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The Journal of Immunology, 2008, 180, 5520 -5529
Copyright © 2008 by The American Association of Immunologists, Inc.
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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 Model1

Honglin Wang*, Daniel Kess*, Anna-Karin B. Lindqvist{dagger}, Thorsten Peters*, Anca Sindrilaru*, Meinhard Wlaschek*, Robert Blakytny{ddagger}, Rikard Holmdahl{dagger} and Karin Scharffetter-Kochanek2,*

* Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany; {dagger} Medical Inflammation Research, University of Lund, Lund, Sweden; and {ddagger} Institute of Orthopaedic Research and Biomechanics, University of Ulm, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 β2 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 disease-associated locus 1 (PSD1). High numbers of CD4+ T cells and TNF-{alpha} producing macrophages were detected both in inflamed skin and joints in these congenic mice, with a complete resolution upon TNF-{alpha} inhibitor therapy or depletion of CD4+ 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Psoriasis 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 initiation (5, 6, 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, 12, 13, 14, 15, 16, 17, 18, 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, 21, 22, 23, 24, 25, 26, 27). In contrast, the mouse PL/J strain carrying a CD18 hypomorphic (CD18hypo) mutation, with reduced expression of the common chain of β2 integrins (CD11/CD18) to 2–16% of wild-type levels, develops a skin disease that closely resembles human psoriasis (28).

CD18 represents the common β2-chain of the β2 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 β2 integrins in human psoriasis and other inflammatory skin diseases is poorly understood (31, 32, 33). Linkage analysis of psoriasis families has identified a 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 x 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 microsatellite-assisted 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
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 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

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).


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  		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

 
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.

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.


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  		Table II. Summary of clinical outcome in PSD1 congenic mice

 
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-{alpha}, 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-{alpha} (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-µm sections of skin and joints were stained with H&E as described (37).

Administration of etanercept

Etanercept (100 µg per mouse) was administrated i.p. every day for a period of 30 days to neutralize TNF-{alpha}. 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 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
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 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).

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 β2 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 disease-susceptible 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 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).


Figure 1
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  		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 ({blacksquare}) and the interval between the selection markers for which inclusion or exclusion is not proven ({square}) 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.

 
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, 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).


Figure 2
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  		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 (*) or p < 0.01 (**).

 
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).

Characterization of the psoriasiform skin disease in congenic mice carrying the PSD1 susceptibility locus

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-{alpha}-releasing macrophages (8, 37), cryosections derived from skin of affected PSD1 congenic and control mice were stained with anti-mouse CD4, anti-mouse F4/80, and anti-TNF-{alpha} mAb. Increased numbers of CD4+ cells, macrophages, and strong staining for TNF-{alpha} 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).


Figure 3
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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, x10). 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, x100). F, Higher magnification of D as indicated by the rectangle revealing a diffuse inflammatory cell infiltrate in the dermis (original magnification, x100). G, H & E staining of a skin section from a control mouse with normal epidermis and dermis (original magnification, x10). 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-{alpha} producing cells in the dermis of PSD1 congenic mice. O, No TNF-{alpha} staining in the skin of control mice. Cell nuclei were counterstained with DAPI (if not otherwise indicated, original magnification was x20). 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.

 
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).


Figure 4
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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-{alpha}-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-{alpha} of inflamed joints derived from PSD1 congenic mice reveals macrophages to be a major source for TNF-{alpha}. Arrows indicate the position of joints with the capsule and bone. Cell nuclei were counterstained with DAPI (original magnification of C and D, x10; E and F, x40; G–J x 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.

 
We earlier reported an increase in macrophage numbers with increased TNF-{alpha} 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-{alpha} in the skin and in the joint capsule, synovia, and bone of the joints in PSD1 carrying mice (Fig. 4H) with almost no TNF-{alpha} 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-{alpha} (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-{alpha} 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-{alpha}-mediated inflammatory skin as well as joint disease, the chromosome 10B congenic (PSD1 congenic) mice were treated with a TNF-{alpha} inhibitor (etanercept), consisting of a recombinant soluble TNF-{alpha} receptor fusion protein, or with anti-CD4 depleting Abs, as described previously for CD18hypo PL/J mice (8, 37). The clinical arthritis score before anti-TNF-{alpha} treatment (5.5 ± 1.73) improved substantially in the PSD1 congenic mice after 4 wk of TNF-{alpha} 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-{alpha} inhibitor treatment (3.75 ± 0.5), and they revealed a substantial improvement after the TNF-{alpha} 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-{alpha}-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.


Figure 5
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  		FIGURE 5. The PSD1 locus on chromosome 10 controls the development of the psoriasiform skin disease and arthritis in a TNF-{alpha}-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 (*, 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 (*, 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).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
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 β2 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 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 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 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 chromosome 10C congenic strains lacking the PL/J region on chromosome 6 had delayed onset of the skin disease when compared with CD18hypo PL/J (N5) mice, which have both chromosome 10 and chromosome 6 genetic context.

Human psoriasis and psoriatic arthritis are interrelated disorders, as up to 40% of psoriasis patients also suffer from psoriatic arthritis (43, 44, 45, 46, 47).

The PSD1 on murine chromosome 10 is syntenic to human chromosome 6q16 and 6q21–q24, as shown in Fig. 6. There are no reports in the literature associating 6q with human psoriasis. All genes identified to date with a known function in the PSD1 interval between 26.7 and 46.5 megabase-pairs are listed (Fig. 6). Importantly, the human chromosome region 6q21 was identified to be one of the major type 1 diabetes mellitus loci, an autoimmune disease (48), and it also harbors susceptibility gene(s) for rheumatoid arthritis (49, 50, 51). The current mouse map (Mouse Genome Database) contains a number of potential candidate genes for the PSD1 region. These include the CD24a gene, which is a genetic modifier for risk and progression of multiple sclerosis (52), experimental autoimmune encephalomyelitis in mice (53), gap junction protein-{alpha}-1 gene, the mutation of which leads to hyperkeratosis in humans (54), and Fyn proto-oncogene, which has been suggested to play a unique role in T cell activation through TCR-mediated signaling (55).


Figure 6
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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.

 
Based on our previous findings (8, 56), an emerging model of psoriasis pathogenesis in humans suggests that dermal macrophages, once activated by T cell cytokines, go on to produce large amounts of TNF-{alpha} that lead to skin changes (8, 9, 56). In fact, we previously observed that, similar to human psoriasis, activated macrophages are significantly increased in the lesional skin as well as in the inflamed skin draining lymph nodes of affected CD18hypo mice and, in addition, they are an important source of TNF-{alpha} in vivo (8). Both depletion of macrophages and neutralization of TNF-{alpha} resulted in a significant alleviation of psoriasiform skin inflammation in CD18hypo PL/J mice (8). In the present report, the findings that activated TNF-{alpha} producing macrophages are increased in numbers, and that the resolution of both psoriasiform skin disease and arthritis occurred upon treatment with TNF-{alpha} inhibitor (etanercept) in PSD1 congenic mice strongly suggest that the PSD1 locus is important for a T cell-induced, and macrophage-sustained, pathogenesis of both the psoriasiform skin disease and arthritis. This is most interesting, as neutralization of TNF-{alpha} in 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 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 β2 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
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-2006-018661 (AutoCure). Back

2 Address correspondence and reprint requests to Dr. Karin Scharffetter-Kochanek, Department of Dermatology and Allergic Diseases, University of Ulm, Maienweg 12, D-89081 Ulm, Germany. E-mail: karin.scharffetter-kochanek{at}uniklinik-ulm.de Back

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. Back

Received for publication August 23, 2007. Accepted for publication February 8, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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