The caninised monoclonal antibody lokivetmab (LKV), directed at interleukin (IL)-31, is very effective at controlling pruritus in most dogs with atopic dermatitis (AD). However, evidence exists that IL-31 is not required for the induction of acute allergic skin inflammation, which might explain why this treatment is less efficacious in some dogs with AD. To compare the comprehensive transcriptome analysis of house dust mite (HDM)-sensitised dogs with and without treatment with LKV to attest our hypothesis that LKV does not majorly affect acute cytokine/chemokine production. Six HDM-sensitised atopic Maltese-beagle dogs. In this cross-over study, the cytokine profiling of acute AD skin lesions was compared by RNA sequencing (RNA-Seq), with or without LKV-induced inhibition of IL-31. Skin biopsies were obtained from each dog at 0, 6, 12, 24, 48, and 96 h after epicutaneous HDM allergen provocation. Macroscopic and microscopic skin lesion scores were not significantly different between the LKV- and nontreatment groups at any time points. Likewise, the results of RNA-Seq analysis revealed no significant difference in the messenger (m)RNA expression of the major cytokines between these two groups. In LKV-treated dogs, IL6, IL9, IL13, IL33, CCL17, and CCL22 were significantly upregulated compared to their baseline expression levels, suggesting that these cytokines are unaffected by IL-31 inhibition. IL-31 inhibition is insufficient to prevent the expression of other proinflammatory mediators in acute AD and these could be considered as other potential therapeutic targets. L'anticorps monoclonal caninisé lokivetmab (LKV), dirigé contre l'interleukine (IL)-31, est très efficace pour contrôler le prurit chez la plupart des chiens atteints de dermatite atopique (DA). Cependant, il semble que l'IL-31 n'est pas nécessairement impliqué dans l'induction d'une inflammation cutanée allergique aiguë, ce qui pourrait expliquer pourquoi ce traitement est moins efficace chez certains chiens atteints de DA. Comparer l'analyse complète du transcriptome de chiens sensibilisés aux acariens de la poussière domestique (HDM) avec et sans traitement au LKV pour vérifier notre hypothèse selon laquelle le LKV n'affecte pas de manière majeure la production aiguë de cytokines/chimiokines. Six chiens beagles atopiques sensibilisés aux HDM. Dans cette étude croisée, le profil des cytokines des lésions cutanées aiguës de la DA est comparé par séquençage d'ARN (ARN-Seq), avec ou sans inhibition de l'IL-31 induite par le LKV. Des biopsies cutanées sont réalisées pour chaque chien à 0, 6, 12, 24, 48 et 96 h après la provocation épicutanée avec l'allergène HDM. Les scores lésionnels cutanés macroscopiques et microscopiques ne sont pas significativement différents entre le groupe LKV et le groupe témoin à aucun moment. De même, les résultats de l'analyse RNA-Seq ne révèle aucune différence significative dans l'expression de l'ARNm des principales cytokines entre ces deux groupes. Chez les chiens traités au LKV, IL6, IL9, IL13, IL33, CCL17 et CCL22 sont significativement surexprimées par comparaison avec leur niveau basal d'expression, ce qui suggère que ces cytokines ne sont pas affectées par l'inhibition de l'IL-31. L'inhibition de l'IL-31 est insuffisante pour empêcher l'expression d'autres médiateurs pro-inflammatoires dans la phase aiguë de la DA et ceux-ci pourraient être considérés comme d'autres cibles thérapeutiques potentielles. el anticuerpo monoclonal caninizado lokivetmab (LKV), dirigido contra la interleucina (IL)-31, es muy eficaz para controlar el prurito en la mayoría de los perros con dermatitis atópica (AD). Sin embargo, existe evidencia de que la IL-31 no es necesaria para la inducción de inflamación cutánea alérgica aguda, lo que podría explicar por qué este tratamiento es menos eficaz en algunos perros con AD. Comparar el análisis completo del transcriptoma de perros sensibilizados con ácaros del polvo doméstico (HDM) con y sin tratamiento con LKV para confirmar nuestra hipótesis de que LKV no afecta en gran medida a la producción aguda de citoquinas/quimioquinas. Seis perros mestizos beagle maltés atópicos sensibilizados con HDM. en este estudio cruzado, se comparó el perfil de citoquinas de las lesiones cutáneas agudas de AD mediante secuenciación de RNA (RNA-Seq), con o sin inhibición de IL-31 inducida por LKV. Se obtuvieron biopsias de piel de cada perro a las 0, 6, 12, 24, 48 y 96 h después de la provocación epicutánea con el alérgeno HDM. Las puntuaciones de lesiones cutáneas macroscópicas y microscópicas no fueron significativamente diferentes entre los grupos con LKV y sin tratamiento en ningún momento. Del mismo modo, los resultados del análisis de RNA-Seq no revelaron diferencias significativas en la expresión del RNA mensajero (m) de las citoquinas principales entre estos dos grupos. En perros tratados con LKV, IL6, IL9, IL13, IL33, CCL17 y CCL22 aumentaron significativamente en comparación con sus niveles de expresión basales, lo que sugiere que estas citoquinas no se ven afectadas por la inhibición de IL-31. La inhibición de la IL-31 es insuficiente para prevenir la expresión de otros mediadores proinflamatorios en la AD aguda y éstos podrían considerarse como otras posibles dianas terapéuticas. Der caninisierte monoklonale Antikörper Lokivetmab (LKV), welcher auf Interleukin (IL)-31 ausgerichtet ist, ist bei der Juckreizkontrolle der meisten Hunde mit atopischer Dermatitis (AD) sehr wirkungsvoll. Es besteht jedoch Evidenz dafür, dass IL-31 zur Auslösung einer akuten allergischen Hautentzündung nicht nötig ist, was erklären könnte, warum diese Behandlung bei manchen Hunden mit AD weniger wirksam ist. Ein Vergleich der umfassenden Transkriptom Analyse von Hausstaubmilben (HDM)-sensibilisierten Hunden mit und ohne Behandlung mit LKV, um unsere Hypothese, dass LKV die akute Zytokin/Chemokin Produktion nicht wesentlich beeinflusst, zu bestätigen. Sechs HDM-sensibilisierte atopische Malteser/Beagle Mischungen wurden eingesetzt. In dieser Cross-Over Studie wurde das Zytokin Profil von akuten AD-Hautreaktionen mittels RNA-Sequenzierung (RNA-Seq) mit und ohne LKV-induzierter Inhibition von IL-31 verglichen. Es wurden Hautbiopsien von jedem Hund zum Zeitpunkt 0, 6, 12, 24, 48 und 96h nach der Provokation mit epikutanem HDM Allergen entnommen. Die makroskopische und mikroskopische Bewertung der Hautveränderungen war zwischen den LKV- und den nicht behandelten Gruppen zu den verschiedenen Zeitpunkten nicht signifikant unterschiedlich. Ebenso zeigten die Ergebnisse der RNA-Seq Analyse keine signifikanten Unterschiede bei der Messenger (m) RNA Exprimierung der wichtigsten Zytokine zwischen den beiden Gruppen. Bei den LKV-behandelten Hunden waren IL6, IL9, IL13, IL33, CCL17 und CCL22 im Vergleich zu den Exprimierungswerten am Anfang (Baseline) signifikant hochreguliert, was darauf hinweist, dass diese Zytokine durch eine IL-31 Inhibition unbeeinflusst sind. Die IL-31 Inhibition reicht nicht aus, um die Exprimierung von proentzündlichen Mediatoren bei akuter AD zu verhindern. Diese könnten als mögliche therapeutische Ziele betrachtet werden. インターロイキン(IL)-31を標的とするイヌ化モノクローナル抗体であるロキベトマブ(LKV)は、ほとんどのアトピー性皮膚炎(AD)の犬の掻痒をコントロールするのに効果的である。しかしながら、IL-31は急性アレルギー性皮膚炎の誘発には必要ないとの証拠が存在し、このことがロキベトマブが一部のADの犬で効果が低い理由を説明しているかもしれない。 本研究の目的は、ハウスダストマイト(HDM)感作犬のLKV投与有無によるトランスクリプトーム解析を比較し、LKVが急性サイトカイン/ケモカイン産生に大きな影響を与えないという我々の仮説を証明することであった。 HDM感作アトピー犬マルチーズビーグル犬6頭。 このクロスオーバー研究では、AD急性皮膚病変のサイトカインプロファイリングを、LKVによるIL-31阻害の有無にかかわらず、RNAシーケンス(RNA-Seq)により比較した。皮下HDMアレルゲン誘発後0、6、12、24、48、96時間に各犬から皮膚生検を実施した。 肉眼的および顕微鏡的な皮膚病変のスコアは、どの時点でもLKV-治療群および非治療群間に有意な差はなかった。同様に、RNA-Seq解析の結果、主要サイトカインのメッセンジャー(m)RNA発現量にこれら2群間で有意差は認められなかった。LKV治療犬では、IL6、IL9、IL13、IL33、CCL17、CCL22がベースラインの発現量と比較して有意に上昇し、これらのサイトカインがIL-31阻害の影響を受けないことが示唆された。 IL-31阻害は、急性ADにおける他の炎症性メディエーターの発現を防ぐには不十分であり、これらは他の潜在的な治療標的として考慮される可能性がある。 针对白细胞介素(IL)-31的犬化单克隆抗体洛基韦单抗(LKV)在控制大多数特应性皮炎(AD)犬的瘙痒方面非常有效。然而，有证据表明，IL-31不是诱导急性过敏性皮肤炎症所必需的，这可能解释了为什么这种治疗对某些AD患犬效果较差。 比较使用和未使用LKV治疗的屋尘螨(HDM)致敏犬的综合转录组分析，以证实我们的假设，即LKV不会主要影响急性细胞因子/趋化因子的产生。 六只HDM致敏特应性马耳他比格犬。 在这项交叉研究中，通过RNA测序(RNA-Seq)比较急性AD皮肤损伤的细胞因子谱，有或无LKV诱导的IL-31抑制。在经皮HDM过敏原激发后0、6、12、24、48和96小时从每只犬身上获取皮肤活检样本。 在任何时间点，LKV治疗组和非治疗组的宏观和微观皮肤病变评分没有显著差异。同样，RNA-Seq分析结果显示这两组之间主要细胞因子的信使(m)RNA表达没有显著差异。在LKV治疗的犬中，与基线表达水平相比，IL6、IL9、IL13、IL33、CCL17和CCL22显著上调，表明这些细胞因子不受IL-31抑制的影响。 IL-31抑制不足以阻止急性AD中其他促炎介质的表达，这些可能被视为其他潜在的治疗靶点。 O anticorpo monoclonal caninizado lokivetmab (LKV), direcionado à interleucina (IL)-31, é muito eficaz no controle do prurido na maioria dos cães com dermatite atópica (DA). Entretanto, existem evidências de que a IL-31 não é necessária para a indução de inflamação cutânea alérgica aguda, o que pode explicar o fato de este tratamento ser menos eficaz em alguns cães com DA. Comparar a ampla a análise transcriptômica de cães sensibilizados a ácaros da poeira doméstica (HDM) com e sem tratamento com LKV para comprovar a nossa hipótese de que LKV não afeta consideravelmente a produção de citoconas/quimiocinas de fase aguda. Seis cães atópicos mestiços maltês-beagle sensibilizados a HDM. Neste estudo cruzado, o perfil de produção de citocinas das lesões agudas de DA foi comparado por sequenciamento de RNA (RNA-Seq) com ou sem inibição de IL-31 induzida por LKV. Biópsias cutâneas foram coletadas de cão em 0, 6, 12, 24, 48 e 96 horas após provocação epicutânea com HDM. Escores de lesões cutâneas macroscópicas e microscópicas não foram significativamente diferentes entre os grupos LKV e sem tratamento em todos os momentos experimentais. De maneira similar, os resultados da análise de RNA-Seq não revelaram diferença significativa na expressão de (m)RNA mensageiro das principais citocinas entre esses dois grupos. Em cães tratados com LKV, IL6, IL9, IL13, IL33, CCL17 e CCL22 foram significativamente aumentados em comparação com seus níveis de expressão basais, sugerindo que essas citocinas não são afetadas pela inibição de IL-31. A inibição da IL-31 é insuficiente para prevenir a expressão de outros mediadores pró-inflamatórios na DA aguda e estes podem ser considerados como outros potenciais alvos terapêuticos. Atopic dermatitis (AD) is a common, relapsing allergic skin disease of humans and dogs associated with mild-to-severe pruritus and characteristic inflammatory skin lesions.1, 2 Interleukin (IL)-31 is now known to represent one of the key cytokines of human and canine AD.3-5 In dogs with AD, a significant reduction in pruritus has been seen following injections of lokivetmab (LKV, Cytopoint; Zoetis), a therapeutic monoclonal antibody (mAb) targeting canine IL-31, thus confirming that this cytokine plays a pivotal role in the genesis of pruritus associated with this disease.6-12 Despite documented therapeutic success in clinical trials, increasing evidence suggests that IL-31 is not required for the induction of acute allergic skin inflammation.13-16 Additionally, it is recognised that LKV is not always clinically effective in practice, especially if chronic skin lesions are present. In our acute model of canine AD, we showed that pretreatment with LKV did not prevent the development of skin lesions after epicutaneous exposure to house dust mite (HDM) allergens despite almost completely inhibiting pruritus.13 Furthermore, using the same model, we failed to detect a correlation between the severity of macroscopic inflammatory skin lesions and the amount of infiltrating IL-31-positive cells, further supporting the minimal role of locally released IL-31 in the development of acute flares of allergic skin inflammation.17 Finally, in our prospective clinical study enrolling client-owned dogs with AD, we observed that half of the dogs treated proactively with LKV monotherapy eventually needed additional anti-allergic medications after two months, indicating other inflammatory mediators, besides IL-31, contribute to flares of AD.13 The objectives of this study were first to compare the comprehensive transcriptome analysis of a group of dogs treated with LKV (LKV group) with an untreated group of dogs (nontreatment control group: NTC group) to attest our hypothesis that LKV does not majorly affect other cytokine/chemokine production. Secondly, in the LKV group, we could identify AD-associated cytokines/chemokines that remained upregulated in the acute phase of canine AD despite LKV therapy by comparing mRNA expression to baseline (before allergen provocation). Such a study should enable a better understanding of the pathobiological mechanism of acute canine AD skin lesions (i.e., AD flares), as well as help identify additional therapeutic target(s) which might be employed to supplement the use of LKV. All procedures were approved beforehand by our university's Institutional Animal Care and Use Committee (IACUC ID nos 17-005-O and 19-825-O). In this experiment, we used an atopic dog model, an inbred line of laboratory Maltese-beagle dogs sensitised to Dermatophagoides farinae HDM, as described previously.18 We have previously demonstrated that IL-31 [messenger (m)RNA and protein] can be identified in the skin and serum after the provocation of these dogs with HDM.17, 19 We determined that at least four dogs would need to be included for this experiment to have a >80% power to find results significant at a p-value of 0.05 (one-sided) using our previously published data.19 Therefore, we included six dogs to avoid underpowering our study. There were five males and one female ranging in age from 3 to 11 years (median age: 11 years old). The study was designed as a randomised, nontreatment-controlled, cross-over study with a 28 weeks washout period. Dogs in the LKV group received a single injection of LKV at 2 mg/kg subcutaneously, 10 days before the epicutaneous HDM allergen provocation. On Day (D)0 of each phase after taking baseline skin biopsies, AD was induced by the epicutaneous application of 20 μL of HDM slurry [25 mg of lyophilised HDM (Greer Laboratories) in 1 mL of mineral oil], as described previously, at five locations on each dog.15 A single biopsy was collected at 6, 12, 24, 48, and 96 h post-HDM provocation from one of these HDM application sites using an 8 mm dermal biopsy punch. Dogs were sedated with intravenous administration of dexmedetomidine (Dexdomitor; Zoetis) and local anaesthesia was provided with subcutaneous injection of 2% lidocaine (Covetrus). Each HDM application site was ≥10 cm from any other to avoid the effect of inflammation induced by the skin biopsy from the previous sampling time points. Blood samples were taken at each of the above time points and serum concentrations of LKV were measured. The detailed study schedule is described in Figure 1 and Appendix S1. Immediately before (0 h) and 6, 12, 24, 48 and 96 h post-HDM provocation, erythematous macules, oedema, papules/pustules, and excoriations were scored as 0 (absent), 1 (faint, mild), 2 (moderate) or 3 (strong, severe) at the site of HDM application by a guest clinician who was blinded to the study protocol. The grades for each lesional score were added to yield a macroscopic skin lesion score with an achievable maximum of 12, as described previously.20 Half of each biopsy sample was snap-frozen and cryosectioned at 5 μm thick, and the sections were stained with haematoxylin & eosin. The intensity of microscopic skin inflammation was evaluated by a scoring system that we developed for this study as follows: (a) The number of infiltrating inflammatory cells in the dermis was subjectively scored as 0 (none), 1 (low), 2 (medium) or 3 (high); and (b) the thickness of the epidermis also was scored as 0 (normal), 1 (mild), 2 (moderate) or 3 (high). The grades for each lesional score were added to yield a microscopic skin lesion score with an achievable maximum score of 6. Because the samples were prepared by the evaluator, it was not a blind evaluation. The other half of each biopsy sample was immediately placed in RNAlater (Qiagen) and frozen at −80°C. The detailed procedures for RNA extraction, RNA purity/integrity assessment and RNA sequencing (RNA-Seq) are described in Appendix S1. We performed the RNA-Seq data analyses and principal component analyses (PCA) using the CLC Genomic Workbench v21.0.3 (Qiagen) with default parameters. The detailed data processing is described in Appendix S1. We conducted the differential expression analysis between the two treatment groups (LKV versus NTC) at each sampling time point or between baseline (0 h) and each sampling time point in the LKV group for the later analysis, using the Differential Expression for RNA-Seq tool of the CLC Genomic Workbench (Qiagen). For establishing significance, we selected an absolute fold-change of >2, a false discovery rate of ≦0.05, and a maximum group mean of reads per kb of exon per million mapped reads of >1. Seven genes (IL6, IL9, IL13, IL31, IL33, CCL17, and CCL22) were selected based on the results of differential expression analysis to detect mRNA expression by either SYBR Green (for IL6, IL31, and CCL22) or TaqMan (for IL9, IL13, IL33, and CCL17) qRT-PCR assay. After normalisation with the housekeeping gene (RPL8), the relative fold-change at each time point compared to the baseline (0 h) expression was determined by the 2−ΔΔCt method. The detailed procedures for qRT-PCR and primer sequences are described in Appendix S1 and Appendix S2, respectively. A Wilcoxon signed-rank test was used to compare macroscopic and microscopic skin lesion scores between LKV and NTC groups at each time point. A Friedman test with a Conover post hoc test was used to compare the macroscopic and microscopic skin lesion scores over time. A p-value ≦ 0.05 was considered significant. All analyses were performed using Excel 16.41 (Microsoft Corporation). There was no significant difference in macroscopic skin lesion scores between the LKV and NTC groups at any time point (p = 0.83, 0.50, 0.83, 0.47, 0.69 and 0.50 at 0, 6, 12, 24, 48 and 96 h, respectively; Figure 2; Appendix S3). Furthermore, significant differences in values were not seen over time within each group (LKV, p = 0.13; NTC, p = 0.49). In concordance with the visible skin lesions, the microscopic skin lesion scores were not significantly different between the LKV and NTC groups at each time point (p = 0.68, 0.27, 0.30, 0.27, 0.58, and 0.27 at 0, 6, 12, 24, 48, and 96 h, respectively; Figure 3; Appendix S3). The median microscopic skin lesion scores increased over time, and significant increases in the scores compared to those at baseline (0 h) were seen after 12 and 48 h in LKV (p = 0.027, 0.0012, 0.0044, and <0.001 at 12, 24, 48, and 96 h, respectively) and NTC (p = 0.002 and <0.001 at 48 and 96 h, respectively; Figure 3), respectively. High LKV titres were measured in the LKV-treated group during the entire study period (Figure S5). We were able to annotate 16,491 genes in each sample. Using PCA, we detected two outlier samples from the same dog (Dog 4) in the LKV group at 0 and 24 h (Figure S1). Because this study was a cross-over study with continuous multiple samplings, we removed all of the sample data for Dog 4 from subsequent analyses, rather than removing only the two sampling time points from this dog. Using our selected criteria for differentially expressed genes DEGs), we identified 211 DEGs (196 downregulated, 15 upregulated genes) comparing the LKV group to the NTC group at each time point (Figure 4). The highest number of DEGs was seen at 48 h, which corresponded to the highest median IL31 mRNA expression at that time. Amongst these DEGs, only one gene (upregulated; IL36G) at 0 h and four genes [one upregulated (IL37), three downregulated (CXCL8, IL9 and CCL17)] at 48 h encoded cytokines, which suggested that the LKV pretreatment did not significantly impact cytokine–gene transcription. We examined fold-changes of key proinflammatory, T helper type 1 (Th1), type 2 (Th2), type 9 (Th9), type 17 (Th17) and type 22 (Th2), and regulatory T cell (Treg) AD-related cytokines and chemokines in the LKV pretreatment group compared to those in the NTC group at each time point (Table 1). As was expected from our previous analysis of DEGs, there were no consistent significant fold-changes in any cytokines and chemokines. In order to further identify cytokines which may be candidates for therapeutic inhibition to complement LKV therapy and prevent flares of canine AD, we performed a differential expression analysis to assess the chronological changes in the expression of these cytokine-encoding genes in the LKV group. This DEG analysis revealed that IL33, IL13, CCL17 and IL9 were statistically significantly upregulated after 24 h compared to the expression at baseline (0 h). IL6 and CCL22 also were significantly upregulated after 24 h, yet no longer so at 96 h (Table 2). Amongst these DEGs, the significant upregulation of IL33, IL13, CCL17 and IL6 was verified by qRT-PCR at several time points, yet IL9 and CCL22 were not confirmed (Figure S2). Although IL1B at 24 h, and IL18 and IL10 at 96 h also were upregulated compared to their baseline expression, we did not consider them valuable targets because they were upregulated at a single time point. IL4, IL17C, IL17F, and IL22 expression trended upward after 12–24 h, yet none of these were significantly upregulated compared to their baseline expression, thus decreasing their interest as targets. Some cytokines were mildly downregulated at multiple time points compared to their baselines (e.g., IFNG, CXCL10, CXCL11, TSLP); however, none of them reached significant difference. The production of IL-31 has been shown to affect the expression of genes associated with the physical and antimicrobial skin barrier in humans in vitro.21, 22 Therefore, we compared the mRNA expression pattern of enzymes and proteins associated with filaggrin metabolism, formation of the cornified envelope, tight junctions, adhesion molecules, desquamation, and antimicrobial peptides, between LKV and NTC groups. However, we did not find any apparent difference in the transcription of these genes between the groups, except for the downregulation of S100A9 and S100A12 at a single time point (48 h; Appendix S4). The effect of IL-31 inhibition on skin barrier function in this model of acute canine AD was minor. In the present study, using an experimental model of acute canine AD, we investigated the effect of LKV-induced IL-31 inhibition on the development of macroscopic, microscopic, and the lesional skin transcriptome at the induction site of acute atopic inflammation. Our aim was to identify upregulated cytokines unaffected by IL-31 inhibition which could be potential concurrent therapeutic target(s) alongside LKV to prevent acute AD flares in dogs. In agreement with our previous study results,17 we did not observe any significant difference in macroscopic and microscopic skin lesion scores between the LKV and NTC groups at any sampling time points, even though the presence of LKV in the systemic circulation was confirmed by high LKV titres in the LKV-treated group during the entire study period. Our findings further support the proposition that IL-31 is not essential for the induction of acute allergic skin inflammation, which could explain why proactive therapy with LKV is insufficient to prevent AD flares in some dogs.17 Several studies of human allergic diseases have reported the direct interaction of IL-31 with other cytokines and chemokines. IL-31 significantly induced the release of proinflammatory cytokines (IL-1β and IL-6) and AD-related chemokines (CXCL1, CXCL8, CCL2, and CCL18) in vitro from eosinophils, especially when they were co-cultured with keratinocytes.23 Human monocyte-derived dendritic cells released several proinflammatory cytokines [tumour necrotising factor (TNF)-α, IL-6, CXCL8, CCL2, CCL5, and CCL22] after IL-31 stimulation.24 IL-31 also increased the gene expression of several cytokines and chemokines (CXCL1, CCL1, CCL4, CCL17, CCL19, CCL22, CCL23, IL20 and IL24) from normal human epidermal keratinocytes in vitro.3, 25 We postulated from these studies that the inhibition of IL-31 by LKV pretreatment would downregulate the transcription of some of these cytokines and chemokines. However, in our study, amongst 211 DEGs, we identified only five cytokine-encoding genes [two upregulated (IL36G and IL37), three downregulated genes (CXCL8, IL9, and CCL17)] of note in samples taken from dogs pretreated with LKV compared to the untreated controls. Because the expression of these genes was only significantly different at a single time point, we could not confirm that it was associated with a direct effect of IL-31 inhibition. Instead, this observation could be interpreted as IL-31 acting independently, or downstream, of other cytokines during the induction of acute canine AD skin lesions. Consequently, the inhibition of IL-31 would not prevent these other cytokines from inducing acute atopic inflammation. This finding is consistent with the result of a human AD skin transcriptome study in which the expression of IL31 was not correlated with that of other cytokine genes that were dysregulated in acute lesional AD skin.26 Based on their active gene expression levels and significant upregulation compared to their expression at baseline, we identified IL-33, IL-13, CCL17, and IL-9, and potentially IL-6 and CCL22, as possible targets for inhibition which might complement the action of LKV. To support this, the upregulation of mRNA or protein expression of IL-33, IL-13, and CCL17 has been reported in the skin or sera of client-owned dogs with AD compared to healthy dogs in multiple studies.27-31 Some of these cytokines and chemokines (e.g., IL-33, CCL17, IL-6, CCL22) are produced at a very early stage of AD acute flare induction, which is likely to be even before Th2 cells produce IL-31.32 Therefore, the expression of mRNA was not likely to have been affected by IL-31 inhibition. All cytokines identified herein already have been investigated as therapeutic targets in human allergic diseases.33-37 Indeed, dupilumab (Dupixent; Regeneron) binds to and inhibits the common receptor of IL-4 and IL-13, which has shown remarkable success in the treatment of human AD. The main effect of this therapy probably results from the inhibition of cutaneous IL-13.38 In vitro studies in humans have shown that IL-31 downregulates the expression of some proteins associated with the physical skin barrier, such as filaggrin, filaggrin-processing enzymes, desmogleins, desmocollins and corneodesmosin, in a dose-dependent manner.21, 22 Furthermore, a significant reduction of transepidermal water loss at some body sites has been reported in dogs with AD after three monthly treatments with LKV compared to that at baseline.39 In this study, however, we did not detect any consistent significant difference in the transcription of these physical skin-barrier-associated genes between the LKV and NTC groups, indicating a lack of effect of IL-31 inhibition on skin barrier function in this acute model over 96 h. There were several limitations to this study. The first limitation was the potentially small sample size. Although we determined the number of dogs based on the sample size calculation estimated from our previous study, we observed greater individual variability than expected in this experiment. Therefore, we might have needed a larger sample size to detect some significant differences. The second limitation of this study was a low expression of IL31 mRNA detected by RNA-Seq and qRT-PCR in these samples even though this canine AD model has been shown to induce IL-31 both at the mRNA and protein levels in previous studies.17, 19 In this study, the IL31 mRNA expression increased 24-fold at 24 h and 14-fold at 48 h in the LKV group (Table 2), and three-fold at 24 h and eight-fold at 48 h in the NTC group (data not shown) as compared to baseline. However, the low median transcripts per million (TPM < 1) indicated that it was not actively expressed throughout the study period.40 Thus, if IL-31 was not actively produced in the skin of our dogs in this study, this may not have been an accurate assessment of the transcriptome profiling with or without IL-31 inhibition by LKV. However, we still believe that IL-31 was produced in the skin of our dogs and that the LKV pretreatment inhibited the IL-31 pathway because the highest number of DEGs in the LKV group was seen at 48 h, which was consistent with the highest median IL31 mRNA expression of all samples. The possible transient expression and fast degradation of canine IL31 mRNA, or an insufficient accuracy of the IL31 sequence in the canine reference genome might explain the low expression of IL31 mRNA determined by RNA-Seq in this study. Indeed, our IL31 mRNA expression levels measured by qRT-PCR did not show any correlation with those obtained from RNA-Seq (r = 0.026; Figure S3). The IL31 mRNA expression by qRT-PCR did not significantly differ between LKC and NTC at each time point (Figure S4). Finally, the skin samples collected in this canine AD model only represent the induction of the earliest stages of acute flares of AD. As a result, we could not assess the role of IL-31 and the complete cytokine milieu that would exist in fully developed or chronic AD skin lesions. Therefore, the targets to supplement LKV treatment, which we identified here, might no longer play a central role in the skin of those patients with chronic AD. Still, our model mirrored the intermittent acute AD relapses that patients experience during the characteristic waxing-and-waning evolution of this disease. It is interesting to note that IL-31-deficient mice exposed chronically to topical HDM showed significantly increased—rather than decreased—numbers of dermal-infiltrating interferon (IFN)-γ-, IL-4- and IL-13-producing cells compared to those of wild-type mice, thus indicating that IL-31 actually might function as a negative regulator of Th1 and Th2 inflammation in chronic AD.16 Therefore, this also could explain some AD flares while dogs continue to be treated with LKV injections. In conclusion, in experimental acute canine AD skin lesions, we demonstrated that the IL-31 pathway does not directly affect major canine AD-relevant cytokine groups. We identified IL-33, IL-13, CCL17, and IL-9, and potentially IL-6 and CCL22, as participating in acute flares of AD skin inflammation in dogs receiving IL-31-blocking treatment with LKV. Inhibiting these cytokines or their receptors, especially in a proactive manner, to help prevent canine AD flares is deserving of further study. Chie Tamamoto-Mochizuki: Conceptualisation; data curation; formal analysis; visualisation; writing—original draft; methodology; investigation; writing—review & editing; project administration. Natalie Crawford: Writing—review & editing; formal analysis; validation. Jordan M. Eder: Formal analysis; writing—review & editing; validation. Andrea J. Gonzales: Conceptualisation; writing—review & editing; supervision; funding acquisition; project administration; resources; validation. Thierry Olivry: Conceptualisation; writing—review & editing; supervision; funding acquisition; project administration; validation. The authors would like to thank the NCSU Laboratory Animal Resource personnel for managing the colony of atopic dogs, and Beth Lubeck and Tyler Jordan for assisting with sample collection and lesion scoring. We are also grateful to the Zoetis Genetics Laboratory for their technical help. Zoetis Inc. CTM has received a PhD scholarship from Zoetis. NC, JME and AG are employees of Zoetis. TO has received research support, as well as lecturing and consulting honoraria, from this company. Appendix S1 Appendix S2 Appendix S3 Appendix S4 Figure S1 Figure S2 Figure S3 Figure S4 Figure S5 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.