Group Title: Arthritis Research & Therapy
Title: Identification of possible candidate genes regulating Sjögren's syndrome-associated autoimmunity : a potential role for TNFSF4 in autoimmune exocrinopathy
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Title: Identification of possible candidate genes regulating Sjögren's syndrome-associated autoimmunity : a potential role for TNFSF4 in autoimmune exocrinopathy
Physical Description: Book
Language: English
Creator: Nguyen, Cuong
Cornelius, Janet
Cooper, Lauren
Neff, Jonathan
Tao, Joann
Lee, Byung
Peck, Ammon
Publisher: Arthritis Research and Therapy
Publication Date: 2008
 Notes
Abstract: INTRODUCTION:Sjögren syndrome (SjS) is a systemic autoimmune disease in which an immunological attack primarily against the salivary and lacrimal glands results in the loss of acinar cell tissue and function, leading to stomatitis sicca and keratoconjunctivitis sicca. In recent years, two genetic regions, one on chromosome 1 (designated autoimmune exocrinopathy 2 or Aec2) and the second on chromosome 3 (designated autoimmune exocrinopathy 1 or Aec1) derived from nonobese diabetic (NOD) mice, have been shown to be necessary and sufficient to replicate SjS-like disease in nonsusceptible C57BL/6 mice.METHODS:Starting with the SjS-susceptible C57BL/6-derived mouse, referred to as C57BL/6.NOD-Aec1Aec2, we generated a large set of recombinant inbred (RI) lines containing portions of Aec2 as a means of identifying more precisely the genetic elements of chromosome 1 responsible for disease development.RESULTS:Disease profiling of these RI lines has revealed that the SjS susceptibility genes of Aec2 lie within a region located at approximately 79 ± 5 cM distal to the centromere, as defined by microsatellite markers. This chromosomal region contains several sets of genes known to correlate with various immunopathological features of SjS as well as disease susceptibility genes for both type 1 diabetes and systemic lupus erythematosus in mice. One gene in particular, tumor necrosis factor (ligand) superfamily member 4 (or Ox40 ligand), encoding a product whose biological functions correlate with both physiological homeostasis and immune regulations, could be a potential candidate SjS susceptibility gene.CONCLUSIONS:These new RI lines represent the first step not only in fine mapping SjS susceptibility loci but also in identifying potential candidate SjS susceptibility genes. Identification of possible candidate genes permits construction of models describing underlying molecular pathogenic mechanisms in this model of SjS and establishes a basis for construction of specific gene knockout mice.
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Research article
Identification of possible candidate genes regulating Sj6gren's

syndrome-associated autoimmunity: a potential role for TNFSF4

in autoimmune exocrinopathy
Cuong Q Nguyen1, Janet G Cornelius2, Lauren Cooper1, Jonathan Neff1, Joann Tao1,
Byung Ha Lee1 and Ammon B Peck1,2,3


1Department of Oral Biology, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610, USA
2Department of Pathology, Immunology & Laboratory Medicine, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610, USA
3Center for Orphan Autoimmune Disorders, College of Dentistry, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610, USA

Corresponding author: Cuong Q Nguyen, Nguyen@pathology.ufl.edu

Received: 26 Aug 2008 Revisions requested: 23 Oct 2008 Revisions received: 27 Oct 2008 Accepted: 25 Nov 2008 Published: 25 Nov 2008

Arthritis Research & Therapy 2008, 10:R137 (doi:10.11 86/ar2560)
This article is online at: http://arthritis-research.com/content/1 0/6/R137
C 2008 Nguyen et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract


Introduction Sj6gren syndrome (SjS) is a systemic autoimmune
disease in which an immunological attack primarily against the
salivary and lacrimal glands results in the loss of acinar cell
tissue and function, leading to stomatitis sicca and
keratoconjunctivitis sicca. In recent years, two genetic regions,
one on chromosome 1 (designated autoimmune exocrinopathy
2 or Aec2) and the second on chromosome 3 (designated
autoimmune exocrinopathy 1 or Aeci) derived from nonobese
diabetic (NOD) mice, have been shown to be necessary and
sufficient to replicate SjS-like disease in nonsusceptible
C57BL/6 mice.

Methods Starting with the SjS-susceptible C57BL/6-derived
mouse, referred to as C57BL/6.NOD-Aec1Aec2, we generated
a large set of recombinant inbred (RI) lines containing portions
of Aec2 as a means of identifying more precisely the genetic
elements of chromosome 1 responsible for disease
development.


Introduction
Sjdgren syndrome (SjS) is a chronic, systemic, human autoim-
mune disease in which an immunological attack initially against
the salivary and lacrimal glands results, respectively, in dry
mouth (stomatitis sicca) and dry eye (keratoconjunctivitis
sicca) diseases) [1-3]. Despite efforts to define the genetic,
environmental, and immunological bases of SjS, the underly-


Results Disease profiling of these RI lines has revealed that the
SjS susceptibility genes of Aec2 lie within a region located at
approximately 79 5 cM distal to the centromere, as defined by
microsatellite markers. This chromosomal region contains
several sets of genes known to correlate with various
immunopathological features of SjS as well as disease
susceptibility genes for both type 1 diabetes and systemic lupus
erythematosus in mice. One gene in particular, tumor necrosis
factor (ligand) superfamily member 4 (or Ox40 ligand),
encoding a product whose biological functions correlate with
both physiological homeostasis and immune regulations, could
be a potential candidate SjS susceptibility gene.
Conclusions These new RI lines represent the first step not only
in fine mapping SjS susceptibility loci but also in identifying
potential candidate SjS susceptibility genes. Identification of
possible candidate genes permits construction of models
describing underlying molecular pathogenic mechanisms in this
model of SjS and establishes a basis for construction of specific
gene knockout mice.


ing etiology of this disease remains ill defined. In attempts to
better define the nature of SjS autoimmunity, a variety of
mouse models exhibiting various aspects of SjS have been
studied extensively [4]. One of the more intensively studied
models of SjS is the nonobese diabetic (NOD) mouse [5-9].
Based on disease profiling of various congenic partners and
gene knockout lines of NOD, we have proposed that the


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Aec: autoimmune exocrinopathy; ANA: anti-nuclear autoantibody; IL: interleukin; INF-y: interferon-gamma; LF: lymphocytic foci; MHC: major histocom-
patibility complex; NOD: nonobese diabetic; PBS: phosphate-buffered saline; QTL: quantitative trait loci; RAR: retinoic acid receptor; RI: recombinant
inbred; RXR: retinoid x receptor; SjS: Sj6gren syndrome; SOAT-1: sterol O-acyltransferase-1; TNFSF4: tumor necrosis factor ligand superfamily
member 4; Treg: T regulatory.







Arthritis Research & Therapy Vol 10 No 6 Nguyen et al.



development and onset of SjS-like disease in these mice can
be divided into at least three distinct consecutive phases [10-
19]. In phase 1, a number of aberrant physiological and bio-
chemical activities, thought to result from a genetically based
retarded salivary gland organogenesis and increased acinar
cell apoptosis, occur prior to and independent of detectable
autoimmunity. In phase 2, believed to result from the glandular
cell injury of phase 1, small numbers of macrophages and den-
dritic cells are attracted to the exocrine gland where these
sentinel cells recruit T and B lymphocytes that form lym-
phocytic foci (LF), some of which histologically appear as ger-
minal centers. In phase 3, the onset of clinical disease as
defined by salivary and lacrimal gland secretary dysfunction
occurs, possibly resulting first from the production of autoan-
tibodies that interfere with the neural-acinar cell signaling path-
ways and then from progressive loss of acinar cell mass
hastened by the action of effector T cells.

A genetic predisposition for development and onset of SjS-like
disease in NOD mice has also been defined. First, SjS-like dis-
ease in these mice appears independent of or only weakly
associated with major histocompatibility complex (MHC) class
I and class II genes [10,20], thus mimicking SjS in humans.
This can be seen by the fact that the congenic strain,
NOD.B10-H2b, in which the NOD MHC I-Ag7Iddl diabetes
susceptibility locus was replaced by the MHC I-Ab locus [20],
continued to show SjS-like disease, including salivary and lac-
rimal gland dysfunction. Second, replacing Idd loci other than
Iddl (for example, Idd9, Idd10, and Idd13) resulted in the
identification of Idd3 on chromosome 3 and Idd5 on chromo-
some 1 as critical genetic regions for development of SjS-like
disease in NOD mice [10]. In a reverse approach, introducing
both Idd3 and Idd5 derived from NOD mice into SjS-nonsus-
ceptible C57BL/6 mice resulted in a severe SjS-like disease,
confirming the contributions of these two genetic loci to the
development and onset of SjS [21]. Furthermore, the preclini-
cal nonimmune aspects manifested in phase 1 of the disease
appeared to associate with the Idd5 locus (referred to as
autoimmune exocrinopathy 2 or Aec2), whereas the immuno-
logical aspects of the disease manifested in phases 2 and 3 of
the disease appeared to associate with Idd3 (referred to as
Aecl). This recently generated mouse strain is referred to as
C57BL/6.NOD-AeclAec2. While the pathophysiological and
immunological aspects may not be linked solely to one or the
other genetic region (as originally proposed [22]), the com-
plete disease profile requires genes within both of these
genetic loci.

For years, identification of candidate genes associated with
autoimmune diseases such as T1 D [23] or systemic lupus ery-
thematosus [24] in animal models has been providing invalua-
ble data on delineating the genetic components of these
diseases, now translating to the human disease. These studies
have formed a template for our current efforts to identify the
SjS susceptibility loci and candidate genes underlying SjS


which, in this respect, have lagged behind many other autoim-
mune diseases. Although our initial work defined the Aeci and
Aec2 genetic regions present in C57BL/6.NOD-AeclAec2
mice as being an approximately 48.5-cM centromeric region
on chromosome 3 and an approximately 73.3-cM telomeric
region on chromosome 1, respectively [10], the size of these
regions precluded identification of candidate genes. Subse-
quently, we shortened Aeci to an approximately 19.2-cM
region in the first studied recombinant inbred (RI) line, C57BL/
6.NOD-AeclR01Aec2 [9]. For the present study, we gener-
ated a set of new RI lines that further demarcate the bounda-
ries of Aec2. These new C57BL/6.NOD-Aec1Aec2R(n) RI
lines identify not only a much shorter Aec2 sublocus at posi-
tion 79 cM of chromosome 1, but also potential candidate SjS
susceptibility genes on which to build hypothetical models that
can be tested for validating possible pathogenic molecular
mechanisms of SjS-like disease.

Materials and methods
Animals
C57BL/6.NOD-Aec1Aec2R(n) and C57BL/6.NOD-
AeclR(n)Aec2R(n) mice were generated by crossing
C57BL/6.NOD-AeclAec2 mice with C57BL/6J mice pur-
chased from The Jackson Laboratory (Bar Harbor, ME, USA).
The F1 heterozygotes were screened for the presence of
crossover events within the Aeci and/or Aec2 genetic regions
by microsatellite marker genotyping. Individual mice indicating
a crossover in Aec2 were bred with a C57BL/6J mouse to pro-
duce Aec2 crossover heterozygous male and female offspring
that were then used to produce F2 generations. Mice of the F2
generations were screened for a male and female homozygous
for the crossover chromosome. Once an appropriate
homozygous recombinant founder pair was identified, the RI
line was maintained via a single line of descent.

All RI lines were bred and maintained under specific pathogen-
free conditions in the animal facility of Animal Care Services of
the University of Florida (Gainesville, FL, USA). Both male and
female mice 4 to 24 weeks of age were used in the following
studies. All mice received water and food ad libitum. Blood
samples were collected while the mice were anesthetized with
isoflurane. Euthanasia was carried out by cervical dislocation
after anesthetization with isoflurane or 100% C02. Studies
described herein were approved by the University of Florida
Institutional Animal Care and Use Committee.

Genotyping
To determine the genetic status of each offspring, DNA was
prepared using the DNeasy Tissue Kit (Qiagen Inc., Valencia,
CA, USA) from a small tail snip taken between 2 and 4 weeks
of age just prior to weaning. Each DNA sample was used as a
template in polymerase chain reaction amplification with D1 mit
primers covering the Aec2 genetic region. Microsatellite mark-
ers that differentiated genes derived from NOD mice from
those derived from C57BL/6J mice were chosen. Primer


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sequences for the microsatellite markers were based on
sequences available from The Jackson Laboratory and pur-
chased from Integrated DNA Technologies (IDT, Coralville, IA,
USA).

Measurement of saliva flow rates
To measure stimulated flow rates of saliva, individual mice
were weighed and given an intraperitoneal injection of 100 1-L
of a mixture containing isoproterenol (0.02 mg/1 mL of phos-
phate-buffered saline [PBS]) and pilocarpine (0.05 mg/1 mL
of PBS). Saliva was collected for 10 minutes from the oral cav-
ity of individual mice using a micropipette starting 1 minute
after injection of the secretagogue. The volume of each saliva
sample was measured. The saliva samples were then frozen at
-80C until analyzed.

Histology
Male and female C57BL/6.NOD-Aec1Aec2R(n) mice were
euthanized at various ages as indicated in the text. Sub-
mandibular and lacrimal glands were surgically removed from
each mouse and placed in 10% phosphate-buffered formalin
for 24 hours. Fixed tissues were embedded in paraffin and
sectioned at 5-iim thickness. Paraffin-embedded sections
were de-paraffinized by immersing in xylene, followed by dehy-
drating in ethanol. The tissue sections were prepared and
stained with hematoxylin and eosin dye (Histology Tech Serv-
ices, Inc., Gainesville, FL, USA). Stained sections were
observed at x 100 magnifications for glandular structure and
leukocyte infiltration. To detect and determine leukocytic infil-
trations in salivary and lacrimal glands, a single histological
section per gland per mouse was examined by two individuals
blinded to the RI lines. LF, defined as aggregates of greater
than 50 leukocytes, were quantified for each section.

Detection of anti-nuclear autoantibodies in the sera
Anti-nuclear autoantibodies (ANAs) in the sera of mice were
detected using an ANA screening kit (Immuno Concepts, Sac-
ramento, CA, USA). Sera were tested at dilutions of 1:40,
1:80, and 1:160. Presented in this paper, however, are data
from testing sera at 1:40 dilutions. In brief, HEp-2 fixed sub-
strate slides were overlaid with the appropriate mouse serum.
Slides were incubated for 30 minutes at room temperature in
a humidified chamber. After three washes for 5 minutes with
PBS, the substrate slides were covered with Alexa 594-conju-
gated goat anti-mouse IgG (H/L) (Invitrogen Corporation,
Carlsbad, CA, USA) diluted 1:50 for 30 minutes at room tem-
perature. After three washes, nuclear fluorescence was
detected by fluorescence microscopy at x 100 magnification.

Modeling of biological pathways using Pathway Studio
To model biological pathways from selected genes located
within the redefined Aec2 genetic region, Pathway Studio ver-
sion 5.0 software (Ariadne Genomics, Rockville, MD, USA)
and the ResNet mammalian database were used. Functions of
selected genes within the two genetic regions and known SjS-


Available online http://arthritis-research.com/content/10/6/R137



related genes were first verified from the ResNet mammalian
database and then imported into Pathway Studio to visually
construct molecular and biological interactions or relation-
ships among the inputted genes.

Statistical analyses
For this study, we have standardized both saliva and tear col-
lections based on the body weight of the individual mice in an
attempt to better control comparisons. We have incorporated
this for mice of the C57BL/6 genetic background because,
first, disease tends to occur in the C57BL/6 genetic back-
ground strains at an earlier age, often necessitating collections
of saliva and tears when the mice are as young as 4 to 6 weeks
of age and are less than half the size of adult mice, and, sec-
ond, there are greater size differences between male and
female mice during the time course studied. Statistical evalua-
tions between saliva collections were determined by using the
unpaired t test generated by GraphPad InStat software
(GraphPad Software, Inc., San Diego, CA, USA). A two-tailed
P value of less than 0.05 was considered significant.

Results
Genetic profiling of the recombinant inbred lines
From an initial mating of C57BL/6J males with C57BL/
6.NOD-AeclAec2 females, we identified 49 unique crosso-
vers in Aec2 of chromosome 1, consisting of 33 lines with a
single crossover in Aec2 and 16 lines with a crossover in both
Aeci and Aec2. In addition, 2 lines were established using
mice with pre-existing double-crossovers in the Aec2 region
(RI lines 02 and 03). During the subsequent inbreeding, we
were successful in generating 39 new homozygous Aec2 RI
lines. To map each genetic segment of the Aec2 region
remaining within each of the 39 newly generated RI lines, we
selected microsatellite markers spaced approximately 4 to 5
cM apart along chromosome 1. As presented in Figure 1,
these new RI lines, taken together, define progressively
smaller genetic segments of Aec2 derived from NOD mice
and permit much finer mapping for SjS susceptibility loci.
Although there are at least two regions on chromosome 1
(around positions 50 and 75 cM) that exhibited higher num-
bers of recombinant events, there do not appear to be any
crossover hotspots.

Disease profiling of the recombinant inbred lines
SjS-like disease in our NOD-derived mouse lines, including
C57BL/6.NOD-AeclAec2, is characterized generally by three
criteria [4], reflecting the objective criteria used to identify SjS
in humans [25]. These are (a) the loss of saliva and tear flow
rates over time, (b) the presence of LF in the salivary and lac-
rimal glands, and (c) the presence of ANAs in sera. To deter-
mine which of the RI C57BL/6.NOD-Aec1Aec2R(n) mice
develop salivary gland dysfunction, temporal changes in saliva
flow rates were determined for both male and female mice at
an early age (7 1 weeks) and then at a later age (22 2
weeks). The number of mice examined for each new RI line


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Arthritis Research & Therapy Vol 10 No 6 Nguyen et al.


Figure 1


C57BL/6.NOD-AeclAec2 C57BL/6.NOD-Aec1Aec2R(x)


*nit302









mit360
mit117


32.8 cM








97
106.1 cM


* (


49.0 1-


*0* 4


63.1 7


92.3
.0 9213




R135 RI105 RI12B RI10 RI16D R121 RI04 RI02 RKI3 R134 RI)8 RI06C R124 RI47 RI09 RO7 RI23 RI29B
R141 RM18 RI33 R126 RI39 RI40 RI46B RI11 R142 RI20 R119
RI29C RI40B RI32 R139B RI38 RI28B
R144 R148 RI30
RI465B


- 10 cM
- 20cM
- 30 cM
- 40 CM
- 50 CM
- 60 cM
- 70 cM
- 80 CM
- 90 CM
- 100 CM
- 110 cM
- 120 cM


Map of chromosome 1 crossover points in C57BL/6.NOD-Aec1Aec2R(n) recombinant inbred (RI) mice. Thirty-nine RI lines are aligned to show the
points of their individual crossovers in the Aec2 region of chromosome 1, as determined by D1 mit microsatellite markers. Crossover frequencies are
higher at approximately 49.7, 74.3, and 79.0 cM but are not considered hotspots for chromosomal 1 crossovers (NS: Not significant, *= p < 0.05,
** = p< 0.01, and *** = p < 0.001).


was dependent on the number of offspring produced in the
first few pregnancies following inbreeding.

Results indicate that the loss of secretary flow rates was
clearly evident for several of the RI lines, thereby retaining the
phenotype of parental C57BL/6.NOD-AeclAec2 mice, while
a number of the RI lines also failed to show a loss of secretary
activities, thereby indicating loss of the SjS-like disease phe-
notype. Selected yet representative data showing differences
in salivary flow rates among the Aec2 RI lines are shown in Fig-
ure 2. For example, both male and female mice of RI lines RI09,
RI33, and RI12, all of which retained the parental Aeci region
but carry various portions of Aec2, exhibited salivary gland
dysfunction as measured by loss of salivary flow rates ranging
generally between 35% and 60% as the mice aged from 8 to
20-24 weeks. These data are consistent with the decreases
of saliva fluid volumes historically observed with NOD,
NOD.B1 0-H2b, and C57BL/6.NOD-AeclAec2 mice
[8,10,16,20,21]. In contrast, male and female RI mice of lines
exhibiting little or no salivary gland dysfunction (for example,
RI34 and RI02) generally showed slightly increased salivary
flow rates over these same time frames, mimicking SjS-non-
susceptible parental C57BL/6J mice.

Although the number of LF present in minor salivary gland
biopsies of SjS patients often does not correlate directly with


disease or severity of disease, both SjS patients and NOD-
derived mice exhibiting SjS-like disease typically present with
LF. As presented in Figures 3 and 4, histological examinations
revealed the presence of LF in the submandibular and extraor-
bital lacrimal glands, starting at 8 to 12 weeks of age in all of
the anticipated disease-susceptible RI strains (for example,
RI06, RI09, RI33, and RI12). In contrast, no LF or at most only
a relatively few, smaller LF were seen in the glands of RI34 and
RI02 mice, correlating with their normal salivary flow rates.
Interestingly, in addition to the lymphocytic infiltrates,
increased levels of lipid deposits could be seen in the sub-
mandibular and lacrimal glands of several RI lines with onset
of disease (data not shown). Quantification of LF in the salivary
and lacrimal glands showing the relative differences in SjS-
susceptible (RI06, RI09, RI1 2, and RI33) versus SjS-nonsus-
ceptible (RI02 and RI34) RI lines is provided in Table 1.

The presence of ANAs, in particular anti-SS-A/Ro and anti-
SS-B/La in the sera of human patients, is one parameter in the
diagnosis of clinical SjS. Concomitantly with the appearance
of mononuclear leukocytes within the salivary and lacrimal
glands of parental C57BL/6.NOD-AeclAec2 mice, increas-
ing numbers and levels of detectable serum autoantibodies
are also detected [26-29]. To identify ANAs in the sera of RI
C57BL/6.NOD-Aec1Aec2R(n) mice, both male and female
mice were serially bled between 6 and 24 weeks of age (until


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1* (








Available online http://arthritis-research.com/content/10/6/R137


Figure 2


P-DC (*


R134 (NS)


12-

8-

fcl ^ 4i


2I-- 8- 20 0--4---i--2--- 0 -
8 20 8 20 24 1 24 8


16-

12-

8-

S4

i o

0 1


U) 12-

8-

4

01


31'R (*)


RI02 (NS)





E-0 &--D


24 8 24 Male

1412 () 0 Female


Age of mice (weeks)

Differences in temporal loss of secretary function in various C57BL/6.NOD-Aec1Aec2R(n) mice. Male and female sibling mice of parental C57BL/
6.NOD-AeclAec2 (P-DC) and C57BL/6.NOD-Aec1Aec2R(n) mice were injected with isoproterenol/pilocarpine, first at 8 weeks of age and then at
20 or 24 weeks of age, to stimulate saliva secretion. Saliva was collected from each mouse for 10 minutes starting 1 minute after injection of the
secretagogue. The volume of each sample was measured and standardized relative to the weight of the mouse. Temporal reductions in saliva secre-
tions, a marker for onset of clinical disease, were used to identify genetic regions containing genes necessary for development of salivary gland dys-
function and Sjogren syndrome. NS, not significant; RI, recombinant inbred.


euthanasia) and the sera were collected and tested on HEp-2
cells. As presented in Figure 5, a number of different patterns
of ANA staining, including speckled/homogenous nuclear,
cytoplasmic/nuclear membrane, speckled cytoplasm, and
cytoplasmic staining, were detected in the sera from different
RI lines. Cytoplasmic with nuclear membrane and cytoplasmic
staining patterns appeared to be more prevalent in sera from
SjS-nonsusceptible RI lines (R102 and R134), whereas sera
from SjS-susceptible RI lines such as RIO06C, RI09, and R146B
produced predominantly speckled/homogenous staining pat-
terns. In general, a majority of sera from mice classified as SjS-
susceptible RI lines produced ANA staining patterns observed
with sera from parental C57BL/6.NOD-AeclAec2 mice and
not NOD mice [9]. This difference between the C57BL/6
background-derived mice versus the NOD and NOD.B10.H2b
mice suggests that the ANA staining pattern is not disease-
specific and that the genetic background plays an important
role in which ANAs are synthesized. Furthermore, the speck-
led pattern of staining in these RI lines appears to be charac-
teristic of the staining observed with anti-SS-A/Ro and anti-
SS-B/La antibodies [30]. At the same time, the cytoplasmic
punctate staining is characteristic of the staining observed
with antibodies against GW bodies [31]. Confirmation of
whether these antibodies are reactive with SS-A/Ro, SS-B/La,
and/or GW bodies is currently ongoing.


Redefining the Sj6gren syndrome susceptibility Aec2
genetic region
Based on the disease profiling data, we are now able to tenta-
tively identify a small segment subregionn) of Aec2 containing
genes essential and sufficient for development and onset of
SjS-like disease associated with NOD and NOD-derived mice.
As shown in Figure 6, the primary (or candidate) SjS suscep-
tibility gene(s) on chromosome 1 lay within a genetic region
around 79 5 cM. SjS susceptibility genes within this sublo-
cus must be coexpressed with the NOD-derived genes of the
Aeci region of chromosome 3 in order to induce a clinical dis-
ease. Not surprisingly, however, this redefined Aec2 subre-
gion contains multiple genes already shown to correlate with
human and mouse SjS as well as several additional autoim-
mune diseases in mice. These genes provide a basis for devel-
oping hypothetical models of molecular mechanisms
underlying SjS, as discussed below.

Discussion
In the present study, in which the specific goal was to redefine
(and narrow) the boundaries of the Aec2 genetic region on
chromosome 1 known to predispose NOD and NOD-derived
lines of mice to SjS, we generated a large set of new RI lines
(n = 39) and examined each line for its SjS-like disease profile.
Disease profiles obtained with the C57BL/6.NOD-


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R109<) Ig.

12-

8

4.


-1 25 27 0


12






20 20 24 24


\_ ____







Arthritis Research & Therapy Vol 10 No 6 Nguyen et al.


Figure 3


Histological characterization of sialadenitis of male and female C57BL/6.NOD-Aec1Aec2R(n) mice. Submandibular glands were freshly explanted
from male and female C57BL/6.NOD-Aec1Aec2R(n) mice euthanized at 20 or 24 weeks of age. The glands were fixed in 10% formalin, embedded
in paraffin, and sectioned and stained with hematoxylin and eosin (H&E) dye. Representative H&E-stained histological sections of submandibular
glands of selected recombinant inbred (RI) lines are presented: (a) RI34, (b) RI02, (c) RI06C, (d) RI09, (e) RI12, and (f) RI33. Original images were
taken at x 100 magnification, with inserts expanded to show structural detail.


AeclAec2R(n) RI lines indicate that the Aec2 genetic region
of C57BL/6.NOD-AeclAec2 mice, postulated to regulate pri-
marily the pathophysiological and biochemical abnormalities
that subsequently result in the activation of the autoimmune
attack against the submandibular and lacrimal glands [10], is
a single subregion mapping to the telomeric portion of chro-
mosome 1 located at approximately 79 5 cM. However, pen-
etrance and severity of SjS-like disease may be further
influenced by genes located within a few centimorgans on the
centromeric side of this region, possibly pointing to SjS-asso-
ciated quantitative trait loci (QTL) genes. Although the size of
the redefined Aec2 region remains relatively large for identifi-
cation of individual candidate SjS susceptibility genes, the
genes residing within this subregion can be grouped into four
functionally clustered sets, each suspected previously of
involvement in SjS susceptibility. These are (a) endogenous
viruses and oncogenic genes, (b) Fas/FasL-associated apop-
tosis, (c) TH 7-associated activities, and (d) fatty acid, lipid,
lipoprotein, and cholesterol homeostasis. However, perhaps
the most obvious aspect is the fact that this redefined Aec2
region contains the QTL-Athl region containing some 10
genes, including tumor necrosis factor ligand superfamily
member 4 (Tnfsf4 or Ox4OL) and Tnfsf6 (Fasi).

Within the first set, several viral/oncogenic genes, such as
Emv38 endogenouss ecotropic MuLV-38), Kras-2-rsl (Kirsten
rat sarcoma oncogene-2, related sequence-1), Xprl (xeno-


tropic/polytropic retrovirus receptor-1), and Abl2 (Abelson
murine leukemia viral oncogene-2), are found in this redefined
Aec2 subregion. In our earlier studies with NOD mice [12], we
observed that high levels of interferon-gamma (INF-y) were
present in the salivary glands of neonate mice, suggesting an
important role for INF-y in the delayed development/prolifera-
tion of acinar tissue observed in the salivary glands of neonate
NOD mice. While it is logical to conclude that induction of INF-
y may be a result of short-term viral infection during the pre-
term and early postpartum periods, what might cause a viral
outbreak at this time point remains unknown. It could be
hypothesized that this occurs due to the changes in maternal
hormone levels at this time. Perhaps more interesting, how-
ever, this region contains the gene Tnfsf6 encoding the proa-
poptotic protein FasL. FasL has numerous functions but is
mainly involved in regulating immune responses, apoptosis,
and retinal cell programmed death [4]. During the early phase
1 period of SjS-like disease in NOD mice, both FasL and Fas
are upregulated at both the gene and protein levels, and this
increased expression of Fas/FasL corresponds to the
observed increase in acinar cell apoptosis within the glands
[32]. However, it remains speculative whether there might be
an association between endogenous/exogenous viral infection
and Fas/FasL activity in the salivary and lacrimal glands.

The redefined Aec2 subregion also contains several genes
involved in autoimmunity and/or tumorgenesis, the latter being


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Available online http://arthritis-research.com/content/10/6/R137


Figure 4


Histological characterization of dacryoadenitis of male and female C57BL/6.NOD-Aec1Aec2R(n) mice. Submandibular and lacrimal glands were
freshly explanted from male and female C57BL/6.NOD-Aec1Aec2R(n) mice euthanized at 20 or 24 weeks of age. The glands were fixed in 10% for-
malin, embedded in paraffin, and sectioned and stained with hematoxylin and eosin (H&E) dye. Representative H&E-stained histological sections of
lacrimal glands of selected recombinant inbred (RI) lines are presented: (a) RI34, (b) RI02, (c) RI06C, (d) RI09, (e) RI1 2, and (f) RI33. Original
images were taken at x 100 magnification, with inserts expanded to show structural detail.



one clinical manifestation of SjS that occurs in a small subset cells [33], and Myoc [myocilin], whose product interacts with
of patients. Of interest, but not thought to be directly involved olfactemedin involved in glaucoma [34]). However, whether
in the development and onset of SjS, is the presence of genes any of these genes are related to SjS susceptibility and lac-
specific to the ocular/lacrimal gland etiology (for example, Pdc rimal gland disease or merely influence the secondary disease
[phosducin], which is a protein of the retinal photoreceptors phenotypes often associated with SjS remains unknown. In


Table 1

Quantification of lymphocytic foci in the salivary and lacrimal glands of mice from several representative C57BL/6.NOD-
AeclAec2R(n) recombinant inbred lines


RI line Age, weeks


Submandibular glands


Number of mice


Positive


Male Female Number


Male and
female


Male Female Number


RI 34 20 and 24 6

RI 02 20 and 24 4


7 3 23%0/o


1.0 + 0.0 6 7 7 54%0/


7 0 0%/o 0.0 +0.0 6 8 4 29%O/o


RI 06 20 4 ND 3 74%/o


RI 09 21 and 22 7


7 11 79%/o


RI 12 24 7 4 5 45%/o


RI 33 20 and 21


9 7 10 63%/o


4.0 +1.7 4 ND 4 100%/o

3.0 + 0.6 7 7 6 43%/o

2.4 +0.7 7 4 10 91%/o

2.2 + 1.0 9 7 13 81o/o


LF, lymphocytic foci; ND, not done; RI, recombinant inbred.


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Average
number of LF


Lacrimal glands


Number of mice


Positive


Average
number of LF


Male and
female


1.7 + 0.4

1.5 +0.3

5.8 + 4.1

2.8 + 0.7

2.8 + 0.6

6.5 + 4.5








Arthritis Research & Therapy Vol 10 No 6 Nguyen et al.


Figure 5


Speckled/homogenous nuclear Cytoplasmic/nuclear membrane Speckled cytoplasm Cytoplasmic
Staining
patterns




RI mice strains

R134 0/8 (0)' 6/8 (75) 0/8 (0) 2/8 (25)
R102 2/13(15) 6/13(46) 4/13(31) 1/8 (8)
Aec2 RI06C 3/4 (75) 1/4 (25) 0/4 (0) 0/4 (0)
R109 5/8 (63) 3/8 (27) 0/8 (0) 0/8 (0)
RI46B 4/9 (44) 2/9 (22) 3/9 (34) 0/9 (0)


Detection of anti-nuclear autoantibodies in sera of C57BL/6.NOD-Aec1RAec2R(n) mice. Serum samples obtained from C57BL/6.NOD-
AeclAec2R(n) mice were diluted 1:40 and incubated with HEp-2 fixed substrate slides for 30 minutes at 25C in a humidified chamber. The slides
were then developed with Alexa 594-conjugated goat anti-mouse IgG and viewed by fluorescence microscopy at x 100 magnification. Examples of
speckled/homogenous staining of the nucleus (left panel), cytoplasmic/nuclear membrane staining (left center panel), speckled/cytoplasmic staining
(right center panel), and cytoplasmic staining (right panel) were observed. The numbers of individual sera tested and the percentages of positive
sera from a sampling of recombinant inbred (RI) lines exhibiting each of the patterns are listed. Number of mice showing positive staining pattern
over total. *Percentage of mice showing positive staining pattern. Aec, autoimmune exocrinopathy.


contrast, one genetic element in this region that has a direct
association with the immunopathology of SjS is the QTL gene
Cypr2 (cytokine production 2) [35]. CYPR-2 is known to reg-
ulate levels of interleukin-10 (IL-10), an important cytokine that
enhances the activity of B lymphocytes, and at the same time
to regulate the functions of TH1 and TH17 cells [36]. Our
recent microarray studies indicate that 1110 is not upregulated


Figure 6


Chromosome


29 7 CM Dlm d 18
32,8CM 01 mdl 302
369cM 01mfl 532
42.0cM Dlmd 181
490"M 0Dmt 13
54.0 cM Dmid 48
59.7cM Dird 135
631cM 01m1 88
70,cM D1mil312
743cM 0lmil348
790cM Dlmil 104
83.4 cM Olnt268
863cM Dlmih I5
92-3 cM D1 ml 270
970cM O1md 355
101.2 CM D0Tn 360
106.1 cM olr 117


- + +
9.....


m1l"s X mlt3W2
32.6 cM








101-2 cM
mlt360X mI117


C57BLJ6.NOD-AeclA.c2


*


R134 R102 R146B R133 RI12B RI09
R138


Redefining the boundaries for the Aec2 Sjogren Syndrome (SjS) sus-
ceptibility genetic locus. Based on the disease profiling of the C57BL/
6.NOD-Aec1Aec2R(n) recombinant inbred (RI) lines, the boundaries
of the Aec2 genetic region containing SjS susceptibility genes have
been temporarily reset to position 79 + 5 cM of chromosome 1
(shaded gray gradient rectangular box). Possible quantitative trait loci
genes may reside a few centimorgans centromeric to this region
unshadedd rectangular box).


during the development of SjS in the C57BL/6.NOD-
AeclAec2 mouse model [32], possibly indicating a lack of
immune regulation by regulatory T (Treg) cells. If so, this lack of
regulation by IL-10 would be consistent with the results of
gene therapy studies in which injections of vectors expressing
recombinant IL-10 reduced or suppressed clinical manifesta-
tions of SjS-like disease in both salivary and lacrimal glands of
mice [37,38].


Maintaining sufficient regulation of immune responses to pre-
vent development of an overt autoimmunity is no doubt
dependent on a physiological balance between TH1, TH2,
TH17, and Treg cell interactions. This cellular interaction
appears to be highly influenced by OX40L encoded by the
Tnfsf4 gene and this gene is located within the redefined Aec2
region. OX40L is expressed by a number of distinct cell pop-
ulations, including activated dendritic cells [39]. OX40L is
capable of functioning as an inhibitor of the maturation of Tregi
cells [40], a regulatory cell population normally producing IL-
10 and INF-y, which (in conjunction with IL-27) can inhibit the
effector CD4+ TH17 cells [41]. Reduced Treg1 cell function,
therefore, results in a positive feedbackfor the generation/acti-
vation of effector CD4+TH17 cells (Figure 7). These effector
TH 7 cells produce predominantly IL-1 7, IL-21, and IL-22 plus
factors like nitric oxide, matrix metalloproteinase, and prostag-
landin E2, each of which is shown to play an important role in
the immunopathophysiology of several autoimmune diseases,
including SjS [41]. Thus, we hypothesize that the presence of
TH17 cells in the salivary and lacrimal glands of C57BL/
6.NOD-AeclAec2 mice, as well as SjS patients [42], indi-
cates an imbalance in the TH 7/Tre 1 ratio favoring the TH 7
populationss. A recent study indicates that retinoic acid can
facilitate an increase in the numbers of Foxp3+ Treg cells and


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Available online http://arthritis-research.com/content/10/6/R137


Figure 7


TGF-p
CYPR2-- IL-10


K)^


INF-y
CYPR2-* IL-10


IgM
B cell
SIL-4
IL-.21

IgG1
B cell


Pathogenic
autoantibodies


IL-17
^ IL-22
MMP
NO
PGE,


Acinar cell
destruction


Proposed model for how OX40L:OX40 promotes autoimmunity in Sjogren syndrome-like disease of C57BL/6.NOD-AeclAec2 mice. Cellular inter-
actions involved in the development of an autoimmune response against the salivary and lacrimal glands leading to loss of acinar tissue are pre-
sented. DC, dendritic cell; IL, interleukin; INF-y, interferon-gamma; MMP, matrix metalloproteinase; NO, nitric oxide, PGE2, prostaglandin E2; TGF-p,
transforming growth factor-beta; Treg, regulatory T (cell).


simultenously inhibit the formation of effector TH 7 cells [43].
Interestingly, we have found that expression of the retinoic
receptors, Rxr retinoidd x receptor) and Rar (retinoic acid
receptor), is downregulated in the lacrimal glands of C57BL/
6.NOD-AeclAec2 mice [44]. This observation is again con-
sistent with a potential problem in cellular homeostasis, espe-
cially at the level of macrophages, dendritic cells, and even
production of the FOXP3+ Treg cell populations whose differ-
entiation and functional maturation are highly dependent on
retinoic and fatty acid stimulation. As presented in Figure 7,
there is a reciprocal maturation of effector CD4+ TH17 and
FOXP3+ Treg cells dependent on the relative balance of IL-6
and transforming growth factor-beta influenced by retinoic
acid. Thus, the intricacy and balance between OX40L, the
retinoids, proinflammatory cytokines, and development of Treg
cells appear to impact the potential development and onset of
autoimmunity, which in SjS appears to favor activation of effec-
tor TH17 cells.

Although several factors encoded by genes in the redefined
Aec2 region may be involved in secondary manifestations of
SjS, OX40L is the one factor that clearly stands out as a pri-
mary candidate gene underlying not only the recognized
immune dysregulation, as presented above, but also the
pathophysiological aberrations associated with chromosome
1 of the C57BL/6.NOD-Aec1Aec2 SjS model. In this rede-
fined region, a common functional cluster of lipid, lipoprotein,


cholesterol, and fatty acid regulatory and processing elements
is found, including Hdlql4 (high-density lipoprotein QLT-14),
Hdlq5 or Apoa2 (high-density lipoprotein QTL-5), Gpa33
(glycoprotein A33), Cql (cholesterol QTL-1), Prdx6 (peroxire-
doxin), and (of special note) Soatl steroll 0-acyltransferase-
1). Involvement of lipids and fatty acids in the pathology of SjS
has become a major focus of SjS research as lipid depositions
[45] and changes in lipid rafts [46] appear to influence the
pathology in both salivary and lacrimal glands. Furthermore,
our recent genomic microarray studies [44,47] (C.Q.
Nguyen, S. Ashok, R.A Mclndoe, J.X. She, B.H. Lee, A.B. Peck,
unpublished data) indicate that multiple genes involved in
fatty acid, lipid, lipoprotein, and cholesterol homeostasis/
transport are differentially expressed, corresponding with lipid
deposits, dysfunctional dendritic cells, and onset of autoimmu-
nity. As illustrated in Figure 8, various relationships between
genes controlling free fatty acid, lipid, and lipoprotein homeos-
tasis are directly or indirectly dependent on the activities of
OX40L. As a consequence, imbalances in this homeostasis
regulated in part by OX40/OX40L can result in widespread
pathology, including inflammation and cell death. Based on dif-
ferential gene expression data, there are major reductions in
the levels of transcripts encoding FDFT-1 (farnesyl diphos-
phate farnesyl transferase-1), ABCA1 (ATP-binding cassette,
subfamily A [ABC1] member 1), and the retinoic acid recep-
tors, RRX and RAR. At the same time, increased levels of tran-
scripts encoding the low- and high-density lipoprotein


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Arthritis Research & Therapy Vol 10 No 6 Nguyen et al.


Figure 8


Farnesyl-
pyrophosphate


__X OL




RANTEs~


Ox-LD1.
ACAV/
LDL


~DL-A~A1

7/.


Proposed genetic predisposition for dysregulated homeostasis/trans-
port of lipid, lipoprotein, cholesterol, and fatty acid metabolism leading
to lipid depositions in the salivary and lacrimal glands of C57BL/
6.NOD-AeclAec2 mice and Sjogren syndrome patients. Accumulation
of free cholesterols (FCs) inside the cells resulted from increased
uptake of low- and high-density lipid receptors. In addition, impairment
of ABCA1 membrane transporter leads to the accumulation of choles-
teryl esters (CEs) metabolized by sterol O-acyltransferase-1 (SOAT-1)
using FCs and free fatty acids (FFAs). ABCA1, ATP-binding cassette,
subfamily A [ABC1 ] member 1; ACAT, acyl-coenzyme A: cholesterol
acyltransferase; ApoE, apolipoprotein E; DC, dendritic cell; Fdft-1, far-
nesyl diphosphate farnesyl transferase-1; HDL, high-density lipid; LDL,
low-density lipid; Lrpr, low-density lipid-related protein receptor; NCEH,
neutral cholesterol esters hydrolase; Ox-LDL, oxidized low-density lipid;
PPAR, peroxisome proliferator activated receptor; RANTES, regulated
on activation normal T cell expressed and secreted; RXR, retinoid x
receptor. Adapted from [48].


receptors, as well as SOAT-1, are observed. We hypothesize,
therefore, that an imbalance occurs in the production of cho-
lesterol and the increased level of cholesterol results in greater
amounts being converted by SOAT-1 to cholesterol esters
that accumulate within the cells due to the downregulation or
dysfunction of the lipid transporter. In addition, the functional
activities of cells whose differentiation and maturation are
dependent on the retinoids and the RXR/RAR-PPARy (RXR/
RAR-peroxisome proliferator activated receptor-gamma) sign-
aling pathways (for example, macrophages and dendritic and
FOXP3+ T cells) will be altered due to altered development,
ultimately affecting antigen presentation and balanced pro-
duction of regulatory cytokines.

Conclusion
Identifying gene products that are differentially expressed in
the Aec2 SjS susceptibility subregion defined by the new RI
lines is moving us closer to identifying specific candidate
genes involved in the onset and development of SjS-like dis-


ease. Based on our current data, our focus is turning to Ox40L
as an effective candidate gene for the development of SjS. The
future application of genetic knockout mice and/or short inter-
fering RNA will permit us to further our understanding of the
potential role of Ox40L in SjS and, more importantly, to trans-
late its relevancy to human SjS.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
ABP performed the genotyping of mice and assisted with the
study design and manuscript preparation. JT carried out histo-
logical analysis. JGC, LC, and JN performed saliva collections
and disease profilings of the mice. BHL helped with manu-
script preparation. CQN participated in the design of the
study, ANA staining, saliva collections, data analyses, and
manuscript preparation. All authors read and approved the
final manuscript.

Acknowledgements
We thank Robert Haynes for the countless hours spent in caring for and
maintaining precise records of these RI lines and Sung Kim for his help
in analyzing the function and pathway associations of selected genes.
This work was supported in part by Public Health Service (PHS) grant
DE01 4344 from the National Institutes of Health (to ABP) and by the
Center for Orphaned Autoimmune Disorders at the University of Florida.
CON was supported by a postdoctoral fellowship from PHS grant T32
DE07200.

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