特应性皮炎犬的IL-17、IL-31和总IgE循环浓度变化

王帆

AlterationsincirculatingconcentrationsofIL-17,IL-31 andtotalIgEindogswithatopicdermatitis

特应性皮炎犬的IL-17、IL-31和总IgE循环浓度变化

翻译：李远奎 校对：王帆

Background – The pathogenesis of canine atopic dermatitis (AD) is complex. Dysregulation of the cutaneous immune system is considered to be an important part of the allergic response. Exploration of association of interleukin-17 (IL-17), IL-31, IgE and leukogram attributes with canine AD could provide novel insights into its immunopathology.

Hypothesis/Objectives – To investigate possible associations of IL-17, IL-3, IgE and leukogram attributes of canine AD.

Animals – 17 dogs diagnosed with AD and six healthy dogs.

Methods and materials – Circulating concentrations of IL-17, IL-31 and total IgE from sera samples were determined using commercial canine-specific quantitative immunoassay kits. Complete blood cell counts were analysed by an automated haematology analyser. Statistical differences between the two groups were determined using an unpaired t-test. The degree of relationship between the IL-17, IL-31, IgE, total leukocyte count (TLC) values and clinical signs scores (Canine Atopic Dermatitis Lesion Index and pruritus Visual Analog Scale pVAS) was determined by Pearson’s r correlation statistic.

Results – Dogs with AD had significantly(P<0.0001) higher circulating concentrations of IL-17, IL-31 and total IgE compared with healthy dogs. Dogs with AD also had significantly higher TLC (P < 0.0002), absolute neutrophils (P < 0.0001) and absolute eosinophils (P <0.0001) counts, and percentage of neutrophils (P < 0.03) and eosinophils (P <0.0001)  compared  with  healthy  controls.  A  significant  positive  correlation (r2 = 0.396; P <0.007) between the pVAS and IL-31 was observed in dogs with AD.

Conclusions and clinical importance – Marked elevation in circulating IL-17, IL-31 and total IgE along with the abnormalities in leukogram may be associated with canine AD and could be possible targets in the therapeutic management of canine AD.

背景-犬特应性皮炎（AD）的发病机制是复杂的。皮肤免疫系统的失调被认为是过敏反应的重要组成部分。探讨白细胞介素17（IL-17）、IL-31、IgE和白细胞计数与犬AD的关系，可以为其免疫病理学研究提供新的思路。

假设/目的-探讨IL-17、IL-3、IgE和白细胞计数与犬AD可能相关性。

动物-17只确诊AD患犬和6只健康犬。

方法与材料-用商业犬用定量免疫分析试剂盒测定血清中IL-17、IL-31和总IgE的循环浓度。用自动血液分析仪分析全血细胞计数。两组间的统计差异用未配对t检验确定。用Pearson's r相关分析法测定IL-17、IL-31、IgE、总白细胞计数（TLC）值与临床症状评分（犬特应性皮炎病变指数和瘙痒视觉模拟评分）的关系程度。

结果-与健康犬相比，AD犬循环中IL-17、IL-31和总IgE浓度显著升高（P<0.0001）。AD组的TLC（P<0.0002）、绝对中性粒细胞（P<0.0001）、绝对嗜酸性粒细胞（P<0.0001）计数、中性粒细胞（P<0.03）、嗜酸性粒细胞（P<0.0001）百分率也明显高于健康对照组。AD犬pVAS与IL-31呈显著正相关（r2=0.396，P<0.007）。

结论和临床意义-循环中IL-17、IL-31、总IgE水平显著升高及白细胞异常可能与犬AD有关，可作为犬AD治疗的靶点。

Introduction

介绍

Canine atopic dermatitis (AD) is a common skin disease with a complex pathogenesis. It has been reported that interleukin-31 (IL-31) is a potent pruritogenic cytokine that plays a key role in pruritic skin conditions in humans and animals.In one study the administration of anti-IL-31 receptor antibody was reported to significantly alleviate pruritus in human patients with AD. In addition to cytokines produced from Th1 and Th2 cells, IL-17, produced from Th17 cells, has been reported to have proinflammatory properties.Interleukin-17 stimulates the production of IL-6 and IL-8 in human keratinocytes and results in increased T-cell migration to the skin.The association of IL-17 with canine AD has not been investigated.

犬特应性皮炎（AD）是一种常见的皮肤病，发病机制复杂。据报道，白细胞介素-31（IL-31）是一种有效的致痒细胞因子，在人类和动物瘙痒性皮肤病中起着关键作用。在一项研究中，抗IL-31受体抗体的应用被报道可显著缓解AD患者的瘙痒。据报道，除了Th1和Th2产生的细胞因子以外，由Th17细胞产生的IL-17具有促炎特性。IL-17刺激人角质形成细胞产生IL-6和IL-8，并导致T细胞向皮肤的迁移增加。IL-17与犬AD的关系尚未研究。

The study of the possible association of IL-17 with IL- 31 and immunoglobulin-E (IgE) may provide insights into canine AD. It also may identify new therapeutic targets for the management of canine AD. Inflammatory cells are known to be involved in the pathogenesis of canine AD. Concurrent investigation of changes in leukograms along- side proinflammatory cytokines may reveal helpful information. The goals of this study were to evaluate circulating concentrations of IL-17, IL-31 and IgE, and leukograms in dogs with clinical AD.

研究IL-17与IL-31和免疫球蛋白E（IgE）的可能联系，可能为研究犬AD提供新的思路。也可能为治疗犬AD找到新的治疗靶点。已知炎性细胞参与了犬AD的发病机制。同时观察白细胞图的促炎细胞因子的变化可能提供有用的信息。本研究的目的是评估临床AD犬循环中IL-17、IL-31和IgE的浓度以及白细胞计数。

Methods and materials

方法和材料

Ethical approval

道德认可

The study was conducted in accordance with ethical standards of the Institutional Animal Ethics Committee of UP Pandit Deen Dayal Upadhyaya Pashu-Chikitsa Vigyan Vishwavidyalaya Evam Go Anu- sandhan Sansthan (DUVASU), Mathura (UP), India.

这项研究是根据北方邦动物伦理委员会的伦理标准进行的，印度马图拉(北方邦)。

Inclusion and exclusion criteria of dogs

犬的纳入和排除标准

Dogs were client-owned dogs and presented to Kothari Hospital of College of Veterinary Science and Animal Husbandry, DUVASU, Mathura, and fulfilled five of eight published criteria for a diagnosis of AD. In addition, dogs were free from flea infestations, none had any skin lesion in the dorsolumbar area and all had a history of receiving regular flea preventative with fipronil and s-methoprene at monthly intervals. Skin scrapings did not detect Demodex or Sarcoptes mites. There also was no response to treatment for mite infestations using amitraz, ivermectin or doramectin. None of the dogs had received glucocorticoids, ciclosporin, oclacitinib, lokivetmab, antihistamines, essential fatty acids or ear products containing glucocorticoids during the last 30 days before the clinical examination. None of the enrolled dogs were receiving allergen-specific immunotherapy.

家养犬就诊于马图拉杜瓦苏兽医科学与畜牧学院科塔里医院，符合八项已公布的诊断AD的标准中的五项。此外，犬没有跳蚤感染，没有腰荐部皮肤病变，所有犬都定期每月一次非泼罗尼和s-甲氧普林预防跳蚤管理。皮肤刮片未发现蠕形螨或疥螨。使用双甲脒、伊维菌素或多拉菌素治疗螨虫感染也没有效果。在临床检查前的30天内，所有犬未使用过糖皮质激素、环孢素、奥拉替尼、洛基维特单抗、抗组胺药、必需脂肪酸或含糖皮质激素的耳药。所有入选的犬均未接受过敏原特异性免疫治疗。

All of the dogs were screened for bacterial pyoderma or yeast overgrowth by cytological examination of skin impression smears. The dogs confirmed to have pyoderma and or yeast overgrowth infections were not included. At the time of the first clinical examination, a Canine Atopic Dermatitis Lesion Index (CADLI) and pruritus Visual Analog Scale (pVAS) based on owner-derived data,were recorded for each atopic dog.

对所有犬进行皮肤压片细胞学检查，筛选出细菌性脓皮病或酵母菌过度增殖。被证实患有脓皮病和或酵母过度增殖感染患犬不包括在内。在第一次临床检查时，记录了每只特应性皮炎病变指数(CADLI)和瘙痒症视觉模拟评分(pVAS)。

Apparently healthy dogs were recruited from dogs presented for routine annual vaccination or wellness examinations. These dogs were free of skin lesions, parasites, bacterial and yeast skin infections and haemo-parasites, as determined by routine diagnostic methods which also included routine analysis of complete blood count and serum biochemistry.

健康的犬是从每年例行疫苗接种或健康检查的犬中招募的。这些犬没有皮肤病变、寄生虫、细菌和酵母菌感染以及血液寄生虫，这是通过常规诊断方法确定的，其中还包括常规的全血计数和血清生化分析。

Collection of blood samples

血样采集

Approximately 5 mL of blood was collected from each dog before the start of any therapy. Two millilitres of blood were collected for routine haematological examination. The remaining 3 mL of blood was transferred into vials containing a clot activator and used for harvesting serum. Harvested serum samples were transferred into cryovials and kept at —20°C until use for evaluation of cytokines and total IgE concentration.

在开始任何治疗之前，从每只犬身上采集大约5毫升的血液。采集两毫升血液进行常规血液学检查。剩余的3毫升血液被转移到装有凝血激活剂的小瓶中，用于采集血清。采集的血清样本转移到冷冻瓶中，并保持在-20°C，直到用于评估细胞因子和总IgE浓度。

Assessment of cytokines and total IgE

细胞因子和总IgE的测定

Circulating concentrations of canine IL-17, IL-31 and total IgE were estimated using commercial quantitative immunoassay ELISA kits that were reported to be canine-specific (E0208Ca-IgE, E0007Ca-IL- 17, E0191Ca-IL-31, Bioassay Technology Laboratory; Shanghai, China) following the procedures as described by the manufacturer. The circulating IL-17 and IL-31 concentrations were expressed as ng/ L and total IgE concentration was expressed as lg/mL. The manufacturer reported  the same inter  and intra assay reproducibility  coefficients of <8% and <10% for IL-17, IL-31 and IgE.

根据制造商描述的程序，使用商业定量免疫分析ELISA试剂盒，测定犬IL-17、IL-31和总IgE的循环浓度。循环IL-17和IL-31浓度表示为ng/L，总IgE浓度表示为lg/mL。制造商报告的IL-17、IL-31和IgE的批间和批内重复性系数分别为<8%和<10%。

Haematological examination

血液学检查

Complete blood cell counts of dogs with AD and the healthy group were analysed by an automated haematology Analyser (BC-2800 Vet Haematology Analyser, Mindray Electronic Co. Ltd; Shenzhen, China).

应用全自动血液分析仪对AD犬和健康组犬的全血细胞计数进行分析。

Statistical                                                

统计

analysis Normality of data distribution was tested using the Shapiro–Wilk test. The data were found to be distributed normally. Statistical differences between the two groups were evaluated using the unpaired t- test on GraphPad Prism 8 (San Diego, CA, USA). Moreover, to deter- mine the degree of relationship between the IL-17, IL-31, IgE and total leukocyte count (TLC) values, and clinical signs scores (CADLI and pVAS), Pearson’s r-correlation statistic was used. Data are presented as mean T standard error (TSE); P-values < 0.05 were con- sidered statistically significant.

数据分布的分析正态性用Shapiro-Wilk检验进行检验。发现数据分布正常。用GraphPad Prism 8的未配对t检验评估两组之间的统计差异。此外，为了确定IL-17、IL-31、IgE和总白细胞计数（TLC）值与临床症状评分（CADLI和pVAS）之间的关系程度，采用Pearson的r相关统计。数据以平均T标准差（TSE）表示，P值<0.05有统计学意义。

Results

结果

Seventeen dogs ranging in age from one to four years of age met the inclusion criteria for canine AD. Six apparently healthy dogs were included in the study. Data on circulating concentrations of IL-31, IL-17 and total IgE in dogs with AD, and healthy control groups are summarized in Figures 1 and 2. Circulating concentrations of IL-17 and IL-31 were found to be significantly (P < 0.0001) elevated in dogs with AD compared to the healthy dogs (Figure 1 and Table 1). Serum concentrations of total IgE also were found to be significantly (P < 0.0001) elevated in dogs with AD compared to that in the healthy dog group (Figure 2 and Table 1).

17只1-4岁的犬符合犬AD的入选标准。6只外表健康的犬被纳入研究。图1和图2总结了AD犬和健康对照组IL-31、IL-17和总IgE的计算浓度。与健康犬相比，AD犬循环中IL-17和IL-31浓度显著升高（P<0.0001）（图1和表1）。与健康犬组相比，AD犬的血清总IgE浓度也显著升高（P<0.0001）（图2和表1）。

The CADLI and pVAS scores (mean T SE) in dogs with AD were calculated to be 22.18±1.37 and 6.47±0.26, respectively. A significant positive correlation (r2 = 0.396; P < 0.007) between IL-31 concentrations and pVAS was present in dogs with AD (Table 2), whereas significant correlation between other parameters was not found (Table 2). Significant alterations in leukogram values were observed between the dogs with AD and healthy controls (Table 1). Data analysis revealed that the dogs with AD had significantly higher TLC (P < 0.0002), absolute neutrophil counts (P < 0.0001), absolute eosinophil counts (P < 0.0001), neutrophils percentage (P < 0.03) and eosinophils percentage (P < 0.0001) compared to healthy dogs.

计算AD犬的CADLI和pVAS评分（平均T SE），分别为22.18±1.37和6.47±0.26。AD犬IL-31浓度与pVAS呈显著正相关（r2=0.396；P<0.007），而其他参数间无显著相关（表2）。在AD犬和健康犬对照组之间，观察到白细胞计数显著变化（表1）。资料分析显示，AD组的TLC（P<0.0002）、中性粒细胞绝对计数（P<0.0001）、嗜酸性粒细胞绝对计数（P<0.0001）、中性粒细胞百分率（P<0.03）、嗜酸性粒细胞百分率（P<0.0001）均显著高于健康组。

Discussion

讨论

In the present study, an elevated circulating total IgE concentration in dogs with AD suggests the possible association of type-1 hypersensitivity with canine AD. Mast cells, upon activation, produce or downregulate secretion of various cytokines, chemokines and arachidonic acid derivatives. Our observation of significant elevation in total circulating IgE concentration in dogs with AD is similar to that observed in clinically affected atopic human patients. Contrary to our findings, no significant difference between the total serum IgE concentrations of dogs with AD and healthy ones has been reported.Although IgE is considered a component in AD, some studies have shown that AD is not always IgE-mediated. Higher production of IgE could be due to percutaneous absorption of allergens into the body resulting in degranulation of mast cells and excessive production of histamine.

在本研究中，AD犬循环总IgE浓度升高提示1型超敏反应可能与AD有关。肥大细胞激活，产生或下调多种细胞因子、趋化因子和花生四烯酸衍生物的分泌。我们对AD犬的总循环IgE浓度显著升高的观察与临床上特应性皮炎人类患者的观察相似。与我们的发现相反，虽然IgE被认为是AD的一个组成部分，但一些研究表明AD并不总是IgE介导的。IgE的高产量可能是由于过敏原经皮吸收到体内，导致肥大细胞脱颗粒和组胺过多产生。

Figure 1. Circulatory concentrations  of pro-inflammatory cytokines of dogs with atopic dermatitis (n = 17) and healthy dogs (n = 6).*Significantly higher (P < 0.0001) than the healthy dogs. Data presented are mean T SE.

图1. 特应性皮炎犬(n=17)和健康犬(n=6)促炎细胞因子的循环浓度。显著高于健康犬(P<0.0001)。 所提供的数据是平均TSE。

                                                      

Figure 2. Circulatory total IgE concentration in dogs with atopic der- matitis (n = 17) and healthy dogs(n = 6).*Significant (P < 0.0001) higher than the healthy dogs. Data presented are mean T SE.

图2. 特应性皮炎(n=17)和健康犬(n=6)循环总IgE浓度。*显著(P<0.0001)高于健康犬。 所提供的数据是平均TSE。

An elevated circulating concentration of IL-17 in dogs with AD was observed in the present study and this may support the possible association of IL-17 with AD in dogs. A previous study reported that the production of IL-17A in the peripheral blood mononuclear cells of dogs was associated with adverse food reaction (AFR). It was suggested that the possible association of IL-17 with concurrent bacterial infection was because IL-17 was not correlated with clinical improvement but rather with the bacterial count. However, in our study, none of the dogs with AD was suffering from concurrent microbial infection. The best-known function of IL-17 is protection against bacterial and fungal infections. Even though we found higher concentrations of IL-17 in dogs with AD than in healthy dogs, the lack of correlation between this interleukin and clinical scores (CADLI and pVAS) leads us to speculate that IL-17 may have a role in the initiation of AD in dogs, rather than in its progression. In line with our observations, an elevated concentration of IL-17 in acute AD lesions and increased percentage of Th17 cells in the peripheral blood and skin tissues of human patients with AD and psoriasis have been reported. The differences between our data in dogs with AD and human patients or dogs with AFR could be due to different inclusion criteria or different population included in the present study. Therefore, further studies are required to explore the possible association of IL-17 with the pathogenesis of canine AD.

本研究观察到AD犬循环中IL-17浓度升高，这可能支持了IL-17与AD的关系。先前的一项研究报道，犬外周血单个核细胞中IL-17A的产生与食物不良反应（AFR）有关。提示IL-17可能与并发细菌感染有关，因为IL-17与临床改善无关，而与细菌有关。然而，在我们的研究中，没有一只患有AD的犬并发微生物感染。IL-17最著名的功能是防止细菌和真菌感染。尽管我们发现在患有AD的犬体内IL-17的浓度比在健康犬体内高，但这种白介素与临床评分（CADLI和pVAS）之间缺乏相关性，我们推测IL-17可能在犬的AD发病中起作用，而不是在其进展中发挥作用。根据我们的观察，在AD和银屑病患者的急性病变中，IL-17浓度升高，外周血和皮肤组织中Th17细胞的百分比增加。AD犬与人类患者或AFR犬数据的差异可能与纳入标准不同或本研究纳入的人群不同有关。因此，IL-17与犬AD发病机制的可能联系有待进一步研究。

Interleukin-31 and its receptor are considered to play key roles in pruritic skin conditions of humans. Marked elevation in IL-31 in pruritic versus nonpruritic human skin conditions and correlation of serum IL-31 concentrations with the severity of disease in adult humans and children with AD has been documented. Similarly, a positive correlation between the severity of canine AD and circulating IL-31 concentrations has been reported.The administration of canine IL-31 to dogs resulted in a significant increase in pruritic behaviour.Therefore, IL-31 seems to play a crucial role in inducing pruritus and may be dysregulated in dogs with AD. Our finding of elevated circulating IL-31 concentration in dogs with AD supports the potential association of IL-31 with canine AD. It also adds support to the observation of IL-31-mediated AD development in dogs.

白介素-31及其受体被认为在人类瘙痒性皮肤病中发挥关键作用。人类瘙痒性皮肤病与非瘙痒性皮肤病中，IL-31显著升高，以及血清IL-31浓度与成人和儿童AD疾病严重程度的相关性已被记录。同样，据报道，犬AD严重程度与循环IL-31的浓度呈正相关性。给犬注射IL-31可显著增加犬的瘙痒行为。因此，IL-31似乎在诱导瘙痒中起着关键作用，并且可能在AD犬中失调。我们发现AD犬循环IL-31浓度升高支持IL-31与AD的潜在联系，同时也为观察IL-31介导的犬AD中的发展提供了支持。

A significant positive correlation between the pVAS and IL-31 concentrations observed in the present study supports a role for IL-31 in canine AD. Estimation of serum IL-31 concentration is considered more informative than the quantification of IL-31 mRNA gene expression because one study failed to detect IL-31 mRNA in the skin of dogs with AD. Our observation of an elevated serum IL-31 concentration in dogs with AD is in agreement with a previous report. Moreover, another study also demonstrated a significant upregulation of the IL-31 cytokine gene in acute canine AD skin lesions.

本研究中观察到的pVAS和IL-31浓度之间的显著正相关，支持了IL-31在犬AD中的作用。血清IL-31浓度的估计被认为比IL-31mRNA基因表达的量化更具信息性，因为一项研究未能检测到IL-31mRNA在犬皮肤中的表达AD.我们对AD犬血清IL-31浓度的观察与先前的报道一致。此外，另一项研究也表明，IL-31细胞因子基因在急性AD皮肤病变中显著上调。

The dogs with AD in the present study revealed significant alterations in leukograms with significant elevations in TLC, absolute neutrophil and eosinophil counts, and percentage of neutrophils and eosinophils. Environmental antigens stimulate peripheral blood mononuclear cells. Therefore, the possibility of the antigen-induced inflammatory response in dogs with AD cannot be ruled out. Elevated values of leukogram attributes in dogs with AD suggest the possible association of AD with the marked inflammatory process. Our observations on altered leukograms support previous reports of significant elevation in neutrophils, eosinophils and TLC in dogs with AD. Elevation in TLC could be due to the cellular and humoral immune response in allergic dermatitis and/or toxins released from tissue damage following inflammation. Significantly elevated eosinophils count and percentage observed in atopic dogs might be attributed to the hypersensitivity reaction which incites release of eosinophils in blood circulation. The atopic dogs participated in the present study were client-owned; therefore, it was not possible to keep all the participating dogs in similar environmental conditions. Furthermore, the numbers of atopic dogs participated in the study are small; hence, studies on a large number of dogs suffering from clinical AD or experimentally induced AD are needed to elucidate the association of IL-17 in the development or pathogenesis of canine AD.

在本研究中，AD患犬显示出明显的白细胞改变，TLC、绝对中性粒细胞和嗜酸性粒细胞计数、中性粒细胞和嗜酸性粒细胞百分比显著升高。环境抗原刺激外周血单个核细胞。因此，不能排除抗原诱导的AD犬炎症反应的可能性。AD犬白细胞计数升高提示AD可能与明显的炎症过程有关。我们对改变的白细胞图的观察支持了先前的报告，即AD犬的中性粒细胞、嗜酸性粒细胞和TLC显著升高。TLC升高可能是由于过敏性皮炎的细胞和体液免疫反应和/或炎症后组织病变释放的毒素。特应性皮炎犬显著升高的嗜酸性粒细胞计数和百分比可能与过敏反应有关，过敏反应刺激了血液循环中嗜酸性粒细胞释放。参与本研究的特应性皮炎患犬均为家养犬。因此，不可能让所有参与研究的犬都处于类似的环境条件下。此外，参与研究的特应性皮炎犬的数量很少。因此，需要对大量临床或实验性AD患犬进行研究，以阐明IL-17与犬AD的发生或发病的关系。

It can be concluded that dogs with AD manifest inflammatory response along with eosinophilia. Marked elevation in circulating IL-17 along with IL-31, IgE and altered leukogram attributes may be associated with canine AD. Therefore, IL-17 along with IL-31 can be considered as the potential targets in the therapeutic management of canine AD.

综上所述，AD犬有明显的炎症反应与嗜酸性粒细胞增多。循环中IL-17、IL-31、IgE和白细胞计数的显著升高可能与犬AD有关，因此IL-17和IL-31可作为犬AD治疗的潜在靶点。

王帆

中国制造的高光时刻~迈瑞的血球仪和中国上海的ELISA试剂盒

曾医生

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