Hiper-IgE sendromunda Th17 farklılaşması; IL-17 salınımı ve ROR?t gösterimi

MÜSBED 2013;3(Suppl. 1):S17-S22 Araştırma / Original Paper DOI: 10.5455/musbed.20130102082754 Th17 Differentiation in Hyper-IgE Syndrome; IL-17 Secretion and RORγt Expression Tunç Akkoç1, İsmail Öğülür1, Ayzer Tevetoğlu1, Ayşegül İzgi1, Özden Hatırnaz-Ng2, Yuk Yin-Ng2, Barış Safa2, Elif Aydıner-Karakoç1 Marmara University Medical Faculty, Pediatric Allergy and Immunology Department, Istanbul - Turkey 2 Institute for Experimental Medicine, Department of Genetics, Istanbul University, Istanbul - Turkey 1 Yazışma Adresi / Address reprint requests to: Elif Karakoc-Aydiner Address: Postane Mh, Billurkent St, F-8 Tuzla, Istanbul - Turkey Elektronik posta adresi / E-mail address: Kabul tarihi / Date of acceptance: 2 Ocak 2013 / January 2, 2013 ÖZET ABSTRACT Hiper-IgE sendromunda Th17 farklılaşması; IL-17 salınımı ve RORγt gösterimi Th17 Differentiation in Hyper-IgE Syndrome; IL-17 Secretion and RORγt Expression Amaç: Hiper-IgE sendromu (HIES), enfeksiyona duyarlılık ve düşük sayıdaki Th17 hücreleri ile karakterize edilir. Th17 hücreleri fungal ve hücre dışı bakteriyel enfeksiyonların eliminasyonunda önemli rol oynarlar. Bu çalışmada amacımız, HIES hastaları ve sağlıklı kontroller arasında interlökin 17 (IL-17) salgılanması ve RAR-bağımlı orphan reseptör gama t (RORγt) ekspresyonunun ölçülmesi ile Th17 hücrelerinin farklılaşmasını araştırmaktır. Yöntem: Çalışmaya 3 adet HIES tanısı almış çocuk ve 4 adet sağlıklı kontrol alındı. HIES skorları değerlendirildi ve hastaların klinik verileri hastane kayıtlarından toplandı. Th17 farklılaşması, ELİSA yöntemi ile IL-17 üretiminin ve gerçek zamanlı PZR yöntemi ile ROR-γt ekpresyonunun ölçülmesiyle değerlendirildi. Bulgular: HIES hastalarında, peripheral kan mononükleer hücreler (PKMH) ve CD45+RA naif T hücreler Th17 farklılaştırma koşullarında kültüre edildiğinde, IL-17 sitokin seviyesinde sağlıklı bireylere göre önemli derecede azalma gözlendi. Buna ek olarak, sağlıklı kontrollerin IL-17 seviyeleri forbol 12-miristat 13-asetat (FMA) ve iyonomisin uyarımlı durumda uyarımsız duruma göre daha yüksek bulundu. Ayrıca PKMH kültürlerinde IL-17 seviyesi FMA ve iyonomisin uyarımlı durumda, HIES hastaları ve sağlıklı kontroller için uyarımsız koşullarla karşılaştırıldığı zaman önemli derecede yüksek gözlendi. HIES hastasında uyarılmış PKMH’lerdeki RORγt ekspresyon seviyesi, sağlıklı kontrolün yarı düzeyinde olarak tespit edildi. Sonuç: IL-17 salgılanması ve ROR-γt ifadesinin değerlendirilmesi mutasyon analizleri için aday olan hastaları belirlemek için yapılmalıdır. Ülkemizde bu adımların uygulanması ve bilinen mutasyonları olmayan HIES hastalarının seçimi, yeni genetik bozuklukların keşfedilmesine ve böylece yeni tedavi yaklaşımlarına olanak sağlayacaktır. Anahtar sözcükler: Th17, interlökin 17 (IL-17), RAR-bağımlı orphan reseptör gama t (ROR-γt), Hiper-IgE sendromu (HIES) Objective: Hyper-IgE syndrome (HIES) is characterized by susceptibility to infection and low number of Th17 cells. Th17 is believed to be critical in the clearance of fungal and extracellular bacterial infections. Present study investigates the differentiation of Th17 cells by evaluation of interleukin 17 (IL-17) secretion and RAR-related orphan receptor gamma t (RORγt) expression in HIES compared with healthy subjects. Method: Three children diagnosed with HIES and 4 healthy subjects were enrolled in the study. HIES scores were evaluated and clinical data of patients were collected from their hospital records. At Th17 polarizing conditions, Th17 differentiation was assessed by the secretion of IL-17 with ELISA and the expression of ROR-γt with real time PCR. Results: HIES (n=3) patients showed significantly lower levels of IL-17 secretion compared to the healthy subjects (n=4) regarding the peripheral blood mononuclear cells (PBMCs) and CD45+RA naive T cells cultured in Th17 differentiating conditions. In addition, phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulated IL-17 levels of healthy group were significantly higher than unstimulated conditions. Moreover, PMA and ionomycin stimulated IL-17 levels of PBMC cultures were significantly higher when compared to unstimulated conditions for both HIES patients and healthy subjects. ROR-γt expression level of stimulated PBMCs for HIES patient was detected nearly half of that of the healthy subject. Conclusion: Evaluation of IL-17 secretion and ROR-γt expression should be performed to determine the patients who are candidates for mutation analyses. Performing these steps and selection of HIES patients without known mutations in our country would provide an opportunity to discover new genetic defects and so new therapeutic approaches in HIES. Key words: Th17, interleukin 17 (IL-17), RAR-related orphan receptor gamma t (ROR-γt), Hyper-IgE syndrome (HIES) INTRODUCTION Th17 cells constitute a recently discovered subset of T helper cells characterized by expression of the transcription factor RORγt and secretion of IL-17 and IL-22 (1). They are instrumental in mucosal immunity by orchestrating antimicrobial peptides expression in epithelial cells as well as by recruiting neutrophils to mucosal tissues (2). Th17 cells Marmara Üniversitesi Sağlık Bilimleri Enstitüsü Dergisi Cilt: 3, Ek Sayı: 1, 2013 / Journal of Marmara University Institute of Health Sciences Volume: 3, Supplement: 1, 2013 - http://musbed.marmara.edu.tr S17 Th17 Differentiation in Hyper-IgE Syndrome; IL-17 Secretion and RORγt Expression are generally considered to be pro-inflammatory and have been shown to mediate autoimmunity in both rodents and humans (3). Furthermore, studies using animal models have demonstrated a role for Th17 cells in mediating protection against several different bacteria and fungi, suggesting dual and context-dependent roles for Th17 cells. Recently, a number of human disorders characterized by susceptibility to infection and low number of Th17 cells have been described, including Hyper-IgE syndrome (HIES) (4-7). HIES is a complex primary immunodeficiency disorder (PID) characterized by dermatitis associated with extremely high serum IgE levels and susceptibility to staphylococcal skin abscesses and pneumonia (8,9). There are two forms of HIES: A dominant form caused by mutations in the signal transducer and activator of transcription 3 (STAT3) (10), and a recessive form caused by mutations in DOCK8 (11). Autosomal dominant (AD) HIES results from dominant negative mutations in STAT3 gene and associated with facial, dental, skeletal, and connective tissue abnormalities (9,12). On the other hand, most cases of autosomal recessive (AR) HIES are caused by mutations in the gene encoding the DOCK8 protein, which maps to the chromosomal locus 9p24.3 (11,13). DOCK8 disruption is associated with a phenotype of severe cellular immunodeficiency characterized by susceptibility to viral infections, atopic eczema, defective T-cell activation and Th17 cell differentiation, and impaired eosinophil homeostasis and dysregulation of Ig (...truncated)