DR [Dermatology Reports 2019; 11:8006] [page 27] Skin tissue expression and serum level of thymic stromal lymphopoietin in patients with psoriasis vulgaris Oki Suwarsa, Hartati Purbo Dharmadji, Endang Sutedja, Lengga Herlina, Putri Reno Sori, Reti Hindritiani, Reiva Farah Dwiyana, Hendra Gunawan Department of Dermatology and Venereology, Faculty of Medicine, Universitas Padjadjaran; Hasan Sadikin General Hospital, Bandung, West Java, Indonesia Abstract Thymic stromal lymphopoietin (TSLP) is known to be associated with allergic dis- eases. It is also suggested that TSLP has a role in autoimmune diseases such as psoria- sis; however, the associated pathways remain unknown. There is currently little information on TSLP in psoriasis vulgaris. We investigated TSLP expressions on lesional and non-lesional skin of psoriasis vulgaris patients using reverse transcrip- tion-polymerase chain reaction. TSLP level was also investigated in serum from psoria- sis vulgaris patients compared to healthy control using enzyme-linked immunosor- bent assay. TSLP expression was higher in lesional skin (1.90) compared to non-lesion- al skin (1.76); however, the difference was not statistically significant (P>0.05). TSLP serum levels were significantly higher in psoriasis patients (287.40 pg/dL) as com- pared to controls (114.70 pg/dL) (P<0.05). This study concluded that TSLP levels in the serum of psoriasis vulgaris patients are higher than controls. TSLP was also found in keratinocyte of psoriasis patients, the expression was higher in the lesional com- pared to non-lesional skin; however, this difference is statistically insignificant. These findings suggest that TSLP may play a role in the pathogenesis of psoriasis vul- garis, but its exact role remains unclear. Introduction Psoriasis is a chronic inflammatory skin disease, with a strong genetic basis, charac- terized by complex alterations in epidermal growth and differentiation, as well as multi- ple biochemical, immunologic, and vascu- lar abnormalities.1 The pathogenesis of this disease is linked to many interactive responses among infiltrating white blood cells, dendritic cells, proinflammatory cytokines, chemokines, and chemical medi- ators produced in the skin.2 Nowadays, it is believed that psoriasis is most likely a T helper-1 (Th1)/ Th17 induced inflammatory disease.3 Thymic stromal lymphopoietin (TSLP) or interleukin (IL)-7-like cytokine has already been associated with atopic der- matitis for its role in inducing a proallergic Th2 response.4 The associated pathways of TSLP in psoriasis remains unknown. There is currently little information on TSLP in psoriasis vulgaris patients.5 In this study, we report the skin tissue expressions on lesion- al and non-lesional skin and serum level of TSLP of psoriasis vulgaris patients. Materials and Methods Patients The study was performed in accordance with the ethical guidelines in the Declaration of Helsinki and was approved by the Health Research Ethics Committee of Hasan Sadikin General Hospital, Bandung, West Java, Indonesia. Oral expla- nation was given about the study and writ- ten informed consent was subsequently obtained from each participant. Seventeen subjects for psoriasis vul- garis group and 17 subjects for control group were recruited from the Dermatology Clinic at Hasan Sadikin General Hospital using a consecutive sampling technique. Exclusion criteria include patients who had systemic and/or topical corticosteroids, antibiotics, and other immunosuppressive agents, such as cyclosporine, methotrexate, azathioprine within the last two weeks prior to the start of the study, as well as history of atopic manifestations, infections, and malignancies. Reverse transcription-polymerase chain reaction for thymic stromal lymphopoietin expression in skin After obtaining informed consent from patients, 2.5-mm punch skin biopsy speci- mens were taken from lesional and non- lesional skin in psoriasis vulgaris patients. Skin samples were collected in Eppendrof tube and immediately stored at -2°C tem- peratures. TSLP transcript expression was analyzed on ribonucleic acid (RNA) extracted from homogenized skin and reverse transcribed using standard proto- cols. Deoxyribonucleic acid (DNA) was analyzed for the expression of human glyc- eraldehyde-3-phosphate dehydrogenase (GAPDH; forward, ATCAGCAATGCCTC- CTGCAC; reverse, TGGCATGGA CTGTGGTCATG) and TSLP (forward, CACCGTCTCT TGTAGCAATCG; reverse, TAGCCT GGGCACCAGATAGC) using Rotor-Gen-Q. Enzyme-linked immunosorbent assay for thymic stromal lymphopoi- etin level in serum Approximately 5 mL of blood were col- lected in a tube without heparin from patients and participants in the control group. Healthy age- and sex-matched con- trol subjects were also included in the study. TSLP were measured using enzyme-linked immunosorbent assay (ELISA) kit, accord- ing to the manufacturer’s instructions (Cloud-Clone Corp, Houston, USA). TSLP level (pg/mL) were used for data analysis. Statistical analysis This study was an observational study, using a cross-sectional method to determine TSLP levels in the serum of psoriasis vul- garis patient and healthy control, as well as TSLP expression in lesions and non-lesions Dermatology Reports 2019; volume 11:8006 Correspondence: Oki Suwarsa, Department of Dermatology and Venereology, Faculty of Medicine, Universitas Padjadjaran; Hasan Sadikin General Hospital, Jl. Pasteur No. 38, Bandung, West Java, 40161, Indonesia. Tel.: +62.22.2032426 (ext. 3449) – Fax: +62.22.2032426. E-mail: oki.suwarsa@unpad.ac.id Key words: Psoriasis vulgaris; Serum; Skin; Thymic stromal lymphopoietin. Acknowledgments: the authors would like to thank the contribution of Health Research Unit, Universitas Padjadjaran, Bandung, West Java, Indonesia, for technical assistance. Contributions: the authors contributed equally. Conflict of interest: the authors declare no potential conflict of interest. Funding: the work was support by an internal grant of Universitas Padjadjaran, Bandung, West Java, Indonesia (Grant No. 2476/UN6C/LT/2018). Received for publication: 1 February 2019. Revision received: 10 May 2019. Accepted for publication: 13 May 2019. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). ©Copyright O. Suwarsa et al., 2019 Licensee PAGEPress, Italy Dermatology Reports 2019; 11:8006 doi:10.4081/dr.2019.8006 No n- co mm er cia l u se on ly [page 28] [Dermatology Reports 2019; 11:8006] skin of psoriasis vulgaris patients. Statistical significance was retained for P- values of less than 0.05. Results Demographic details Seventeen subjects from each group were reviewed and analyzed descriptively. Patients with psoriasis vulgaris included 13 males and 4 females, with age ranges between 10 and 68 years old (Table 1). The healthy control group with no complaint of any dermatological disorder comprised of 13 males and 4 females, their age ranges between 12 and 67 years old. Psoriasis area severity index Psoriasis area severity index was used to assess the degree of severity of disease in this study. In general, 58.8% of participants in this study had a mild form of the disease. Thymic stromal lymphopoietin expression in psoriasis skin We analyzed the expression of TSLP in lesional and non-lesional psoriasis skin. Samples from 17 patients were assessed and were all positive for TSLP (Table 1). As shown in Table 2, the lesional skin (medi- an=1.90; range=1.51-2.21) showed eleva- tion of TSLP expression compared to non- lesional skin (median=1.76; range=1.66- 2.17). Statistical analysis showed that the difference between TSLP expression in lesional and non-lesional skin of psoriasis patients was statistically insignificant (P>0.05). Thymic stromal lymphopoietin serum level To determine whether TSLP could be involved in pathogenesis psoriasis vulgaris, we analyzed the serum level of TSLP in psoriasis vulgaris patients and normal con- trols. Sample from 17 patients and 17 con- trols were assessed (Table 1). TSLP serum levels in subjects with psoriasis vulgaris patients (median=286.30 pg/mL; range=71.90-695.1 pg/mL) were signifi- cantly higher than normal controls (medi- an=114.70 pg/mL; range=50.0-206.1 pg/mL) (P<0.05) (Table 3). Discussion TSLP is a cytokine expressed mainly in the epithelial cells of the lungs, gastroin- testinal tract, thymus, tonsils, and epidermal keratinocytes.6 TSLP will bind to the a-chain IL-7 receptors (IL-7Ra) and TSLP receptor (TSLPR),6,7 which are expressed in various hematopoietic cells, such as in den- dritic cells, T cells, B cells, mast cells, and macrophages.6 TSLP has been associated with atopic dermatitis,8 asthma,9,10 and allergic rhinitis8 for its role in inducing a proallergic Th2 response.8 Here we report that TSLP is also produced in patients with psoriasis vulgaris, a Th1-related autoimmune disease. Our findings add to the existing complexity of the cytokine and inflammatory networks in patients with psoriasis and suggest that TSLP might have different immunomodula- tory roles, depending on the type of inflam- matory environment. In addition, it can also explain that one cytokine may have differ- ent functions. In the pathogenesis of psoriasis, when precipitating factors from the environment were exposed to a person with genetic pre- disposition, this will cause keratinocytes to release IL-1 and TNF-α cytokines which will induce dendritic cells in the skin as antigen presenting cell. It is also known that keratinocytes in psoriasis patients express TSLP that bind to TSLPR and IL-7Rα receptors in dendritic cells.5 Maturation of dendritic cells will produce cytokines such as IL-12 and IL-23 which will induce differ- entiation of naive T cells into Th1/Th17 cells. Th1/Th17 cell differentiation will result in the secretion of cytokines that con- tribute to keratinocyte proliferation and inflammatory responses.4 In our study, TSLP was observed in serum and skin of psoriasis patients, indi- cating that their production and function are linked to the pathogenesis of the disease. The current study shows that TSLP is pro- duced by keratinocytes in patients with pso- riasis and that TSLP, in this context, primes for IL-23 production by dendritic cells.5 Article Table 1. Thymic stromal lymphopoietin expression in serum and skin biopsy specimens from patients with psoriasis. No. Sex Age (y) PASI score TSLP in serum TSLP in serum TSLP in TSLP in psoriasis (pg/mL) control (pg/mL) lesional non-lesional A F 31 10.2 359.1 204.3 2.05 1.71 B M 48 8.4 223.6 149.9 1.90 1.92 C M 28 18.7 215.9 58.1 1.95 1.76 D M 45 2.7 286.3 153.4 2.03 1.80 E M 56 2.6 288.5 74.7 2.06 1.73 F F 51 2 338.3 144.7 1.90 1.67 G M 42 2.7 279.1 79.3 1.57 1.72 H M 24 3 209.6 122.4 1.86 1.72 I M 62 15.7 260.1 69.9 1.70 1.70 J F 64 4.3 149.2 68.8 1.89 1.66 K M 37 6.2 505.8 204.8 2.21 1.87 L M 49 9.9 438.0 206.1 1.67 1.83 M M 43 13.9 695.1 50.0 1.83 1.75 N F 20 2.1 643.7 155.4 1.51 2.17 O M 10 3.3 292.2 90.0 1.78 2.15 P M 68 10.6 544.0 160.2 1.95 2.01 Q M 52 3.0 179.0 149.9 2.05 1.83 PASI, Psoriasis Area Severity Index; TSLP, thymic stromal lymphopoietin; F, female; M, male. No n- co mm er cia l u se on ly From a pathological perspective, it will be important to establish the links between TSLP and other cytokines involved in path- ogenic inflammatory networks in patients with diverse autoimmune diseases. In patients with psoriasis, TSLP could serve as a therapeutic target to reduce dendritic cell activation and production of pathogenic IL- 23.4 This study also found that the differ- ence in TSLP expression in lesional and non-lesional skin was insignificant. Several factors that can affect the result in various studies on cytokine levels include different standardization, reagent sensitivity, exami- nation material, sample handling, and vari- ous biological factors such as age, race, and genetic polymorphism.11 In 2010, Tindall et al.12 found genetic polymorphisms in the IL-23A gene in South African populations and European populations. In this study, 33 different DNA variations were identified, and the suspected functional variation of IL-23A will have a significant impact on the patient’s immune response. To date, there is no data available that explains the existence of TSLP polymorphisms in psoriasis vul- garis. However, several researches have been conducted on Graves’ disease. In 2012, Tsai et al.13 from Taiwan found TSLP polymorphisms in Graves’ disease which can be used to estimate the prognosis and target therapy. The results yielded in this study were different from that of previous research conducted by Volpe et al. in 2014 in Paris.5 That study, TSLP were assessed with reverse transcription-polymerase chain reaction (RT-PCR), found that TSLP expression was higher in lesional than in non-lesional skin of psoriasis vulgaris patients and normal individuals.5 Genetic and environmental factors are thought to play a significant role in the pathogenesis of psoriasis vulgaris, due to the fact that these conditions often affect the results of psoria- sis research, and yielded varying results in each country. The limitation of this study was we could not investigate TSLP expression in normal skin control and the correlation between serum and skin TSLP directly. There was no report about serum TSLP level in previous studies. As TSLP is a cir- culating mediator, we assumed that there might be indirect correlation between serum and skin TSLP and further studies are required to obtain further knowledge on this correlation. Conclusions This study found that TSLP level in the serum of psoriasis vulgaris patients are higher than controls, while TSLP expres- sion was higher in the lesional skin com- pared to the non-lesional skin of psoriasis vulgaris patients; however, this difference is statistically insignificant. These findings suggest that TSLP may play a role in the pathogenesis of psoriasis, but its exact role remains unknown. References 1. Gudjonsson JE, Elder JT. Psoriasis. In: Wolff K, Goldsmith LA, Katz SI, et al., eds. Fitzpatrick’s dermatology in gener- al medicine. 8th ed. New York: McGraw-Hill; 2012. pp 197-231. 2. Lowes MA, Bowcock AM, Krueger JG. Pathogenesis and therapy of psoriasis. Nature 2007;445:866-73. 3. Coimbra S, Figueiredo A, Castro E, et al. The roles of cells and cytokines in the pathogenesis of psoriasis. Int J Dermatol 2012;51:389-98. 4. Blauvelt A. New concept in the patho- genesis and treatment of psoriasis: key roles for IL-23, IL-17A and TGF-β. Expert Rev Dermatol 2007;2:69-75. 5. Volpe E, Pattarini L, Martinez- Cingolani C, et al. Thymic stromal lym- phopoietin links keratinocytes and den- dritic cell-derived IL-23 in 209 patients with psoriasis. J Allergy Clin Immunol 2014;134:373-81. 6. He R, Geha RS. Thymic stromal lym- phopoietin. Ann Acad Sci 2010;1183: 13-24. 7. Ziegler SF, Roan F, Bell BD, et al. The biology of thymic stromal lymphopoi- etin (TSLP). Adv Pharmacol 2013;66:129-55. 8. Soumelis V, Reche P, Kanzler H, et al. Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat Immunol 2002;3:673-80. 9. Zhou B, Comeau MR, De Smedt T, et al. Thymic stromal lymphopoietin as a key initiator of allergic airway inflam- mation in mice. Nat Immunol 2005;6:1047-53. 10. Al-Shami A, Spolski R, Kelly J, et al. A role for TSLP in the development of inflammation in an asthma model. J Exp Med 2005;202:829-39. 11. Banks RE. Measurements of cytokine in clinical samples using immunoassay: problem and pitfalls. J Rev Clin Lab Sci 2000;37:131-82. 12. Tindall EA, Hayes VM. Comprehensive sequence analysis of the human IL23A gene defines new variation content and high rate of evolutionary concervation. DNA Res 2010;17:117-22. 13. Tsai KH, Tsai FJ, Lin HJ, et al. Thymic stromal lymphopoietin gene promoter polymorphisms and expression levels in Graves disease and Graves ophtalmopa- thy. BMC Med Gen 2012;13:1-7. Article Table 2. Thymic stromal lymphopoietin expression on lesional and non-lesional skin of psoriasis vulgaris patients. Variable Group P-value* Lesional skin n=17 Non-lesional skin n=17 TSLP 0.234 Mean ± Std 1.87±0.186 1.82±0.156 Median 1.90 1.76 Range (min-max) 1.51-2.21 1.66-2.17 TSLP, thymic stromal lymphopoietin. *P-value by Wilcoxon test analysis. Statistical significant was accepted at P<0.05. Table 3. Comparison of thymic stromal lymphopoietin serum level in psoriasis patients and control group. Variable Group P-value** Psoriasis n=17 Control n=17 TSLP 0.000 Mean ± Std 332.18±170.531 121.11±53.501 Median 286.30 114.70 Range (min-max) 71.90-695.10 50.00-206.10 TSLP, thymic stromal lymphopoietin. **P-value by t test analysis. Statistical significant was accepted at P<0.05. [Dermatology Reports 2019; 11:8006] [page 29] No n- co mm er cia l u se on ly