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A 9-Centimorgan Interval of Chromosome 10
Controls the T Cell-Dependent Psoriasiform
Skin Disease and Arthritis in a Murine
Psoriasis Model
Honglin Wang, Daniel Kess, Anna-Karin B. Lindqvist,
Thorsten Peters, Anca Sindrilaru, Meinhard Wlaschek,
Robert Blakytny, Rikard Holmdahl and Karin
Scharffetter-Kochanek
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2008 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2008; 180:5520-5529; ;
doi: 10.4049/jimmunol.180.8.5520
http://www.jimmunol.org/content/180/8/5520
The Journal of Immunology
A 9-Centimorgan Interval of Chromosome 10 Controls the T
Cell-Dependent Psoriasiform Skin Disease and Arthritis in a
Murine Psoriasis Model1
Honglin Wang,* Daniel Kess,* Anna-Karin B. Lindqvist,† Thorsten Peters,*
Anca Sindrilaru,* Meinhard Wlaschek,* Robert Blakytny,‡ Rikard Holmdahl,†
and Karin Scharffetter-Kochanek2*
P
soriasis is a chronic disease of unresolved pathogenesis
affecting 3% of the general population of which 10 – 40%
also develop arthritis (1). It presents with erythematous,
scaling skin lesions (2) and can manifest as psoriatic arthritis
(PsA),3 an inflammatory joint disease resulting in extensive bone
resorption and joint destruction (3). Genome-wide linkage studies
have noted overlapping regions of significance for human skin
psoriasis and PsA within and outside of the MHC region (4). Different cell types have been proposed to be the primary triggers in
the pathogenesis of psoriasis (2), with T cells being essential for its
*Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany; †Medical Inflammation Research, University of Lund, Lund, Sweden; and
‡
Institute of Orthopaedic Research and Biomechanics, University of Ulm, Germany
Received for publication August 23, 2007. Accepted for publication February 8, 2008.
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 by the German Research Foundation (DFG) within the
SFB 497 “Signals and Signal Processing during Cellular Differentiation”, and individual research grants from the German Research Foundation (DFG) (SCHA 411/
12-1, -2), the Strategic Research Foundation, and the EU project LSHB CT-2006018661 (AutoCure).
2
Address correspondence and reprint requests to Dr. Karin ScharffetterKochanek, Department of Dermatology and Allergic Diseases, University of Ulm,
Maienweg 12, D-89081 Ulm, Germany. E-mail: karin.scharffetter-kochanek@
uniklinik-ulm.de
3
Abbreviations used in this paper: PsA, psoriatic arthritis; CD18hypo, CD18 hypomorphic; cM, centimorgan; Mbp, megabase-pair; PASI, psoriasis area and severity
index; PSD1, psoriasiform skin disease-associated locus 1; PSORS, psoriasis susceptibility; QTL, quantitative trait locus.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
initiation (5–7), whereas macrophages maintain its inflammatory
state (8, 9). Both genetic and environmental factors contribute to
its manifestation. Psoriasis is frequently inherited between successive generations, but it does not follow a classical autosomal Mendelian profile. Type I psoriasis has an early onset, before the age of
40 years, and is suggested to be heritable (10). Type II psoriasis is
characterized by a late onset and its heritability is not entirely
clear.
To identify genetic factors underlying this skin disorder, several
linkage analysis studies have been performed in humans. Although
at least six different psoriasis susceptibility loci, designated
PSORS1–PSORS6, have been mapped by using genome-wide
scans, the cause of human psoriasis remains unknown (11–19).
Research into the pathogenesis of human psoriasis has profited
from suitable animal models. Most of these, however, display only
a single or a few aspects resembling human psoriasis (20 –27). In
contrast, the mouse PL/J strain carrying a CD18 hypomorphic
(CD18hypo) mutation, with reduced expression of the common
chain of ␤2 integrins (CD11/CD18) to 2–16% of wild-type levels,
develops a skin disease that closely resembles human psoriasis
(28).
CD18 represents the common ␤2-chain of the ␤2 integrin family,
with four heterodimeric molecules (CD11a/CD18, CD11b/CD18,
CD11c/CD18, and CD11d/CD18) being exclusively expressed on
hematopoietic cells. Among several possibilities, reduced CD18
expression may cause a disrupted formation of the immunological
synapse resulting in the generation and persistence of autoreactive
cells (29, 30). The pathogenic role of ␤2 integrins in human psoriasis and other inflammatory skin diseases is poorly understood
(31–33). Linkage analysis of psoriasis families has identified a
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Psoriasis is a complex genetic disease of unresolved pathogenesis with both heritable and environmental factors contributing to
onset and severity. In addition to a disfiguring skin inflammation, approximately 10 – 40% of psoriasis patients suffer from
destructive joint involvement. Previously, we reported that the CD18 hypomorphic PL/J mouse carrying a mutation resulting in
reduced expression of the common chain of ␤2 integrins (CD11/CD18) spontaneously develops a skin disease that closely resembles
human psoriasis. In contrast, the same mutation on C57BL/6J background did not demonstrate this phenotype. By a genome-wide
linkage analysis, two major loci were identified as contributing to the development of psoriasiform dermatitis under the condition
of low CD18 expression. Using a congenic approach, we now demonstrate that the introduction of a 9-centimorgan fragment of
chromosome 10 derived from the PL/J strain into the disease-resistant CD18 hypomorphic C57BL/6J was promoting the development of psoriasiform skin disease and notably also arthritis. We therefore designated this locus psoriasiform skin diseaseassociated locus 1 (PSD1). High numbers of CD4ⴙ T cells and TNF-␣ producing macrophages were detected both in inflamed skin
and joints in these congenic mice, with a complete resolution upon TNF-␣ inhibitor therapy or depletion of CD4ⴙ T cells. For the
first time, we have identified a distinct genetic element that contributes to the T cell-dependent development of both psoriasiform
skin disease and associated arthritis. This congenic model will be suitable to further investigations of genetic and molecular
pathways that cause psoriasiform dermatitis and arthritis, and it may also be relevant for other autoimmune diseases. The
Journal of Immunology, 2008, 180: 5520 –5529.
The Journal of Immunology
5521
Table I. Location of an insert originating from the 129/SvEv strain in both PL/J CD18hypo and C57BL/6J CD18hypo parental
strains for congenic breeding
Wild-Type Strainsd
Parental Strainse
cMb
Mbpc
C57BL/6J
PL/J
129/SvEve
C57BL/6Jhypo
PL/J CD18hypo
D10mit213
D10mit86
D10mit214
D10mit126
D10mit38
D10mit194
D10mit196
D10mit299
D10mit31
D10mit175
D10mit207
D10mit42
D10mit261
D10mit230
D10mit66
D10mit67
D10mit231
D10mit95
D10mit68
D10mit41
D10mit119
D10mit12
D10mit96
D10mit178
D10mit70
D10mit133
D10mit162
D10mit233
D10mit266
D10mit14
D10mit102
D10mit271
11
17
19
21
26.8
29
31
32
36
41.8
42
44
47
49
49
49
52
51
51.5
52.5
53
56
56
59
59
59
59
62
62
65
69
70
21.1
24.3
25.3
26.7
43.8
46.8
61.1
66.2
68
78.1
79.3
82.2
85.2
89.7
⬃
90.2
90.5
92
92.4
93.6
95.6
99
99.1
99.7
103.5
104.7
106.5
114
114.6
118.2
120.5
124
147
156
126
129
166
82
200
184
152
110
164
183
113
115
143
152
120
180
107
248
146
239
151
137
144
141
105
128
91
193
149
115
134
150
132
123
178
93
203
177
153
108
164
194
111
142
137
156
115
191
88
245
129
218
149
133
160
139
108
98
84
186
143
93/101
150
150
132
123
178
82
200
181
148/152
108
164/160
194
117
115
143
152/165
120
180
93
239
144
239
153
142/136
147/149
139/147
90/91
106
84
192
149
115
147
156
126
129
166
82
200
181
148/152
108
164
194
117
115
143
152
120
180
93
239
144
239
153
142/136
147/149
139/147
90/91
128
91
192
149
115
134
150
132
123
178
93
203
181
148/152
108
164
194
117
115
143
152
120
180
93
239
144
239
153
142/136
147/149
139/147
90/91
98
84
186
143
101
a
Markers used for identification of an insert originating from the 129/SvEv strain on chromosome 10.
Genetic position according to Mouse Genome Informatics (http://www.informatics.jax.org).
Physical position according to Ensembl Mouse Genome Server; the physical position of D10mit66 is not available (http://www.ensembl.org/
Mus_musculus).
d
Genotypes of wild-type mice for C57BL/6J and PL/J stains given as size of PCR products in base pairs (bp).
e
Genotypes of parental CD18hypo strain for congenic breeding and 129/SvEv strain used as embryonic stem cells for the generation of CD18hypo
mutation, all given as size of PCR products in base pairs (bp). PCR products for inserts originating from the 129/SvEv strain in both PL/J CD18hypo and
C57BL/6J CD18hypo parental strains are listed in bold.
b
c
region on chromosome 17 that includes the ICAM-2 locus, an
important ligand of the CD11/CD18 heterodimers (13).
The CD18hypo PL/J model is of particular interest, because the
development of psoriasiform skin disease is highly dependent on
the genetic background; that is, the psoriasiform dermatitis develops only in CD18hypo PL/J mice, but not in C57BL/6J or 129/SvEv
mice with the same CD18hypo mutation (28). Homozygous mutant
mice of a (PL/J ⫻ C57BL/6J)F1 background did not develop the
disease, despite the CD18hypo mutation, suggesting recessive susceptibility for PL/J alleles or a dominant suppressing C57BL/6J
inheritance. Backcross analysis indicates that, in addition to CD18,
a small number of other genes determine susceptibility to this disease (28).
Performing a genome-wide linkage analysis, we identified two
quantitative trait loci (QTL) on chromosome 10 with significant
linkage to the development of psoriasiform skin disease and one
QTL on chromosome 6 with linkage to its early onset (34). To
further investigate whether loci on chromosome 10 (PSD1, or psoriasiform skin disease-associated locus 1) and/or chromosome 6
are causal for the development of psoriasiform skin disease, we
have developed speed congenic strains by means of microsatelliteassisted selection (35).
We demonstrate herein that the CD18 hypomorphic C57BL/6J
mice, resistant to development of psoriasiform skin disease,
could be made susceptible to the disease by introducing a 9-centimorgan (cM) fragment of chromosome 10 derived from the
PL/J strain. This chromosome 10 congenic strain developed
both the psoriasiform skin disease and severe arthritis, which
strongly resembled human psoriasis and associated PsA. The
congenic strain for the chromosome 6 locus did not develop
skin and/or joint disease.
These data identify the PSD1 locus on chromosome 10 as a
verified genetic element contributing to both the inflammatory skin
disease and arthritis in the CD18hypo murine model. Our model is
highly suitable to further dissect genes and molecular pathways
underlying inflammatory skin and joint diseases.
Materials and Methods
Genotyping
DNA was prepared from tail biopsies by a quick alkaline lysis protocol
(36). PCR was performed with 10 ng of tail DNA according to published
procedures (34). PCR products were analyzed on a MegaBACE 1000 (Amersham Biosciences) according to the manufacturer’s protocol.
Selection of markers for congenic screening
Eighty-six microsatellite markers, previously identified as being polymorphic between the PL/J and 129/SvEv strains (34), were genotyped in the
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Markera
5522
A LOCUS CONTROLS THE PSORIASIFORM DERMATITIS AND ARTHRITIS
CD18hypo PL/J (N5) and CD18hypo C57BL/6J (N10) (The Jackson Laboratory) strains, which have been used as parental strains for the construction of the congenics. As screening markers for the speed congenic procedure, an additional 91 microsatellite markers were selected for
backcrossing. The 91 markers, with an average marker distance of 15.5
cM, covered all chromosomes and could identify contaminating PL/J-derived fragments present in the CD18hypo PL/J (N5) donor strain to enable
elimination of these fragments in recipient mice during the congenic
breeding.
Generation of congenic mouse strains
Assessment of the psoriasiform skin disease and arthritis
The severity of skin disease and arthritis was assessed twice a week for an
observation period of up to 30 wk using an adapted psoriasis area and
severity index (PASI) (34) and an adapted arthritis score (39). For
CD18hypo mice, the PASI score was modified accordingly: 0, no signs; 1,
erythema of the ears, scaling of the tail; 2, hair loss in addition to the signs
for 1; 3, hair loss, isolated or widespread slight scaling; 4, moderate scaling
on a large area of the body or strong scaling at a few small or large regions.
The clincal arthritis score was adapted from a described scoring system
(39) as follows: 0, no symptoms; 1, slight swelling and/or erythema; 2,
pronounced edematous swelling; 3, deformed paw or joint with ankylosis
and joint rigidity. The sum of all four paws was scored twice a week with
a maximal score of 12 per mouse.
The skin disease and arthritis were assessed in homozygous congenic
mice. As clinical appearance and histology of the skin and joints are identical in PSD1 congenic and chromosome 10C congenic mice containing the
PSD1 region, we used the PSD1 congenic strain for further analysis. When
we investigated the incidence of psoriasiform skin disease and arthritis,
mice littermates to the different congenic mice from the intercrossing of the
N6 backcross generation were used as controls. All control mice were
heterozygous PL/J and C57BL/6J in the regions of interest. We assumed
that heterozygous mice were suitable as controls in evaluation of the incidence of the phenotypes since our previous data have shown that mice
heterozygous for the PSD1 region do not develop psoriasiform skin disease
(34). These control mice are specified in Table II. Additionally, CD18hypo
PL/J mice at N5 generation were used for phenotyping to assess the time
course of the disease. All experiments were done in compliance with the
German Law for Welfare of Laboratory Animals.
FIGURE 1. Generation of C57BL/6J congenic strains and identification
of a major quantitative susceptibility locus for the psoriasiform skin disease
and arthritis on chromosome 10. To determine which locus is causally
involved in the development of psoriasiform skin disorder, loci of interest
derived from the PL/J strain were selected for congenic breeding with the
psoriasiform skin disease-resistant CD18 hypomorphic C57BL/6J (B6) inbred background strain. Map of chromosome 10 showing microsatellite
markers used for breeding chromosome 10A, chromosome 10B, chromosome 10C, and chromosome 10D congenic mice strains. The scale indicates positions in centimorgans according to Ensembl mouse genome assembly National Center for Biotechnology Information 32 (http://www.
ensembl.org/Mus_musculus/). The interval selected for inclusion of homozygous PL/J alleles in the congenic strain (f) and the interval between
the selection markers for which inclusion or exclusion is not proven (䡺)
are indicated. The interval originally derived from 129/SvEv strain that is
present in both PL/J CD18hypo and C57BL/6J CD18hypo mice is shown (red
bar). Location of the psoriasiform skin disease-associated locus (PSD1) is
indicated on chromosome 10 (D10mit126 –D10mit194). The position of the
CD18 hypomorphic mutation is indicated. The dotted lines indicate the
border of selected regions for congenic breedings.
Immunofluorescence staining
Frozen cryosections of skin or joints from both male and female mice at 5
mo of age with the indicated phenotype were fixed in ice-cold acetone for
10 min before staining. To stain keratinocytes, CD4 T cells, macrophages,
and TNF-␣, we used rabbit anti-K14 (Covance) in conjugation with Alexa488
(Caltag Laboratories), Alexa488-conjugated rat anti-mouse CD4 (Caltag
Laboratories, clone RM4 –5), Alexa488-conjugated rat anti-mouse F4/80
(Caltag Laboratories, clone CI:A3–1), and anti-mouse TNF-␣ (BD Pharmingen, clone MP6-XT22) in conjunction with streptavidin Cy3 conjugate
(Dianova) mAb. DAPI (4⬘,6-diamidino-2-phenylindole) (Fluka) was used
for staining of nuclei. All Abs were diluted in an Ab diluent (Dako).
Histological processing
Skin and paw joints from the PSD1 congenic and control mice were fixed
in 4% paraformaldehyde solution for 24 h, and in the case of joints these
were decalcified for 3 wk in 15% EDTA solution, dehydrated, and embedded in paraffin. Paraffin-embedded 5-␮m sections of skin and joints were
stained with H&E as described (37).
Administration of etanercept
Etanercept (100 ␮g per mouse) was administrated i.p. every day for a
period of 30 days to neutralize TNF-␣. In parallel, injection of 0.9% NaCl
was used as control. Effects of etanercept on the severity of arthritis and the
psoriasiform dermatitis were assessed by the adapted arthritis score and the
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Congenic mice for the regions of interest on chromosomes 10 and 6 were
produced by breeding the PL/J genomic fragments onto C57BL/6J background. Speed congenic procedure was used as previously described (35).
The donor strain was a CD18hypo PL/J (N5) strain originating from the
CD18hypo PL/J (N4) mice previously described (37), but with an additional
generation of backcrossing onto the PL/J inbred strain. Theoretically,
⬃3.125% of the genome in this CD18hypo PL/J (N5) strain still originates
from 129/SvEv (38). For the selection of one donor mouse, male PL/J
CD18hypo (N5) mice with severe psoriasiform phenotype were genotyped
with 86 microsatellite markers polymorphic between the PL/J and 129/
SvEv inbred strains. The PL/J CD18hypo (N5) male with the least contamination of 129/SvEv alleles (3 out of 86 microsatellite markers, beside the
129/SvEv-derived CD18 insert on chromosome 10) was selected as the
donor for construction of the congenics (Table I).
The selected CD18hypo PL/J (N5) male was mated with two females of
the CD18hypo C57BL/6J inbred strain. The CD18hypo C57BL/6J inbred
strain is available at The Jackson Laboratory (www.jax.org) as well as
results from 10 generations of backcrossing. The CD18hypo C57BL/6J
(N10) mice were genotyped and no 129-derived fragments were identified
using the 86 microsatellite markers. Although we cannot exclude the presence of 129-derived fragments outside of the PSD1 region in the genome
of N10 CD18hypo C57BL/6J mice, this did not affect the finding of the
PSD1 involvement in the development of the psoriasiform skin disease and
arthritis. Six generations of backcrossing were performed onto CD18hypo
C57BL/6J (N10) background, where the donor for each generation was
selected based on its genotype according to the speed congenic procedure
(35) to keep the donor fragment in the region of interest and to eliminate
donor fragments such as those derived from like PL/J, as well as from129/
SvEv, in the rest of the genome. N6 mice harboring PL/J fragments of
interest on N10 CD18hypo C57BL/6J background were intercrossed to generate the homozygous congenic strains for each congenic fragment, respectively. To monitor the psoriasiform skin disease clinically and histologically, we used littermate mice from the intercross of N6 or from the N6
backcross generation as control strains. All the control mice for this phenotypic characterization were homozygous wild-type C57BL/6J in the
PSD1 region. We have specified these controls in the figure legends.
The Journal of Immunology
5523
this study the PL/J CD18hypo strain has been further backcrossed.
The CD18hypo mutation on chromosome 10 originates from 129/
SvEv (34), and thus PL/J CD18hypo and C57BL/6J CD18hypo still
contain genomic DNA fragments from the 129/SvEv strain surrounding the CD18 gene on chromosome 10. To determine the
exact position of 129/SvEv inserting in the genome of PL/J
CD18hypo and C57BL/6J CD18hypo, respectively, a set of microsatellite markers for chromosome 10 was used for genotyping. In
the PL/J CD18hypo (N5) mice, the 129/SvEv genomic DNA fragments on chromosome 10 surrounding the CD18hypo mutation
were determined to be 27 cM long, between markers D10mit299
and D10mit162 (Table I). For the C57BL/6J CD18hypo inbred
strain (N10, The Jackson Laboratory), the 129/SvEv genomic fragment was determined to be 30 cM long, between markers
D10mit194 and D10mit162 (Table I).
adapted PASI score. Mice from the N6 backcross generation lacking the
PL/J-derived congenic fragment were used as controls.
Statistical analysis
Quantitative results are presented as mean values ⫾ SD. Mean values were
tested by means of a two-tailed heteroscedastic Student’s t test.
Results
Identification of 129/SvEv alleles in PL/J CD18hypo and
C57BL/B6J CD18hypo parental strains
In the present study we continued to use the CD18hypo strains
previously reported (34) for the generation of congenic strains. In
Construction of congenic strains for PL/J alleles by
marker-assisted selection
We have previously identified two loci on chromosome 10 and one
locus on chromosome 6 as being significantly linked to the psoriasiform skin disease and earlier onset of disease in the most severely affected mice, respectively (34). These loci were identified
in a cross where both parental strains carried a mutation resulting
in reduced expression of the ␤2 integrin chain CD18, the CD18hypo
mutation. The two loci on chromosome 10 were shown to interact
in an additive fashion in disease development. To confirm these
loci and to further investigate their role in the development of
psoriasiform skin disease, each locus, derived from the diseasesusceptible CD18hypo PL/J (N5) strain, was backcrossed onto the
resistant CD18hypo C57BL/6J inbred strain to produce congenic
strains. Consequently, the congenic mice produced in the present
study all carry the CD18hypo mutation, and the effect of the congenic fragment on disease development was evaluated under the
condition of low expression of CD18. This is relevant since the
loci were identified under the same conditions (34).
Ninety-one informative markers covering the genome with an
average marker distance of 15.5 cM were genotyped in each successive generation to define the introgressed regions of interest
derived from CD18hypo PL/J and to enable C57BL/6J purity in the
Table II. Summary of clinical outcome in PSD1 congenic mice
Straina
Chromosome
Chromosome
Chromosome
Chromosome
Chromosome
Control
PL/J
(CD18hypo)g
10A
10B
10C
10D
6
Psoriasiform Skin Disease
Incidence (%)b,c
PASI Score
(mean ⫾ SD)c
0/19 (0)
7/8 (87.5)
14/14 (100)
0/10 (0)
0/9 (0)
0/32 (0)
9/10 (90%)
0
2.75 ⫾ 1.58
3.21 ⫾ 0.80
0
0
0
3.30 ⫾ 1.19
pd
Arthritis Incidence (%)e,f
Arthritis Score
(mean ⫾ SD)f
—f1
ⴱⴱf2
ⴱⴱf3
—f4
0/19 (0)
6/8 (75.0)
11/14 (78.6)
0/10 (0)
0/9 (0)
0/32 (0)
0/10 (0)
0
5.16 ⫾ 1.83
3.43 ⫾ 2.34
0
0
0
0
—f5
—f6
pd
—f7
ⴱⴱf8
ⴱⴱf9
—f10
ⴱⴱf11
ⴱⴱf12
a
N6 congenic mice were genotyped by microsatellite markers, which can be retrieved from http://www.ensembl.org. Thirty-two heterozygote littermates at different target
regions (8 mice per group) served as controls. The data from these 32 control mice were pooled together since none of control mice developed the psoriasiform skin disease and/or
arthritis.
b
Onset and severity of the psoriasiform skin disease were estimated by an adapted PASI score every 2 wk for 30 wk in each group.
c
Adapted PASI scores were calculated for affected and unaffected mice at 5 mo of age and are presented as group means with standard errors. Mice that had to be sacrificed
due to severe disease were given a score of 7.
d
The p values calculated with the Newman-Keuls test were considered significant at p ⬍ 0.01 (ⴱⴱ). f1–f4, The p value was calculated from the comparison of adapted PASI
score between control mice and chromosome 10A or chromosome 10B or chromosome 10C or chromosome 10D congenic mice; f5, f6, the p value was calculated from the
comparison of adapted PASI score between PL/J CD18hypo mice and chromosome 10B or chromosome 10C congenic mice; f7–f10, the p value was calculated from
the comparison of arthritis score between control mice and chromosome 10A or chromosome 10B or chromosome 10C or between chromosome 10D congenic mice; f11, f12,
the p value was calculated from the comparison of arthritis score between PL/J CD18hypo mice and chromosome 10B or chromosome 10C congenic mice. The p value for
chromosome 6 congenic mice was not listed. —, no significance.
e
Arthritis developed in 6 out of 8 chromosome10B mice that showed severe psoriasiform skin disease at 5 mo of age.
f
Adapted clinical arthritis scores were calculated for affected and unaffected mice at 5 mo of age and all are presented as group means with standard errors.
g
N5 PL/J CD18hypo mice were typed by Southern blot for assessment of the psoriasiform skin phenotype and arthritis.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 2. Frequency of the psoriasiform skin disease in chromosome
10B and chromosome 10C congenic strains and N5 CD18hypo PL/J mice.
To assess the time course for the development of psoriasiform skin disease
in PL/J CD18hypo mice and chromosome 10B and chromosome 10C congenic mice, the onset of psoriasiform skin disease was routinely monitored
using an adapted PASI score every 2 wk for 18 wk in each group. Statistical significant differences in the time of onset of the psoriasiform skin
disease were found between CD18hypo PL/J mice and chromosome 10C
congenic mice at 6 wk, and between CD18hypo PL/J mice and chromosome
10B congenic mice or chromosome 10C congenic mice at 8 wk. p values
calculated with the Newman-Keuls test were considered significant for p ⬍
0.05 (ⴱ) or p ⬍ 0.01 (ⴱⴱ).
5524
A LOCUS CONTROLS THE PSORIASIFORM DERMATITIS AND ARTHRITIS
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FIGURE 3. The congenic interval PSD1 on chromosome 10 is responsible for the development of psoriasiform skin disease. A, A representative male
chromosome 10B congenic mouse carrying the PSD1 locus derived from CD18hypo PL/J parent developed a progressive psoriasiform skin disease at 5 mo
of age, characterized by erythema, scale, and crust formation on the back. B, Higher magnification of A as indicated by the red rectangle with severe scale
and crust formation. C, A littermate control resulting from heterozygote crosses of N6 C57BL/6J congenic strains that were confirmed for the absence of
the PSD1 region derived from PL/J CD18hypo mouse did not develop the skin disease. D, H&E staining of skin sections from PSD1 congenic mice with
hyperkeratosis and epidermal hyperplasia, together with a dense inflammatory cell infiltration being observed in the dermis (original magnification, ⫻10).
E, Higher magnification of the area indicated by the asterisk of D revealed subcorneal microabscesses with an accumulation of neutrophils below and in
the hyperorthokeratosis (original magnification, ⫻100). F, Higher magnification of D as indicated by the rectangle revealing a diffuse inflammatory cell
infiltrate in the dermis (original magnification, ⫻100). G, H&E staining of a skin section from a control mouse with normal epidermis and dermis (original
magnification, ⫻10). H, Immunostaining with Abs against keratin K14 indicates a significant increase in epidermal thickness of the affected skin from PSD1
congenic mice, compared with the epidermis of control mice (I), which consists of only two cell layers. J, A marked increase in numbers of CD4⫹ T cells
infiltrating the affected skin was observed in the dermis of PSD1 congenic mice. K, In contrast, virtually no CD4⫹ T cells were found in the skin of control
mice. L, In the dermis of PSD1 congenic mice high numbers of F4/80⫹ macrophages were observed. M, Almost no macrophages were found in the skin
of control mice. N, Strong infiltration of TNF-␣ producing cells in the dermis of PSD1 congenic mice. O, No TNF-␣ staining in the skin of control mice.
Cell nuclei were counterstained with DAPI (if not otherwise indicated, original magnification was ⫻20). e, Epidermis; d, dermis; h, hair follicle; k,
hyperorthokeratosis; m, pigment loaded macrophages. Dotted lines indicate the border between epidermis and dermis. One representative experiment out
of four is shown.
rest of the genome. Six generations of marker-assisted backcrossing were performed using the speed congenic approach (35). Congenic mice were established for the chromosome 10 regions (Fig.
1) and the chromosome 6 regions (data not shown).
In total, five congenic strains were generated. The congenic
mouse strains were named according to the congenic intervals on
chromosomes 6 and 10: chromosome 6 (D6mit274–D10mit14, 50
cM) (data not shown), chromosome 10A (D10mit75–D10mit126,
The Journal of Immunology
5525
19 cM), chromosome 10B (D10mit126–D10mit38, 6 cM), chromosome 10C (D10mit75–D10mit271, 70 cM) and chromosome
10D (D10mit233–D10mit271, 8 cM) (Fig. 1).
The PSD1 locus, a 9-cM genetic element of chromosome 10,
controls the susceptibility for the psoriasiform skin disease
and arthritis
Phenotypic evaluation of the psoriasiform skin disease was performed in homozygous congenic mice. Littermate mice heterozygous at the PL/J-originated congenic fragment for each locus were
used for controls, which were generated as littermates to the homozygous congenics in the intercross breeding (see Materials and
Methods). Using littermates as controls, we were able to eliminate
the potential influence of contaminating fragments from the donor
CD18hypo PL/J (N5) in the rest of the genome, PL/J- as well as
129/SvEv-derived fragments.
The psoriasiform skin disease developed at the age of 16 wk in
7 out of 8 mice of the chromosome 10B congenic mice and in all
chromosome 10C congenic mice (Fig. 2). Interestingly, all congenic mice carrying the PSD1 fragment, namely the chromosome
10B and chromosome 10C congenic mice, also developed psoriatic
arthritis in 6 out of 8 chromosome 10B congenic mice and 11 out
of 14 chromosome 10C congenic mice (Table II). The chromosome 10A and 10D congenic mice, however, did not show any
signs of psoriasiform skin disorder and/or arthritis. None of the
littermate control mice heterozygous at the target regions developed any signs of psoriasiform skin disorder or arthritis (Table II).
Since the presence of the congenic fragment in the chromosome
10B mice is enough to make the otherwise resistant C57BL/6J
CD18hypo susceptible for both the psoriasiform skin disease and
arthritis, these data clearly indicate that a susceptibility locus common both for psoriasiform skin disease and arthritis is located
within the 19.8-megabase-pair (9-cM) interval (D10mit126–
D10mit194) on chromosome 10 (Fig. 1). We therefore designated
this locus psoriasiform skin disease-associated locus 1 (PSD1).
The chromosome 10B mice are referred to as the PSD1 congenic
mice. Interestingly, 9 out of 10 PL/J CD18hypo mice (N5) developed the severe psoriasiform skin disease; however, none developed arthritis at 5 mo of age (Table II).
Interestingly, and in line with our previous observation that the
locus on chromosome 6 promotes earlier onset of the psoriasiform
skin disease (34), the onset of disease in chromosome 10B and
chromosome 10C congenic strains, which did not carry chromosome 6 region, was later than the onset in CD18hypo PL/J (N5)
mice (Fig. 2). No signs of psoriasiform skin disorder and/or
arthritis were observed in the congenic mice carrying the chromosome 6 fragment alone (Table II).
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FIGURE 4. The congenic interval PSD1 on
chromosome 10 is responsible for the development
of arthritis in PSD1 congenic mice. A, A representative female chromosome 10B congenic mouse
carrying PSD1 locus derived from CD18hypo PL/J
parents is shown with psoriasiform skin disease, as
well as reddened swollen ankle and paw joints indicative of arthritis (white arrows) (left mouse)
compared with normal ankle and paw joints (white
arrows) of the littermate control wild-type
C57BL/6J in the PSD1 region derived from the
intercross of the N6 generation (right mouse). B,
Representative H&E staining of sections from
metatarsals of the arthritic paw of a PSD1 congenic
mouse and (C) from a normal paw of a littermate
control mouse. D, Higher magnification of the
boxed area in B shows bone erosion (black arrows)
with numerous osteoclasts. E, Higher magnification of the area indicated by the asterisk in B reveals pannus growing over the cartilage with cartilage damage (arrows). F, A marked macrophage
(F4/80) infiltrate was found both in the dermis, in
the joint capsule, and in the synovia of PSD1 congenic mice, compared with almost no staining in
control mice (G). H, Abundant staining for TNF␣-producing cells was found in the dermis as well
as the joint capsule, synovia, and adjacent bone of
PSD1 congenic mice, compared with almost no
staining in the dermis and the joint capsule of a
littermate control mouse without the PSD1 fragment (I). J, Double staining for F4/80 and TNF-␣
of inflamed joints derived from PSD1 congenic
mice reveals macrophages to be a major source for
TNF-␣. Arrows indicate the position of joints with
the capsule and bone. Cell nuclei were counterstained with DAPI (original magnification of C and
D, ⫻10; E and F, ⫻40; G–J ⫻ 20). e, Epidermis;
b, bone marrow; c, capsule with synovia; d, dermis.
Dotted lines indicate the border between epidermis
and dermis. One representative experiment out of
four is shown.
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A LOCUS CONTROLS THE PSORIASIFORM DERMATITIS AND ARTHRITIS
Characterization of the psoriasiform skin disease in congenic
mice carrying the PSD1 susceptibility locus
Characterization of psoriatic arthritis in mice carrying the
PSD1 congenic locus
Similar to human psoriasis patients, the chromosome 10B and
chromosome 10C mice carrying the PSD1 locus developed both
the psoriasiform skin disease and an arthritis with swelling and
erythema of ankle joints as well as the tarsometatarsal and metatarsophalangeal paw joints (Fig. 4, A, left mouse, indicated by
white arrows), compared with a littermate control (Fig. 4, A, right
mouse). H&E staining revealed marked arthritic changes in the
joints of PSD1 congenic mice (Fig. 4B), compared with normal
paw joints of the littermate control mice (Fig. 4C). Higher magnification of Fig. 4B showed infiltration of bones by numerous
osteoclasts (Fig. 4D) and pannus formation with cartilage damage
(Fig. 4E).
We earlier reported an increase in macrophage numbers with
increased TNF-␣ release in the psoriasiform skin of CD18hypo
PL/J mice (8). Notably, in the present study we observed significantly increased numbers of macrophages not only in the dermis,
but also in the joint capsule and synovia of the joints in PSD1
carrying mice (Fig. 4F) compared with a virtual absence of macrophages in control mice (Fig. 4G). A striking number of inflammatory cells revealed strong staining for TNF-␣ in the skin and in
the joint capsule, synovia, and bone of the joints in PSD1 carrying
mice (Fig. 4H) with almost no TNF-␣ staining in control mice
(Fig. 4I). Double staining confirmed that virtually all F4/80⫹ macrophages in the inflamed joints, including the capsule and synovia
of PSD1-carrying mice, produced TNF-␣ (Fig. 4J). These data
provide strong evidence that C57BL/6J CD18hypo mice congenic
for a 9-cM genetic element of chromosome 10 from the PL/J strain
(PSD1 locus) develop not only the psoriasiform skin disease but
also a severe psoriatic arthritis.
TNF-␣ inhibitor treatment and CD4-depleting Abs result in the
resolution of psoriasiform dermatitis and arthritis in PSD1
congenic mice
To confirm that the PSD1 locus is responsible for the T cell-dependent and TNF-␣-mediated inflammatory skin as well as joint
FIGURE 5. The PSD1 locus on chromosome 10 controls the development of the psoriasiform skin disease and arthritis in a TNF-␣-dependent
fashion. A, A significant decrease in the adapted arthritis score, indicative
of clinical improvement of the arthritis, was observed after treatment with
etanercept of affected PSD1 congenic mice (ⴱ, p ⫽ 0.04, Student’s t test),
(B) but not after treatment with 0.9% NaCl of PSD1 congenic mice (p ⫽
0.10, Student’s t test). C, A significant decrease in adapted PASI score
indicative of clinical resolution of the psoriasiform dermatitis was seen in
affected PSD1 congenic mice after 30 days of treatment with etanercept
(ⴱ, p ⫽ 0.04, Student’s t test), (D) while control injection with 0.9% NaCl
did not result in any improvement (p ⫽ 0.65, Student’s t test).
disease, the chromosome 10B congenic (PSD1 congenic) mice
were treated with a TNF-␣ inhibitor (etanercept), consisting of a
recombinant soluble TNF-␣ receptor fusion protein, or with antiCD4 depleting Abs, as described previously for CD18hypo PL/J
mice (8, 37). The clinical arthritis score before anti-TNF-␣ treatment (5.5 ⫾ 1.73) improved substantially in the PSD1 congenic
mice after 4 wk of TNF-␣ inhibitor treatment (2.25 ⫾ 0.5, p ⫽
0.04) (Fig. 5A), while no improvement of the arthritis scores was
observed in PSD1 congenic mice treated with 0.9% NaCl ( p ⫽
0.1) (Fig. 5B). The adapted PASI scores increased before TNF-␣
inhibitor treatment (3.75 ⫾ 0.5), and they revealed a substantial
improvement after the TNF-␣ inhibitor treatment with etanercept
(1.5 ⫾ 0.58, p ⫽ 0.04) (Fig. 5C). In contrast, no significant
changes of the adapted PASI score were observed in PSD1 mice
treated with 0.9% NaCl ( p ⫽ 0.65) (Fig. 5D). These data unequivocally show that the PSD1 interval controls TNF-␣-dependent skin
and joint inflammation. Similar data were obtained after 4 wk of
CD4 T cell depletion (data not shown), further suggesting that both
the psoriasiform skin disease and the associated arthritis share a
CD4⫹ T cell-dependent pathogenesis.
Discussion
In the present study we confirmed that a locus on chromosome 10,
previously identified by us (34), drives the development of psoriasiform skin disease as well as arthritis in the presence of low
expression of the common chain of ␤2 integrins (CD18). We show
that a congenic strain containing the 9-cM PL/J element on chromosome 10 (D10mit126 to D10mit194), designated as chromosome 10B PSD1 congenic strain, on the resistant C57BL/6J
CD18hypo background developed psoriasiform skin disease, and
most notably also a severe arthritis of joints. The PL/J background
in the presence of low CD18 expression drives the development of
psoriasiform skin disease but rarely in combination with arthritis.
In contrast, the two produced congenic strains harboring fragments
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All chromosome 10B and chromosome 10C congenic mice carrying the PSD1 locus developed a progressive psoriasiform skin disease, characterized by severe erythema and scale and crust formation on the back (Fig. 3, A and B). In contrast, control mice did not
show any pathological skin phenotype (Fig. 3C). The psoriasiform
skin disease in mice carrying the PSD1 locus showed an identical
clinical picture as observed in CD18hypo PL/J mice (8, 28). Histological analysis of skin sections from PSD1 congenic mice revealed hyperplasia of the epidermis (Fig. 3D), hyperorthokeratosis
(Fig. 3, D and E), subcorneal microabscesses (Fig. 3E), and a
diffuse inflammatory cell infiltrate in the dermis (Fig. 3F), as compared with normal epidermis and dermis in control mice (Fig. 3G).
Keratin 14 staining confirmed epidermal hyperplasia in the lesional skin from PSD1 congenic mice (Fig. 3H) compared with
control mice (Fig. 3I). As the psoriasiform skin disease critically
depends on the presence of CD4⫹ T cells and activated TNF-␣releasing macrophages (8, 37), cryosections derived from skin of
affected PSD1 congenic and control mice were stained with antimouse CD4, anti-mouse F4/80, and anti-TNF-␣ mAb. Increased
numbers of CD4⫹ cells, macrophages, and strong staining for
TNF-␣ were observed in the dermis of PSD1 congenic mice (Fig.
3, J, L, and N), but not in sections derived from control mice (Fig.
3, K, M, and O).
The Journal of Immunology
5527
telomeric and centromeric of the 9-cM PSD1 fragment (chromosomes 10A and 10D) did not develop the psoriasiform skin disease
and/or arthritis within an observation period of 10 mo.
These data indicate that a gene (or genes) within the PSD1 locus
contribute(s) to pathogenic events common to the psoriasiform
skin disease and arthritis in this context under the condition of low
expression of CD18.
Since the chromosome 10C mice, congenic for a larger fragment
of the chromosome 10 including the PSD1 locus, developed a phenotype identical to the PSD1 congenic (chromosome 10B), we
conclude that the PSD1 locus is the major locus within that region.
In our previous study, we found another locus on the lower part of
chromosome 10 that was also linked to the psoriasiform skin disease and that interacted with PSD1 locus in an additive fashion in
the disease development (34). Herein, our data demonstrate that
chromosome 10C congenic mice containing two susceptible loci
reveal a slightly earlier onset of disease when compared with chromosome 10B congenic only carrying PSD1 region on chromosome
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FIGURE 6. Potential
candidate
genes in the PSD1 interval of chromosome 10 in congenic mice compared with a syntenic map of human
chromosome 6q. The black bar to the
left of the map is the PSD1 segment
with scale indicating positions in centimorgans. Gene names, symbols, and
gene positions of both mice and humans are listed on the right site. The
PSD1 region was annotated according
to the Ensembl Genome database
(http://www.ensembl.org). Ensembl
is a database of automatic annotation
on selected eukaryotic genomes.
10. However, the difference is not statistically significant, and further dissection of potentially interacting genes is required in future
analyses.
Based on many observations, including the fact that human psoriasis is inducible in SCID mouse xenograft models by the injection of T cells from human skin lesions, it is largely accepted that
psoriasis is a T cell-dependent disease (40, 41). Also, CD4⫹ T
cells are crucial for the induction and development of the psoriasiform skin disease in the CD18hypo PL/J psoriasis model (37, 42).
The fact that depletion of CD4⫹ T cells in the PSD1 congenic
strain resulted in the resolution of both the psoriasiform skin disease and the arthritis verifies the PSD1 element as causally contributing to the manifestation of a T cell-dependent psoriasiform
inflammation in the two organs (data not shown).
These data unequivocally indicate that the PSD1 genetic element harbors important modifier genes that are causally involved
in a shared T cell-dependent pathogenesis of skin and joint inflammation. CD18hypo PL/J mice rarely reveal the combined clinical
5528
A LOCUS CONTROLS THE PSORIASIFORM DERMATITIS AND ARTHRITIS
resistant strain. The pathogenic pathways controlled by the gene(s)
in this locus need further elucidation. The PSD1 locus harbors 46
published genes. Continuous backcrossing will be performed to
further dissect the PSD1 region into smaller genetic intervals for
further identification of modifier genes, as well as their function
and interaction with the ␤2 integrins in psoriasiform skin and joint
disease. The PSD1 congenic mouse strain qualifies as a valuable
tool for preclinical studies and the identification of novel treatment
strategies.
Acknowledgments
We are grateful to Heidi Hainzl for technical assistance with the immunohistochemistry, and Solveig Borgehammar and Malin Neptin for help with
genotyping.
Disclosures
The authors have no financial conflicts of interest.
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picture of psoriasiform skin disease and arthritis in a backcross to
the PL/J background for six generations. This discrepancy indicates that either other PL/J loci are resistance loci for arthritis, or
that the C57BL/6J genome harbors susceptibility loci for arthritis
apart from the PSD1 region and the CD18hypo mutation. Crossing
the PSD1 congenics to the parental CD18hypo C57BL/6J mice to
observe whether an F1 at the PSD1 locus with homozygous
C57BL/6J background would be sufficient for arthritis, or creating
an F1 with the N5 CD18hypo PL/J donor to study whether the
homozygosity across the PSD1 interval in setting of background
heterozygosity would be permissive for arthritis can be further
ways to dissect the genetic modifier(s) of the arthritis.
In addition to chromosome 10 regions, a locus on chromosome
6 was identified to predominantly influence the time point of disease development (34). In the present study, congenic strain carrying the PL/J element on chromosome 6 did not develop the psoriasiform skin disorder, however, chromosome 10B and
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Human psoriasis and psoriatic arthritis are interrelated disorders,
as up to 40% of psoriasis patients also suffer from psoriatic arthritis
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The PSD1 on murine chromosome 10 is syntenic to human
chromosome 6q16 and 6q21– q24, as shown in Fig. 6. There are no
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one of the major type 1 diabetes mellitus loci, an autoimmune
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Database) contains a number of potential candidate genes for the
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humans (54), and Fyn proto-oncogene, which has been suggested
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human psoriasis successfully treats both skin and joint inflammation (57).
In the present study we identified the 9-cM PSD1 genetic element on chromosome 10 that distinctly contributes to the susceptibility to psoriasiform skin disease and arthritis in an otherwise
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