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of June 15, 2017.
X-linked Foxp3 (Scurfy) Mutation
Dominantly Inhibits Submandibular Gland
Development and Inflammation Respectively
through Adaptive and Innate Immune
Mechanisms
Rahul Sharma, Umesh S. Deshmukh, Lingjie Zheng, Shu
Man Fu and Shyr-Te Ju
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Copyright © 2009 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2009; 183:3212-3218; Prepublished online 31
July 2009;
doi: 10.4049/jimmunol.0804355
http://www.jimmunol.org/content/183/5/3212
The Journal of Immunology
X-linked Foxp3 (Scurfy) Mutation Dominantly Inhibits
Submandibular Gland Development and Inflammation
Respectively through Adaptive and Innate Immune Mechanisms1
Rahul Sharma,* Umesh S. Deshmukh,* Lingjie Zheng,† Shu Man Fu,2*† and Shyr-Te Ju2,3*†
S
jögren’s syndrome is an autoimmune disease affecting the
salivary and lacrimal glands, causing dry eyes and dry
mouth (1). The mechanism that triggers leukocyte infiltration into these organs remains largely unknown. We observed
Sjögren’s syndrome-like disease in Il2r␣⫺/⫺ mice and Il2⫺/⫺
mice; both are partially deficient in the naturally occurring CD4⫹
Foxp3⫹ regulatory T cells (Treg)4 (2– 4). These mice display inflammation in the submandibular gland (SMG) with impaired salivation function (2). Paradoxically, the SMG of Scurfy (Sf) mice
that carry the Foxp3sf mutant gene and are totally devoid of Treg
is not inflamed (2). Added to the complexity are the observations
that transfer of Sf CD4⫹ T cells into Rag1⫺/⫺ recipients induced
strong inflammation in SMG, and that oral application of LPS
induced SMG inflammation in Sf mice (2).
Mouse SMG contains three major components: acini, striated
ducts, and granular convoluted tubules (GCT). The early stages of
SMG development occur soon after birth and are dominated by the
formation of acini, whereas GCT development occurs 2 wk later
*Center for Inflammation, Immunity, and Regenerative Medicine, Department of
Medicine, and †Department of Microbiology, University of Virginia, Charlottesville,
VA 22908
Received for publication December 29, 2008. Accepted for publication July 5, 2009.
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.
1
This work was supported in part by National Institutes of Health Grants AR-051203
(to S.-T.J.), DE-017579 (to S.-T.J.), AR-045222 (to S.M.F.), AR-047988 (to S.M.F.),
AR-049449 (to S.M.F.), and AR051391 (to U.S.D.), and a grant from Sjogren’s Syndrome Foundation (to U.S.D.).
2
S.M.F. and S.-T.J. are co-senior authors who contributed equally to this study.
and continues to adulthood (5). GCT development is sexually dimorphic, i.e., dominantly expressed in male soon after GCT development begins. Dominant GCT expression in female or castrated male can be induced by testosterone (6). Because Foxp3sf
mutation effect is X linked (7), we address whether the SMG development in Sf mice is blocked as a result of reproductive organ
damage and what is the consequence of the SMG growth arrest
with respect to inflammation attack.
In this study, we report an unusual regulation of organ-specific
development and T cell-mediated inflammation uniquely associated with not only the SMG of Sf mice, but also the long-lived
Sf.Il2⫺/⫺, Sf.Il2r␣⫺/⫺, and Sf.Faslpr/lpr mice. We observed that
the SMG development based on GCT expression was severely
blocked in these mice. Importantly, GCT expression in male
Il2⫺/⫺ and Il2r␣⫺/⫺ mice was moderately inhibited, but introducing Foxp3sf gene severely blocked their development and rendered
their SMG resistant to inflammation, whereas SMG in Sf.Rag1⫺/⫺
mice developed normally and became inflamed upon transfer of Sf
lymph node cells. Atrophy and inflammation were observed in the
accessory reproductive organs in long-lived Sf.Faslpr/lpr mice. We
could restore GCT dominance by treating Sf.Faslpr/lpr mice with
testosterone, but their SMG remained free of inflammation. Conversely, daily oral application of LPS to Sf.Faslpr/lpr mice induced
SMG chemokine production and inflammation, but the GCT
growth arrest remained. Thus, the X-linked Foxp3sf gene dominantly inhibits both SMG development and inflammation, but by
different mechanisms through adaptive and innate immunity, respectively. The significance of the study to the etiology, mechanism of action, and the developmental process of SMG inflammation is discussed.
3
Address correspondence and reprint requests to Dr. Shyr-Te Ju, Room 5777, 5th
Floor, Old Medical School building, Hospital Drive, University of Virginia, Charlottesville, VA 22908-0412.
Materials and Methods
Mice
4
Abbreviations used in this paper: Treg, regulatory T cell; GCT, granular convoluted
tubule; Sf, Scurfy; SMG, submandibular gland.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804355
C57BL/6 (B6), B6.Faslpr/lpr, B6.Il2⫹/⫺, B6.Il2r␣⫹/⫺, B6.Cg-Foxp3sf/⫹/J,
and B6.129S7-Rag1tm/Mom/J (Rag1⫺/⫺) mice were obtained from The
Jackson Laboratory. B6.Il2⫺/⫺, B6.Il2r␣⫺/⫺, Sf mice (Foxp3sf/Y), and
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Scurfy (Foxp3Sf/Y), Il2ⴚ/ⴚ, and Il2r␣ⴚ/ⴚ mice are deficient in CD4ⴙFoxp3ⴙ regulatory T cells (Treg), but only the latter two develop
inflammation in the submandibular gland (SMG), a critical target of Sjögren’s syndrome. In this study, we investigated the reason that
SMG of Scurfy (Sf), Sf.Il2ⴚ/ⴚ, Sf.Il2r␣ⴚ/ⴚ, and the long-lived Sf.Faslpr/lpr mice remained free of inflammation, even though their lymph
node cells induced SMG inflammation in Rag1ⴚ/ⴚ recipients. A strong correlation was observed between the development of the
granular convoluted tubules (GCT) of the SMG in these mice and SMG resistance to inflammation. Moreover, GCT development in
Sf.Rag1ⴚ/ⴚ mice was not impeded, indicating a role of adaptive immunity. In the Sf.Faslpr/lpr mice, this block was linked to atrophy and
inflammation in the accessory reproductive organs. Testosterone treatment restored GCT expression, but did not induce SMG inflammation, indicating GCT is not required for inflammation and additional mechanisms were controlling SMG inflammation. Conversely,
oral application of LPS induced SMG inflammation, but not GCT expression. LPS treatment induced up-regulation of several chemokines in SMG with little effect on the chemokine receptors on CD4ⴙ T cells in Sf mice. Our study demonstrates that Sf mutation affects
SMG development through adaptive immunity against accessory reproductive organs, and the manifestation of SMG inflammation in
Sf mice is critically controlled through innate immunity. The Journal of Immunology, 2009, 183: 3212–3218.
The Journal of Immunology
Sf.Il2⫺/⫺ mice were generated, as previously described (8, 9). Mice
(Sf.Il2r␣⫺/⫺) carrying both Il2r␣⫺/⫺ and Foxp3sf/Y genes were generated
by breeding B6.Il2r␣⫹/⫺ males with B6.Il2r␣⫹/⫺Foxp3sf/⫹ females. Mice
carrying both of the Foxp3sf and Rag1⫺/⫺ genes (Sf.Rag1⫺/⫺) were generated by breeding Rag1⫺/⫺ male with B6.Cg-Foxp3sf/⫹/J mice, followed
by breeding Rag1⫹/⫺Foxp3sf/⫹ female progeny with Rag1⫺/⫺ male. Presence of the Il2⫺/⫺, Il2r␣⫺/⫺, Foxp3sf, Faslpr/lpr, and Rag1⫺/⫺ mutation was
confirmed by PCR, as detailed in The Jackson Laboratory’s website. Mice
were examined twice weekly for clinical signs of disease, including skin
inflammation, body weight loss, wasting, etc. All animal experiments were
approved by the Animal Care and Use Committee of the University of
Virginia.
Treatment of mice
Histology
Tissues/Organs were fixed with 10% neutral buffered formalin (Fisher Scientific). Sections of paraffin-embedded samples were stained with H&E
and examined under microscope.
Semiquantification of inflammation and GCT expression
The extent of leukocyte infiltration in tissue sections was examined and
categorized into five groups, from 0 to 4, to indicate, respectively, the
degree of inflammation as none (0 infiltration lesion), mild (1 lesion),
moderate (2 lesions), strong (3– 4 lesions), and severe (⬎4 lesions). The
overall inflammation index of samples of a group of mice was calculated as mean ⫾ SD. For quantification of GCT, images of H&E-stained
SMG sections were acquired using Olympus BX51 microscope
equipped with a digital camera at ⫻100 magnifications. Images of two
random optical fields from at least three different mice of each strain
were captured. The images were then analyzed using Kodak 1D image
analysis software (Eastman Kodak), and the area occupied by the GCT
was marked using the region of interest tool. The total area occupied by
the GCT in each image was expressed as percentage of area occupied
by GCT.
Chemokines and chemokine receptor analyses
SMG were removed from Sf mice treated either with LPS or PBS and
collected in RNA later reagent (Qiagen). Pieces of SMG were suspended
in RLT buffer (Qiagen) and homogenized using TissueLyser system (Qiagen). Total RNA from homogenate was purified using RNEasy minikit
(Qiagen) followed by first-strand cDNA synthesis using the QuantiTect
reverse transcription kit (Qiagen). The expression levels of CCL2, CCL3,
CCL4, CCL5, CCL9, CXCL2, CXCL10, and CXCL12 were determined by
real-time PCR on MyiQ machine (Bio-Rad) using the Taqman gene expression assays (Applied Biosystems). A RNA sample from the SMG of a
normal C57BL/6 mouse was used as a calibration control. This sample was
chosen because its cytokine expression pattern closely represents the average pattern obtained from five normal samples. The data are represented
as fold change in gene expression over the B6 calibrator. Statistical significance of variance was determined by the nonparametric Mann-Whitney
U test using GraphPad Prism software. A value of p ⬍ 0.05 was considered
statistically significant.
Flow cytometric analysis
Flow cytometric staining for various chemokine receptors was conducted
on the draining lymph nodes of SMG in Sf mice treated with PBS and LPS.
Age-matched, male B6 mice were included for comparison. Lymphocytes
were stained with anti-CD4 mAb along with Abs specific to CCR3 (TG14/
CCR3), CCR5 (HM-CCR5), CXCR2 (TG11/CXCR2), and CXCR3
(CXCR3-173) (Biolegend). The expression of chemokine receptors on
gated CD4⫹ T cells was presented.
Results
Foxp3Sf gene dominantly suppressed SMG inflammation in male
Il2⫺/⫺ and Il2r␣⫺/⫺ mice
Il2⫺/⫺ and Il2r␣⫺/⫺, but not Sf mice developed spontaneous inflammation in SMG even though Sf mice contained T cells capable
of inducing SMG inflammation in Rag1⫺/⫺ recipients (2). To determine whether the resistance is inherently associated with
Foxp3sf/Y mice, we introduced Foxp3sf gene into male Il2⫺/⫺ and
Il2r␣⫺/⫺ mice to generate Sf.Il2⫺/⫺ and Sf.Il2r␣⫺/⫺ mice, respectively. In all cases, these mice lived significantly longer than Sf mice,
but their SMG remained free from inflammation (8). By contrast,
inflammation in colon normally observed in Il2⫺/⫺ and Il2r␣⫺/⫺
mice remained (Fig. 1A).
We transferred lymph node cells from Sf and Sf.Il2⫺/⫺ mice
to determine whether they contained competent cells capable of
inducing SMG inflammation in Rag1⫺/⫺ recipients. In addition,
we also tested Sf.Faslpr/lpr mice because these mice lived more
than 15 wk and their SMG remained free of inflammation (see
Fig. 4A). All induced moderate/strong inflammation in the SMG
of the recipients (Fig. 1B). Taken together, these results indicate
that the SMG resistance to inflammation in Sf, Sf.Il2⫺/⫺, and
Sf.Faslpr/lpr mice occurs even in the presence of competent inflammation-inducing T cells, and this resistance is inherently
and dominantly associated with Foxp3sf in male mice in an
organ-specific manner, i.e., inflammation is inhibited in SMG,
but not colon.
Dominant resistance coincided with inhibition of SMG
development
We compared the SMG expression level between male and female
Il2⫺/⫺ and Il2r␣⫺/⫺ mice that were 8 wk old using the semiquantification method. B6 mice were used as control. As shown in Fig.
2A, a strong age-dependent expression of GCT was observed in B6
male as opposed to the weak expression of GCT with less granule
content in B6 female. The SMG of Il2⫺/⫺ and Il2r␣⫺/⫺ mice also
displayed sexual dimorphism, but the GCT in male Il2⫺/⫺ and
Il2r␣⫺/⫺ mice was less than age-matched B6 male. This could be
caused by SMG inflammation that induces GCT atrophy (Fig. 2B)
(2). By contrast, the SMG of Sf.Il2⫺/⫺ and Sf.Il2r␣⫺/⫺ mice was
severely underdeveloped with fewer and smaller GCT than male
Il2⫺/⫺ and Il2r␣⫺/⫺ mice (Fig. 2B). Indeed, highly statistically
significant differences in GCT expression levels between Il2⫺/⫺
and Sf.Il2⫺/⫺ mice and between Il2r␣⫺/⫺ and Sf.Il2r␣⫺/⫺ mice
were observed (Fig. 2C). Unlike Il2⫺/⫺ and Il2r␣⫺/⫺ mice, the
SMG growth arrest in Sf.Il2⫺/⫺ and Sf.Il2r␣⫺/⫺ mice occurred in
the absence of SMG inflammation, indicating that the inhibition of
GCT growth in this case is not caused by local inflammation, but
rather the consequence of the early systemic inflammatory response. In support of this, we observed SMG sexual dimorphism
between Sf.Rag1⫺/⫺ male and Sf.Rag1⫺/⫺ female; both lacked a
functional adaptive immune response (Fig. 2D). Moreover, transfer of Sf lymph node cells into male Sf.Rag1⫺/⫺ recipients readily
induced SMG inflammation (Fig. 2E). These results indicate that
Foxp3sf/Y indirectly controls SMG development and SMG inflammation through the adaptive immune system.
Foxp3 expression has been demonstrated in the epithelial cells
of mammary and prostate glands (10). Foxp3 expression in the
SMG has not been determined. The facts that SMG of Sf.Rag1⫺/⫺
mice displays sexual dimorphism for GCT expression and becomes inflamed upon transfer of Sf lymph node cells indicate that
Foxp3, if expressed in SMG per se, does not play a role in the
regulation of SMG development and the SMG resistance to inflammation-inducing T cells.
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Adoptive transfer experiments were conducted by i.v. injection of 15 ⫻ 106
lymph node cells into adult Rag1⫺/⫺ male recipients. Various organs and
tissues were harvested at 4 –12 wk after transfer. Testosterone treatment
was conducted on 4-wk-old Sf.Faslpr/lpr mice by twice weekly s.c. injection
of 25 ␮l of testosterone oenanthate (Sigma-Aldrich) solution (2 mg/ml in
corn oil) or vehicle control. Various organs and tissues were harvested after
3 wk of treatment. Sf.Faslpr/lpr mice and control littermates (4 wk old) were
also treated by daily oral feeding of LPS or poly(I:C) solution (10 ␮l of
1 mg/ml, one in the morning and one in the evening; Sigma-Aldrich).
Various organs and tissues were harvested 3 wk later, H&E stained, and
examined under microscope.
3213
3214
ORGAN (SMG)-SPECIFIC DEVELOPMENT AND INFLAMMATION
Sf mice display inflammation damage in reproductive organs
Whether GCT growth arrest was due to a response of SMG to the
systemic inflammation or lack of testosterone resulting from the
inflammation of the reproductive organs was investigated. The latter possibility was considered because Sf.Faslpr/lpr, despite living
beyond 15 wk, remained reproductive incompetent. We examined
the gross structure and histological features of the reproductive
organ of 6- to 8-wk-old Sf.Faslpr/lpr mice with age-matched B6
male (Fig. 3). In gross examination, we observed tremendous atrophy in the various accessory reproductive organs of the Sf.
Faslpr/lpr mice, but not of the B6 male. These included coagulation
glands/seminal vesicle (black arrows, top panel), preputial glands
(red arrowheads), epididymis (black arrowheads), and prostate
(Fig. 3, row 3). Histological examination confirmed the atrophy of
these organs, displayed as shrunken glands and empty lumens.
This was accompanied with a prominent presence of leukocytic
infiltrates in the periglandular regions (green arrowheads). Leukocytic infiltrates could also be observed in the interstitial regions of the testis and in the regions containing the interstitial
cells of the Leydig between the seminiferous tubules (green
arrowheads). The data suggest that the inflammation-induced
atrophy of the reproductive organs was responsible for the arrest of GCT development. Because testosterone is the major hormone responsible for the development of GCT and reproductive organs, we treated Sf.Faslpr/lpr mice with the long-lasting testosterone
oenanthate, as described in Materials and Methods (6, 11). Testosterone successfully restored the growth of the accessory reproductive organs of Sf.Faslpr/lpr mice (Fig. 3, right column), but the
leukocytic infiltrates in the reproductive organs were still present.
Thus, the potential anti-inflammatory effect of testosterone is unlikely the reason for the restoration of growth and development of
inflamed organs. The data suggest that atrophy of the reproductive
organs was caused by the lack of testosterone resulted from inflammation in the reproductive organs. However, restoration of
organ development by the injected testosterone could not reverse
the inflammation.
Dissociation of GCT growth from SMG inflammation
Testosterone treatment also fully restored the dominant GCT expression in the SMG of Sf.Faslpr/lpr mice (Fig. 4A). Both the number of GCT and the granule content were restored to the level of
normal B6 male. The observation supports the interpretation that
the lack of testosterone from the atrophic reproductive organs was
responsible for the GCT growth arrest. Despite a fully restored
SMG, the SMG remained free of inflammation, suggesting GCT is
not required for SMG inflammation. The treatment did not inhibit
inflammation in other organs normally observed in Sf.Faslpr/lpr
mice, such as skin, lungs, and the accessory reproductive organs
(Fig. 3) (8).
In the second approach, we treated 4-wk-old Sf.Faslpr/lpr mice
with LPS that has been shown to induce SMG inflammation in Sf
mice (2). The latter experiment treated 1-wk-old Sf mice, and
SMG inflammation was determined 2 wk later, i.e., at 3 wk old
when normal GCT expression was not obvious. In the present experiment, SMG inflammation was determined at 8 wk old. In addition, we also treated these mice with a different TLR agonist
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FIGURE 1. Foxp3sf/Y dominantly suppressed SMG inflammation in Il2⫺/⫺ and Il2r␣⫺/⫺ mice. A, Sf, Il2⫺/⫺, Il2r␣⫺/⫺, Sf.Il2⫺/⫺, and Sf.Il2r␣⫺/⫺ mice
were generated, as previously described (8). Samples of SMG and colon were processed, H&E stained, and examined under microscope. Normal B6 male
were included as control. Samples were obtained from 6- to 10-wk-old mice, except that of Sf mice were obtained from 21-day-old mice. The extent of
leukocyte infiltration in tissue sections was examined and categorized into five groups, from 0 to 4, to indicate, respectively, the degree of inflammation
as none (0 infiltration lesion), mild (1 lesion), moderate (2 lesions), strong (3– 4 lesions), and severe (⬎4 lesions). The overall inflammation index of samples
of a group of mice (3– 6 mice) was calculated as mean ⫾ SD. B, Adoptive transfer (i.v.) of 15 ⫻ 106 lymph node cells from Sf, Sf.Il2⫺/⫺, and Sf.Faslpr/lpr
mice induces inflammation in the SMG of Rag1⫺/⫺ recipients examined 4 wk later. The data presented are representative of two experiments; each involved
two to three recipients. Arrowheads indicate areas of inflammation.
The Journal of Immunology
3215
poly(I:C). A strong inflammation was induced by both treatments,
but the GCT expression of the treated mice remained inhibited
(Fig. 4B and data not shown). The two approaches provided reciprocal evidence that dissociates GCT growth inhibition from resistance to inflammation attack.
Several chemokine receptors/integrins are strongly up-regulated
on the CD4⫹ T cells in Sf mice (12). Yet, these T cells failed to
enter the SMG. We hypothesize that LPS can directly act on SMG
by increasing their chemokine production. This change sensitizes
the SMG to the inflammation-inducing T cells already present in
the Sf mice. We analyzed the chemokine expression pattern of the
SMG of control Sf mice and those treated with LPS using quantitative RT-PCR. We chose eight chemokines, including those that
are known to be up-regulated upon LPS stimulation (13). Four
chemokines, CCL3, CCL5, CXCL2, and CXCL10, were significantly increased in the LPS-treated Sf mice as compared with
PBS-treated control (Fig. 4C). Although the expression levels of
CCL2, CCL4, CCL9, and CXCL12 were not significantly up-reg-
ulated, additional samples are needed to firmly determine whether
this is really the case with CCL4. These data demonstrated that
LPS induced a direct change in the SMG of Sf mice and the underdeveloped SMG in Sf mice is capable of responding to LPS by
producing chemokines.
We then analyzed CD4⫹ T cells of the SMG-draining lymph
nodes of these mice for the expression of chemokine receptors
specific to the up-regulated chemokines. The expression of chemokine receptors targeted by CCL3, CCL5, CXCL2, and CXCL10
showed considerable variability, and the differences between PBStreated and LPS-treated Sf mice were not significant (Fig. 4D).
Although a strong trend of increase in their expression was observed when compared with B6 controls, the difference was not
significant, except for CXCR2. Apparently, the overall mild increase in these trafficking/retention receptors on CD4⫹ T cells of
Sf mice was not sufficient to induce inflammation unless increase
in chemokine production in SMG was achieved by LPS treatment.
These observations provided evidence that LPS acted on SMG
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FIGURE 2. Dominant SMG resistance to inflammation coincided with inhibition of sexual dimorphic GCT dominance. A, The SMG of 3- and 8-wk-old
B6 mice, both male and female, were H&E stained and examined for GCT expression to determine the sexual dimorphic expression of GCT. GCT (gct),
but not acini (Ac), were eosinophilic and strongly stained pinkish. An age-dependent and dominant GCT display in 8-wk-old male was observed. B, The
SMG of 8-wk-old Il2⫺/⫺, Il2r␣⫺/⫺, Sf.Il2⫺/⫺, and Sf.Il2r␣⫺/⫺ mice was examined for GCT expression. The sexual dimorphic display of GCT in Il2⫺/⫺
and Il2r␣⫺/⫺ mice was not as strong as that in B6, but GCT expression in Sf.Il2⫺/⫺ and Sf.Il2r␣⫺/⫺ mice was severely inhibited in comparison. Arrowheads
indicate areas of inflammation. The data presented are representative of three to six mice for each group. C, GCT expression levels in various groups of
mice were semiquantified, as described in Materials and Methods. Statistical analyses between groups using Student’s t test were shown. D, Foxp3sf/Y does
not directly affect SMG development. The SMG of 10-wk-old Sf.Rag1⫺/⫺ mice was H&E stained and examined under microscope. Dominant GCT
expression was observed in male over female (n ⫽ 3). E, The SMG of Sf.Rag1⫺/⫺ male is not resistant to inflammation. Adult Sf.Rag1⫺/⫺ male (n ⫽ 4)
were transferred i.v. with 15 ⫻ 106 lymph node cells from Sf mice. Various organs/tissue were collected 6 wk later, stained, and examined under
microscope. Severe SMG inflammation was observed after transfer. Inflammation was also observed in skin, lung, liver, stomach, small intestine, and colon
(data not shown) (12).
3216
ORGAN (SMG)-SPECIFIC DEVELOPMENT AND INFLAMMATION
chemokine production and induced inflammation without affecting
GCT development.
Discussion
Our study provides evidence for the following scheme for SMG
development and inflammation response. The genetic defect in
Sf mice results in the inhibition of two critical components of
the process: one required for GCT expression, and one for the
susceptibility to inflammation; both depended on a functional
immune system. A consequence of the severe and early systemic inflammatory response in Sf mice is the inhibition of
GCT-inducing agents such as testosterone by the adaptive immunity. Testosterone treatment induced GCT overexpression,
but not the component controlling susceptibility to inflammation-inducing T cells. The reciprocal result, obtained with LPS
and poly(I:C) treatments, demonstrated that TLR agonist induced SMG inflammation, but not GCT development in Sf
mice. Evidence provided strongly suggests that the inflammation in the underdeveloped SMG is limited by its innate immune
response that can be overcome by TLR agonists through chemokine induction.
We have previously shown that daily LPS application induces
SMG inflammation in Sf mice (2). In the present study, daily LPS
application to the long-lived Sf.Faslpr/lpr mice was also able to
induce SMG inflammation, indicating that although the key defect
in the induction of SMG inflammation is long-lasting, the ability to
break it by LPS remained as well. In addition, an agonist for a
different TLR also induced SMG inflammation in these mice. Although multiple i.p. injections of these agonists into adult (New
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FIGURE 3. Inflammation in accessory reproductive organs. Accessory
reproductive organs from B6, Sf.Faslpr/lpr, and testosterone-treated Sf.
Faslpr/lpr were removed and photographed (top panels). Black arrows,
black arrowheads, and red arrowheads indicate coagulation glands/seminal
vesicles, epididymis, and preputial glands, respectively. H&E-stained sections of seminal vesicles, prostate, epididymis, and testis were examined
under microscope (⫻200). Green arrowheads indicate areas of leukocytic
infiltrate.
Zealand White ⫻ New Zealand White)F1 mice activate innate immunity in the SMG (14), we chose oral applications to neonates
for ease of application and less injury. It has been shown that LPS
directly affects various organs through circulation (15). We
showed that LPS treatment up-regulated the production of several
chemokines in the SMG of Sf mice, thereby indicating that oral
applications of LPS can act on the SMG of Sf mice and induce
biological changes. In contrast, the corresponding chemokine receptors on CD4⫹ T cells were not up-regulated upon LPS treatment. These observations strongly suggest that the neonatal and
underdeveloped SMG in Sf mice is limited in its ability to produce
sufficient chemokines required for the attraction of inflammationinducing CD4⫹ T cells to cause SMG pathology and salivation
dysfunction.
As compared with normal B6 male, the SMG of the sex- and
age-matched Il2⫺/⫺ and Il2r␣⫺/⫺ mice are underdeveloped, but
not severely growth arrested as the SMG of Sf.Il2⫺/⫺ and
Sf.Il2r␣⫺/⫺ mice. Unlike Sf or Sf.Faslpr/lpr mice, Il2⫺/⫺ and
Il2r␣⫺/⫺ mice still contain a significant level of Treg. In addition,
they lack IL-2 and IL-2R␣, respectively. Thus, the inflammation
process in Il2⫺/⫺ and Il2r␣⫺/⫺ mice is reduced and delayed as
compared with the corresponding Sf.Il2⫺/⫺ and Sf.Il2r␣⫺/⫺ mice.
Like Sf.Il2⫺/⫺ and Sf.Il2r␣⫺/⫺ mice, Il2⫺/⫺ and Il2r␣⫺/⫺ male
are reproductive incompetent with light body weight and probably
lack normal level of testosterone, but they live significantly longer
than Sf.Il2⫺/⫺ and Sf.Il2r␣⫺/⫺ mice. These factors could account
for the weak inhibition of SMG development in male Il2⫺/⫺ and
Il2r␣⫺/⫺ mice. In this regard, the relatively more advanced SMG
development in these mice with appropriate environmental
changes may allow sufficient chemokine production to attract inflammation-inducing CD4⫹ T cells to SMG and induce inflammation as the mice lived beyond weaning to adult age.
Several hormones can induce dominant GCT overexpression
in female or castrated male (5, 6). We examined the long-lived
Sf.Faslpr/lpr mice and observed dramatic atrophy and significant
inflammation in many of the accessory reproductive organs.
Specifically, severe atrophy was observed in the seminal vesicle, prostate, and epididymis with varying degree of inflammation. Spontaneous inflammation in multiple male accessory reproductive organs is rare, and our study showed for the first
time that it does indeed develop in Sf.Faslpr/lpr mice that lived
beyond weaning and reach adult age. This finding strongly suggests that testosterone is the main reason that Sf mice fail to
display a normal male dominant presentation of the GCT. Indeed, our testosterone treatment fully restored GCT development, but the SMG remained free of inflammation. Conversely,
testosterone treatment also restored the development of the accessory reproductive organs, but the inflammation remained.
This result dissociates the testosterone-dependent organ development from inflammation induced by Treg deficiency in both
directions, i.e., inflammation in accessory reproductive organs
or lack of inflammation in SMG was maintained regardless of
testosterone-induced organ development. This result also suggests that lacking GCT is not the reason for the inability of Sf
mice to develop SMG inflammation.
Despite totally lacking Treg, many of the organs/tissues such
as CNS, joints, endocrine, and mucosal organs are free of inflammation in Sf mice despite the fact they have a large functional inflammatory repertoire (16, 17). In this regard, the
present study has implication toward the genetic control of regulatory mechanisms involved in the multiorgan inflammation in
Sf mice. In contrast to the Foxp3Sf/y-dependent regulation of
SMG inflammation in Sf.Il2⫺/⫺ mice, the Il2⫺/⫺ defect conversely inhibited the skin and lung inflammation in Sf mice, but
The Journal of Immunology
3217
not the liver inflammation and colitis associated with Il2⫺/⫺
mice, i.e., Sf.Il2⫺/⫺ mice do not develop skin and lung inflammation (12). This apparent organ-specific control of inflammation is in fact mediated by different mechanisms. It turns out
that IL-2 is required for the expression of a panel of receptors
involved in the trafficking/homing and retention of the pathogenic CD4⫹ T cells to skin and lungs. Among the receptors that
are highly up-regulated are cysteinyl leukotriene receptor 1,
leukotriene B4 receptor 1, CCR8, CXCR6, IL-1R-like1, and
CD103 of the ␣E␤7 (12). We have extensively characterized the
role of IL-2 in the up-regulation of CD103 of the ␣E␤7 integrin
on CD4⫹ T cells and their retention in the skin and lungs. The
fact that these pathogenic CD4⫹ T cells failed to infiltrate SMG
of Sf mice suggests either TLR agonists induce the ligands for
these receptors for the infiltration of the T cells or that a different set of chemokine receptors and/or integrins is involved.
The result of LPS treatment suggests that the latter case is most
likely a possible mechanism. However, whether LPS induces
the specific ligands for each of these up-regulated receptors has
not been exhaustively determined. Additional study is needed to
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FIGURE 4. Dissociation of GCT
expression from SMG inflammation
and the effect of LPS treatment on
chemokine production and chemokine
receptor expression. A, Sf.Faslpr/lpr
mice (4 wk old) were treated with testosterone oenanthate by weekly s.c.
injection of 25 ␮l of testosterone oenanthate solution, as described in Materials and Methods. Various organs
and tissues were harvested 3 wk later,
H&E stained, and examined under
microscope. GCT expression was
strongly induced, but SMG remained
free of inflammation. B, Sf.Faslpr/lpr
mice (4 wk old) and littermates were
fed with 10 ␮l of LPS or poly(I:C), as
described in Materials and Methods.
Various organs were collected 1 day
after the last application. They were
stained and examined under microscope. Only the data of SMG were
presented. The inflammation status of
other organs/tissues was not changed
by the treatment. The data presented
are representative of two experiments,
and three mice were used in each experiment. C, Chemokine expression
patterns in the SMG of LPS-treated or
PBS-treated Sf mice (n ⫽ 4) were
determined by quantitative RT-PCR,
as described in Materials and Methods. D, Flow cytometric analysis of
chemokine receptor expression on
CD4⫹ T cells from the SMG-draining lymph nodes of LPS-treated and
PBS-treated Sf mice (n ⫽ 3). Agematched B6 male mice were included for comparison. ⴱ, Indicates
p ⬍ 0.02; ⴱⴱ, p ⬍ 0.05.
establish the exact mechanism by which SMG inflammation is
induced by LPS treatment.
Acknowledgments
We thank C. Abaya and Angela Ju for their technical assistance and Dr. K.
Tung for his critical review of the manuscript.
Disclosures
The authors have no financial conflict of interest.
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