特应性皮肤综合征患猫嗜酸性斑块中选定的细胞因子和趋化因子的转 ...

王帆

Transcriptome analysis of selected cytokine and chemokines in the eosinophilic plaques of cats with atopic skin syndrome

特应性皮肤综合征患猫嗜酸性斑块中选定的细胞因子和趋化因子的转录组分析

作者：Cheryl Vargo | Elizabeth W. Howerth | Frane Banovic

 

翻译：罗雪

 

Abstract

摘要

Background: Previous evaluations of cytokine and chemokine gene expressions [messenger (m)RNA] in the skin of allergic cats were mostly unsuccessful in detecting the T-helper 2 (Th2) pathway, which is associated with the major effector cytokines interleukin (IL)-4, IL-5 and IL-13.

背景：先前对过敏猫皮肤中细胞因子和趋化因子基因表达[信使(m)RNA]的评估大多未能检测到辅助型T细胞2（Th2）通路，该通路与主要效应细胞因子白细胞介素（IL）-4、IL-5和IL-13相关。

Hypothesis/Objective: To evaluate differences in the mRNA expression in eosinophilic plaques of cats diagnosed with feline atopic skin syndrome (FASS) compared to healthy controls.

假设/目标：评估诊断为特应性皮肤综合征（FASS）患猫的嗜酸性斑块中mRNA表达，与健康对照猫的差异。

Animals: Four client-owned cats with FASS with eosinophilic plaques and five healthy control cats.

动物：4只客户拥有的FASS伴有嗜酸性斑块的患猫和5只健康的对照猫。

Materials and Methods: Gene expressions (mRNA) of 14 cytokines and chemokines from eosinophilic plaque skin of cats with FASS and site-matched skin samples from healthy controls were analysed using quantitative reversetranscription PCR analysis.

材料和方法：采用定量逆转录PCR分析FASS患猫嗜酸性斑块皮肤和健康对照组皮肤样本中14种细胞因子和趋化因子的基因表达（mRNA）。

Results: Eosinophilic plaques were characterized by upregulation of Th2 cytokines IL-4 (p ≤0.01), IL-5 (p ≤0.01) and IL-13 (p ≤0.01) and Th2-attracting chemokine CCL17 (p ≤0.05). Moreover, there was higher expression of S100 calcium-binding protein A 8 (p ≤0.01) as well as C-X-C Motif chemokine ligand 10 (CXCL10; p ≤0.01), IL-10 (p ≤0.05) and the Th17 cytokine IL-17A (p ≤0.01) in lesional skin compared to healthy samples. There was no difference in gene expressions of IL-12A, IL-31, IL-33, thymic stromal lymphopoietin (TSLP), tumour necrosis factor-α (TNF-α) or CCL5.

结果：嗜酸性斑块的特征是Th2细胞因子IL-4（p≤0.01）、IL-5（p≤0.01）和IL-13（p≤0.01）和Th2趋化因子CCL17（p≤0.05）上调。此外，与健康样本相比，病变皮肤的S100钙结合蛋白A 8（p≤0.01）和C-X-C Motif趋化因子配体10（CXCL10；p≤0.01）、IL-10（p≤0.05）和Th17细胞因子IL-17A（p≤0.01）的表达更高。IL-12A、IL-31、IL-33、胸腺基质淋巴生成素（TSLP）、肿瘤坏死因子-α（TNF-α）和CCL5的基因表达均无差异。

Conclusions: Results demonstrate that eosinophilic plaques feature dominant Th2 and IL-17A inflammatory responses in the skin. Further larger-sample transcriptome studies are needed to advance our understanding of the pathogenesis of different skin lesions in FASS.

结论：结果表明，嗜酸性斑块皮肤中主要是Th2和IL-17A炎症反应。需要进一步的更大样本转录组研究来推进我们对FASS中不同皮肤病变的发病机制的理解。

 

INTRODUCTION

介绍

 

Feline atopic skin syndrome (FASS) is a pruritic and inflammatory allergic skin disease induced by environmental allergens (e.g. dust mites, pollens and moulds). Cats with FASS typically exhibit one or more of the following clinical lesion patterns: miliary dermatitis (MD), self-induced symmetrical alopecia (SIA), facial head neck pruritus with excoriations and erosions (HNP) and eosinophilic granuloma complex(EGC).Eosinophilic plaques are grouped within the eosinophilic granuloma complex (EGC) and are characterized as an elevated, intensively pruritic, erythematous, and often eroded or ulcerative lesion most commonly found on the ventral abdomen and medial thighs.

猫特应性皮肤综合征（FASS）是一种由环境过敏原（如尘螨、花粉和霉菌）引起的瘙痒性炎性过敏性皮肤病。患有FASS的猫典型地表现出以下一种或多种临床病变模式：粟粒性皮炎（MD）、自我诱导的对称性脱毛（SIA）、面部头颈部瘙痒伴抓痕和糜烂（HNP）和嗜酸性肉芽肿复合物（EGC）。嗜酸性斑块归类于嗜酸性肉芽肿复合物（EGC），其特征是凸起的、强烈瘙痒、发红和经常糜烂或溃疡性病变，最常见于腹侧和大腿内侧。

 

Only a few studies have reported the type of inflammatory mediators in the different skin lesions (e.g. MD, SA, HNP and EGC) of cats with FASS.Two large gene expression studies involving 38 allergic cats showed no apparent difference in several pro-inflammatory and pro-allergic cytokine messenger RNA (mRNA) transcript expressions in lesional skin compared to healthy controls.Based on these results,the authors proposed that cats with allergic dermatitis lack the common allergy-associated T-helper 2 (Th2) immunopathogenesis observed in other species (e.g. human, canine and equine).Further understanding of the pathogenesis and immunological pathways in skin lesions of FASS cats is warranted to develop mechanism-based therapy for this disease.

只有少数研究报道了在FASS猫的不同皮肤病变（如MD、SA、HNP和EGC）中炎症介质的类型。两项涉及38只过敏猫的大型基因表达研究显示，与健康对照组相比，病变皮肤中几种促炎和促过敏细胞因子信使RNA（mRNA）转录表达没有明显差异。基于这些结果，作者提出，患有过敏性皮炎的猫缺乏在其他物种（如人、犬和马）中观察到的常见的过敏相关辅助型T细胞2（Th2）免疫发病机制。进一步了解FASS猫皮肤病变的发病机制和免疫途径对于开发基于机制的治疗方法是有必要的。

 

In this pilot study, we hypothesized that the gene expression (mRNA) in the skin of cats with FASS with eosinophilic plaques would demonstrate upregulation of the Th2 pathway with the elevation of interleukin (IL)-4, IL-5 and IL-13 compared to control skin samples of healthy cats.

在这项初步研究中，我们假设，与健康猫的对照组皮肤样本相比，伴有嗜酸性斑块的FASS患猫皮肤中的基因表达（mRNA）随着白细胞介素（IL）-4、IL-5和IL-13的升高，Th2通路表达上调。

 

METHODS AND MATERIALS

方法和材料

Animals

动物

All aspects of the study were approved and conducted in accordance with the Institutional Animal Care and Use Committee (IACUC). Signed informed consent was obtained from all owners.

该研究的所有方面都按照机构动物护理和使用委员会（IACUC）的批准和进行。所有宠主均签署了知情同意书。

 

Four cats diagnosed with FASS and eosinophilic plaques(e.g. raised pruritic, erythematous, erosive to ulcerated lesions with skin cytological results revealing eosinophilic inflammation without bacteria) were prospectively enrolled at the University Veterinary Teaching Hospital between January 2019 and June 2020. Cats were diagnosed with FASS based on history, clinical examination findings, exclusion of other pruritic dermatoses and fulfilment of published diagnostic criteria for FASS.

2019年1月至2020年6月期间，在大学兽医教学医院登记了4只诊断为FASS和嗜酸性斑块（如凸起的瘙痒、发红、溃疡，皮肤细胞学结果显示无菌性嗜酸性炎症）。猫诊断为FASS基于病史、临床检查结果、排除其他瘙痒性皮肤病且符合已发表的FASS诊断标准。

 

All allergic cats were deemed systemically healthy except for the clinical signs of FASS. All cats diagnosed with FASS showed no clinical improvement after receiving ectoparasite control for at least three months. In addition, all cats underwent a minimum 8-week elimination diet trial, as described previously, without clinical improvement. Clinical lesions and pruritus were assessed in all enrolled cats by Scoring Feline Allergic Dermatitis (SCORFAD) and a 10cm-long Visual Analog Scale (VAS).

除FASS的临床症状外，所有过敏的猫均被认为全身状态健康。所有诊断为FASS的猫在接受体外寄生虫控制至少3个月后没有显示出临床改善。此外，如前所述，所有的猫都进行了至少8周的食物排除试验，没有临床改善。使用猫过敏性皮炎（SCORFAD）评分和10cm长的视觉模拟量表（VAS）对所有入组猫的临床病变和瘙痒进行评估。

 

Five privately owned cats without any history and evidence of recent or chronic medical conditions were enrolled as controls; these cats were classified as healthy based on history, physical examination, complete blood count and serum biochemical evaluation results.

5只没有任何病史和近期或慢性疾病证据的私有猫作为对照入组；根据病史、体格检查、全血计数和血清生化评估结果，这些猫认为是健康的。

 

This number of cats was deemed sufficient for this experiment to have a 100% power to detect a significant twofold difference in values (mRNA transcription) between healthy and FASS skin.

这些数量的猫被认为足以让实验有100%的能力检测出健康猫和FASS皮肤之间的值（mRNA转录）的显著双重差异。

 

In order to limit the influence of previously administered medications on mRNA expression of cytokines/chemokines in the skin of enrolled cats with FASS and healthy cats, drug withdrawal times for all cats were two weeks for all immunomodulating drugs (e.g. antihistamines, glucocorticoids and ciclosporin).

为了控制先前使用的药物对入组的FASS患猫和健康猫皮肤中细胞因子/趋化因子mRNA表达的影响，所有猫全部免疫调节药物的停药时间为两周（如抗组胺药、糖皮质激素和环孢素）。

 

Sample collections

样本收集

All cats were sedated at the discretion of the clinician with either dexmedetomidine hydrochloride (5 μg/kg, Dexdomitor, Zoetis) or a combination of dexmedetomidine hydrochloride (5 μg/kg), ketamine (3 mg/kg, Dechra Veterinary products) and butorphanol (0.3 mg/kg, Torbugesic™; Zoetis); all drugs were administered intramuscularly. An injection of lidocaine hydrochloride 2% (Hospira Inc.) was administered subcutaneously at the biopsy site to provide

additional local anaesthesia.

所有猫均镇静，药物由临床医生选择使用盐酸右美托咪定(5 μg/kg，多咪静，硕腾）或盐酸右美托咪定（5 μg/kg）与氯胺酮(3 mg/kg，德克罗，兽用产品）联合，布托啡诺（0.3 mg/kg，Torbugesic™；硕腾）；所有药物均肌肉注射。在活检部位皮下注射2%盐酸利多卡因（爱默生公司）以提供额外的局部麻醉。

 

One 8mm skin biopsy sample was taken from lesional skin of eosinophilic plaques and bisected: one half was placed in 10% neutral buffered formalin for paraffin embedding and routine histopathological evaluation, and the second half was immediately placed in RNALater solution (Ambion) and stored at −80°C for subsequent transcriptome analysis. Two 6mm skin biopsy samples from site-matched areas (e.g. abdomen and lateral thorax) were obtained from healthy cats and immediately placed in RNALater solution for subsequent transcriptome analysis.

从嗜酸性斑块的病变皮肤中取一个8mm的皮肤活检样本并平分：一半被放置在10%中性缓冲福尔马林中，并石蜡包埋，进行常规组织病理学评估，另一半立即放置在RNALater溶液中（Ambion），保存在−80°C中用于后续转录组分析。从健康猫身上对应区域（如腹部和胸侧壁）获取两个6mm的皮肤活检样本，并立即放置在RNALater溶液中，用于随后的转录组分析。

 

Histopathological evaluation

组织病理学评价

Formalin-fixed paraffin-embedded skin biopsy samples were routinely processed and stained with haematoxylin and eosin. Histological preparations were evaluated subjectively by two of the authors (CV and EH) blinded to the type of gross lesion sampled. The entire skin section was scanned under low magnification and then scored over approximately three ×40 magnification fields. Epidermal changes were evaluated for the presence of crusts, hyperplasia, spongiosis and exocytosis. The dermal infiltrate was subjectively scored as follows: mild, 0–100 cells per field; moderate, 100–200 cells per field; and severe, >200 cells per field. Cell types were listed in the area of highest cellular density, and dermal inflammation was noted to be predominantly superficial dermis, deep dermis or superficial-to-deep dermis.

对福尔马林固定石蜡包埋的皮肤活检样本进行常规处理，并用苏木精和伊红染色。两位作者（CV和EH）在未见获取的病变类型时对组织学样本进行主观评估。整个皮肤切片在低倍镜下扫查，然后在大约3×40放大视野进行评分。表皮的变化有结痂、增生、海绵样水肿和细胞外排。真皮浸润的主观评分如下：轻度，每个视野0-100个细胞；中度，每个视野100-200个细胞；重度，每个视野>200个细胞。在细胞密度最高的区域罗列了细胞类型，记录真皮炎症主要是浅表真皮、深层真皮或浅表至深层真皮。

 

RNA extraction and quantitative reversetranscription (qRT)-PCR) analysis

RNA提取和定量逆转录（qRT）-PCR)分析

Total mRNA was extracted using the miRNeasy Mini Kit (Qiagen), and RNA quantification was evaluated by spectrophotometry (NanoDrop 2000/2000c, Thermo Fisher Scientific). All RNA samples had a 260/280 ratio of ~1.8–2.0 and RNA quality evaluation revealed an average ribosomal integrity number (RIN) for samples of 8.1, indicating high-quality RNA for gene expression data.

使用miRNeasy Mini Kit（Qiagen）提取总mRNA，并采用分光光度法（NanoDrop 2000/2000c，赛默飞世尔科技）对RNA的进行定量评估。所有RNA样本的260/280比值为-1.8-2.0，RNA质量评估显示样本的平均核糖体完整性数（RIN）为8.1，表明基因表达数据具有高质量的RNA。

 

The qRT-PCR analysis was performed as described previously with slight modifications.Total RNAs from skin biopsy samples were reverse-transcribedinto complementary DNA (cDNA) by using the qScript cDNA SuperMix (Quantabio) and the PTC-200 Gradient Thermal Cycler (MJ Research). The cDNA of selected genes using forward and reverse primers (Integrated DNA Technologies) was amplified with PerfaCTa SYBR Green FastMix (Quantabio) in accordance with the instructions of the manufacturer. The list of utilized primers is available in Table S1.

qRT-PCR分析如前所述，但略有改动。使用qScript cDNA SuperMix（Quantabio）和PTC-200梯度热循环器（MJ Research）将皮肤活检样本中的总RNA逆转录成互补DNA（cDNA）。按照制造商的说明，使用正向和反向引物(整合DNA技术)用PerfaCTa SYBR Green FastMix (Quantabio)扩增选定基因的cDNA。使用的引物列表见表S1。

 

The selected genes evaluated, IL-4, IL-5, IL-10, IL-12A, IL-13, IL-17A, IL-31, IL-33, S100 calcium-binding protein A8 (S100A8), C-C motif chemokine ligand (CCL)17, CCL5, tumour necrosis factor-α (TNF-α), CX-C motif chemokine ligand 10 (CXCL10) and thymic stromal lymphopoietin (TSLP), were chosen based on pathogenesis studies that evaluated the mRNA expression of these molecules in the skin of humans and dogs with atopic dermatitis (AD).

选择基因的评估，IL-4、IL-5、IL-10、IL- 12A、IL-13、IL-17A、IL-31、IL-33、S100钙结合蛋白A8（S100A8）、C-C基序趋化因子配体（CCL）17、CCL5、肿瘤坏死因子-α（TNF-α）、CX-C基序趋化因子配体10（CXCL10）和胸腺基质淋巴生成素（TSLP）根据发病机理研究被选择，该研究评估了这些分子在患有特应性皮炎(AD)的人和犬的皮肤中的mRNA表达。

 

All of the primers were validated, and confirmation of the correct size of the PCR product for all primers was performed by agarose gel electrophoresis. For each primer pair, the PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and sequenced. Using the ABI Sequencing analysis software, sequences were aligned, matched and confirmed using Blast. The

qRT-PCRs were performed in a total volume of 50μL with 5μL cDNA used per reaction; wells with no cDNA served as negative controls.

对所有引物进行验证，并通过琼脂糖凝胶电泳确认所有引物的PCR产物的正确的大小。对每对引物，PCR产物用QIAquick PCR纯化试剂盒（Qiagen）进行纯化并测序。使用ABI测序分析软件，使用Blast对序列进行比对、匹配和确认。实时荧光定量PCR在总体积为50μL中进行，每次反应使用5μL cDNA；无cDNA的孔作为阴性对照。

 

Cycle threshold (Ct) values were standardized to the normalizing gene ribosomal protein L17 (RPL17), which has been documented to be a stably expressed gene in feline skin and then converted to fold change using the 2−ΔΔCT formula.

将循环阈值(Ct)标准化为正常基因核糖体蛋白L17 (RPL17)，该基因已被证明是在猫皮肤中稳定表达的基因，然后使用2−ΔΔCT 公式将其转换为倍数变化。

 

Statistical analysis

统计分析

Statistical analysis was performed using Prism v8.0 (GraphPad Software). As the data were not normally distributed, the Wilcoxon–Mann–Whitney (rank-sum) U-test was used to compare values from cats affected by eosinophilic plaques with those from healthy control cats. All analyses were performed as two-tailed tests, and the level of significance was p ≤0.05. Fold changes are expressed as median values.

使用Prism v8.0（GraphPad软件）进行统计学分析。由于数据不是正态分布，我们使用Wilcoxon-Mann-Whitney（秩和）u检验来比较有嗜酸性斑块的猫与健康对照猫的值。所有分析均采用双尾检验，显著性水平为p≤0.05。倍数变化以中值表示。

 

RESULTS

结果

Animals

动物

The signalment and clinical scores for cats affected by FASS and healthy cats are provided in Table S2. Four client-owned cats diagnosed with FASS and eosinophilic plaques were enrolled in this study (Figure 1a). The ages of the cats with FASS ranged from 1.5 to 10years (mean 5.5years). Two cats were spayed females, and two cats were neutered males. Breeds included three domestic short hair (DSH) and one Siamese cat. The ages of the five privately owned healthy control cats ranged from 0.8 to 9.8years (mean 4.5years). Three cats were neutered males, and two were spayed females; all were DSH cats.

表S2提供了FASS患猫和健康猫的特征和临床评分。本研究纳入了4只被诊断为FASS伴有嗜酸性斑块的客户拥有的猫（图1a）。FASS猫的年龄为1.5~10岁（平均5.5岁）。两只绝育的母猫，两只绝育的公猫。品种包括三只家养短毛猫（DSH）和一只暹罗猫。5只私人拥有的健康对照猫的年龄范围为0.8岁到9.8岁（平均4.5岁）。三只是绝育的公猫，两只是绝育的母猫；都是DSH猫。

 

 

FIGURE 1 Clinical images of well-demarcated, erythematous ulcerative and alopecic eosinophilic plaque in cat 1 (a). (b) Microscopic section reveals a focal erosion, epidermal hyperplasia with compact hyperkeratosis and spongiosis (asterisk) and dermal infiltration of mast cells, eosinophils and lymphocytes (arrowhead). Haematoxylin and eosin stain; x20 magnification.

图1猫1 (a).的边界清楚的、发红的溃疡的脱毛的嗜酸性斑块的临床照片。(b)显微镜下切片显示局灶性糜烂，表皮增生伴致密性角化过度和海绵样水肿（星号），真皮肥大细胞、嗜酸性粒细胞和淋巴细胞浸润（箭头）。苏木精和伊红染色；x20倍。

 

Histopathological evaluation

组织病理学评估

The histological scoring and cell types for all samples are listed in Table S3. All samples had mild orthokeratotic hyperkeratosis with mild-to-moderate epidermal hyperplasia and spongiosis (Figure 1b). In two of the four cats with FASS, the epidermis had extensive areas of ulceration. Dermal inflammation extended into the deep dermis in three cats with FASS. Numbers of cells infiltrating the dermis varied amongst the FASS cases, yet consistently was composed of mast cells, eosinophils and lymphocytes. Flame figures were absent in all skin sections examined.

所有样本的组织学评分和细胞类型列于表S3。所有样本都有轻度的正角化性角化过度，伴有轻度至中度的表皮增生和海绵样水肿(图1b)。在患有FASS的四只猫中的两只中，表皮有大面积的溃疡。有三只患有FASS的猫真皮炎症扩展到真皮深层。在FASS病例中，渗入真皮的细胞数量各不相同，但始终由肥大细胞、嗜酸性粒细胞和淋巴细胞组成。在检查的所有皮肤切片中都没有火焰状形态。

 

qRT-PCR analysis

qRT-PCR分析

Expression of mRNAs for all transcripts was detected in all skin biopsies. Eosinophilic plaques from FASS cats were characterized by upregulation of mRNA expression of cytokines (Figures 2 and 3) belonging to Th2 markers IL-4 (125.3-fold, p  ≤0.01), IL-5 (57-fold, p  ≤0.01), IL-13 (3.7-fold, p  ≤0.01) and Th2-attracting chemokine CCL17 (23.5-fold, p ≤0.05). Moreover, there was higher expression of S100 calcium-binding protein A 8 (3977.3-fold, p ≤0.01) as well as CXCL10 (6.8-fold,p ≤0.01), IL-10 (5.9-fold, p ≤0.05) and the Th17 cytokine IL-17A (116.4-fold, p  ≤0.01) in lesional skin compared to healthy samples. There was no significant change in mRNA expression in the following six transcripts (IL-12A, p = 0.28; IL-31, p = 0.28; IL-33, p = 0.91; CCL5, p = 0.92; TNF-α, p = 0.92; TSLP, p = 0.99).

在所有皮肤活检中检测到所有转录物的mRNAs表达。FASS猫的嗜酸性斑块的特征在于属于Th2标记物IL-4 (125.3倍，p ≤0.01)、IL-5 (57倍，p ≤0.01)、IL-13 (3.7倍，p ≤0.01)和Th2-趋化因子CCL17 (23.5倍，p ≤0.05)的细胞因子的mRNA表达上调(图2和3)。此外，与健康样本相比，病变中S100钙结合蛋白A 8 (3977.3倍，p ≤0.01)以及CXCL10 (6.8倍，p ≤0.01)、IL-10 (5.9倍，p ≤0.05)和Th17细胞因子IL-17A (116.4倍，p ≤0.01)的表达更高。在以下六种转录物中，mRNA表达没有显著变化(IL-12A，p = 0.28；IL-31，p = 0.28；IL-33，p = 0.91；CCL5，p = 0.92；TNF-α，p = 0.92；TSLP，p = 0.99)。

 

 

FIGURE 2 Gene expression delta cycle threshold values (ΔCt) of selected markers in lesional eosinophilic plaques of cats with feline atopic skin syndrome (FASS) were assessed by quantitative reverse-transcription PCR and normalized to the expression of the RPL17 gene; healthy feline skin served as control (*, p≤0.05; **, p≤0.01).

图2采用定量逆转录PCR检测猫特应性皮肤综合征（FASS）的嗜酸性斑块病变中选定标记物的基因表达德尔塔循环阈值（ΔCt），标准化为RPL17基因的表达；健康猫皮肤为对照（*，p≤0.05；**，p≤0.01）。

 

FIGURE 3 Median fold changes of quantitative reverse-transcription PCR analysis for selected markers in eosinophilic plaque skin of cats diagnosed with feline atopic skin syndrome (FASS). CCL5, C-C chemokine ligand 5; CCL17, C-C chemokine ligand 17; CXCL10, C-X-C motif chemokine ligand 10; IL, interleukin; S100A8, S100 calcium-binding protein; TNF-α, tumour necrosis

factor-alpha; TSLP, thymic stromal lymphopoietin. *p≤0.05.

图3被诊断为猫特应性皮肤综合征（FASS）的猫的嗜酸性斑块皮肤中选定标记物的定量逆转录PCR分析的中位数倍数变化。CCL5、C-C趋化因子配体5；CCL17、C-C趋化因子配体17；CXCL10、C-X-C基序趋化因子配体10；IL、白细胞介素；S100A8、S100钙结合蛋白；TNF-α，肿瘤坏死因子-阿尔法；TSLP，胸腺间质淋巴生成素。*p≤0.05.

 

DISCUSSION

讨论

The results of our study show that eosinophilic plaques in cats with FASS exhibit a predominantly Th2 inflammatory response in the skin. Our study findings expand from the conclusions of two previous large transcriptome studies where a Th2 skewing of cytokines in lesional skin of allergic cats was absent or minimally observed. These contrasting results could reflect the different protocols in inclusion criteria (e.g. diagnostic criteria, withdrawal times for medication effects, the severity of disease, including both feline atopic syndrome and cats with FASS, FASS clinical phenotypes) and the processing of the skin biopsies for RNA isolation. Both previous transcriptome studies evaluated mRNA expressions without providing any data on the quality of RNA (i.e. RIN), with one study utilizing formalin-fixed, paraffin-embedded samples that are susceptible to RNA degradation. In addition, elimination diet trials were not performed in the majority of allergic cats in one study and the other investigation enrolled samples from allergic cats based on histological descriptions without detailed clinical phenotypes.Compared to the two previous studies,we evaluated a larger number of cytokines in the lesional skin of cats with FASS, the results of which build upon the limited body of knowledge regarding the immunological pathways in skin lesions of cats with FASS. Furthermore, to the best of the authors' knowledge, our study provides the most comprehensive assessment to date of cytokine and chemokine expression of a single clinical phenotype of FASS.

我们的研究结果表明，FASS患猫的嗜酸性斑块在皮肤中表现出主要的Th2炎症反应。我们的研究结果扩展了之前两个大型转录组研究的结论，在这两个研究中，在过敏猫的皮肤病变中不存在或很少观察到Th2细胞因子异常。这些对比结果可以反映纳入标准(例如，诊断标准、药物影响的停药时间、疾病的严重程度，包括猫特应性综合征和FASS患猫、FASS临床表型)和用于RNA提取的皮肤活检的处理的不同方案。之前的两项转录组研究都评估了mRNA表达，但没有提供任何关于RNA质量的数据(即RIN)，其中一项研究使用的是使RNA降解的福尔马林固定石蜡包埋的样本。此外，在一项研究中，没有在大多数过敏猫中进行食物排除试验，另一项研究根据组织学描述招募了过敏猫的样本，而没有详细的临床表型。与之前的两项研究相比，我们评估了FASS患猫病变中大量的细胞因子，其结果建立在有限的关于FASS猫皮肤病变的免疫途径的知识体系上。此外，据作者所知，我们的研究提供了迄今为止对FASS单一临床表型的细胞因子和趋化因子表达最全面的评估。

 

Human and canine AD are no longer strictly considered a Th2/Th1 disease. Nonlesional and lesional skin of human and canine AD feature a heterogeneous inflammatory profile with upregulation of multiple Tcell subsets (e.g. Th2/Th22/Th17/Th1) compared to healthy controls. In human AD, the major Th1, Th2 and Th17 responses are progressively heightened from nonlesional to acute and then chronic AD skin lesions.Although a limited number of eosinophilic plaque samples and selected immune markers were evaluated in our study, the results of mRNA expression in our study demonstrated Th2 polarization (elevated IL-4, IL-5, IL-13) and Th17 inflammatory responses.

人类和犬AD不再被严格地认为是一种Th2/Th1疾病。与健康对照组相比，人类和犬AD的非病变和病变皮肤具有多种炎症特征，多个T细胞亚群（如Th2/Th22/Th17/Th1）表达上调。在人类AD中，主要的Th1、Th2和Th17反应从没有病变到急性再到慢性皮肤病变是逐渐加重的。虽然在我们的研究中评估了有限数量的嗜酸性斑块样本和选定的免疫标记物，但在我们的研究中，mRNA表达的结果显示Th2极化（IL-4、IL-5、IL-13升高）和Th17炎症反应。

 

Interleukin-4 and IL-13 represent the two key Th2 cytokines that have been shown to be important in the development of human AD via several mechanisms such as the increased expression of chemoattractants for eosinophils, basophils and Th2 cells, inhibition of keratinocyte production of antimicrobial peptides, impairment of skin barrier function, promotion of lipid abnormalities in the epidermis, stimulation of itch-sensory neurons in skin and skin remodelling. Both cytokines IL4 and IL-13, in conjunction with CCL17, a potent chemoattractant for Th2 cells, were elevated in skin lesions of cats with FASS in this report.Interleukin 17A has multiple physiological functions. In epithelium, IL-17A stimulates human keratinocytes to secrete IL-8 and increase the production of antimicrobial peptides and S100 family of proteins, which triggers keratinocyte inflammatory responses, and induces migration and activation of neutrophils.These IL-17A-driven epithelial responses are part of the innate cutaneous immune responses against pathogens. In our study and per inclusion criteria, eosinophilic plaques were ulcerated and the increased IL-17A response could represent activated innate immune responses when the skin barrier is defective. The S100 proteins have calcium-binding capacity and can induce a variety of inflammatory responses. S100A8, produced by keratinocytes, neutrophils, monocytes and macrophages, possesses both antimicrobial and inflammatory properties. Expression levels of S100A8 were strongly upregulated in the eosinophilic plaques of this study; elevated S100A8 transcripts have been reported in human and canine lesional AD skin.

白细胞介素-4和IL-13代表了两种关键的Th2细胞因子，已表明它们通过几种机制在人类AD的发展中起重要作用，例如增加嗜酸性粒细胞、嗜碱性粒细胞和Th2细胞的趋化因子的表达，抑制角质形成细胞产生抗微生物肽，损害皮肤屏障功能，促进表皮中的脂质异常，刺激皮肤中的瘙痒感觉神经元和改变皮肤结构。在该报告中，细胞因子IL-4和IL-13连同CCL 17(Th2细胞强效的趋化因子)在患有FASS的猫的皮肤病变中升高。白细胞介素17A具有多种生理功能。在上皮中，IL-17A刺激人角质形成细胞分泌IL-8，并增加抗微生物肽和S100蛋白家族的产生，这能触发角质形成细胞炎症反应，并诱导中性粒细胞的迁移和活化。这些IL-17A驱动的上皮反应是针对病原体的先天皮肤免疫反应的一部分。在我们的研究中，根据纳入标准，当皮肤屏障有缺陷时，嗜酸性斑块发生溃疡，增强的IL-17A反应相当于激活的先天免疫反应。S100蛋白具有钙结合能力，并能诱导多种炎症反应。S100A8由角质形成细胞、中性粒细胞、单核细胞和巨噬细胞产生，具有抗菌和抗炎特性。在这项研究中，S100A8的表达水平在嗜酸性斑块中严重上调；S100A8表达物在人类和犬的AD病变皮肤中升高已有报道。

 

Interleukin-5 is recognized as a key mediator for all stages of eosinophil development (growth, differentiation, maturation) and recruitment of eosinophils to areas of allergic inflammation in various organs, where they act as important effector cells. Eosinophils are the predominant cell type in eosinophilic plaques; however, a heterogeneous cell population could be observed depending on the age of biopsied skin lesions.Interleukin-5 was increased 57-fold in the eosinophilic plaques analysed in this study, corresponding to the increased eosinophilic infiltrate observed on histopathological examination.

白细胞介素-5被认为是所有阶段的嗜酸性粒细胞发育(生长、分化、成熟)以及嗜酸性粒细胞募集到各种器官的过敏性炎症区域的关键介质，在那里它们充当重要的效应细胞。嗜酸性粒细胞是嗜酸性斑块中的主要细胞类型；然而，根据活检皮肤病变的病程，可以观察到多样化的细胞群。在这项研究分析的嗜酸性斑块中，白细胞介素-5增加了57倍，这与组织病理学检查中观察到的嗜酸性粒细胞浸润增加相对应。

 

In conclusion, this study demonstrates that eosinophilic plaques in cats with FASS feature dominant Th2 inflammatory responses in the skin. The main limitations of our study are the small number of samples, the limited number of cytokines and chemokines that were analysed, and the lack of evaluation of protein expressions. Further larger-sample transcriptome and proteomic studies are needed to advance our understanding of the pathogenesis of different clinical phenotypes in FASS.

总之，这项研究表明，FASS患猫的嗜酸性斑块的特征是皮肤中以Th2炎症反应为主。我们研究的主要局限性是样本数量少，分析的细胞因子和趋化因子数量有限，缺乏对蛋白质表达的评估。需要进一步的更大样本转录组和蛋白质组研究来推进我们对FASS不同临床表型发病机制的理解。

 

 

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