马来酸奥拉替尼长期治疗犬特应性皮炎的免疫调节作用

王帆

Immunomodulatory effect of long-term oclacitinib maleate therapy in dogs with atopic dermatitis

马来酸奥拉替尼长期治疗犬特应性皮炎的免疫调节作用

作者：Guilherme De Caro Martins等人

翻译：王帆

Background – Canine atopic dermatitis (cAD) is a chronic disease characterised by hypersensitivity to environmental allergens. Oclacitinib maleate selectively inhibits pro-inflammatory mediators associated with cAD. However, the impact of chronic oclacitinib use on immunocompetence requires further investigation.

Objectives – Herein, we examined the potential immunomodulatory effects of prolonged oclacitinib treatment in dogs.

Animals – Thirteen privately owned dogs with cAD, treated with 0.4–0.6 mg/kg oclacitinib for 12 months.

Methods and materials – Pruritus level was evaluated using a pruritus Visual Analog Scale (pVAS) and the canine atopic dermatitis extent and severity index, 4th iteration (CADESI IV). Peripheral blood samples were collected for routine laboratory assays and lymphocyte subtypes were analysed using flow cytometry. Antigenspecific intracellular cytokine production from CD4+ and CD8+ T lymphocytes was analysed following in vitro stimulation by Dermatophagoides farinae antigens.

Results – Oclacitinib treatment significantly reduced pVAS and CADESI-04 scores, by 51% and 86.7%, respectively. Flow cytometric analysis revealed increased CD4+ and CD14+ lymphocyte populations. The cytokine profile at 360 days after treatment initiation was similar to that before treatment and was not associated with clinical relapse.

Conclusion – Oclacitinib, when administered at the currently labelled dose for one year, is associated with a significant increase in circulating CD4+ T cells, but does not alter cytokine production from antigen-stimulated T cells. The results reported do not support evidence for immunosuppression mediated by the mechanisms evaluated in this study.

摘要

背景 – 犬特应性皮炎(cAD)是一种慢性疾病，特征为对环境过敏原过敏。奥拉替尼选择性抑制与cAD相关的促炎介质。然而，需要进一步研究长期使用奥拉替尼对免疫力的影响。

目的 – 为此，我们检查了长期接受奥拉替尼治疗的犬的潜在免疫调节作用。

动物 – 13只患有cAD的宠物犬，接受0.4-0.6 mg/kg奥拉替尼治疗12个月。

方法和材料 – 使用瘙痒视觉模拟量表(pVAS)评价瘙痒水平，采集样本进行常规实验室分析，并使用流式细胞术分析淋巴细胞亚型。在粉尘螨抗原体外刺激后，分析了CD4 + 和CD8 + T淋巴细胞产生的抗原特异性细胞内细胞因子。

结果 – 奥拉替尼治疗显著降低pVAS和CADESI-04评分，分别降低51%和86.7%。流式细胞分析显示CD4 + 和CD14 + 淋巴细胞群增加。治疗开始后360天的细胞因子谱与治疗前相似，与临床复发无关。

结论 – 以当前标签剂量给药1年，奥拉替尼显著提高循环CD4 + T细胞，但不改变抗原刺激T细胞的细胞因子生成。本研究的报告结果通过机制评估，未发现免疫抑制的证据。

Introduction

介绍

Atopic dermatitis (AD) is a chronic inflammatory dermatopathy that affects approximately 10% of the canine population worldwide. The most frequently observed clinical sign is intense pruritus, which significantly affects the quality of life of both animals and their owners. The diagnosis is clinical, based on the history and characteristic clinical signs associated with the exclusion of other pruritic dermatopathies.

特应性皮炎(AD)是一种慢性炎症性皮肤病，全世界约10%犬患病。最常见的临床症状是严重瘙痒，这严重影响了动物及其主人的生活质量。诊断方法为临床基于病史和临床特征，以及排除其他瘙痒性皮肤病。

The severity of pruritus and cutaneous lesions associated with canine atopic dermatitis (cAD) can be managed through allergen-specific immunotherapy, controlling dysbiosis, correcting stratum corneum barrier function and controlling inflammation with immunomodulatory drugs.Oclacitinib maleate mitigates the clinical effects of cAD through its action on the signalling pathways of Janus kinase 1 (JAK1) enzymes, which modify production of several interleukins (IL), including IL-2 and IL-6 (proinflammatory), IL-4 and IL-13 (pro-allergenic), and IL-31 (pruritogenic).The ability of oclacitinib to rapidly and effectively reduce pruritus has been demonstrated in dogs, and it is currently indicated to control the acute and chronic clinical signs of cAD. However, the effects associated with the long-term inhibition of IL signalling pathways have not been clarified adequately.

通过过敏原特异性免疫治疗、控制菌群失调、纠正角质层屏障功能、免疫调节药物控制炎症等方法，可以控制犬特应性皮炎(cAD)的严重瘙痒和皮肤病变。马来酸奥拉替尼通过对Janus激酶1 (JAK1)酶信号通路的作用减轻cAD的临床效应，JAK1酶可以调节几种白介素(IL)的产生，包括IL-2和IL-6(促炎因子)，IL-4和IL-13(促过敏因子)和IL-31(致痒因子)。已证明奥拉替尼可迅速有效地减轻犬的瘙痒，目前可用于控制cAD的急慢性临床症状。然而，与IL信号通路长期抑制的相关作用还没有被充分阐明。

In a previous study, increased numbers of CD4+ T cells and an elevated CD4+/CD8+ T-cell ratio were shown to be hallmarks of cAD.In another study, the effect of oclacitinib on peripheral blood mononuclear cells (PBMC) obtained from dogs with cAD demonstrated that when it is used at the currently recommended dose, it does not significantly affect antigen-specific proliferation of T cells. However, the effect of chronic oclacitinib use on canine T cells is unknown. Therefore, the objective of the present study was to evaluate the clinical efficacy of oclacitinib in the setting of chronic cAD, and to assess its immunomodulatory effects during long-term use. We hypothesised that oclacitinib is immunomodulatory but not immunosuppressive, and is therefore safe for longterm use.

在之前的一项研究中，CD4+ T细胞数量的增加和CD4+/CD8+ T细胞比值的升高被证明是cAD的特征。在另一项研究中，从cAD患犬上获得的奥拉替尼对外周血单个核细胞(PBMC)的影响表明，当以目前推荐的剂量使用时，它不会显著影响T细胞的抗原特异性增殖。然而，长期使用奥拉替尼对犬T细胞的影响尚不清楚。因此，本研究的目的是评估奥拉替尼在慢性CAD患犬中的临床疗效，并评估其在长期使用中的免疫调节作用。我们假设，奥拉替尼具有免疫调节作用而非免疫抑制作用，因此长期使用是安全的。

Methods and materials

材料和方法

Study site and animals

研究地点和动物

This project was approved by the Committee on Animal Research and Ethics of the Federal University of Minas Gerais (CEUA UFMG) under protocol no. 57/2016. Owners of the participating dogs signed an informed consent form. The study included 13 dogs diagnosed with cAD.

该项目由米纳斯吉拉斯州联邦大学动物研究和伦理委员会根据2016年第57号协议批准。参与计划的宠主须签署知情同意书。这项研究包括了13只确诊cAD患犬。

The diagnosis of cAD was based on histories and characteristic clinical signs using the criteria described by Favrot et al., following a two month period on a commercial hydrolysed protein diet to rule out cutaneous adverse food reactions. A 30 day wash-out period free from any medications with immunomodulatory potential was required for enrollment. All dogs were treated with oral oclacitinib maleate (Apoquel, Zoetis; Kalamazooo, MI, USA) at a dose of 0.4– 0.6 mg/kg twice daily for 14 days, then 0.4–0.6 mg/kg once daily for one year. No other immunomodulatory medications were allowed during the study period.

cAD的诊断是基于病史和Favrot等人所描述的标准的典型临床症状，然后在两个月的商业化水解蛋白日粮中排除皮肤食物不良反应。入组需要30天洗脱期，不使用任何具有免疫调节潜力的药物。所有犬口服马来酸奥拉替尼，0.4-0.6 mg/kg，每日两次，连续14天，然后0.4-0.6 mg/kg，每日一次，连续一年。在研究期间不允许使用其他免疫调节药物。

Efficacy of maleate oclacitinib

马来酸奥拉替尼有效性

Drug efficacy was evaluated on day (D)0, D15, D30, D60, D90, D120, D150, D180, D210, D240, D270, D300, D330 and D360 by examining two parameters: lesional skin using the Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04), and an owner’s assessment of pruritus using a validated pruritus Visual Analog Scale (pVAS). Three investigators performed each CADESI-04 assessment in an attempt to reduce the subjectivity of the analyses. To assess pruritus, the owners were asked to mark a vertical line on the pVAS based on their perception of the intensity of pruritus exhibited.

药物疗效评估(D) 0天,D15, D30, D60, D90, D120, D150, D180, D210, D240, D270, D300, D330和D360通过检查两个参数:使用犬特应性皮炎严重程度指数,第四版(CADESI-04)评估皮肤病变,和宠主使用瘙痒视觉模拟量表(pVAS)评估瘙痒程度。3名研究人员进行了CADESI-04评估，试图减少分析的主观性。为了评估瘙痒程度，要求宠主在pVAS上根据他们对瘙痒程度的感知画一条垂直线。

Leucocyte evaluation following oclacitinib maleate treatment

使用马来酸奥拉替尼治疗后对白细胞进行评估

For flow cytometric analysis of PBMC, jugular venipuncture was performed to collect peripheral blood into tubes containing heparin sodium before treatment and every two months during the study period. Evaluation of leucocytes was performed as described by Martins et al.Initially, 50 µL peripheral whole blood was collected in a tube containing heparin sodium and transferred to three tubes, one containing 2.5 µL CD3-FITC/ CD4+-PE/ CD8+-AlexaFluor 647 anti-dog antibody (Bio-Rad Laboratories Inc.; Hercules, CA, USA), another containing 20 µL CD14-AlexaFluor 647 anti-human antibody (BioRad) + 1.0 µL CD21-PE anti-dog antibody (Bio-Rad), and a third without any antibody. The samples were incubated for 30 min at room temperature (RT) away from light. Next, osmotic red cell lysis was performed, the tubes were centrifuged and the supernatant was removed. The samples were washed twice with phosphate-buffered saline (PBS)-W [PBS with 0.5% bovine serum albumin (BSA) and 0.1% sodium azide, pH 7.2] and centrifuged again at 240 g at 18°C for 7 min. Then, 100 µL fixative solution (Facs Lysing Solution, BD Bioscience; San Jose, CA, USA) was added to the pellet obtained. The FACSCanthoII-BD flow cytometer and the DIVA software (BD Bioscience) were used for data acquisition, with 30,000 total events obtained for tubes containing CD3+, CD4+, CD8+, CD21+ and CD14+, and 5,000 events at the CD14 gate. FLOWJO software (v7.6.1, Flow Cytometry Analysis Software; Ashland, OR, USA) was used for data analysis.

治疗前及研究期间每2个月颈静脉穿刺收集外周血放置含肝素钠管中进行PBMC流式细胞分析。根据Martins等人的描述，对白细胞进行评估。最初，收集50 µL的外周全血，置于一个含有肝素钠的试管中，并转移到三个试管中，一个试管中含有2.5µL CD3-FITC/ CD4+-PE/ CD8+-AlexaFluor 647抗犬抗体，另一种含有20µL CD14-AlexaFluor 647抗人抗体 + 1.0µL CD21-PE抗犬抗体，第三种不含任何抗体。样品在室温下避光孵育30分钟。接下来，进行渗透性红细胞裂解，离心管，取上清。用磷酸盐缓冲盐水(PBS)-W (PBS加0.5%牛血清白蛋白(BSA)和0.1%叠氮化钠，pH 7.2)洗涤2次，18℃，240 g离心7 min。添加到获得的颗粒中。使用FACSCanthoII-BD流式细胞仪和DIVA软件进行数据采集，CD3+、CD4+、CD8+、CD21+和CD14+的试管共获得30000个项目，CD14门处共获得5000个项目。FLOWJO软件进行数据分析。

Antigen stimulation assays were performed as described by Martins et al. Initially, total peripheral blood was collected in a tube containing heparin sodium from five animals with cAD and seven control animals. Next, RPMI supplemented with 10% inactivated fetal bovine serum, L-glutamine 2 mM, and penicillin G 100 U/mL (pH 7.2) were added to the blood. The cultures were divided into three groups: (1) no stimuli; (2) stimulated with phorbol myristate acetate (PMA, 25 ng/mL) and ionomycin (1 lg/mL); and (3) stimulated with Dermatophagoides farinae antigens (25 ng/mL). Brefeldin (20 µL) was added to all samples and incubated for 4 h at 37°C and under 5% CO2, followed by the addition of 200 lL ethylenediaminetetraacetic acid (EDTA, Sigma; St Louis, MO, USA), which was maintained for 10 min at RT. Then, 3 mL PBS-W was added, and centrifugation was performed at 240 g for 10 min at 18°C. The supernatant was removed, the pellet was re-suspended in PBS-W, and 100 µL cell suspension was transferred to tubes containing specific antibodies for the cell populations of interest: (1) CD4-PE anti-dog antibody, (2) CD4-FITC anti-dog antibody, (3) CD8+-PE anti-dog antibody, (4) CD8+-FITC anti-dog antibody and (5) unlabelled. The samples were incubated for 30 min at RT and protected from light. Next, osmotic erythrocyte lysis was performed with 3 mL FACS Lysis Solution for 10 min. The cells were centrifuged (1,300 rpm, 18°C, for 7 min), the supernatant was removed, resuspended in PBS-W and centrifuged. The supernatant was removed, and the pellet was resuspended in 500 µL PBS-W and 3 mL PBS-P (PBS-W and 0.5% saponin), followed by centrifugation and supernatant removal. Next, 20 µL anti-cytocin antibody [IL-4-PE and interferon (IFN)-c-FITC] were added, incubated for 30 min, washed with PBS-W, and centrifuged at 1,300 rpm at 18°C for 7 min. Next, 100 µL fixative solution was added to the pellet. For data acquisition, 30,000 total events were obtained using the FACSCanthoIIBD flow cytometer and DIVA software. FLOWJO software v7.6.1 (Flow Cytometry Analysis Software) was used for data analysis, and the percentage of IL-4 and IFN-c produced by CD4+ and CD8+ lymphocytes was determined as described by Martins et al.

根据Martins等人的描述，进行抗原刺激试验。最初，从5只CAD动物和7只对照组动物采集总外周血放入肝素钠试管中。然后，在血液中加入添加10%灭活胎牛血清的RPMI, l -谷氨酰胺2 mM，青霉素G 100 U/mL (pH 7.2)。将培养物分为三组:(1)无刺激;(2)用肉豆蔻酸醋酸佛蓬(PMA, 25 ng/mL)和离子霉素(1 lg/mL)刺激;(3)粉尘螨抗原(25 ng/mL)刺激。在所有样品中加入20 L Brefeldin，在37℃和5% CO2下孵育4 h，然后加入200 lL乙二胺四乙酸(EDTA),保持10分钟rt,然后,3毫升PBS-W补充道,并完成了240 g离心10分钟在上层清液被18 c,颗粒在PBS-W 中重新悬浮,和100 L细胞悬液被转移到管包含特定抗体的细胞群:(1) CD4-PE抗犬抗体，(2)CD4-FITC抗犬抗体，(3)CD8+-PE抗犬抗体，(4)CD8+-FITC抗犬抗体，(5)未标记的。样品在室温下孵育30分钟，避光。然后，用3 mL FACS裂解液进行渗透红细胞裂解10分钟。细胞离心(1300 rpm, 18℃，7分钟)，取上清液，重悬于PBS-W中，离心。取上清液，将颗粒重悬于500 L PBS-W和3 mL PBS-P (PBS-W和0.5%皂苷)中，离心取上清液。加入抗细胞素抗体[IL-4-PE and干扰素(IFN)-c-FITC] 20 L，孵育30 min, PBS-W洗涤，18℃1300 rpm离心7 min。加入100 L固定液。在数据采集方面，使用FACSCanthoIIBD流式细胞仪和DIVA软件获得了30000个项目。采用FLOWJO软件v7.6.1 (Flow Cytometry Analysis software)进行数据分析，测定CD4+和CD8+淋巴细胞产生IL-4和IFN-c的百分比，如Martins等所述。

Statistical analysis

统计学分析

The variables were evaluated over time using the generalised equation estimating (GEE) method16 to analyse the correlation between repeated measures.17 Log-linear marginal regressions with constant variance and the mean proportional variance were adjusted for each variable, with time as the explanatory variable. R software (v3.3.2), was used for analyses, and values were considered statistically significant at P ≤ 0.05.

随着时间的推移，使用广义方程估计(GEE)方法对变量进行评估，以分析重复测量之间的相关性对每个变量调整具有恒定方差和平均比例方差的对数线性边际回归，以时间作为解释变量。R软件(v3.3.2)进行分析，在p≤0.05时认为值有统计学意义。

Results

结果

Characterisation of animals

动物特征

The cAD group consisted of 13 dogs with atopic dermatitis (seven males, six females) with ages ranging between 2 and 10 years (median age 4.6 years). Of these, five were shih tzus (38.4%) and two (15.38%) were of mixed breed heritage. One dog represented each of the following breeds: Maltese, English bulldog, French bulldog, Pekingese, schnauzer and poodle. Five of the 13 atopic dogs were selected randomly for additional testing. The demographis and clinical aspects of participants are summarised in Table 1.

cAD组由13只特应性皮炎患犬组成(7只雄性，6只雌性)，年龄在2到10岁之间(中位年龄4.6岁)。其中西施犬5只(38.4%)，杂交犬2只(15.38%)。每只犬犬种分别为:马尔济斯犬、英国斗牛犬、法国斗牛犬、北京犬、雪纳瑞和贵宾犬。13只特应性皮炎患犬中随机选取5只进行补充测试。表1总结了研究动物的人口统计和临床方面的情况。

Efficacy of oclacitinib maleate

马来酸奥拉替尼的有效性

CADESI-04 and pVAS data for each dog (assessed every two months) are described in Tables 2 and 3. The treatment had a significant effect (P < 0.05) on the pVAS variable, with 51% reduction during the period (Table 4). Oclacitinib significantly affected the CADESI-04 variable (P < 0.0001), with 86.7% reduction during the period (Table 4).

表2和表3描述了每只犬的CADESI-04和pVAS数据(每两个月评估一次)。治疗对pVAS变量有显著影响(P < 0.05)，期间降低51%(表4)。奥拉替尼对CADESI-04变量有显著影响(P < 0.0001)，期间降低86.7%(表4)。

Immunophenotyping

免疫表型

The mean CD4+ percentage level increased by 1.4% each month, presenting a significant treatment effect over time (P = 0.002). Moreover, a higher frequency (P < 0.047) of CD4+ T cells was observed after 12 months of treatment when compared with that before treatment (Figure 1). Additionally, a significant treatment effect was observed in terms of the mean CD14+ monocyte levels (P = 0.001), which were increased by 3% each month. The percentage of CD8+ T cells presented a tendency to increase, but demonstrated no statistical difference over time. The CD4+:CD8+ ratio remained reasonably constant. There was no change detected in CD21+ levels (Figure 1).

CD4+百分比平均每月上升1.4%，治疗效果显著(P = 0.002)。此外,更高的频率(P < 0.047)的CD4 + T细胞观察12个月的治疗后与治疗前相比(图1)。此外,一个重要的疗效观察是CD14 +单个核细胞水平平均值(P = 0.001),每个月增加了3%。CD8+ T细胞百分比呈上升趋势，但随时间变化无统计学差异。CD4+:CD8+比值保持相当稳定。

Antigen stimulation

抗原刺激

During the 12-month oclacitinib treatment period, no significant treatment effect was observed in terms of IL-4 production by CD4+ cells in antigen-stimulated T-cell cultures (Figure 2). A general pattern of reduced (P < 0.05) production of IL-4 by CD8+ T cells after antigenic stimulation (at D240 and D300 compared to D360) and reduced (P < 0.05) production of IFN-c by both CD4+ T cells (at D120, D240 and D300 compared to D360, and at D240 and D300 compared to D0) and CD8+ T cells (at D240 and D300 compared to D0) was noted after antigenic stimulation of cultures (Figure 2). In fact, at D0 the median frequency of intracytoplasmic T-cell cytokines in control cultures (CD8+ IL-4+ : 2.07; CD8+ IFN-c+ : 4.89; CD4+ IL-4+ : 1.57; CD4+ IFN-c+ : 10.10) and after antigenic stimulation (CD8+ IL-4+ : 1.78; CD8+ IFN-c+ : 4.86; CD4+ IL-4+ : 2.66; CD4+ IFN-c+ : 13.40) were similar (P > 0.05) to their respective culture conditions at D360 (control cultures – CD8+ IL-4+ : 2.05; CD8+ IFN-c+ : 4.31; CD4+ IL-4+ : 2.81; CD4+ IFN-c+ : 10.40; stimulated cultures – CD8+ IL-4+ : 4.06; CD8+ IFN-c+ : 4.75; CD4+ IL-4+ : 3.33; CD4+ IFN-c+ : 11.70)].

在12个月的奥拉替尼治疗期间，在抗原刺激的T细胞培养中CD4+细胞产生IL-4方面没有观察到显著的治疗效果(图2)。在抗原刺激后的培养中，CD8+ T细胞产生IL-4减少(D240和D300与D360相比)(P＜0.05)，CD4 + T细胞产生的IFN-c减少(D120, D240和D300 与D360相比,以及D240 和D300与D0相比)(P＜0.05)和CD8 + T细胞产生的IFN-c减少(D240和D300 与D0相比)（P＜0.05）(图2)。事实上,在D0时，对照组胞浆内T细胞细胞因子的平均频率(CD8+IL-4 +:2.07;CD8+IFN-c+:4.89;CD4+IL-4+:1.57;CD4+IFN-c+:10.10)和抗原刺激后CD8+IL-4+: 1.78;CD8+IFN-c+: 4.86;CD4+IL-4+:2.66;CD4+IFN-c+:13.40)在各培养条件下在D360时相似（P＞0.05）(对照组CD8+IL-4+: 2.05;CD8+IFN-c+: 4.31;CD4+IL-4+:2.81;CD4+IFN-c+:10.40;CD8+IL-4+:4.06;CD8+IFN-c+:4.75;CD4+IL-4+:3.33;CD4+IFN-c+:11.70)]。

Detailed results of all laboratory tests performed for each dog are available in Supporting information.

对每只犬进行的所有实验室测试的详细结果可在补充信息中获得。

Discussion

讨论

This study evaluated pruritus levels and the immunomodulatory effect of oclacitinib maleate treatment in dogs with AD. The treatment significantly reduced pVAS and CADESI-04 scores. In addition, flow cytometric analysis demonstrated that cytokine profiles at D360 after treatment initiation had returned to levels similar to those noted before treatment, but this effect was not associated with clinical relapse.

本研究评估了马来酸奥拉替尼治疗AD犬的瘙痒程度和免疫调节作用。治疗显著降低pVAS和CADESI-04评分。此外，流式细胞仪分析显示，治疗开始后D360细胞因子谱已恢复到与治疗前相似的水平，但这一效应与临床复发无关。

Of the 13 dogs enrolled in this study, only two were of mixed breed origin. Breed predispositions are known in cAD, including for those breeds represented in our study. The shih tzu is a common breed in Brazil in which the prevalence of cAD is very high, so it is perhaps not surprising that they represented a large proportion of the study population (five of 13).

在参与这项研究的13只犬中，只有两只是杂交品种。在cAD中，已知有品种倾向性，包括我们研究中的那些品种。在巴西，西施犬是一种常见的品种，而巴西的cAD患病率非常高，因此，它们在研究动物中占很大比例(13只中有5只)也就不足为奇了。

Oclacitinib consistently reduced pruritus in line with previous reports. After D180, we observed a 40.7% reduction in the pVAS; after D360 days, a 51% decrease demonstrated efficacy for controlling cAD-associated pruritus for long periods of time. In terms of skin lesions evaluated using CADESI-04, the results also corroborated previous reports. After D180, a 78.5% reduction in the average CADESI-04 score was observed; after D360 days, an 86.7% decrease was noted in the average score. This indicated the skin’s gradual recovery after the pruritic stimulus had ceased. Therefore, it is necessary to allow sufficient time for the skin to recover without stimuli, such as self-trauma, which contribute to the appearance of these lesions.

与以前的报告一致，奥拉替尼持续减轻瘙痒。D180后，我们观察到pVAS下降40.7%;D360天后，长时间控制CAD相关瘙痒的有效性下降51%。在CADESI-04评估的皮肤病变方面，结果也证实了之前的报道。D180后，CADESI-04平均评分下降78.5%;第360天后，平均评分下降86.7%。这表明瘙痒刺激停止后，皮肤逐渐恢复。因此，有必要让皮肤有足够的时间在没有刺激的情况下恢复，如导致这些病变出现的自我损伤。

According to the manufacturer, oclacitinib can modulate the immune system and could increase susceptibility to infection, as well as exacerbate neoplastic conditions.Recent studies have revealed no significant difference in the number of tumours diagnosed in animals with AD treated with or without oclacitinib. This was a primary motivation for undertaking this study, as oclacitinib is commonly used for long-term control of the clinical signs of cAD.

根据制造商的说法，奥拉替尼可以调节免疫系统，并可能增加感染的易感性，以及加剧肿瘤疾病。最近的研究表明，在接受或不接受奥拉替尼治疗的AD动物中，诊断出的肿瘤数量没有显著差异。这是开展这项研究的主要动机，因为奥拉替尼通常用于cAD临床症状的长期控制。

Studies of normal lymphocyte subpopulations in healthy dogs demonstrate that ranges may vary according to several factors, including breed, age and use of different protocols for immunophenotyping, including source and clones of monoclonal antibodies. However, data regarding the immunophenotypic profile of circulating leucocytes, particularly distinguishing lymphocyte subpopulations in dogs treated with oclacitinib, remained unavailable. The analysis of lymphocyte subpopulations is crucial for evaluating immunocompetence, the influence of drugs on the immune system, and the progression of infectious diseases. One weakness of this study is the limited sample size and breed distribution.

对健康犬的正常淋巴细胞亚群的研究表明，淋巴细胞亚群的范围可能因几个因素而异，包括品种、年龄和使用不同的免疫表型协议，包括来源和克隆单克隆抗体。然而，关于循环白细胞免疫表型的数据，特别是在使用奥拉替尼治疗的犬中区分淋巴细胞亚群，仍然没有数据。淋巴细胞亚群的分析对于评估免疫能力、药物对免疫系统的影响以及感染性疾病的进展至关重要。本研究的一个缺点是样本量和品种分布有限。

As oclacitinib specifically inhibits an enzyme that interacts with cytokine-specific receptors, unlike less selective drugs such as glucocorticoids, a decrease in lymphocyte subpopulation values was not expected over time. Anecdotal evidence has indicated a higher prevalence of demodicosis in animals treated with oclacitinib for a prolonged period. These findings can be attributed to reduced CD4+ cell counts, as it has been reported that animals with demodectic mange present decreased CD4+ values. However, our study population revealed increased CD4+ T cells, with no changes recorded in the CD4+:CD8+ ratio, which remained reasonably constant.Likewise, increasing CD4+ T-cell values in the skin may be explained by the course of the allergic disease itself, triggered by allergen-challenged skin.The expression of the chemokine receptor CCR4 in circulating and skin T cells has been associated with skin-homing T cells in cAD. In fact, cAD was associated with increased CD4+ T cells in comparison to healthy dogs.Likewise, oclacitinib did not demonstrate any in vitro proliferative effect in treated canine T cells. Furthermore, another in vitro evaluation has reported that oclacitinib inhibits JAK1- dependent cytokines, an important mechanism to induce IL-2 production and a critical cytokine that triggers the lymphoproliferative effect.

由于奥拉替尼特异性抑制与细胞因子特异性受体相互作用的酶，不像选择性较低的药物，如糖皮质激素，淋巴细胞亚群值不会随着时间的推移而下降。坊间证据表明，在长期接受奥拉替尼治疗的动物中，蜱虫病的流行率较高。这些发现可以归因于CD4+细胞计数的减少，因为据报道，患有蠕形螨病的动物出现CD4+值的下降。然而，我们的研究动物显示CD4+ T细胞增加，CD4+:CD8+比例没有变化，保持相当稳定。同样，皮肤中CD4+ T细胞值的增加可能是由过敏性疾病本身的病程所引起的，是由过敏性皮肤触发的。趋化因子受体CCR4在循环和皮肤T细胞中的表达与cAD中的皮肤归巢T细胞有关。事实上，与健康犬相比，cAD与CD4+ T细胞增加有关。同样，在治疗过的犬的T细胞中，奥拉替尼也没有显示出任何体外增殖效应。此外，另一项体外评估报告称，奥拉替尼抑制依赖JAK1的细胞因子，这是诱导IL-2产生的重要机制，也是触发淋巴增殖效应的关键细胞因子。

The cytokine profile is considered to be an important biomarker to determine the pro-inflammatory and/or immunosuppressive effect on the canine immune system, and could contribute to the analysis of the immune response pattern in cAD. In blood circulation, free cytokine concentrations typically are low and the reported values vary considerably. Furthermore, enzymelinked immunosorbent assay (ELISA), the immunoenzymatic test used in this analysis, does not identify the cell source producing cytokines. In this study, flow cytometry was used to evaluate cellular cytokine production rapidly and simultaneously. The present work is the first description of IL-4 and IFN-γ production as intracytoplasmic cytokines from T cells during oclacitinib treatment of cAD.

细胞因子谱被认为是确定犬免疫系统促炎和/或免疫抑制作用的重要生物标志物，并可能有助于分析cAD的免疫反应模式。在血液循环中，游离细胞因子的浓度通常很低，报告的值相差很大。此外，本分析中使用的酶联免疫吸附试验(ELISA)不能识别产生细胞因子的细胞源。在本研究中，流式细胞仪被用来快速和同时评估细胞因子的产生。本研究首次描述了在奥拉替尼治疗CAD期间，T细胞胞浆内产生细胞因子是IL-4和IFN-γ。

To date, no data regarding the influence of oclacitinib on IL-4 and IFN-γ production by CD4+ and LT CD8+ T lymphocyte subtypes have been available in the literature. A previous study investigated the impact of oclacitinib on T cells cultivated using a peripheral blood mononuclear cell culture from dogs, and the recommended therapeutic dose of oclacitinib did not affect ConA-stimulated T-cell proliferation or cytokine production (IL-2, IL-10, IL-15, IL18, IFN-γ and TNF-α). In the present study, a reduction in the frequency of CD8+IL-4+ and CD8+IFN-γ+, in addition to CD4+IFN-γ + T cells, was observed at D240 and D300 after oclacitinib treatment, with the frequency restored at D360 after treatment. Interestingly, an in vitro analysis using canine T cells treated with oclacitinib at 1 μM (corresponding to the conventional treatment for dogs) did not result in immunomodulatory (IL-10) or inflammatory (TNF-α, IFN-γ) cytokine production. By contrast, our data showed a prominent inflammatory cytokine reduction (IFN-γ) by CD4+ and CD8+ T cells, in addition to a reduction in IL-4 levels (by CD4+ and CD8+ T cells) until D300 after treatment. The cytokine profile observed at D360 after treatment, which was similar to that documented before treatment, was not associated with clinical relapse. Our data could indicate an adaptation by the canine immune system to long-term exposure to the oclacitinib, with restoration of cytokine production to levels observed before treatment initiation.

迄今为止，尚无关于奥拉替尼对CD4+和LT CD8+ T淋巴细胞亚型产生IL-4和IFN-γ的影响的文献资料。之前的一项研究调查了奥拉替尼对使用犬外周血单个核细胞培养的T细胞的影响，推荐的治疗剂量并不影响ConA刺激的T细胞增殖或细胞因子的产生(IL-2、IL-10、IL-15、IL18、IFN-γ和TNF-α)。在本研究中，在奥拉替尼治疗后的D240和D300，除CD4+IFN-γ+ T细胞外，CD8+IL-4+和CD8+IFN-γ+ T细胞的频率均有所降低，治疗后的D360频率恢复。有趣的是，一项体外分析使用1 μM的奥拉替尼治疗的犬的T细胞(与犬的常规治疗相对应)，并没有导致免疫调节细胞因子(IL-10)或炎症细胞因子(TNF-α, IFN-γ)的产生。相比之下，我们的数据显示，直到D300治疗后，CD4+和CD8+ T细胞显著降低炎症细胞因子(IFN-γ)， IL-4水平降低(CD4+和CD8+ T细胞)。治疗后D360观察到的细胞因子谱与治疗前相似，与临床复发无关。我们的数据可能表明犬的免疫系统对长期暴露于奥拉替尼的适应性，细胞因子的产生恢复到治疗开始前观察到的水平。

Conclusions

结论

In the current study, the prolonged use of oclacitinib maleate for up to 12 months at recommended doses during treatment of cAD did not negatively influence lymphocyte subpopulations. Therefore, under the conditions studied in this group of dogs, oclacitinib maleate can be considered a safe option for control of clinical signs associated with cAD.

在目前的研究中，在CAD治疗期间，以推荐剂量使用马来酸奥拉替尼长达12个月，并未对淋巴细胞亚群产生负面影响。因此，在本组犬的研究条件下，马来酸奥拉替尼可以被认为是控制cAD相关临床症状的安全选择。