Journal of Dermatology 2015; 42: 874–880
doi: 10.1111/1346-8138.12961
ORIGINAL ARTICLE
Psoriatic T cells reduce epidermal turnover time and affect cell proliferation contributed from differential gene expression Junqin LI,1 Xinhua LI,1 Ruixia HOU,1 Ruifeng LIU,1 Xincheng ZHAO,1 Feng DONG,2 Chunfang WANG,3 Guohua YIN,1 Kaiming ZHANG1 1
Institute of Dermatology, Taiyuan City Center Hospital, 3Laboratory Animal Center, Shanxi Medical University, Taiyuan, Department of Dermatology, Changzhi City Second People’s Hospital, Changzhi, China
2
ABSTRACT Psoriasis is mediated primarily by T cells, which reduce epidermal turnover time and affect keratinocyte proliferation. We aimed to identify differentially expressed genes (DEG) in T cells from normal, five pairs of monozygotic twins concordant or discordant for psoriasis, to determine whether these DEG may account for the influence to epidermal turnover time and keratinocyte proliferation. The impact of T cells on keratinocyte proliferation and epidermal turnover time were investigated separately by immunohistochemistry and cultured with 3H-TdR. mRNA expression patterns were investigated by RNA sequencing and verified by real-time reverse transcription polymerase chain reaction. After co-culture with psoriatic T cells, the expression of Ki-67, c-Myc and p53 increased, while expression of Bcl-2 and epidermal turnover time decreased. There were 14 DEG which were found to participate in the regulation of cell proliferation or differentiation. Psoriatic T cells exhibited the ability to decrease epidermal turnover time and affect keratinocyte proliferation because of the differential expression of PPIL1, HSPH1, SENP3, NUP54, FABP5, PLEKHG3, SLC9A9 and CHCHD4.
Key words:
differentially expressed genes, epidermal turnover time, keratinocyte, psoriasis, T cells.
INTRODUCTION Psoriasis is a chronic inflammatory disease that imposes a significant burden on patients’ physical and mental health.1 Abnormal keratinocyte proliferation and epidermal turnover time (ETT) are the main pathological features of psoriasis and are closely related to the course and recurrence of this disease.2 Immune abnormalities in T cells play important roles in the pathogenesis of psoriasis. In psoriasis, the level of the Thelper 1 cytokine is enhanced in T cells directly differentiated from psoriatic bone marrow CD34+ cells in vitro, and the number and function of regulatory T cells is decreased.3,4 Moreover, expression of the T-cell receptor variable region is predominant in peripheral blood T-cell of psoriasis.5 Additionally, it was found that psoriatic T cells influence keratinocyte proliferation.6 However, the abnormality of T cells which results in the influence on keratinocyte proliferation and ETT was unknown. In our previous studies, we have investigated the incidence of psoriasis in monozygotic (MZ) twins discordant for psoriasis (MZ-DP) who lived in the same environment after birth. Additionally, our studies demonstrated that the onset of psoriasis in MZ twins concordant for psoriasis (MZ-CP) varied from 1 month to 3 years. Thus, our data have suggested that concordance for psoriasis is not 100% among MZ twins. Consistent with this, previous studies have demonstrated that concordance can be
less than 72%.7 For the specificity of MZ twins, it may be the best choice to study abnormality of T cells in psoriasis. Ki-67 is a nuclear protein that is expressed in all active phases of the cell cycle, but is not present in non-proliferating cells.8 Similarly, c-Myc plays a pivotal role in cell cycle progression.9,10 Additionally, Bcl-2 and p53 function together to regulate apoptosis.11–13 Here, we first studied the impact of psoriatic peripheral blood T cells on ETT and cell proliferation (i.e. Ki-67, c-Myc, p53 and Bcl-2) before and after stimulation with streptococcal superantigen (Strep-SAg). For the purpose of discovering how the molecular abnormality of T cells contributed to the impact, we also identify differentially expressed genes (DEG) in T cells from normal individuals, MZ-CP, MZ-DP, and those with atopic dermatitis (AD) and sporadic psoriasis.
METHODS Specimen source Fifteen psoriasis and 15 normal subjects who were not MZ (Table S1) were studied to determine the effects of peripheral blood T cells on ETT and expression of Ki-67, c-Myc, p53 and Bcl-2. Normal and psoriasis patients were also age- and sexmatched. Aside from the above subjects, five pairs of MZ-CP, five pairs of MZ-DP and five patients with AD were also enrolled in the study of gene expression profiles and real-time
Correspondence: Kaiming Zhang, M.D., Institute of Dermatology, Taiyuan City Center Hospital, 1 Dong San Dao Xiang, Jiefang Road, Taiyuan, Shanxi 030009, China. Email: [email protected] Received 25 March 2015; accepted 20 April 2015.
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reverse transcription polymerase chain reaction (RT–PCR, Table S1). MZ twins were matched for age and sex, and none of them had smoked. The normal MZ-DP patients had not suffered from psoriasis after 2 years of sampling. Normal individuals presented with no systemic or autoimmune diseases. Psoriasis and AD were diagnosed according to clinical and histological parameters. Patients had not received corticosteroids or immunosuppressants within 6 months of enrollment. All patients provided informed consent for participation in the study. The protocol was approved by the medical ethics committee of Taiyuan City Center Hospital and followed the Declaration of Helsinki protocols.
Peripheral blood T-cell proliferation and stimulation Peripheral blood T cells were cultured, stimulated and identified as described in our previous study.14 Strep-SAg was prepared as previously described.15 T cells were seeded in 24well culture plates (Corning, Corning, NY, USA). Some cells were stimulated with Strep-SAg and collected after 24 h of culture. Some cells were isolated using a cell sorter for analysis of CD4+ and CD8+ T cells (CD4 and CD8 magnetic beads; Invitrogen, Carlsbad, CA, USA).
clearing and mounting, the sections were analyzed under an Olympus microscope (Olympus, Tokyo, Japan).
RNA isolation Total RNA was extracted using TRIzol reagent (Invitrogen) and treated with DNase (Invitrogen). mRNA was purified using oligo (dT) magnetic beads (Invitrogen), and purified mRNA was used to synthesize complementary DNA (Takara, Dalian, China), which was used for sequencing and amplification by real-time RT–PCR.
Tag preparation, sequencing, gene annotation and screening DEG Complementary DNA obtained from one pair of MZ-CP, one pair of MZ-DP, five patients with AD, five patients with psoriasis and five normal patients were treated with NlaIII and MmeI to generate 21-bp tags. These tags were sequenced, and the genes were annotated by referring to the National Center for Biotechnology Information genetic database (ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/human.rna.fna.gz). Differentially expressed gene screening was performed according to our previous study.14
Real-time RT–PCR Skin culture The skin was cleaned with Hank’s balanced salt solution (Invitrogen) and cut. The epidermal skin was obtained with 3-mm punches and cultured in Netwell 3-D 12-well culture plates (Corning) with semipermeable membranes, which were used to ensure contact between the skin and Dulbecco’s modified Eagle’s medium (Invitrogen). One hundred microliters of T cell (1 9 106 cells/mL) or Dulbecco’s modified Eagle’s medium was added to the culture medium. Cell culture was continued for 3 days after treatment with 3% trypsin (Invitrogen).
Test of ETT
Real-time RT–PCR was performed as previously described.14 Primers for real-time RT–PCR are shown in Table S2.
Statistical analysis The differences in gene expression obtained from real-time RT–PCR were analyzed by the 2DDCt method.16 The means of indicated groups were compared using a two-tailed unpaired Student’s t-test. Results were considered statistically significant at P < 0.05.
RESULTS
Skin was cultured in Dulbecco’s modified Eagle’s medium with 3 H-TdR (5 lCi/mL; China Institute of Atomic Energy, Beijing, China) for 40 min, then washed and cultured with Dulbecco’s modified Eagle’s medium without 3H-TdR. Skin was fixed and sliced at different times. Nuclear-4 emulsion (China Institute of Atomic Energy) was applied on the skin in a darkroom. The skin was developed with D-19b developer (China Lucky Group, Hebei, China), fixed with F-5 acid hardening fixer (China Lucky Group), stained with hematoxylin–eosin, dehydrated and mounted. Cells exhibiting isolated nuclei and strong refraction of silver staining under an oil immersion lens were considered labeled cells. The time was monitored when the labeled cells moved from the basal layer to the stratum granulosum; this was considered the ETT. After culture for 6 days, skin was fixed and sectioned.
Peripheral blood T cells from psoriasis exhibited changes in Ki-67, c-Myc, p53 and Bcl-2 expression in the epidermis
Immunohistochemistry
Effects of psoriatic peripheral blood T cells stimulated with Strep-SAg on the expression of Ki67, c-Myc, p53 and Bcl-2 in the epidermis
Mouse antihuman antibodies targeting Ki-67, c-Myc, p53 and Bcl-2 (Santa Cruz Biotechnology, Dallas, TX, USA) were used in this study. Immune complexes were visualized using peroxidaseconjugated secondary antibodies (Santa Cruz Biotechnology), according to the manufacturer’s protocol. After dehydration,
© 2015 Japanese Dermatological Association
We examined the expression of Ki-67, c-Myc, p53 and Bcl-2 in psoriatic lesions before and after the effects of psoriatic peripheral blood T cells in vitro. We found significant upregulation of Ki-67, c-Myc and p53 in psoriatic lesions (Fig. 1). However, Bcl-2 expression was rarely observed in these tissues. In contrast, expression of these proteins was normal in uninvolved skin from psoriasis. Interestingly, Ki-67, c-Myc and p53 expression were increased in normal skin and uninvolved psoriatic skin after co-culture with psoriatic peripheral blood T cells. Additionally, Bcl-2 expression was decreased in uninvolved and normal skin after co-culture with psoriatic peripheral blood T cells.
The expression of these proteins was similar in normal and uninvolved psoriatic skin after co-culture with unstimulated and Strep-SAg-stimulated normal T cells (Fig. 1). Compared with co-
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Normal skin
Normal skin cocultured with psoriaƟc CD8+ T cell
Uninvolved psoriaƟc skin
Normal skin cocultured with psoriaƟc T cell sƟmulated by Strep-SAg
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Normal skin cocultured with psoriaƟc T cell
Uninvolved psoriaƟc skin cocultured with psoriaƟc T cell
Uninvolved psoriaƟc skin cocultured with normal T cell sƟmulated by Strep-SAg
Normal skin cocultured with psoriaƟc CD4+ T cell
Uninvolved psoriaƟc skin cocultured with psoriaƟc T cell sƟmulated by Strep-SAg
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Normal skin
Normal skin cocultured with psoriaƟc CD8+ T cell
Uninvolved psoriaƟc skin
Normal skin cocultured with psoriaƟc T cell sƟmulated by Strep-SAg
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Uninvolved psoriaƟc skin cocultured with psoriaƟc T cell
Normal skin cocultured with psoriaƟc T cell
Normal skin cocultured with psoriaƟc CD4+ T cell
Uninvolved psoriaƟc skin cocultured with normal T cell sƟmulated by Strep-SAg
Uninvolved psoriaƟc skin cocultured with psoriaƟc T cell sƟmulated by Strep-SAg
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Normal skin
Normal skin cocultured with psoriaƟc CD8+ T cell
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Normal skin cocultured with psoriaƟc T cell sƟmulated by Strep-SAg
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Uninvolved psoriaƟc skin cocultured with psoriaƟc T cell
Normal skin cocultured with psoriaƟc T cell
Normal skin cocultured with psoriaƟc CD4+ T cell
Uninvolved psoriaƟc skin cocultured with normal T cell sƟmulated by Strep-SAg
Uninvolved psoriaƟc skin cocultured with psoriaƟc T cell sƟmulated by Strep-SAg
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Normal skin cocultured with psoriaƟc CD8+ T cell
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Normal skin cocultured with psoriaƟc T cell sƟmulated by Strep-SAg
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Normal skin cocultured with psoriaƟc T cell
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Uninvolved psoriaƟc skin cocultured with normal T cell sƟmulated by Strep-SAg
Normal skin cocultured with psoriaƟc CD4+ T cell
Uninvolved psoriaƟc skin cocultured with psoriaƟc T cell sƟmulated by Strep-SAg
Figure 1. 3,30 -Diaminobezidine staining of epidermal samples from the treatment and control groups for assessing the expression of Ki-67, c-Myc, p53 and Bcl-2 in epidermis influenced by psoriatic peripheral blood T cells. (a) Expression of Ki-67 (original magnification 9200). (b) Expression of c-Myc (9200). (c) Expression of p53 (9200). (d) Expression of Bcl-2 (9200). Strep-Sag, streptococcal superantigen. culture with normal T cells, co-culture with Strep-SAg-stimulated psoriatic T cells caused significant increases in the expression of Ki-67, c-Myc and p53 in normal and uninvolved psoriatic skin. While no differences in gene expression were observed following
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co-culture with unstimulated or Strep-SAg-stimulated normal T cells, the expression of Bcl-2 was significantly decreased in normal and uninvolved psoriatic skin after co-culture with StrepSAg-stimulated psoriatic T cells.
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Normal skin unstimulated control (psoriatic T cell)
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Normal skin unstimulated control (normal T cell) Normal skin cocultured with normal T cell Uninvolved psoriatic skin unstimulated control
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Uninvolved psoriatic skin cocultured with psoriatic T cell Psoriatic lesion
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Figure 2. Epidermal turnover time of psoriatic lesions, normal skin and uninvolved psoriatic skin after co-culture with psoriatic peripheral blood T cells. Note: Compared with normal skin and uninvolved skin, the epidermal turnover time (ETT) of psoriatic lesions was shortened. After co-culture with psoriatic T cells, the ETT of uninvolved psoriatic skin was significantly shortened compared with unstimulated control. After co-culture with psoriatic T cells, the ETT of normal skin was significantly shortened compared with those of the unstimulated control and normal skin co-cultured with normal T cells. *P < 0.005, **P < 0.001.
Different effects of psoriatic CD4+ and CD8+ T cells on the expression of Ki-67, c-Myc, p53 and Bcl-2 in normal epidermis The above-mentioned results revealed that T cells from psoriasis affected the expression of Ki-67, c-Myc, p53 and Bcl-2 in the epidermis. To determine the effects of specific subsets of T cells, we investigated the expression of these proteins in normal epidermis following co-culture with CD4+ or CD8+ T cells. Interestingly, Ki-67, c-Myc and p53 levels were significantly increased, while Bcl-2 expression was significantly decreased when co-cultured with psoriatic CD4+ T cells compared with unstimulated epidermis and epidermis co-cultured with normal CD4+ T cells (Fig. 1). The same trends were observed when co-cultured with psoriatic CD8+ T cells. However, there were no differences in expression between unstimulated, co-cultured with normal CD4+ T cells and co-cultured with normal CD8+ T cells. The expression of Bcl-2 was decreased while the other three protein expressions were significantly increased when co-cultured with psoriatic CD4+ T cells compared with psoriatic CD8+ T cells. Linear correlation analysis revealed that there was a positive correlation between the expression of Ki-67 and c-Myc co-cultured with psoriatic CD4+ T cells, while there was no correlation when co-cultured with psoriatic CD8+ T cell.
11.5 3.8 days, P > 0.5). After co-culture with psoriatic T cells, the ETT of normal skin (6.9 3.1 days) was significantly shortened compared with those of the unstimulated control and normal skin co-cultured with normal T cells (P < 0.001 and P < 0.005, respectively). After co-culture with psoriatic T cells, there was no significant difference between normal skin and uninvolved skin from psoriasis (P > 0.05).
DEG in psoriasis and controls For the purpose of discovering molecular abnormality of T cells which contribute to the influence of keratinocyte proliferation and ETT, we also identified DEG in T cells. The DNA typing concordance of twins in this study was 99.7%–99.9%. DEG specific to psoriasis were expected to have the same profiles in MZ-CP and different profiles in MZ-DP. We screened 5438 genes with expression ratios below 2 in MZ-CP (Fig. 3a), and identified 1114 of 5438 genes with expression ratios of more than 2 in MZ-DP (Fig. 3b). In total, 214 DEG were founded in sporadic psoriasis as compared with controls. Additionally, of these 214 genes and the above-mentioned 1114 genes, 72 were common DEG. To confirm further the specificity of these DEG to psoriasis, we
(a)
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Peripheral blood T cells from psoriasis decreased ETT Peripheral blood T cells from psoriasis influenced the proliferation of keratinocytes, which play an important role in ETT. Therefore, we investigated the ETT of normal skin, psoriatic lesions and uninvolved skin from psoriasis (Fig. 2). Compared with normal skin (11.5 3.8 days) and uninvolved skin (8.7 3.2 days), the ETT of psoriatic lesions (4.5 2.1 days) was shortened (both P < 0.001). There was no significant difference between normal skin and uninvolved skin from psoriasis (P > 0.1). Additionally, we investigated the ETT of whole skin samples co-cultured with peripheral blood T cells. After co-culture with psoriatic T cells, the ETT of uninvolved psoriatic skin was significantly shortened compared with unstimulated control (5.7 3.8 vs 8.7 3.2 days, P < 0.001). Co-culture with normal T cells did not affect the ETT of normal skin (10.6 4.1 vs
© 2015 Japanese Dermatological Association
Figure 3. Scatter plots showing the 1114 genes that were similarly expressed in monozygotic (MZ) twin pairs concordant for psoriasis (MZ-CP) and discordant for psoriasis (MZ-DP). (a) The 1114 genes in MZ-CP twin pairs. (b) The 1114 genes in MZ-DP twin pairs.
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investigated the 72 genes between psoriasis and AD. Only 14 of these genes were differentially expressed by twofold between psoriasis and AD (Table 1).
Verification of DEG by real-time RT–PCR Next, we examined the expression of eight randomly selected genes in five pairs of MZ-CP and MZ-DP (Fig. 4a,b). Consistent with gene expression profiling, PPIL1, HSPH1, SENP3, NUP54, FABP5, PLEKHG3, SLC9A9 and CHCHD4 transcripts were equivalent in internal MZ-CP, but were different in MZDP. Real-time RT–PCR demonstrated that PPIL1, HSPH1, SENP3, NUP54, FABP5 and PLEKHG3 expression were increased in affected twins. Moreover, SLC9A9 and CHCHD4 transcripts were reduced in psoriasis. To exclude interference of factors unrelated to psoriasis, expression of the aforementioned eight genes were also assessed in 15 sporadic psoriasis and controls using real-time RT–PCR. Consistent with gene expression profiling, expression of PPIL1, HSPH1, SENP3, NUP54 and FABP5 were increased, whereas PLEKHG3 and SLC9A9 expression were decreased in psoriasis. Additionally, profiling showed that CHCHD4 expression was significantly increased (5.09-fold) in psoriasis, whereas real-time RT–PCR demonstrated that the expression was not different (Fig. 4c).
DISCUSSION Growing evidence has supported that abnormalities in T cells are important for invasion and maintenance of psoriasis.17 However, the etiology of psoriasis remains unclear. In our study, we found that the proteins expression related to cell proliferation (i.e. Ki-67, c-Myc and p53) were altered, suggesting that psoriatic T cells may influence keratinocyte proliferation. Ki-67 is a nuclear protein that is expressed in all active phases of the cell cycle, but is not present in non-proliferating cells.8 Thus, our data showed that psoriatic T cells, but not normal T cells, enhanced the expression of Ki-67 in keratinocytes, suggesting that psoriatic T cells induced hyperprolif-
eration of keratinocytes. Similarly, c-Myc plays a pivotal role in cell growth, cell cycle progression, and also induces growth inhibition or apoptosis through both p53-dependent and p53independent mechanisms.9,10 Additionally, Bcl-2 and p53 function together to regulate apoptosis.11–13 Therefore, psoriatic T cells may influence the expression of c-Myc, p53 and Bcl-2 to regulate cell proliferation or apoptosis. From these data, we can conclude that psoriatic T cells influence keratinocyte proliferation and apoptosis, causing disorders in epidermal dynamics. Because the expression of Ki-67, c-Myc, p53 and Bcl-2 were similar in uninvolved psoriatic and normal skin co-cultured with psoriatic T cells, it suggested that pathogenic T cells rather than keratinocyte may be critical for the initiation of psoriasis. Our results showed that both psoriatic CD4+ and CD8+ T cells caused changes in the expression of these proteins, indicating that both of them contributed to the disorder of epidermal kinetics in psoriasis. Nickoloff and Gilhar found that CD4+ T cells may play a more direct role than CD8+ T cells in the pathogenesis of psoriasis.18,19 Our data supported this hypothesis, as we observed that CD4+ T cells exerted a stronger influence on the expression of cell proliferation-related proteins than CD8+ T cells. The telomerase activity, proliferation of and cytokine secretion by psoriatic peripheral blood T cells have been shown to be different from normal.20 Therefore, we investigated the effects of Strep-SAg stimulation on the activity of peripheral blood T cells from psoriasis. Our results showed that psoriatic T cells stimulated by Strep-SAg promoted the proliferation of keratinocytes, while normal T cells did not, suggesting that the activity of stimulated T cells was different in psoriasis. Finally, Lin et al.21 revealed that particular T-cell receptors are preferentially upregulated in psoriasis. Consistent with this, our previous data showed that the T-cell receptor beta variable is predominantly expressed in psoriatic T cells.5 Therefore, it is reasonable to presume that the activity of psoriatic T-cell subsets is important in the induction of epidermal dynamic disorders and pathogenesis of psoriasis. Accordingly, Strep-SAg may induce the activation of a special T-cell subset by polyclonal stimulation.
Table 1. Differentially expressed genes Gene
Genes symbol
P vs N
P vs AD
E1 vs E2
F1 vs F2
GenBank link
10808 51645 4542 53371 131474 2171 55720 79026 23092 51540 26030 26168 285195 26155
HSPH1 PPIL1 MYO1F NUP54 CHCHD4 FABP5 TSR1 AHNAK TTLL5 SCLY PLEKHG3 SENP3 SLC9A9 NOC2L
3.568037 3.234122 0.396925 6.721225 5.088009 2.909693 2.506837 0.407554 0.439926 3.391292 0.167329 6.670165 0.307512 3.557612
2.41782 2.44378 0.422139 4.306759 6.068208 3.784806 2.647467 0.389839 0.491695 2.897563 0.207546 3.624609 0.442367 4.807591
0.809309 0.698722 1.112289 0.710798 0.558728 0.889284 0.578587 1.40498 1.206332 1.153511 1.537285 0.619876 0.763096 0.610407
3.655485 2.657172 0.467622 4.079268 5.173267 2.541443 2.917203 0.323246 0.330847 3.693734 0.128349 5.792453 0.250091 2.881828
http://www.ncbi.nlm.nih.gov/gene/?term=10808 http://www.ncbi.nlm.nih.gov/gene?term=51645 http://www.ncbi.nlm.nih.gov/gene/?term=4542 http://www.ncbi.nlm.nih.gov/gene?term=53371 http://www.ncbi.nlm.nih.gov/gene?term=131474 http://www.ncbi.nlm.nih.gov/gene?term=2171 http://www.ncbi.nlm.nih.gov/gene?term=55720 http://www.ncbi.nlm.nih.gov/gene?term=79026 http://www.ncbi.nlm.nih.gov/gene?term=243092 http://www.ncbi.nlm.nih.gov/gene?term=51540 http://www.ncbi.nlm.nih.gov/gene?term=26030 http://www.ncbi.nlm.nih.gov/gene?term=26168 http://www.ncbi.nlm.nih.gov/gene?term=285195 http://www.ncbi.nlm.nih.gov/gene?term=26155
AD, atopic dermatitis; CP, concordant for psoriasis; DP, discordant for psoriasis; E1, psoriasis of MZ-CP; E2, other psoriasis of MZ-CP; F1, psoriasis of MZ-DP; F2, normal of MZ-DP; MZ, monozygotic; N, normal; P, sporadic psoriasis.
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Figure 4. Real-time reverse transcription polymerase chain reaction (RT–PCR) verification of the differentially expressed genes. (a) Real-time RT–PCR verification of differentially expressed genes in five monozygotic twin pairs concordant for psoriasis (MZ-CP). (b) Real-time RT–PCR verification of differentially expressed genes in five monozygotic twin pairs discordant for psoriasis (MZ-DP). (c) Gene expression profiling and real-time RT–PCR analysis of the genes of patients with sporadic psoriasis. Epidermal turnover time depends on cell number and proliferation.22 Interestingly, we found that psoriatic T cells, but not normal T cells, decreased ETT in both uninvolved psoriatic skin and normal skin.
© 2015 Japanese Dermatological Association
Our data suggested that peripheral blood T cells played a significant role in the epidermal dynamic disorders in psoriasis by influencing cell proliferation and ETT. In order to discover the molecular reason for the influence, the DEG in psoriatic T cells were also studied. In this study, we investigated the gene expression of psoriatic T cells from MZ-CP and MZ-DP living in the same environment, as well as T cells from sporadic psoriasis in order to improve our understanding of psoriatic T cells. We found 14 DEG in a few of MZ-DP and sporadic psoriasis, including FABP5, HSPH1, PPIL1, SENP3 and NUP54. FABP5 has been shown to modulate differentiation in psoriatic keratinocytes by enhancing the transcriptional activity of peroxisome proliferator-activated receptors.23,24 HSPH1 is a molecular chaperone that guides the normal folding, intracellular disposition and proteolytic turnover of many of the key regulators of cell growth, differentiation and survival.25,26 HSPH1 is overexpressed in various cancers, but is expressed at low levels in many normal tissues. The constitutive overexpression of HSP105 in cancer cells protects tumor cells from apoptosis by suppressing activation of caspase-3 and caspase-9 and inducing expression of HSP70.27–29 The upregulation of HSPH1 observed in this study might have resulted in the abnormal keratinocyte proliferation in psoriasis. PPIL1, another upregulated gene, is also important in the proliferation of cancer cells30 and may mediate the pathogenesis of psoriasis through the same mechanism as HSPH1. SENP3 is also elevated in psoriasis. The manifestation of vascular proliferation in psoriatic lesions31 may be a result of increased SENP3, subsequently enhancing the expression of hypoxia-inducible factor-1a-regulated vascular endothelial growth factor, which is critical for vascular development.32 The expression of NUP54 is increased, which is an essential protein present in a multiprotein complex that is primarily required for nuclear protein import.33 The abnormal expression of NUP54 could inhibit the proliferation of human pulmonary arterial smooth muscle cells by opening the KATP channel.34 In psoriatic T cells, increased expression of NUP54 may influence the cell proliferation by affecting nuclear protein import. These genes were not different in MZ-CP. Because the majority of the DEG were found to be related to cell proliferation and differentiation, we expect that these genes were involved in the pathogenesis of psoriasis by affecting keratinocyte proliferation and ETT. However, the exact mechanism of the DEG contribution to the influence need to be further studied.
ACKNOWLEDGMENTS: This project was supported by the National Natural Science Foundation of China (81271768 and 81401360). CONFLICT OF INTEREST:
There are no conflicts of interest
to declare.
REFERENCES 1 Mahler R, Jackson C, Ijacu H. The burden of psoriasis and barriers to satisfactory care: results from a Canadian patient survey. J Cutan Med Surg 2009; 13: 283–293.
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2 Man XY, Yang XH, Ca SQ, Bu ZY, Zheng M. Overexpression of vascular endothelial growth factor (VEGF) receptors on keratinocytes in psoriasis: regulated by calcium independent of VEGF. J Cell Mol Med 2008; 12: 649–660. 3 Zhang K, Li X, Yin G, Liu Y, Niu X, Hou R. Functional characterization of CD4+CD25+ regulatory T cells differentiated in vitro from bone marrow-derived haematopoietic cells of psoriasis patients with a family history of the disorder. Br J Dermatol 2008; 158: 298–305. 4 Zhang K, Li X, Yin G, Liu Y, Tang X. Functional characterization of T cells differentiated in vitro from bone marrow-derived CD34+ cells of psoriatic patients with family history. Exp Dermatol 2010; 19: e128–e135. 5 Yin G, Li X, Li J et al. Screening of differentially expressed genes and predominant expression of variable region of T cell receptor in peripheral T cells of psoriatic patients. Eur J Dermatol 2011; 21: 938–944. 6 Li X, Fan X, Zhang K, Yin G, Liu Y. Influence of psoriatic peripheral blood CD4+T and CD8+T lymphocytes on C-myc, Bcl-xL and Ki-67 gene expression in keratinocytes. Eur J Dermatol 2007; 17 (5): 392– 396. 7 Lønnberg AS, Skov L, Skytthe A et al. Heritability of psoriasis in a large twin sample. Br J Dermatol 2013; 169 (2): 412–416. 8 Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984; 133: 1710–1715. 9 Meyer N, Penn LZ. Reflecting on 25 years with MYC. Nat Rev Cancer 2008; 8: 976–990. 10 Zhang Q, Spears E, Boone DN, Li Z, Gregory MA, Hann SR. Domainspecific c-Myc ubiquitylation controls c-Myc transcriptional and apoptotic activity. Proc Natl Acad Sci U S A 2013; 110: 978–983. 11 Kluck RM, Bossy-Wetzel E, Green DR, Newmever DD. The release of cytochrome c from mitochondria: a primary site for bcl-2 regulation of apoptosis. Science 1997; 275: 1132–1136. 12 Green DR, Kroemer G. Cytoplasmic functions of the tumour suppressor p53. Nature 2009; 458: 1127–1130. 13 Feng Z, Levine AJ. The regulation of energy metabolism and the IGF-1/mTOR pathways by the p53 protein. Trends Cell Biol 2010; 20: 427–434. 14 Li X, Li J, Yang Y et al. Differential gene expression in peripheral blood T cells from patients with psoriasis, lichen planus, and atopic dermatitis. J Am Acad Dermatol 2013; 69: e235–e243. 15 Nooh MM, Nookala S, Kansal R, Kotb M. Individual genetic variations directly effect polarization of cytokine responses to superantigens associated with streptococcal sepsis: implications for customized patient care. J Immunol 2011; 186: 3156–3163. 16 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402–408. 17 Boniface K, Guignouard E, Pedretti N et al. A role for T cell-derived interleukin 22 in psoriatic skin inflammation. Clin Exp Immunol 2007; 150: 407–415. 18 Gilhar A, David M, Ullmann Y, Berkutski T, Kalish RS. T-lymphocyte dependence of psoriatic pathology in human psoriatic skin grafted to SCID mice. J Invest Dermatol 1997; 109: 283–288. 19 Nickoloff BJ, Wrone-Smith T. Injection of pre-psoriatic skin with CD4 + T cells induces psoriasis. Am J Pathol 1999; 155: 145– 158. 20 Wu K, Higashi N, Hansen ER, Lund M, Bang K, Thestrup-Pedersen K. Telomerase activity is increased and telomere length shortened
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in T cells from blood of patients with atopic dermatitis and psoriasis. J Immunol 2000; 165: 4742–4747. Lin WJ, Norris DA, Achziger M, Kotain BL, Tomkinson B. Oligoclonal expansion of intraepidermal T cells in psoriasis skin lesions. J Invest Dermatol 2001; 117: 1546–1553. Paulrud CO. Basic concepts of the bovine teat canal. Vet Res Commun 2005; 29: 215–245. Ogawa E, Owada Y, Ikawa S et al. Epidermal FABP (FABP5) regulates keratinocyte differentiation by 13(S)-HODE-mediated activation of the NF-jB signaling pathway. J Invest Dermatol 2011; 131: 604– 612. Dallaglio K, Marconi A, Truzzi F et al. E-FABP induces differentiation in normal human keratinocytes and modulates the differentiation process in psoriatic keratinocytes in vitro. Exp Dermatol 2013; 22: 255–261. Zhang XS, Lue YH, Guo SH et al. Expression of HSP105 and HSP60 during germ cell apoptosis in the heat-treated testes of adult cynomolgus monkeys (Macaca fascicularis). Front Biosci 2005; 10: 3110–31121. €ttner P, Mosig S, Lechtermann A, Funke H, Mooren FC. Exercise Bu affects the gene expression profiles of human white blood cells. J Appl Physiol 2007; 102: 26–36. Hosaka S, Nakatsura T, Tsukamoto H, Hatayama T, Baba H, Nishimura Y. Synthetic small interfering RNA targeting heat shock protein 105 induces apoptosis of various cancer cells both in vitro and in vivo. Cancer Sci 2006; 97: 623–632. Yamagishi N, Ishihara K, Saito Y, Hatayama T. Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells. Exp Cell Res 2006; 312: 3215–3223. Yamagishi N, Goto K, Nakagawa S, Saito Y, Hatayama T. Hsp105 reduces the protein aggregation and cytotoxicity by expanded-polyglutamine proteins through the induction of Hsp70. Exp Cell Res 2010; 316: 2424–2433. Obama K, Kato T, Hasegawa S, Satoh S, Nakamura Y, Furukawa Y. Overexpression of peptidyl-prolyl isomerase-like 1 is associated with the growth of colon cancer cells. Clin Cancer Res 2006; 12: 70–76. Chua RA, Arbiser JL. The role of angiogenesis in the pathogenesis of psoriasis. Autoimmunity 2009; 42: 574–579. Bawa-Khalfe T, Yeh ETH. SUMO losing balance: SUMO proteases disrupt SUMO homeostasis to facilitate cancer development and progression. Genes Cancer 2010; 1: 748–752. Grandi P, Schlaich N, Tekotte H, Hurt EC. Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p, and a novel protein Nup57p. EMBO J 1995; 14: 76–87. Yang MX, Liu ZX, Zhang S et al. Proteomic analysis of the effect of iptakalim on human pulmonary arterial smooth muscle cell proliferation. Acta Pharmacol Sin 2009; 30: 175–183.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Table S1. Specimen sources Table S2. Primers for real time reverse transcription polymerase chain reaction (RT–PCR) Table S3. Differentially expressed genes
© 2015 Japanese Dermatological Association
doi: 10.1111/1346-8138.12961
ORIGINAL ARTICLE
Psoriatic T cells reduce epidermal turnover time and affect cell proliferation contributed from differential gene expression Junqin LI,1 Xinhua LI,1 Ruixia HOU,1 Ruifeng LIU,1 Xincheng ZHAO,1 Feng DONG,2 Chunfang WANG,3 Guohua YIN,1 Kaiming ZHANG1 1
Institute of Dermatology, Taiyuan City Center Hospital, 3Laboratory Animal Center, Shanxi Medical University, Taiyuan, Department of Dermatology, Changzhi City Second People’s Hospital, Changzhi, China
2
ABSTRACT Psoriasis is mediated primarily by T cells, which reduce epidermal turnover time and affect keratinocyte proliferation. We aimed to identify differentially expressed genes (DEG) in T cells from normal, five pairs of monozygotic twins concordant or discordant for psoriasis, to determine whether these DEG may account for the influence to epidermal turnover time and keratinocyte proliferation. The impact of T cells on keratinocyte proliferation and epidermal turnover time were investigated separately by immunohistochemistry and cultured with 3H-TdR. mRNA expression patterns were investigated by RNA sequencing and verified by real-time reverse transcription polymerase chain reaction. After co-culture with psoriatic T cells, the expression of Ki-67, c-Myc and p53 increased, while expression of Bcl-2 and epidermal turnover time decreased. There were 14 DEG which were found to participate in the regulation of cell proliferation or differentiation. Psoriatic T cells exhibited the ability to decrease epidermal turnover time and affect keratinocyte proliferation because of the differential expression of PPIL1, HSPH1, SENP3, NUP54, FABP5, PLEKHG3, SLC9A9 and CHCHD4.
Key words:
differentially expressed genes, epidermal turnover time, keratinocyte, psoriasis, T cells.
INTRODUCTION Psoriasis is a chronic inflammatory disease that imposes a significant burden on patients’ physical and mental health.1 Abnormal keratinocyte proliferation and epidermal turnover time (ETT) are the main pathological features of psoriasis and are closely related to the course and recurrence of this disease.2 Immune abnormalities in T cells play important roles in the pathogenesis of psoriasis. In psoriasis, the level of the Thelper 1 cytokine is enhanced in T cells directly differentiated from psoriatic bone marrow CD34+ cells in vitro, and the number and function of regulatory T cells is decreased.3,4 Moreover, expression of the T-cell receptor variable region is predominant in peripheral blood T-cell of psoriasis.5 Additionally, it was found that psoriatic T cells influence keratinocyte proliferation.6 However, the abnormality of T cells which results in the influence on keratinocyte proliferation and ETT was unknown. In our previous studies, we have investigated the incidence of psoriasis in monozygotic (MZ) twins discordant for psoriasis (MZ-DP) who lived in the same environment after birth. Additionally, our studies demonstrated that the onset of psoriasis in MZ twins concordant for psoriasis (MZ-CP) varied from 1 month to 3 years. Thus, our data have suggested that concordance for psoriasis is not 100% among MZ twins. Consistent with this, previous studies have demonstrated that concordance can be
less than 72%.7 For the specificity of MZ twins, it may be the best choice to study abnormality of T cells in psoriasis. Ki-67 is a nuclear protein that is expressed in all active phases of the cell cycle, but is not present in non-proliferating cells.8 Similarly, c-Myc plays a pivotal role in cell cycle progression.9,10 Additionally, Bcl-2 and p53 function together to regulate apoptosis.11–13 Here, we first studied the impact of psoriatic peripheral blood T cells on ETT and cell proliferation (i.e. Ki-67, c-Myc, p53 and Bcl-2) before and after stimulation with streptococcal superantigen (Strep-SAg). For the purpose of discovering how the molecular abnormality of T cells contributed to the impact, we also identify differentially expressed genes (DEG) in T cells from normal individuals, MZ-CP, MZ-DP, and those with atopic dermatitis (AD) and sporadic psoriasis.
METHODS Specimen source Fifteen psoriasis and 15 normal subjects who were not MZ (Table S1) were studied to determine the effects of peripheral blood T cells on ETT and expression of Ki-67, c-Myc, p53 and Bcl-2. Normal and psoriasis patients were also age- and sexmatched. Aside from the above subjects, five pairs of MZ-CP, five pairs of MZ-DP and five patients with AD were also enrolled in the study of gene expression profiles and real-time
Correspondence: Kaiming Zhang, M.D., Institute of Dermatology, Taiyuan City Center Hospital, 1 Dong San Dao Xiang, Jiefang Road, Taiyuan, Shanxi 030009, China. Email: [email protected] Received 25 March 2015; accepted 20 April 2015.
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reverse transcription polymerase chain reaction (RT–PCR, Table S1). MZ twins were matched for age and sex, and none of them had smoked. The normal MZ-DP patients had not suffered from psoriasis after 2 years of sampling. Normal individuals presented with no systemic or autoimmune diseases. Psoriasis and AD were diagnosed according to clinical and histological parameters. Patients had not received corticosteroids or immunosuppressants within 6 months of enrollment. All patients provided informed consent for participation in the study. The protocol was approved by the medical ethics committee of Taiyuan City Center Hospital and followed the Declaration of Helsinki protocols.
Peripheral blood T-cell proliferation and stimulation Peripheral blood T cells were cultured, stimulated and identified as described in our previous study.14 Strep-SAg was prepared as previously described.15 T cells were seeded in 24well culture plates (Corning, Corning, NY, USA). Some cells were stimulated with Strep-SAg and collected after 24 h of culture. Some cells were isolated using a cell sorter for analysis of CD4+ and CD8+ T cells (CD4 and CD8 magnetic beads; Invitrogen, Carlsbad, CA, USA).
clearing and mounting, the sections were analyzed under an Olympus microscope (Olympus, Tokyo, Japan).
RNA isolation Total RNA was extracted using TRIzol reagent (Invitrogen) and treated with DNase (Invitrogen). mRNA was purified using oligo (dT) magnetic beads (Invitrogen), and purified mRNA was used to synthesize complementary DNA (Takara, Dalian, China), which was used for sequencing and amplification by real-time RT–PCR.
Tag preparation, sequencing, gene annotation and screening DEG Complementary DNA obtained from one pair of MZ-CP, one pair of MZ-DP, five patients with AD, five patients with psoriasis and five normal patients were treated with NlaIII and MmeI to generate 21-bp tags. These tags were sequenced, and the genes were annotated by referring to the National Center for Biotechnology Information genetic database (ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/human.rna.fna.gz). Differentially expressed gene screening was performed according to our previous study.14
Real-time RT–PCR Skin culture The skin was cleaned with Hank’s balanced salt solution (Invitrogen) and cut. The epidermal skin was obtained with 3-mm punches and cultured in Netwell 3-D 12-well culture plates (Corning) with semipermeable membranes, which were used to ensure contact between the skin and Dulbecco’s modified Eagle’s medium (Invitrogen). One hundred microliters of T cell (1 9 106 cells/mL) or Dulbecco’s modified Eagle’s medium was added to the culture medium. Cell culture was continued for 3 days after treatment with 3% trypsin (Invitrogen).
Test of ETT
Real-time RT–PCR was performed as previously described.14 Primers for real-time RT–PCR are shown in Table S2.
Statistical analysis The differences in gene expression obtained from real-time RT–PCR were analyzed by the 2DDCt method.16 The means of indicated groups were compared using a two-tailed unpaired Student’s t-test. Results were considered statistically significant at P < 0.05.
RESULTS
Skin was cultured in Dulbecco’s modified Eagle’s medium with 3 H-TdR (5 lCi/mL; China Institute of Atomic Energy, Beijing, China) for 40 min, then washed and cultured with Dulbecco’s modified Eagle’s medium without 3H-TdR. Skin was fixed and sliced at different times. Nuclear-4 emulsion (China Institute of Atomic Energy) was applied on the skin in a darkroom. The skin was developed with D-19b developer (China Lucky Group, Hebei, China), fixed with F-5 acid hardening fixer (China Lucky Group), stained with hematoxylin–eosin, dehydrated and mounted. Cells exhibiting isolated nuclei and strong refraction of silver staining under an oil immersion lens were considered labeled cells. The time was monitored when the labeled cells moved from the basal layer to the stratum granulosum; this was considered the ETT. After culture for 6 days, skin was fixed and sectioned.
Peripheral blood T cells from psoriasis exhibited changes in Ki-67, c-Myc, p53 and Bcl-2 expression in the epidermis
Immunohistochemistry
Effects of psoriatic peripheral blood T cells stimulated with Strep-SAg on the expression of Ki67, c-Myc, p53 and Bcl-2 in the epidermis
Mouse antihuman antibodies targeting Ki-67, c-Myc, p53 and Bcl-2 (Santa Cruz Biotechnology, Dallas, TX, USA) were used in this study. Immune complexes were visualized using peroxidaseconjugated secondary antibodies (Santa Cruz Biotechnology), according to the manufacturer’s protocol. After dehydration,
© 2015 Japanese Dermatological Association
We examined the expression of Ki-67, c-Myc, p53 and Bcl-2 in psoriatic lesions before and after the effects of psoriatic peripheral blood T cells in vitro. We found significant upregulation of Ki-67, c-Myc and p53 in psoriatic lesions (Fig. 1). However, Bcl-2 expression was rarely observed in these tissues. In contrast, expression of these proteins was normal in uninvolved skin from psoriasis. Interestingly, Ki-67, c-Myc and p53 expression were increased in normal skin and uninvolved psoriatic skin after co-culture with psoriatic peripheral blood T cells. Additionally, Bcl-2 expression was decreased in uninvolved and normal skin after co-culture with psoriatic peripheral blood T cells.
The expression of these proteins was similar in normal and uninvolved psoriatic skin after co-culture with unstimulated and Strep-SAg-stimulated normal T cells (Fig. 1). Compared with co-
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Uninvolved psoriaƟc skin cocultured with psoriaƟc T cell sƟmulated by Strep-SAg
Figure 1. 3,30 -Diaminobezidine staining of epidermal samples from the treatment and control groups for assessing the expression of Ki-67, c-Myc, p53 and Bcl-2 in epidermis influenced by psoriatic peripheral blood T cells. (a) Expression of Ki-67 (original magnification 9200). (b) Expression of c-Myc (9200). (c) Expression of p53 (9200). (d) Expression of Bcl-2 (9200). Strep-Sag, streptococcal superantigen. culture with normal T cells, co-culture with Strep-SAg-stimulated psoriatic T cells caused significant increases in the expression of Ki-67, c-Myc and p53 in normal and uninvolved psoriatic skin. While no differences in gene expression were observed following
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co-culture with unstimulated or Strep-SAg-stimulated normal T cells, the expression of Bcl-2 was significantly decreased in normal and uninvolved psoriatic skin after co-culture with StrepSAg-stimulated psoriatic T cells.
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Normal skin unstimulated control (psoriatic T cell)
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Uninvolved psoriatic skin cocultured with psoriatic T cell Psoriatic lesion
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epidermal turnover time
Figure 2. Epidermal turnover time of psoriatic lesions, normal skin and uninvolved psoriatic skin after co-culture with psoriatic peripheral blood T cells. Note: Compared with normal skin and uninvolved skin, the epidermal turnover time (ETT) of psoriatic lesions was shortened. After co-culture with psoriatic T cells, the ETT of uninvolved psoriatic skin was significantly shortened compared with unstimulated control. After co-culture with psoriatic T cells, the ETT of normal skin was significantly shortened compared with those of the unstimulated control and normal skin co-cultured with normal T cells. *P < 0.005, **P < 0.001.
Different effects of psoriatic CD4+ and CD8+ T cells on the expression of Ki-67, c-Myc, p53 and Bcl-2 in normal epidermis The above-mentioned results revealed that T cells from psoriasis affected the expression of Ki-67, c-Myc, p53 and Bcl-2 in the epidermis. To determine the effects of specific subsets of T cells, we investigated the expression of these proteins in normal epidermis following co-culture with CD4+ or CD8+ T cells. Interestingly, Ki-67, c-Myc and p53 levels were significantly increased, while Bcl-2 expression was significantly decreased when co-cultured with psoriatic CD4+ T cells compared with unstimulated epidermis and epidermis co-cultured with normal CD4+ T cells (Fig. 1). The same trends were observed when co-cultured with psoriatic CD8+ T cells. However, there were no differences in expression between unstimulated, co-cultured with normal CD4+ T cells and co-cultured with normal CD8+ T cells. The expression of Bcl-2 was decreased while the other three protein expressions were significantly increased when co-cultured with psoriatic CD4+ T cells compared with psoriatic CD8+ T cells. Linear correlation analysis revealed that there was a positive correlation between the expression of Ki-67 and c-Myc co-cultured with psoriatic CD4+ T cells, while there was no correlation when co-cultured with psoriatic CD8+ T cell.
11.5 3.8 days, P > 0.5). After co-culture with psoriatic T cells, the ETT of normal skin (6.9 3.1 days) was significantly shortened compared with those of the unstimulated control and normal skin co-cultured with normal T cells (P < 0.001 and P < 0.005, respectively). After co-culture with psoriatic T cells, there was no significant difference between normal skin and uninvolved skin from psoriasis (P > 0.05).
DEG in psoriasis and controls For the purpose of discovering molecular abnormality of T cells which contribute to the influence of keratinocyte proliferation and ETT, we also identified DEG in T cells. The DNA typing concordance of twins in this study was 99.7%–99.9%. DEG specific to psoriasis were expected to have the same profiles in MZ-CP and different profiles in MZ-DP. We screened 5438 genes with expression ratios below 2 in MZ-CP (Fig. 3a), and identified 1114 of 5438 genes with expression ratios of more than 2 in MZ-DP (Fig. 3b). In total, 214 DEG were founded in sporadic psoriasis as compared with controls. Additionally, of these 214 genes and the above-mentioned 1114 genes, 72 were common DEG. To confirm further the specificity of these DEG to psoriasis, we
(a)
(b)
Peripheral blood T cells from psoriasis decreased ETT Peripheral blood T cells from psoriasis influenced the proliferation of keratinocytes, which play an important role in ETT. Therefore, we investigated the ETT of normal skin, psoriatic lesions and uninvolved skin from psoriasis (Fig. 2). Compared with normal skin (11.5 3.8 days) and uninvolved skin (8.7 3.2 days), the ETT of psoriatic lesions (4.5 2.1 days) was shortened (both P < 0.001). There was no significant difference between normal skin and uninvolved skin from psoriasis (P > 0.1). Additionally, we investigated the ETT of whole skin samples co-cultured with peripheral blood T cells. After co-culture with psoriatic T cells, the ETT of uninvolved psoriatic skin was significantly shortened compared with unstimulated control (5.7 3.8 vs 8.7 3.2 days, P < 0.001). Co-culture with normal T cells did not affect the ETT of normal skin (10.6 4.1 vs
© 2015 Japanese Dermatological Association
Figure 3. Scatter plots showing the 1114 genes that were similarly expressed in monozygotic (MZ) twin pairs concordant for psoriasis (MZ-CP) and discordant for psoriasis (MZ-DP). (a) The 1114 genes in MZ-CP twin pairs. (b) The 1114 genes in MZ-DP twin pairs.
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investigated the 72 genes between psoriasis and AD. Only 14 of these genes were differentially expressed by twofold between psoriasis and AD (Table 1).
Verification of DEG by real-time RT–PCR Next, we examined the expression of eight randomly selected genes in five pairs of MZ-CP and MZ-DP (Fig. 4a,b). Consistent with gene expression profiling, PPIL1, HSPH1, SENP3, NUP54, FABP5, PLEKHG3, SLC9A9 and CHCHD4 transcripts were equivalent in internal MZ-CP, but were different in MZDP. Real-time RT–PCR demonstrated that PPIL1, HSPH1, SENP3, NUP54, FABP5 and PLEKHG3 expression were increased in affected twins. Moreover, SLC9A9 and CHCHD4 transcripts were reduced in psoriasis. To exclude interference of factors unrelated to psoriasis, expression of the aforementioned eight genes were also assessed in 15 sporadic psoriasis and controls using real-time RT–PCR. Consistent with gene expression profiling, expression of PPIL1, HSPH1, SENP3, NUP54 and FABP5 were increased, whereas PLEKHG3 and SLC9A9 expression were decreased in psoriasis. Additionally, profiling showed that CHCHD4 expression was significantly increased (5.09-fold) in psoriasis, whereas real-time RT–PCR demonstrated that the expression was not different (Fig. 4c).
DISCUSSION Growing evidence has supported that abnormalities in T cells are important for invasion and maintenance of psoriasis.17 However, the etiology of psoriasis remains unclear. In our study, we found that the proteins expression related to cell proliferation (i.e. Ki-67, c-Myc and p53) were altered, suggesting that psoriatic T cells may influence keratinocyte proliferation. Ki-67 is a nuclear protein that is expressed in all active phases of the cell cycle, but is not present in non-proliferating cells.8 Thus, our data showed that psoriatic T cells, but not normal T cells, enhanced the expression of Ki-67 in keratinocytes, suggesting that psoriatic T cells induced hyperprolif-
eration of keratinocytes. Similarly, c-Myc plays a pivotal role in cell growth, cell cycle progression, and also induces growth inhibition or apoptosis through both p53-dependent and p53independent mechanisms.9,10 Additionally, Bcl-2 and p53 function together to regulate apoptosis.11–13 Therefore, psoriatic T cells may influence the expression of c-Myc, p53 and Bcl-2 to regulate cell proliferation or apoptosis. From these data, we can conclude that psoriatic T cells influence keratinocyte proliferation and apoptosis, causing disorders in epidermal dynamics. Because the expression of Ki-67, c-Myc, p53 and Bcl-2 were similar in uninvolved psoriatic and normal skin co-cultured with psoriatic T cells, it suggested that pathogenic T cells rather than keratinocyte may be critical for the initiation of psoriasis. Our results showed that both psoriatic CD4+ and CD8+ T cells caused changes in the expression of these proteins, indicating that both of them contributed to the disorder of epidermal kinetics in psoriasis. Nickoloff and Gilhar found that CD4+ T cells may play a more direct role than CD8+ T cells in the pathogenesis of psoriasis.18,19 Our data supported this hypothesis, as we observed that CD4+ T cells exerted a stronger influence on the expression of cell proliferation-related proteins than CD8+ T cells. The telomerase activity, proliferation of and cytokine secretion by psoriatic peripheral blood T cells have been shown to be different from normal.20 Therefore, we investigated the effects of Strep-SAg stimulation on the activity of peripheral blood T cells from psoriasis. Our results showed that psoriatic T cells stimulated by Strep-SAg promoted the proliferation of keratinocytes, while normal T cells did not, suggesting that the activity of stimulated T cells was different in psoriasis. Finally, Lin et al.21 revealed that particular T-cell receptors are preferentially upregulated in psoriasis. Consistent with this, our previous data showed that the T-cell receptor beta variable is predominantly expressed in psoriatic T cells.5 Therefore, it is reasonable to presume that the activity of psoriatic T-cell subsets is important in the induction of epidermal dynamic disorders and pathogenesis of psoriasis. Accordingly, Strep-SAg may induce the activation of a special T-cell subset by polyclonal stimulation.
Table 1. Differentially expressed genes Gene
Genes symbol
P vs N
P vs AD
E1 vs E2
F1 vs F2
GenBank link
10808 51645 4542 53371 131474 2171 55720 79026 23092 51540 26030 26168 285195 26155
HSPH1 PPIL1 MYO1F NUP54 CHCHD4 FABP5 TSR1 AHNAK TTLL5 SCLY PLEKHG3 SENP3 SLC9A9 NOC2L
3.568037 3.234122 0.396925 6.721225 5.088009 2.909693 2.506837 0.407554 0.439926 3.391292 0.167329 6.670165 0.307512 3.557612
2.41782 2.44378 0.422139 4.306759 6.068208 3.784806 2.647467 0.389839 0.491695 2.897563 0.207546 3.624609 0.442367 4.807591
0.809309 0.698722 1.112289 0.710798 0.558728 0.889284 0.578587 1.40498 1.206332 1.153511 1.537285 0.619876 0.763096 0.610407
3.655485 2.657172 0.467622 4.079268 5.173267 2.541443 2.917203 0.323246 0.330847 3.693734 0.128349 5.792453 0.250091 2.881828
http://www.ncbi.nlm.nih.gov/gene/?term=10808 http://www.ncbi.nlm.nih.gov/gene?term=51645 http://www.ncbi.nlm.nih.gov/gene/?term=4542 http://www.ncbi.nlm.nih.gov/gene?term=53371 http://www.ncbi.nlm.nih.gov/gene?term=131474 http://www.ncbi.nlm.nih.gov/gene?term=2171 http://www.ncbi.nlm.nih.gov/gene?term=55720 http://www.ncbi.nlm.nih.gov/gene?term=79026 http://www.ncbi.nlm.nih.gov/gene?term=243092 http://www.ncbi.nlm.nih.gov/gene?term=51540 http://www.ncbi.nlm.nih.gov/gene?term=26030 http://www.ncbi.nlm.nih.gov/gene?term=26168 http://www.ncbi.nlm.nih.gov/gene?term=285195 http://www.ncbi.nlm.nih.gov/gene?term=26155
AD, atopic dermatitis; CP, concordant for psoriasis; DP, discordant for psoriasis; E1, psoriasis of MZ-CP; E2, other psoriasis of MZ-CP; F1, psoriasis of MZ-DP; F2, normal of MZ-DP; MZ, monozygotic; N, normal; P, sporadic psoriasis.
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Twins concordant for psoriasis Δ CT
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Normals of twins discordant for psoriasis gene expression profiling real-time RT-PCR
Expression change fold (Psoriasis/Normal)
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Figure 4. Real-time reverse transcription polymerase chain reaction (RT–PCR) verification of the differentially expressed genes. (a) Real-time RT–PCR verification of differentially expressed genes in five monozygotic twin pairs concordant for psoriasis (MZ-CP). (b) Real-time RT–PCR verification of differentially expressed genes in five monozygotic twin pairs discordant for psoriasis (MZ-DP). (c) Gene expression profiling and real-time RT–PCR analysis of the genes of patients with sporadic psoriasis. Epidermal turnover time depends on cell number and proliferation.22 Interestingly, we found that psoriatic T cells, but not normal T cells, decreased ETT in both uninvolved psoriatic skin and normal skin.
© 2015 Japanese Dermatological Association
Our data suggested that peripheral blood T cells played a significant role in the epidermal dynamic disorders in psoriasis by influencing cell proliferation and ETT. In order to discover the molecular reason for the influence, the DEG in psoriatic T cells were also studied. In this study, we investigated the gene expression of psoriatic T cells from MZ-CP and MZ-DP living in the same environment, as well as T cells from sporadic psoriasis in order to improve our understanding of psoriatic T cells. We found 14 DEG in a few of MZ-DP and sporadic psoriasis, including FABP5, HSPH1, PPIL1, SENP3 and NUP54. FABP5 has been shown to modulate differentiation in psoriatic keratinocytes by enhancing the transcriptional activity of peroxisome proliferator-activated receptors.23,24 HSPH1 is a molecular chaperone that guides the normal folding, intracellular disposition and proteolytic turnover of many of the key regulators of cell growth, differentiation and survival.25,26 HSPH1 is overexpressed in various cancers, but is expressed at low levels in many normal tissues. The constitutive overexpression of HSP105 in cancer cells protects tumor cells from apoptosis by suppressing activation of caspase-3 and caspase-9 and inducing expression of HSP70.27–29 The upregulation of HSPH1 observed in this study might have resulted in the abnormal keratinocyte proliferation in psoriasis. PPIL1, another upregulated gene, is also important in the proliferation of cancer cells30 and may mediate the pathogenesis of psoriasis through the same mechanism as HSPH1. SENP3 is also elevated in psoriasis. The manifestation of vascular proliferation in psoriatic lesions31 may be a result of increased SENP3, subsequently enhancing the expression of hypoxia-inducible factor-1a-regulated vascular endothelial growth factor, which is critical for vascular development.32 The expression of NUP54 is increased, which is an essential protein present in a multiprotein complex that is primarily required for nuclear protein import.33 The abnormal expression of NUP54 could inhibit the proliferation of human pulmonary arterial smooth muscle cells by opening the KATP channel.34 In psoriatic T cells, increased expression of NUP54 may influence the cell proliferation by affecting nuclear protein import. These genes were not different in MZ-CP. Because the majority of the DEG were found to be related to cell proliferation and differentiation, we expect that these genes were involved in the pathogenesis of psoriasis by affecting keratinocyte proliferation and ETT. However, the exact mechanism of the DEG contribution to the influence need to be further studied.
ACKNOWLEDGMENTS: This project was supported by the National Natural Science Foundation of China (81271768 and 81401360). CONFLICT OF INTEREST:
There are no conflicts of interest
to declare.
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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Table S1. Specimen sources Table S2. Primers for real time reverse transcription polymerase chain reaction (RT–PCR) Table S3. Differentially expressed genes
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