands of C57BL/6 and RORgt Tg mice. Five mice per group were analyzed and representative data are shown. (B) Mean number of CD44hi
CD62Llo, CD44loCD62Lhi, and CD44hiCD62Lhi cells gated on CD4+ cells from spleen and cLN of C57BL/6 and RORgt Tg mice. Four mice per group
were analyzed and representative data of two independent experiments with consistent results are shown. (C) Quantitative PCR analysis of mRNA expression in splenic CD4+ T cells isolated from C57BL/6 and RORgt Tg mice. The experiment was performed in duplicate. Data were normalized to the
expression of the reference gene, Hprt. (D) Purified splenic CD4+ T and CD42 cells (1 3 107 cells) were inoculated into Rag22/2 mice. At 22 wk
postinoculation, the pathology of salivary glands was analyzed. Salivary glands were sectioned at 4 mm, and each section was stained with H&E. Scale bars,
30 mm. Data are representative of four tissue samples with similar results. (E) Histological score of inflammatory lesions in salivary glands of RORgt Tg
CD4+ or CD42→Rag22/2 mice at 22 wk postinoculation. *p , 0.05 (Mann–Whitney U test). N.D., not detected.
The Journal of Immunology
61
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FIGURE 4. Detection of Th1, Th2, Th17, and Tfh cell–related factors in inflammatory lesions. (A and B) Quantitative PCR analysis of Rorc, Il17a, Il21,
Il23r, Ccl20, Ifng, Il2, Il12, Tnf, Il4, Tbx21, Gata3, and Bcl6 mRNA expression in salivary glands of C57BL/6 and RORgt Tg mice. The experiment was
performed in duplicate. (C) Quantitative PCR analysis of mRNA expression in CD4+ T cells isolated from the spleen and salivary glands (SG) of RORgt Tg
mice. Data were normalized to the expression of the reference gene, Hprt. (D) Flow cytometry analysis of infiltrating CD4+ T cells in salivary glands of
RORgt Tg mice after stimulation with PMA and ionomycin for 4 h. Four mice per group were analyzed and representative data are shown. *p , 0.05
(Mann–Whitney U test).
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A PATHOLOGICAL ROLE OF RORgt IN SJÖGREN’S SYNDROME
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FIGURE 5. Spontaneous development of sialadenitis in IL-172/2/Tg mice. (A) H&E-stained sections of salivary glands from RORgt Tg and IL-172/2/RORgt
Tg (IL-172/2/Tg) mice. Scale bars, 70 mm. Data are representative from one of the four tissue samples with similar results. (B) Histological score of
inflammatory lesions in the salivary glands of animals described in (A). (C) Saliva volume of RORgt Tg and IL-172/2/Tg mice. (D) Immunofluorescence
analysis of salivary glands from RORgt Tg and IL-172/2/Tg. Data are representative of four tissue samples with similar results. Original magnification 3100. (E) Flow cytometric analysis of lymphocyte gated cells (15–20%) stained with CD19, CD3, CD4, and CD8 on CD3+ subsets in infiltrated cells
isolated from salivary glands of RORgt Tg and IL-172/2/Tg mice at 12 wk of age. Data are representative of two independent experiments with four mice
per group. (F) Quantitative PCR analysis of mRNA expression level of Il17a, Il17f, Ccr6, Tnf, Il6, Il22, Rorc, Tbx21, and Gata3 in salivary glands of RORgt
Tg and IL-172/2/Tg mice. The experiment was performed in duplicate. Data were normalized to the expression of the reference gene, Hprt. *p , 0.05
(Mann–Whitney U test).
The Journal of Immunology
Reduced CD4+CD25+Foxp3+ Treg cells in RORgt Tg mice
Development of sialadenitis via reduced Treg cells
To evaluate the effect of reduction of Treg cells on prevention
of sialadenitis, we coinoculated CD4+CD252GFP2 cells (as
Teff cells) and Treg cells isolated from C57BL/6-Foxp3GFP or
RORgt-Foxp3GFP mice into Rag22/2 mice. Such inoculation was
associated with the lack of development of sialadenitis in Teff
(C57BL/6)→Rag22/2 mice, in contrast to the appearance of sialadenitis in Teff (Tg)→Rag22/2 mice (Fig. 7A). Infiltrated cells
were not detected in the salivary glands of Teff (Tg) + Treg (Tg,
5 3 105 cells)→Rag22/2 mice, as well as in those of Teff (Tg) +
Treg (C57BL/6, 5 3 105 cells)→Rag22/2 mice, and the histological
score of inflammatory lesions in the salivary glands of Teff (Tg) +
Treg (Tg)→Rag22/2 mice was similar to score of Teff (Tg) +
Treg (C57BL/6)→Rag22/2 mice (Fig. 7B). These data showed
that abundant Treg cells from RORgt Tg mice could inhibit the
development of sialadenitis. However, the reduced Treg cells
could not inhibit sialadenitis in Teff (Tg) + 1:10 Treg (Tg, 5 3
104 cells)→Rag22/2 mice (Fig. 7B). These findings indicated
that Teff cells of RORgt Tg mice was required for induction of
sialadenitis, and the reduction of Treg cells in RORgt Tg mice
did not prevent the development of sialadenitis, suggesting its
involvement in the pathogenesis of SS.
Discussion
Although IL-17 and IL-23 produced by Th17 cells are associated
with the pathogenesis of SS, the pathological role of Th17 cells
remains obscure (11–15). The results of the present study shed
light on the function of RORgt in the development of sialadenitis.
Infiltrating mononuclear cells in the salivary glands of RORgt Tg
mice were mainly CD4+ T cells at the early stage of the inflammation whereas B cells gradually increased with age. These
changes resemble lymphocyte infiltration in the labial salivary
glands of patients with SS. The results provide evidence for the
role of RORgt overexpression in CD4+ T cells in the development
of spontaneous sialadenitis-like SS through downregulation of
CD4+CD25+Foxp3+ Treg cells.
Experimental evidence suggests that IL-17 produced from CD4+
T cells is directly involved in the onset of SS-like disease (11, 22).
However, we demonstrated in the present study the development
of sialadenitis in IL-17–deficient RORgt Tg mice, indicating that
IL-17 is not essential for the development of sialadenitis in RORgt
Tg mice. Our results showed expression of other inflammatory
cytokines (Tnf and Il6) and Th1-, Th2-, and Tfh-related factors in
the salivary glands of IL-172/2/Tg mice and RORgt Tg mice.
These results suggest that various cytokines are involved in the
inflammation of sialadenitis in RORgt Tg mice. Previous work
provides evidence that although Th1 and Th2 effector memory
cells could not be converted into Th17 cells, Th17 cells could be
converted into Th1 and Th2 cells via the upregulation of Tbet and
Gata3 expression in presence of IL-12 or IL-4 (23, 24). We
detected the higher expression of Tbet, Gata3, and Bcl6 in CD4+
T cells of salivary glands than those in splenic CD4+ T cells of
RORgt Tg mice. Therefore, RORgt-overexpressed CD4+ T cells
might convert into various T cells subsets (Th1, Th2, and Tfh) in
salivary glands despite having the Th17-like phenotype in lymphoid tissue.
In CD4+ T cells of RORgt Tg mice, high expression of Ccr6,
which is induced by RORgt (17, 25), and Ccl20 in the salivary
glands of RORgt Tg mice was noted, which might be involved in
migration of RORgt highly expressing CD4+ T cells into salivary
glands. The high expression of Ccl20 in the salivary glands of
patients with SS may be triggered by previous bacterial or viral
infections in the local regions (26–29).
RORgt is also crucial role for the development and function of
lymphoid tissue inducer (LTi) cells, which share proinflammatory
cytokines with Th17 cells, as well as their requirement for RORgt
(30). CD4+ LTi cells from spleen of postnatal mice express both
IL-17 and IL-22 (31). Because IL-17 was not involved in the
generation of sialadenitis, and CD4+ LTi cells were not detected in
the inflammatory lesions of RORgt Tg mice (data not shown),
CD4+ LTi cells may not engage in the pathogenesis of sialadenitis
in our model. However, the expression of Il22 was not suppressed
in salivary glands of IL-172/2/Tg mice. Further analysis of participation of IL-22 producer cells (including Th17, LTi cells) in
the sialadenitis is needed.
Our findings showed the presence of activated CD4+ T cells and
CD4+CD25+Foxp3+ cells that expressed CTLA4, GITR, and
CD103. These findings are thought to be related to dysfunctional
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Our data showed that IL-17 is not involved in the development of
sialadenitis in RORgt Tg mice (Fig. 5). We then examined whether
overexpression of RORgt resulted in inhibition of Foxp3 expression in CD4+CD25+ T cells, because CD4+CD25+Foxp3+ Treg
cells are known to inhibit onset of autoimmune disease. To answer
this question, we first analyzed the expression of Foxp3 in CD4+
CD25+ cells from RORgt Tg mice. The expression of Foxp3 and
the proportion of Treg cells were significantly lower in the thymus, spleen, and cLN of RORgt Tg mice compared with C57BL/6
mice (Fig. 6A, 6B). Second, the expression of CD25, a Treg cell
signature gene, was significantly lower in Treg cells of RORgt Tg
mice compared with that in the same cells of C57BL/6 mice.
Alternatively, the staining intensities of CTLA4, GITR, and
CD103 were higher in RORgt Tg mice than in C57BL/6 mice
(Fig. 6C, 6D), which was probably secondary to ongoing inflammation in RORgt Tg mice. In Treg cells of RORgt Tg mice,
expression of RORgt was also significantly higher than in those of
C57BL/6 mice (Fig. 6C, 6D). Moreover, we examined the role of
IL-2 signaling in downregulation of Foxp3 expression, because the
above results showed downregulation of IL-2 receptor CD25 expression in Treg cells of RORgt Tg mice. Phosphorylation of STAT5
was significantly lower and the expression level of Socs1 mRNA was
significantly increased in IL-2–stimulated Treg cells in RORgt Tg
mice compared with those in C57BL/6 mice (Fig. 6E, 6F). Alternatively, TGF-b–induced smad2/3 phosphorylation was not inhibited
in Treg cells of RORgt Tg mice (Supplemental Fig. 4A).
We also investigated the effect of RORgt overexpression on the
inhibitory function of Treg cells using Foxp3-GFP reporter mice
(C57BL/6-Foxp3GFP, RORgt-Foxp3GFP mice). The Treg cells are
known to be unresponsive to TCR stimulation and do not produce effector cytokines in vitro (21). We found that RORgtoverexpressing CD4+CD25+GFP+ cells produced large amounts
of IL-17 and IL-10 compared with C57BL/6-Foxp3GFP mice
(Fig. 6G). Moreover, we also tested the ability of Treg cells
to suppress effector cell proliferation in vitro by comparing
the function of CD4+CD25+GFP+ cells isolated from RORgtFoxp3GFP, C57BL/6-Foxp3GFP mice in inhibiting the proliferation
of CD4+CD252 GFP2 cells from C57BL/6-Foxp3GFP mice. The
suppressive activity of CD4+CD25+GFP+ cells isolated from
RORgt-Foxp3GFP mice was equal to that of the same cells isolated
from C57BL/6-Foxp3GFP mice (Fig. 6H, 6I). In contrast, the
suppressive activity of CD4+CD25+GFP2 cells, which were significantly higher in RORgt Tg mice, on effector T (Teff) cells
was less than CD4 + CD25+ GFP+ cells in RORgt Tg mice
(Supplemental Fig. 4B, 4C). These figures showed that RORgt
overexpression in Treg cells has no effect on regulatory function.
63
64
A PATHOLOGICAL ROLE OF RORgt IN SJÖGREN’S SYNDROME
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FIGURE 6. Downregulation of Foxp3 expression in CD4+CD25+ Treg cells. (A) Flow cytometric analysis of Foxp3 expression in CD4+CD25+ cells in
thymus, spleen, and cLN of C57BL/6 and RORgt Tg mice. Data are representative of two independent experiments with four mice per group. (B) Proportion of Foxp3+ cells gated on CD4+CD25+ and CD4+CD25+Foxp3+ cells in thymus, spleen, and cLN of C57BL/6 and RORgt (Figure legend continues)
The Journal of Immunology
65
Treg cells (32). Based on this finding, we hypothesized that
RORgt overexpression inhibits the expression of Foxp3 in CD4+
CD25+ cells. Foxp3 can suppress Th17 cell differentiation through
antagonism of RORgt activity (33), and its interaction has been
reported (34). Our results showed that the expression of Foxp3
was significantly decreased in CD4+CD25+ cells, indicating that
downregulation of Foxp3 expression in these cells is the culprit
mechanism in sialadenitis. Deficiency of Treg cells is probably one
of the underlying mechanisms of SS, because massive leukocyte
infiltration was observed in the salivary glands of IL-2Ra2/2,
IL-22/2, and Rag12/2 mice inoculated with lymph node cells from
scurfy mice and other mice with impaired Treg function (32, 35,
36). In adoptive transfer models, such as CD4+CD45RBhi naive
T cells without Treg cells in Rag-deficient mice, sialadenitis was
also observed (37). Previous studies demonstrated the presence of
significantly lower CD4+CD25+ Treg cell count in peripheral blood
of patients with SS compared with healthy controls. However, the
inhibitory function of these cells in SS was not different from that
of the control (39). Recent studies have reported a close correlation
between the proportion of Foxp3+ Treg cells in the salivary glands
of patients with SS and the severity of inflammation as well as
certain risk factors for lymphoma development (39, 40). In the
present study, suppressive activity of Treg cells in RORgt Tg mice
was equal to that of the same cells in C57BL/6 mice, and sialadenitis was not observed in Rag22/2 mice inoculated with effector
cells plus Treg cells from RORgt Tg mice. However, we found
sialadenitis in Rag22/2 mice inoculated with effector cells plus
decreased numbers of Treg cells from RORgt Tg mice. These data
provide strong support to the notion that the reduction of Treg cells
in RORgt Tg mice might play a role in the development of sialadenitis.
How can overexpression of RORgt result in suppression of
Foxp3 expression? Judging from the low level of CD25 expression
and the inhibition of IL-2-STAT5 signaling in Treg cells from
Tg mice. Four mice per group were analyzed and representative data of two independent experiments with consistent results are shown. (C) Flow cytometric
analysis of CD25, CTLA4, GITR, CD103, and RORgt in splenocytes from C57BL/6 and RORgt Tg mice. These cells were gated on CD4+CD25+Foxp3+
cells. Data are representative of two independent experiments with four mice per group. (D) Mean fluorescence intensity (MFI) of CD4+CD25+Foxp3+
cells. Data are representative of two independent experiments with four mice per group. (E) Detection of phosphorylation of STAT5 after stimulation with
1 mg/ml IL-2 in CD4+CD25+Foxp3+ cells from C57BL/6-Foxp3GFP or RORgt Tg-Foxp3GFP mice by flow cytometry. The bar graph shows the MFI in CD4+
CD25+Foxp3+ cells. Data are representative of two independent experiments with four mice per group. (F) Quantitative PCR analysis of mRNA expression
level of Socs1 in IL-2–stimulated Treg cells of C57BL/6 and RORgt Tg mice. The experiment was performed in duplicate. Data were normalized to the
expression of the reference gene, Hprt. (G) Cytokine secretion (IFN-g, IL-17, IL-10) in CD4+CD25+Foxp3+ cells from C57BL/6-Foxp3GFP or RORgt TgFoxp3GFP mice stimulated for 72 h with anti-CD3/28 beads. Data are representative of three independent experiments with four mice per group. (H) CFSElabeled CD4+CD25-Foxp32 cells from C57BL/6-Foxp3GFP mice cultured with or without CD4+CD25+Foxp3+ cells from C57BL/6-Foxp3GFP or RORgt
Tg-Foxp3GFP mice and stimulated with anti-CD3/28 beads for 72 h. Data are CFSE gated and representative of three independent experiments with four
mice per group. (I) The bar graph shows the rate of inhibition. *p , 0.05 (Mann–Whitney U test). N.D., detected.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 7. Function of RORgt-overexpressing Treg cells in inhibition of sialadenitis. (A) CD4+CD252GFP2 cells (Teff) (2 3 106 cells) isolated from
C57BL/6-Foxp3GFP or RORgt Tg-Foxp3GFP mice were inoculated into Rag22/2 mice. *p , 0.05 (Mann-Whitney U test). (B) CD4+CD252GFP- cells
(Teff) (2 3 106 cells) isolated from RORgt Tg-Foxp3GFP mice were inoculated into Rag22/2 mice with CD4+CD25+GFP+ cells (Treg) (5 3 105 cells) of
C57BL/6-Foxp3GFP mice or CD4+CD25+GFP+ cells (Treg) (5 3 105 or 5 3 104 [1:10] cells) of RORgt Tg-Foxp3GFP mice. The bar graph shows the
histological score of inflammatory lesions in the salivary glands. At 22 wk postinoculation, the pathology of salivary glands was analyzed. Salivary glands
were sectioned at 4 mm, and each section was stained with H&E. Scale bars, 50 mm. Data are representative of four tissue samples with similar results.
Histological score of inflammatory lesions in the salivary glands of the experiment described. *p , 0.05 (Tukey t test).
66
A PATHOLOGICAL ROLE OF RORgt IN SJÖGREN’S SYNDROME
Disclosures
The authors have no financial conflicts of interest.
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