Chapter 2.4

Expression of thymic stromal lymphopoietin in canine atopic dermatitis

Jolanta Klukowska-Rötzler*, Ludovic Chervet†‡, Eliane J. Müller†§, Petra Roosje‡§, Eliane Marti*§ and Jozef Janda*

*Department of Clinical Research-VPH, Vetsuisse Faculty, University of Bern, Länggassstrasse 124, CH-3012 Bern, Switzerland

†Molecular Dermatology, Institute for Animal Pathology, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, CH-3012 Bern, Switzerland

‡Division of Clinical Dermatology, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, Länggassstrasse 128, CH-3012 Bern, Switzerland

§DermFocus, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, CH-3012 Bern, Switzerland

Correspondence: Jozef Janda, Department of Clinical Research-VPH, Vetsuisse Faculty, University of Bern, Länggassstrasse 124, CH-3012 Bern, Switzerland. E-mail: jozef.janda@vetsuisse.unibe.ch

Background – In humans, thymic stromal lymphopoietin (TSLP) plays a central role in the development of allergic inflammation, such as atopic dermatitis (AD), but it is unknown whether it is involved in the pathogenesis of canine AD (CAD).

Hypothesis/Objectives – Our aim was to characterize canine TSLP and to assess its expression in CAD.

Methods – Canine TSLP was identified based on sequence homology with human TSLP and the complementary DNA (cDNA) cloned by RT-PCR. Real-time quantitative RT-PCR was established to assess the expression of canine TSLP in cultured canine keratinocytes and in skin biopsy specimens from lesional and nonlesional skin of 12 dogs with CAD and eight healthy control dogs.

Results – Partial canine TSLP cDNA was cloned and characterized. It contained four exons that shared 70 and 73% nucleotide identity with human and equine TSLP, respectively, encoding the signal peptide and full-length secreted protein. We found significantly increased TSLP expression in lesional and nonlesional skin of dogs with CAD compared with healthy control dogs (P < 0.05), whereas no difference was measured between lesional and nonlesional samples. In cultured primary canine keratinocytes, we found increased TSLP expression after stimulation with house dust mite allergen extract or Toll-like receptor ligands lipopolysaccharide and poly I:C.

Conclusions and clinical importance – Increased TSLP expression in the skin of dogs with CAD supports an involvement of TSLP in the pathogenesis of CAD similar to that in humans. Further studies should elucidate the function and therapeutic potential of TSLP in CAD.

Introduction

Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine, which plays a key role in differentiation of T-helper 2 (Th2) cells and development of allergic inflammation.¹ The expression of TSLP is increased in the skin of human patients with atopic dermatitis² and in the lungs of human asthma patients.³ Results from mouse models support the central role of TSLP in allergic diseases, because tissue-specific overexpression of TSLP leads to the development of local allergic inflammation accompanied by a systemic Th2 response.^4,5 Conversely, mice lacking the TSLP receptor are more resistant to sensitization to inhaled allergens.⁵ Thymic stromal lymphopoietin acts on dendritic cells (DCs), which then undergo maturation, express co-stimulatory molecules and efficiently present antigens.² In contrast to DCs stimulated by pathogens, human DCs stimulated by TSLP do not produce interleukin (IL)-12, which is the major T-helper 2 (Th1)-polarizing cytokine. Instead, TSLP-stimulated DCs induce differentiation of naive T cells into inflammatory Th2 cells, which produce IL-13, IL-4 and tumour necrosis factor-α.² Thus, TSLP may act as a master switch in allergic inflammation.¹

Canine atopic dermatitis (CAD) is a common inflammatory skin disease of dogs, which shares several features with human atopic dermatitis (HAD), including predilection sites, genetic predisposition, age of onset, epidermal alterations⁶ and immunopathological mechanisms.⁷ Both HAD and CAD are characterized by imbalances in lymphocyte populations and their cytokines. Increased activities of both Th1 and Th2 cytokines have been observed in CAD, although results for individual cytokines differ among the studies. Nuttall et al.⁸ observed overexpression of IL-4, interferon-γ, tumour necrosis factor-α and IL-2 and reduced expression of transforming growth factor-β1 in lesional skin of dogs with CAD. Another study⁹ extended the analysis to include cytokines and transcription factors involved in T-helper polarization and showed increased expression of signal transducer and activator of transcription 4 (STAT4), IL-13, suppressor of cytokine signaling 3 (SOCS3) and IL-10 and decreased expression of IL-12p40 and GATA-3 in lesional skin. The Th2 cytokines, such as IL-4 and IL-13, drive production of allergen-specific IgE antibodies,¹⁰ which are a central feature in the pathogenesis of CAD.¹¹ Epicutaneous allergen challenge of sensitized dogs leads to an early increase in the expression of IL-6 and IL-13.¹² In analogy to humans and mice, TSLP could play an important role in Th2 polarization in CAD. However, canine TSLP has not been characterized and its role in CAD is not known.

The aim of this study, therefore, was to identify and characterize the canine TSLP gene and to establish real-time quantitative RT-PCR (RT-qPCR) protocols to assess the expression of TSLP in lesional and nonlesional skin of dogs with CAD and in cultured canine keratinocytes.

Materials and methods

Animals

This study was performed using 12 dogs diagnosed with CAD (five females and seven males; age range 3–12 years, mean 6.25 years and median 6 years) and eight healthy control dogs (five females and three males; age range 1.5-10 years, mean 6.7 years and median 6 years) of the following breeds: Labrador retriever, golden retriever, beagle, boxer, Jack Russell terrier, German shepherd, Hovawart, bull terrier, German Wachtelhund, American cocker spaniel and Newfoundland. These dogs were used and characterized in a previous study.⁶ The diagnosis of CAD was based on a combination of history and clinical signs,¹³ with exclusion of other pruritic differential diagnoses. Skin biopsy specimens (8 mm) were obtained from both lesional and nonlesional skin in the dogs with CAD. Normal skin tissue was obtained from eight age-matched and, if possible, breed-matched dogs with no known history or clinical signs of CAD. These dogs were euthanized for reasons unrelated to our study, and samples were taken immediately after euthanasia. The study protocol was approved by the local ethical committee on the use of animals, Canton Bern, Switzerland (BVET 84/05).

Isolation and culture of primary canine keratinocytes

Canine footpad keratinocytes were isolated from 10-week-old beagle dogs and cultured in monolayers as described.^14,15 Cells were maintained in William’s E medium (BioConcept, Allschwill, Switzerland) including antibiotics (Gibco Antibiotic-Antimycotic 100X liquid; Gibco, Invitrogen, Carlsbad, CA, USA), 10% fetal calf serum (Gibco), 2 mmol/L L-glutamine (Gibco), 10⁻¹⁰ mol/L cholera toxin (Sigma-Aldrich, St Louis, MO, USA) and 10 ng/mL of human epidermal growth factor (EGF, E9644; Sigma-Aldrich). Monolayer culture cells were incubated at 34°C in air supplemented with 5% CO₂, passaged after trypsin digestion (5x Trypsin/EDTA PBS; BioConcept) and subcultured before they reached confluence. Cells between passages 12 and 20 were used for these experiments. For stimulation experiments, keratinocytes were incubated in complete medium lacking cholera toxin. Keratinocytes were stimulated for 4 h with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL; Sigma-Aldrich) and ionomycin (1 μmol/L; Sigma-Aldrich) and for 18 h with concanavalin A (ConA, 5 μg/mL; Sigma-Aldrich), the Toll-like receptor (TLR)-4 ligand lipopolycaccharide (LPS, 1 μg/mL, derived from Esherichia coli 0128:B12; Sigma-Aldrich), the TLR-3 ligand polyinosinic:polycytidylic acid (poly I:C, 5 μg/mL; Sigma-Aldrich), the TLR-7 ligand imiquimod (5 μg/mL; Invivogen, San Diego, CA, USA), the TLR-2 ligand Pam3Cys (10 μg/mL; EMC microcollections, Tübingen, Germany), house dust mite extract (HDM, Dermatophagoides farinae, 5 μg/mL; Heska, Fribourg, Switzerland) and heat-inactivated HDM (5 μg/mL). Heat inactivation was performed at 99°C for 10 min in order to inhibit enzymatic activity of HDM. The TLR ligands were used at concentrations previously shown to induce activation of canine dendritic cells.¹⁶ Cells were lysed in RLT buffer (Qiagen, Basel, Switzerland), and RNA was isolated as described in the next subsection.

Isolation of RNA and synthesis of cDNA

Total RNA was isolated from cultured keratinocytes using RNeasy Mini Kit (Qiagen) and from skin biopsy specimens using RNeasy Fibrous Tissue Kit (Qiagen) according to the manufacturer’s instructions. Contaminating genomic DNA was removed by on-column DNase treatment. Total RNA was quantified spectrophotometrically at 260 nm (NanoDrop 1000; Thermo Scientific, Reinach, Switzerland), and samples were stored at -80°C until used.

Reverse transcription of total RNA was performed using Superscript III RT (Invitrogen) to generate complementary DNA (cDNA), which was used as a template for PCR. The cDNA synthesis reaction consisted of 500 ng RNA and 1 mmol/L random primers (Promega, Dübendorf, Switzerland), 0.5 mmol/L of each dNTP, 40 U RNase inhibitor (Promega) and 100 U SuperScript III reverse transcriptase (Invitrogen), in a total reaction volume of 40 μL. The RNA samples, random primers and dNTPs were incubated at 65°C for 5 min. Reverse transcriptase was added and the incubation continued at 25°C for 5 min followed by 50°C for 50 min. The reaction was terminated by heating at 70°C for 15 min. The cDNA samples were stored at -20°C until further processing.

Cloning of canine TSLP

To clone the canine TSLP cDNA, sequences homologous to human TSLP (GenBank accession NM_033035.4) were identified in Canis familiaris genome build 3.2 using BLASTN (blast.ncbi.nih.gov). From a skin biopsy specimen obtained from a dog affected with CAD, RNA was isolated and cDNA produced. Primers were designed using Primer3¹⁷ based on homologies with human and equine¹⁸ (Genbank NM_001164063) TSLP. The cloned primary sequence was determined by direct sequencing. Alignment of human and canine TSLP nucleotide and deduced amino acid sequences was performed using ClustalW.¹⁹

Real-time quantitative RT-PCR

Expression of TSLP mRNA was assessed by RT-qPCR with primers designed to span the exon 1 and 2 junction using Primer3 online software¹⁶ (forward, 5′-AGT ACA CGG GGT GGC TGA-3′; reverse, 5′-GTC ATT TAC CAA GCC CTG GA-3′; and probe, 5′-FAM-TCC GTG CTC CTG GTC CCA TCC ATG TAT T-TAMRA-3′). The PCR consisted of 2 μL cDNA, primers (300 nmol/L each), fluorescence-labelled probe (200 nmol/L) and TaqMan Universal PCR Mastermix (Applied Biosystems, Austin, TX, USA). The PCRs were performed in a total volume of 25 μL and carried out in a 7300 Real-Time PCR System (Applied Biosystems). The PCR consisted of an initial 2 min at 50°C, 10 min denaturation at 95°C, followed by 45 cycles of 15 s denaturation at 95°C and 1 min annealing and elongation at 60°C. Reactions were performed in duplicates, and no-template and RT-negative controls were included in each run. Efficiencies of the PCRs were calculated using a relative standard curve derived from a cDNA pool. Gene expression was quantified using the ΔΔCt method.²⁰ Expression of TSLP was normalized to the expression of the housekeeping gene 18S ribosomal RNA (18S rRNA; Applied Biosystems).

Statistical analysis

Statistical analyses were carried out with the software package NCSS 2001 (NCSS, Kaysville, UT, USA). The Mann-Whitney U-test was used to compare dogs withCAD with healthy control dogs. The Wilcoxon signed rank test was used for comparison of paired data (lesional versus nonlesional). Student’s two-sample t test (unpaired) was used to compare stimulated and nonstimulated keratinocytes.

Results

Characterization of canine TSLP

A partial cDNA of canine TSLP was generated by RT-PCR from an RNA sample of lesional skin from a dog with CAD, cloned and sequenced and the sequence deposited in GenBank (accession JQ698664). The cDNA sequence contained 465 nucleotides and was organized in four exons (Figure 1). The exon-intron boundaries were in accord with the splicing scheme for human TSLP gene reported previously by Quentmeier et al.,²¹ whose numbering scheme was followed by the investigators. The splice donor and acceptor sites were consistent with the GT/AG rule. The canine TSLP gene was located on chromosome 3 (1.503-1.507 Mbp), which corresponds to human chromosome 5 and equine chromosome 14. When the coding sequence of canine TSLP cDNA was aligned with the orthologous human and equine sequences (GenBank accession numbers NM_033035.4 and NM_001164063.1), the nucleotide identity was 70 and 73%, respectively, and covered the sequence encoding the signal peptide and full-length mature protein of human and equine TSLP. Alignment of canine and human nucleotide sequences is shown in Figure 2a.

Figure 1. Canine thymic stromal lymphopoietin (TSLP) nucleotide sequence and the deduced amino acid sequence. Exon-exon boundaries are marked by filled squares. The predicted signal peptide cleavage site is marked by ||.

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