TNF- α and TH2 Cytokines Induced Canine Atopic Dermatitis–like Morphologic and Molecular Characteristics in the Canine Epidermal Organoid Culture System

TNF- α and TH2 Cytokines Induced Canine Atopic Dermatitis–like Morphologic and Molecular Characteristics in the Canine Epidermal Organoid Culture System | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article TNF- α and TH2 Cytokines Induced Canine Atopic Dermatitis–like Morphologic and Molecular Characteristics in the Canine Epidermal Organoid Culture System Bo Chen, Ronald Francis Slocombe, Oluwadamilola Samuel Omotainse, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6308044/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Atopic Dermatitis (AD) is a chronic inflammatory and pruritic skin disease that affects both human and veterinary patients. Canine atopic dermatitis is prevalent in 27% of the canine population. This frequently encountered disease leads to discomfort and reduced quality of life in affected animals. Our methodology utilizes a well-controlled model system of the canine primary epidermal organoids (cPEOs) derived from normal canine keratinocytes and exhibits morphological characteristics and key marker proteins consistent with normal canine skin. We investigated the direct impact of specific immune mediators, namely IL-4, IL-13 (Th2 cytokines), and TNF-α (a pro-inflammatory cytokine), both individually and in combination, on skin barrier components using this model system. The results demonstrated that cytokines induce CAD-like morphological and molecular characteristics in the canine epidermal organoid system, including epidermal spongiosis and reduced suprabasal epidermal differentiation. Th2 cytokines increased epidermal proliferation and TNF-α appeared to induce cellular apoptosis. These findings indicate that the canine epidermal organoid system holds promise as a valuable tool for understanding the pathogenesis of AD in both humans and veterinary patients and can become a potential platform for assessing individual treatment options or screening drug candidates for canine atopic cases. Biological sciences/Cell biology Health sciences/Pathogenesis canine atopic dermatitis canine primary epidermal organoids Th2 cytokines skin barrier molecular signature Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Canine Atopic Dermatitis (CAD) is a genetically predisposed allergic skin disease characterized by inflammation and itching, primarily triggered by IgE antibodies directed against environmental allergens 1 . Similar to Atopic Dermatitis (AD) in humans, the pathogenesis of CAD is believed to encompass intricate interactions between genetic factors and environmental influences that alter both the immune response and skin barrier function 2 . Also, in most cases, skin barrier dysfunction and abnormal immune responses are interlinked aspects of this disease, exacerbating each other. This complexity impedes the identification of the primary event that initiates the cascade, thereby hindering the understanding of the critical events in the pathogenesis. A carefully controlled in vitro model, free of complex interactions, is required to study the key events leading to clinical CAD. Recently, there has been a growing number of research publications utilizing the organoid system to investigate diseases of veterinary importance 3 . Organoids are in vitro structures resembling organs, which faithfully recapitulate various characteristics of a specific organ, and have the capability of prolonged expansion of these mini-organs within chemically inducible systems 4 . These organoids can be expanded without major chromosomal aberrations or loss of differentiation potential, proving them to be an excellent model in disease investigation. Simple epidermal organoids mimicking epidermis were generated from canine keratinocytes and this epidermal organoid also expressed key marker proteins consistent with normal canine skin 5 , 6 . The pathogenesis of atopic dermatitis in both humans and dogs has been strongly suggested to be associated with T-helper 2-polarized (Th2) immune responses triggered by various cytokines 7 , 8 . A dysregulation of immune responses in CAD manifested heightened levels of Th2, Th17 and regulatory T (Treg) cells 9 . Canine atopic dermatitis manifests both acute and chronic phases 10 . During the acute phase, atopic skin overexpresses Th2 cytokines. IL-4 and its homologous cytokine IL-13 play crucial roles as primary drivers of the Th2 immune response, while in chronic lesions, there is a more complex immune profile characterized by the presence of Th2, Th1, and Treg cells 10 . In this chronic stage, pro-inflammatory cytokines such as TNF-α are recognized to play a pivotal role in the development of itch sensation and inflammation. The aim of current study was to tease out the specific roles of various cytokines that play in the development of morphologic and molecular changes occurring in CAD skin, using a well-controlled model, without secondary infections. Focus was placed on the epidermal differentiation and barrier proteins. Specifically, we generated canine primary epidermal organoids (cPEOs) by utilizing epidermal stem cells sourced directly from the canine epidermis, with the culture medium initially supplemented with recombinant canine IL-4, IL-13 (Th2 cytokines) and TNF-a (pro-inflammatory cytokine) singly or a combination during the 8-day culture. This model enables assessment of the controlled induction of the effects of various cytokines on the formation of the epidermal barrier. Results 1. Canine Primary Epidermal Organoids (cPEOs) Recapitulate the Epidermis Canine primary epidermal organoids were generated from keratinocytes isolated from normal canine skin which are grown in Matrigel supplemented with various growth factors. The multicellular spherical structures were easily visible by phase contrast microscopy by 3 days of culture. The organoids achieved maximum growth by 8 days in culture (Fig. 1 a). In this model, the basal cells are present at the periphery of the organoid and as the cells grow inward, they display differentiation and keratinization at the centre of the organoid. In order to determine the structure and protein expression pattern of cPEOs and to investigate the similarities cPEOs with normal canine skin, Hematoxylin and Eosin (H&E) staining and immunohistochemistry (IHC) were performed (Fig. 1 b). The histologic structure and expression of proteins in various layers of the organoids and normal canine skin suggest that the in vitro cPEOs closely mimic normal canine skin. The organoids appear as round, densely cellular structures. The outermost cell layer exhibits a basaloid appearance, akin to the stratum basale, while the central cells are larger with enlarged nuclei, reminiscent of the more differentiated layers of the epidermis, such as the stratum spinosum and stratum granulosum. In certain organoids, structures resembling keratohyalin granules of the stratum granulosum were visible, and some organoids displayed a cornified core, consistent with keratinised stratified squamous epithelium observed in a healthy epidermis. The cPEOs also displayed a PAS-positive membrane at the periphery, suggesting the presence of a basement membrane, further resembling normal canine epidermis. In normal epidermis, keratinocytes in the basal layer express Keratin 5 (K5), while Keratin 10 (K10) is expressed in the suprabasal layer. Similarly, in cPEOs, K5 expression was unevenly distributed, with stronger signals at the periphery, while K10 was primarily found in the central areas. In cPEOs, the expression of Ki67 and p63 indicated proliferating cells in the outermost cell layer, which is consistent with seen in the basal layer of normal canine skin. Additionally, structural proteins Filaggrin (FLG), Involucrin (IVL), and Loricrin (LOR), which are essential components of cornified envelope in stratum corneum, were present in the innermost layers of the organoid. This pattern aligns with a differentiation process from the outer to the inner layers, similar to the formation of a 'basal-out' epidermis in healthy skin. Cornification is the final stage in stratum corneum formation, representing the endpoint of epidermal differentiation and cell death 12 . However, apoptosis, highlighted by caspase-3, was absent in the superficial stratum corneum of the skin and the center of cPEOs, instead showing scattered, randomly distributed signals. 2. Cytokines Lead to Spongiosis and Detachment of Desmosomal Contacts of cPEOs Our next experiments were aimed at developing an in vitro model for canine atopic dermatitis. Th2 cytokines and TNF-α are cytokines known to be involved in canine atopic dermatitis 10 . In order to mimic the histologic features of atopic skin, we have grown organoids in the presence of IL-4, IL-13, TNF-α either individually or in combination as described in the methods section. On day 8, the organoids were collected and processed for histologic evaluation. The representative phase contrast images of the organoids (Fig. 2 ) revealed that the impact of cytokine treatments on the size of the organoids was not significantly perceivable. However, organoid size was found to be strongly influenced by organoid density (data not shown). Debris surrounding the organoids in the TNF-α group was more readily observed. Histologic examination of cPEOs revealed that after exposure to TNF-α and/or Th2 cytokines, they exhibited increased intercellular spaces between the cells (Fig. 2 b), a characteristic feature known as spongiosis, commonly seen in atopic skin. The induced spongiosis was confirmed at transmission electron microscopy (TEM), which displayed widening of intercellular spaces between keratinocytes of cPEOs with cytokine treatment (Fig. 3 ). Detachment of desmosomal contacts was observed in spongiotic regions because of enlarged intercellular spaces (denoted by double arrow in Fig. 3 b). In contrast, desmosomal contacts remained intact in areas without perceivable spongiosis when stimulated with TNF-α and/or Th2 cytokines. Lengths of intercellular spaces were measured in the spongiotic areas in the layer equivalent to stratum spinosum for each treatment group. Comparing the control group, significant enlargement in the lengths of intercellular spaces was observed in the induced spongiotic areas in the treatment group by statistical analysis. The results were also consistent with the observations made with H&E staining. The most significant effect was observed with the combination of IL-4, IL-13, and TNF-α. Additionally, the combined treatment with IL-4 and IL-13 showed larger intercellular spaces compared to individual treatments, highlighting their synergistic effect when used together. Furthermore, TNF-α also exacerbates the outcomes of Th2 immune responses. However, the mechanism triggered by TNF-α may differ from that of IL-4 and IL-13, as indicated by the increased presence of apoptotic structures among keratinocytes treated with TNF-α observed by TEM (Fig. 3 c). 3. Cytokines Decreased the Epidermal Suprabasal Differentiation Histologic analysis of cPEOs showed that after exposures to cytokine stimulations, especially Th2 cytokines, cPEOs exhibited decreased production of keratohyalin granules (hypogranulosis) and reduced formation of cornified cores, indicative of reduced differentiation, and potential alterations in the balance between proliferation and terminal differentiation. To investigate for the effect on keratinocyte differentiation at the molecular level, the expression of a panel of genes, which are differentially expressed in the various layers of the epidermis was analysed at the mRNA level using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The fold gene expression levels of each gene of interest, relative to the control, are presented in Fig. 4 . Compared to the control group of cPEOs, the transcriptional expression levels of K10, FLG, IVL, and LOR were significantly diminished following exposure to IL-4, IL-13, and TNF-α individually or in combination. Following TNF-α treatments, the mean fold gene expressions for FLG, IVL, and LOR showed a relatively smaller decrease while for K10, there was a marked reduction in gene expression. This finding potentially indicates different mechanisms between Th2 cytokines and TNF-α affecting suprabasal differentiations. Transcriptional changes of K10, FLG, IVL, and LOR were validated by evaluating protein expression by IHC. K10 showed immunolabelling in the suprabasal layers towards the centre of the cPEOs. As cornified envelope proteins, FLG, IVL, and LOR were strongly expressed in the layer equivalent to the stratum corneum. The expression levels of these markers appeared to decrease in intensity and area following each cytokine treatment (Fig. 5 ). Compared to the control group of cPEOs, the transcriptional expression levels of K5, CLD1, and OCD were not significantly changed or showed any trends after IL-4, IL-13 and/or TNF-α treatments. This suggests that these cytokines might not directly influence basal layer markers or impair the tight junction composition. 4. The Effect of Cytokines on Proliferation and Apoptosis In the present study, an increase in Ki67 relative expression compared to the control was observed following exposure to Th2 cytokines, either individually or in combination with IL-4 and IL-13 (Fig. 6 a). This can also be significantly detected when measuring immunopositive pixels per organoid area (Fig. 6 b). With TNF-α treatment, either alone or in combination with Th2 cytokines, changes in the Ki67 relative expressions were less noticeable or varied inconsistently across different animals. Apoptosis signals, highlighted by Caspase 3 through IHC, showed that TNF-α appeared to increase apoptosis, as positive signals were more easily observed in each organoid structure in the groups involving TNF-α (Fig. 6 c). This observation is consistent with the findings from phase contrast and TEM images of TNF-α group (Figs. 2 a and 3 c). Discussion We have successfully developed a canine epidermal organoid system to mimic the morphological changes observed in the canine skin. Using this model, we demonstrated the effect of Th2 cytokines and TNF-α on epidermal barrier function and epidermal differentiation. As the concentrations of IL-4, IL-13, and TNF-α in canine atopic skin tissue remain unknown, the concentration in our study was set according to published data on atopic research on human 3D skin models 13 – 16 . We selected relatively high concentrations of cytokines, aiming to induce discernible changes effectively. The treatment period was determined based on the day-gradient experiments (4-, 6-, and 8-day treatments, data not shown). The 8-day treatment revealed the most significant spongiotic changes in cPEOs, closely mimicking the atopic skin morphology. Canine atopic dermatitis manifests as a vicious cycle of an allergic immune response leading to an altered skin biome and permeability, increasing exposure to multiple antigens. In vivo , such alterations, which signify impairment of the skin barrier, become responsible for activation of the immune system because they promote penetration of pathogens or allergens, thereby creating the vicious circle for the development of atopic dermatitis. Our results demonstrated the roles of Th2 cytokines (IL-4 and IL-13) and the proinflammatory cytokine (TNF-α) in the morphological alterations such as tissue spongiosis, hypogranulosis, and hyperproliferation. Epidermal barrier damage, which are featured by reduced differentiations identified by altered mRNA and protein expression levels of suprabasal markers (including cornified envelope proteins), represent one of the hallmarks of canine atopic dermatitis–like characteristics in our study. Spongiosis that describes the presence of intercellular oedema is a characteristic histopathologic appearance in diseases of the skin, such as atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, certain types of psoriasis and pemphigus. Spongiosis in the epidermis are characterized by impairment or loss of cohesion between keratinocyte and the influx of fluid 17 . In our investigation, we found that IL-4, IL-13, and TNF-α each induce spongiosis independently. The mechanism of spongiosis remains unclear. A study revealed that IL-4, IL-13, and IFN-γ are capable of inducing epidermal spongiosis accompanied by heightened hyaluronic acid (HA) accumulation and reduced E-cadherin expression 18 . This indicates a potential osmotic flow of water into the intercellular compartment of the epidermis due to hyaluronan accumulation and weakened adherens junctions. Apoptosis may also contribute to the formation of spongiosis, particularly evident when exposed to TNF-α, as evidenced by the frequent presence of apoptotic structures in our study. TNF-induced apoptosis as an extrinsic apoptotic pathway via TNF-R1 may explain the spongiosis caused by apoptosis-driven cell separation and subsequent fluid accumulation in the epidermis 19 . These findings suggests that exact mechanisms leading to spongiosis can vary or be multifactorial depending on the underlying disease. Furthermore, TNF-α has a myriad of pro-inflammatory effects on the skin, different from atopy-associated cytokines, and which is a major mediator in the pathogenesis of many other inflammatory disorders, e.g. rheumatoid arthritis, Crohn’s disease, and psoriasis in humans. In the chronic phase of CAD, TNF-α also plays a role involved in the general inflammatory reaction. TNF-α is reported to have a mild inhibitory effect on keratinocyte proliferation 20 , which was not identified in our study, probably the effect depends on the concentration of TNF-α used in various experiments. Epidermal differentiation and turnover are a complex process 21 , 22 . Keratinocytes originate from proliferative cells in the basal layer of the epidermis and mature into flattened, dead cells in the outermost layer. Keratinocytes in the basal layer express K5 and in the suprabasal layer express K10. Keratohyalin granules, composed of keratin and keratin-binding proteins like FLG or IVL or LOR, form in the stratum granulosum. The stratum granulosum cell layer is sealed by functional tight junctions in the epidermis. Proteins within tight junctions, such as CLD1 and OCD, are associated with CAD, showing decreased expression and abnormal distribution patterns in atopic dogs 23 , 24 . However, in our study, the transcriptional expressions of CLD1 and OCD did not change significantly following exposure to Th2 cytokines and/or TNF-α. This observation is consistent with the findings by using a 3D model using normal human skin biopsies 25 . The mechanisms behind the reduced expression of tight junction molecules in the atopic epidermis have not been fully elucidated, which was found associated to other cytokines and Staphylococcus species-dominant microbiome dysbiosis 26 . The stratum corneum, most crucial for the epidermal barrier, consists of terminally differentiated keratinocytes (‘corneocytes’) embedded in an extracellular lipid-rich matrix, known as the "bricks and mortar" structure 27 . That the critical role of the stratum corneum works as air-fluid barrier in the skin barrier is increasingly recognized as the primary focus in AD, and also in CAD, too. Cornified envelope, assembled by terminally differentiated keratinocytes, comprises proteins like FLG, IVL, LOR, periplakin, envoplakin, and the small proline-rich protein family (SPRRs) 12 , 22 . It was reported reduced expressions of CE proteins occur in both lesional and non-lesional skin of atopic dogs 28 . Similarly in our research, Th2 cytokines and TNF-α decrease the expression of CE proteins (FLG, IVL, and LOR). Th2 cytokines more significantly decreased the expression of cornified envelope (CE) proteins (FLG, IVL, and LOR) with increased proliferation compared to TNF-α, suggesting distinct underlying influenced pathway. In contrast, markers of basal cells (K5) and tight junction proteins (CLD1 and OCD) in stratum granulosum did not exhibit significant changes or trends following exposure to Th2 cytokines or TNF-α. These findings indicate that after the induction of Th2 cytokines and TNF-α, keratinocytes specifically modify the suprabasal structural components and/or the barrier function, particularly in the stratum corneum, which is consistent with the finding in patients with AD 29 . In our study, we did not observe any significant change in the size of organoids following the administration of Th2 cytokines. This finding did not correspond with epidermal hyperplasia and hyperkeratosis in atopic skin 30 . The inconsistency may be partially due to the fact that the 8-day culture period of the organoid model is likely insufficient to recapitulate the real epidermal turnover, which typically occurs over around 30 days in native human skin 31 . The simplicity of this epidermal model, consisting of only keratinocyte clusters, also limits its ability to fully mimic the function and structure of real epidermis. Furthermore, the current epidermal organoid model is a basal-out model, and does not form cornified layers facing the surrounding environment directly. Thus, barrier function tests such as transepidermal water loss and permeability assays are hard to achieve in this model system. In CAD, less dense lipid matrix is also a prominent feature in the damaged skin barrier, leading to a fragile and dry epidermis, which was not investigated in the present study. Although IL-4 and IL-13 are not major factors in inducing apoptosis nor directly involved in the apoptotic pathway, a study by Kamsteeg et al. (2011) found that IL-4 and IL-13 triggered DNA fragmentation and cell death in keratinocytes using a human skin equivalent model. In our study, after performing IHC staining for caspase-3, no perceivable differences were observed between the Th2 cytokine groups and the control. We observed only a few apoptotic cells in our cPEOs, carrying low sensitivity. However, in TNF-α group, an increased number of positive cells were identified. These findings suggest that increased apoptosis of keratinocytes in CAD is primarily driven by factors other than IL-4 and IL-13. These limitations and uncertainties are anticipated to be addressed in the future research. Pathophysiological concepts underlying the development of CAD are multifactorial and skin barrier defects in CAD result from the dysregulation of multiple pathways. Nevertheless, our epidermal organoid system derived from normal skin samples offers a robust in vitro model to tease out roles of each cytokine in the disease progression. To our knowledge, this is the first study to investigate the effects of cytokines using epidermal organoids, even within the broader field of human medicine. Moreover, as in humans, CAD is a group of heterogeneous diseases caused by genetic variants that can differ among individuals, leading to unpredictable responses to therapy 8 . For example, loss-of-function mutations in FLG, the gene encoding profilaggrin and filaggrin, is a significant genetic predisposing factor for AD [33] but implicated in only some atopic dogs [8]. Given the unclear genetic background of each CAD case, we assume the canine epidermal organoid system derived from patient skin samples preserves the genetic information and has the great potential to be a platform for testing therapeutic options tailored to each individual dog. However, obtaining atopic skin samples is challenging because CAD diagnosis is typically made by excluding other diseases, without necessarily requiring a biopsy. Further research is planned to artificially create a model with FLG knockout mimicking clinical CAD cases and also to test the feasibility of epidermal organoids as a validated tool to screen CAD treatment options. In conclusion, it is suggested that the present model system can be used to study CAD, recognising the critical role of the stratum corneum plays as the maintenance of the air-fluid barrier and which appears to be a primary defect in the development of CAD. Th2 cytokines and TNF-α elicit a CAD-like phenotype including epidermal spongiosis, and reduced epidermal differentiation especially in stratum corneum, Th2 cytokines increased epidermal proliferation and TNF-α appeared to induce cellular apoptosis. It is anticipated cytokine-supplemented cPEOs could serve as an excellent candidate for the testing of prospective therapies for CAD. Materials & Methods Isolation and culture of canine primary keratinocyte Skin samples with no visible abnormalities were obtained from freshly scavenged tissues that were submitted for biopsy. Tissue scavenging was approved by the Animal Ethics Committee (AEC) of the University of Melbourne (Ethics ID #22006). All methods were performed in accordance with the University of Melbourne’s policies on the use of animals for research, in compliance with the Australian Code for the Care and Use of Animals for Scientific Purposes. A representative skin sample was processed for histology to ensure that there were no other significant pathological changes. Keratinocytes were isolated by the two-step digestion. Isolated keratinocytes were collected and immediately flash frozen. Organoid culture and cytokine treatment Organoids were cultured as previously described with some modifications 5 . Keratinocytes within soluble Matrigel were plated on the surface of previously layered Matrigel in the organoid culture medium with/without cytokines. Organoid media formulations were as indicated in the Supplementary Table S1 online. The cytokine concentrations and combinations for six groups are A) Control; B) 30 ng/mL IL-4; C) 30 ng/mL IL-13; D) 5 ng/mL TNF-α; E) 30 ng/mL IL-4, 30 ng/mL IL-13; F) 30 ng/mL IL-4, 30 ng/mL IL-13 and 5 ng/mL TNF-α. The day of seeding cells was defined as Day 0. Fresh culture medium with cytokines was replaced every 3 days until Day 8. On Day 7, phase contrast images were captured by an inverted microscope with camera (CKX-53 and DP23; Olympus). Hematoxylin and Eosin (HE) Staining and Immunohistochemistry (IHC) Organoids were directly fixed in 4% paraformaldehyde (PFA) at room temperature for 2 h after removing the medium. Fixed samples were then embedded in a solid agarose pad followed by paraffin. Paraffin-embedded samples were cut at 3 µm and slides were stained with H&E and PAS after deparaffinization. As routine IHC methods, tissue sections were deparaffinized and rehydrated in xylene and a graded series of ethanol solutions, and then microwaved for antigen retrieval. After endogenous peroxidase block and incubation with antibodies, the slides were incubated with secondary antibody REAL Envision Detection System Peroxidase/DAB+, mouse/rabbit (Dako) according to the manufacturer’s instructions. All antibodies used are listed in Supplementary Table S2 online. Negative controls included incubation with non-immune goat serum instead of primary antibody. Dog skin was used as the positive control. Images were obtained using a light microscope with camera (BX-41 and DP74; Olympus). RNA Extraction and Real-Time RT-qPCR Organoid samples were lysed by TRIzol reagent (Invitrogen) and RNA was extracted following the manufacturer’s instructions. The RNA purity and integrity were measured using NanoPhotometer N120 (IMPLEN). A260/280 were in the desired range (1.80–2.20) for all tested samples. The extracted RNA was reverse transcribed using GoScript™ Reverse Transcriptase Mix, Oligo(dT) (Promega) and used for first-strand complementary DNA (cDNA) synthesis as per manufacturer’s instructions. Quantitative real-time PCR for the selected gene and calculations of cycle threshold (Ct) values were performed using Rotor-Gene Q (Qiagen) with a system using GoTaq® Flexi DNA polymerase (Promega, USA) according to the manufacturer’s protocol. The primers used are listed in Supplementary Table S3 online. All primers were validated by generating standard curves with R2 value >99%. As a stably expressed gene in canine skin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is selected as the reference gene. The mRNA expression levels was normalized to GAPDH expression based on the ΔCt method and calculated based on fold gene change (2^-(∆∆Ct)) 11 . The experiment was performed in duplication while differences between two Ct values in the same repetition should be less than 0.5. Ultrastructural Study: Transmission Electron Microscopy (TEM) Organoid samples for transmission electron microscopy were fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer according to the standard procedures and embedded in epon resin. Ultrathin sections (70–85 nm) were cut, stained with uranyl acetate and lead citrate, and observed with a FEI Talos L120C cryo electron microscope (Thermo Fisher Scientific) at 60 kV and a CCD (1024 x 1024 pixel) camera was used for image recording. Statistical Analysis Statistical analysis was all conducted by GraphPad Prism version 10.2.0. For the analysis of intercellular spaces measured under TEM, five representative photos (11000×) for each group were captured to measure the lengths of intercellular spaces. A two-way ANOVA was performed, followed by Tukey's multiple comparisons test to compare each treatment group with the control. For gene expression analysis using fold gene change (2^-(∆∆Ct)), an unpaired Student’s t-test was performed to compare each treatment group with the control. For the IHC results for Ki67, five organoids with the most advanced differentiation and similar size were selected from each group. The labelling index was determined by calculating the average percentage of Ki67 immunopositive pixels relative to the total area pixels of the organoid, with pixel data obtained using ImageJ. Relative Ki67 immunopositive pixels were statistically analyzed using an unpaired Student’s t-test (two tailed) to compare each treatment group with the control. A P-value of ≤ 0.05 was considered statistically significant. Data from at least three independent experiments are presented as the mean ± standard deviation (SD). Declarations Ethics Approval Statement Tissue scavenging was approved by the Animal Ethics Committee (AEC) of the University of Melbourne (Ethics ID #22006) before the investigation. However, AEC approval for activities involving scavenged tissue is not required because no animals were harmed or killed for the purpose of this study. All experiments were conducted in accordance with the University of Melbourne’s policies on the use of animals for research, in compliance with the Australian Code for the Care and Use of Animals for Scientific Purposes. Availability of Data and Materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding The work was supported by the China Scholarship Council (CSC) – University of Melbourne Ph.D. Scholarship organized by CSC (File No. 202008320396) and University of Melbourne. Authors' Contributions BC, RFS, and SRG conceptualized and designed the study. BC was responsible for data acquisition, analysis, and interpretation. BC, OSO, and MB contributed to the investigation and methodology. BC prepared the initial draft of the manuscript. All authors reviewed, revised, and approved the final version of the manuscript. Acknowledgements We are grateful for support from the Asia-Pacific Centre for Animal Health group (Melbourne Veterinary School, Faculty of Science, the University of Melbourne) and Ian Holmes Imaging Centre (Bio21, the University of Melbourne). We thank Dr. Dijina Swaroop (Department of Medicine, Monash University) for sharing scientific expertise. Declaration of generative AI in scientific writing In this manuscript, generative AI tools (ChatGPT by OpenAI and Grammarly) were only used to improve readability and language. All scientific concepts, conclusions, and interpretations were developed by the authors. References Halliwell, R. Revised nomenclature for veterinary allergy. Vet. Immunol. 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Res. 311 , 131–140. 10.1007/s00403-018-1883-z (2019). van Drongelen, V. et al. Explant cultures of atopic dermatitis biopsies maintain their epidermal characteristics in vitro. Cell. Tissue Res. 361 , 789–797. 10.1007/s00441-015-2162-3 (2015). Trautmann, A., Akdis, M., Klunker, S., Blaser, K. & Akdis, C. A. in International archives of allergy and immunology Vol. 124 230–232S. Karger, Switzerland, (2001). Ohtani, T. et al. Increased Hyaluronan Production and Decreased E-Cadherin Expression by Cytokine-Stimulated Keratinocytes Lead to Spongiosis Formation. J. Invest. Dermatology . 129 , 1412–1420. https://doi.org/10.1038/jid.2008.394 (2009). Kerstan, A., Bröcker, E. B. & Trautmann, A. Decisive role of tumor necrosis factor-α for spongiosis formation in acute eczematous dermatitis. Arch. Dermatol. Res. 303 , 651–658. 10.1007/s00403-011-1149-5 (2011). Song, W. J. et al. Canine adipose tissue-derived mesenchymal stem cells pre-treated with TNF-alpha enhance immunomodulatory effects in inflammatory bowel disease in mice. Res. Vet. Sci. 125 , 176–184. https://doi.org/10.1016/j.rvsc.2019.06.012 (2019). Kubo, A., Nagao, K. & Amagai, M. Epidermal barrier dysfunction and cutaneous sensitization in atopic diseases. J. Clin. Invest. 122 , 440–447. 10.1172/JCI57416 (2012). Matsui, T. & Amagai, M. Dissecting the formation, structure and barrier function of the stratum corneum. Int. Immunol. 27 , 269–280. 10.1093/intimm/dxv013 (2015). Roussel, A. J., Bruet, V., Marsella, R., Knol, A. C. & Bourdeau, P. J. Tight junction proteins in the canine epidermis: a pilot study on their distribution in normal and in high IgE-producing canines. Can. J. Vet. Res. 79 , 46–51 (2015). Olivry, T. & Dunston, S. M. Expression patterns of superficial epidermal adhesion molecules in an experimental dog model of acute atopic dermatitis skin lesions. Vet. Dermatol. 26 , 53–56. 10.1111/vde.12188 (2015). e-17-58. Donetti, E. et al. Th2 Cytokines Affect the Innate Immune Barrier without Impairing the Physical Barrier in a 3D Model of Normal Human Skin. J. Clin. Med. 12 (2023). https://mdpi-res.com/d_attachment/jcm/jcm-12-01941/article_deploy/jcm-12-01941-v2.pdf?version=1677723588 Blicharz, L. et al. The Influence of Microbiome Dysbiosis and Bacterial Biofilms on Epidermal Barrier Function in Atopic Dermatitis—An Update. Int. J. Mol. Sci. 22 , 8403 (2021). Rosso, J. D., Zeichner, J., Alexis, A., Cohen, D. & Berson, D. Understanding the Epidermal Barrier in Healthy and Compromised Skin: Clinically Relevant Information for the Dermatology Practitioner: Proceedings of an Expert Panel Roundtable Meeting. J Clin Aesthet Dermatol 9, S2-S8 (2016). Theerawatanasirikul, S. et al. Differential Expression Patterns of Proteins Involved in Epidermal Proliferation and Differentiation in Canine Atopic Dermatitis. Thai J. Veterinary Med. 42 , 287–296 (2012). Omori-Miyake, M., Yamashita, M., Tsunemi, Y., Kawashima, M. & Yagi, J. In Vitro Assessment of IL-4- or IL-13-Mediated Changes in the Structural Components of Keratinocytes in Mice and Humans. J. Invest. Dermatology . 134 , 1342–1350. https://doi.org/10.1038/jid.2013.503 (2014). Bizikova, P. et al. Clinical and histological manifestations of canine atopic dermatitis. Vet. Dermatol. 26 , 79–e24. 10.1111/vde.12196 (2015). Review. Iizuka, H. Epidermal architecture that depends on turnover time. J. Dermatol. Sci. 10 , 220–223. https://doi.org/10.1016/0923-1811(95)00407-J (1995). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6308044","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":442284860,"identity":"ab39a7c3-2b31-4951-9eef-0731eb69b8ce","order_by":0,"name":"Bo Chen","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Chen","suffix":""},{"id":442284862,"identity":"3251a80d-0244-4917-9c6a-d3298314ed18","order_by":1,"name":"Ronald Francis Slocombe","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Ronald","middleName":"Francis","lastName":"Slocombe","suffix":""},{"id":442284864,"identity":"3eef7709-b640-4b9d-8a86-f0352e999a10","order_by":2,"name":"Oluwadamilola Samuel Omotainse","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Oluwadamilola","middleName":"Samuel","lastName":"Omotainse","suffix":""},{"id":442284866,"identity":"4ce11aec-618d-4874-bdc2-d4b3e8e4affb","order_by":3,"name":"Mirjana Bogeski","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Mirjana","middleName":"","lastName":"Bogeski","suffix":""},{"id":442284867,"identity":"78c509f4-88d9-47b5-bc8c-d49dabe53533","order_by":4,"name":"Smitha Rose Georgy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYDADfiA+wMBgkQCkDYjTItkA1iJBghaDA2CKCC3yDewXH3zcU5u4+UbuwUM3KiTyGNibt0kw1BzGYzhPseGMZ8cTt93ISzicc0aimIHnWJkEwzE8Whh40qR5DhwDaskxOJzbJpHYIJFjJsHAhluLfANP+m+Qls0zQFr+AbXIvwFq+YdbC8MB9mPMPAdqEjdIgLQ0gGzhMZNgbMPjsMM8zJIzDhwwnnHmjcHhnGMSxWw8acUWiX3puB3W3v7ww4cDdbL97TnGn3NqbPL42Q9vvPHhmzVuhzHzgGLhsGMDTIANRCTg1gAE7A+ARJ09XjWjYBSMglEwsgEAuQlZDJs4VikAAAAASUVORK5CYII=","orcid":"","institution":"University of Melbourne","correspondingAuthor":true,"prefix":"","firstName":"Smitha","middleName":"Rose","lastName":"Georgy","suffix":""}],"badges":[],"createdAt":"2025-03-26 03:08:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6308044/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6308044/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80714013,"identity":"2a9c8bd5-4520-4c5f-914f-2686d05e0ec9","added_by":"auto","created_at":"2025-04-16 09:27:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4234198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCharacteristics of canine primary epidermal organoid system.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea. Growth of canine primary epidermal organoid system. Representative serial phase contrast images of canine epidermal organoids growing at the indicated time points. (Bar = 40 μm)\u003c/p\u003e\n\u003cp\u003eb. H\u0026amp;E, PAS, and immunohistochemical staining of paraffin-embedded skin tissue compared with organoids at the terminated time points (Day 8) illustrating the similarities in the morphological and molecular levels. Arrows point to scattered apoptotic keratinocytes in the canine skin in the Caspase-3 panel. (Bar = 50 μm).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6308044/v1/def68f436b786420f0e4d008.png"},{"id":80712637,"identity":"7b0b94cf-69fe-46f4-b267-0b1140498230","added_by":"auto","created_at":"2025-04-16 09:19:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2867654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMorphological changes in organoids after cytokine treatments.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea. Representative phase contrast images of canine epidermal organoids in control and treatment groups on Day 7; b. Representative histologic staining of untreated and IL-4, IL-13, and/or TNF-α supplemented canine epidermal organoids. (Bar = 40 μm).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6308044/v1/ca6cc044d9e9f1856b55ea3a.png"},{"id":80712633,"identity":"ac8096f1-8a8e-49e9-8ea4-2cdcd26319e9","added_by":"auto","created_at":"2025-04-16 09:19:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3268723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEffects of cytokine stimulation on the intercellular spaces evaluated via transmission electron microscopy (TEM).\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCanine epidermal organoids were either unstimulated (a) or stimulated with a combination of 30 ng/mL IL-4, 30 ng/mL IL-13, and 5 ng/mL TNF-α (b), or 5 ng/mL TNF-α alone (c). C, cell; A, apoptotic cell. The mean length of intercellular spaces in the control group and treatment groups were measured from transmission electron microscope images (d). Statistical analysis was performed using the two-way ANOVA and the significance is denoted as *** p \u0026lt; 0.001 and **** p \u0026lt; 0.0001 compared to the control.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6308044/v1/b9f50a373e2a0019262c176f.png"},{"id":80714014,"identity":"d9315937-b7d9-4782-af4e-1384e8ea2d19","added_by":"auto","created_at":"2025-04-16 09:27:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":477020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCytokines altered differentiation markers but did not affect basal marker K5 or tight junction proteins.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRelative gene expression of Keratin 5, Keratin 10, Filaggrin, Involucrin, and Loricrin, Claudin 1, and Occludin of cPEO treated with various cytokines in comparison with control organoids. The data are displayed as means ± standard deviation (SD), and individual values. The data were analysed using Student’s t-test and the statistical significance in denoted as \u0026nbsp;* p \u0026lt; 0.05, ** p \u0026lt; 0.01, and # p \u0026lt; 0.0001 compared to the control.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6308044/v1/7eb3816f6b90e16749861a2b.png"},{"id":80712638,"identity":"4108b6d7-8a13-484e-b41f-a81b70a95a0a","added_by":"auto","created_at":"2025-04-16 09:19:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4109909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe impact of cytokines on the differentiation of canine primary epidermal organoids (cPEOs).\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eReduced differentiation was confirmed by immunohistochemistry stainings for Keratin 10, Filaggrin, Involucrin, and Loricrin, as brown signals indicating positivity exhibited the visible decrease in the area proportion and/or intensity. (Bar = 200 μm).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6308044/v1/58600e1f2b8e681e17939d82.png"},{"id":80714015,"identity":"79754f44-429b-44a5-987c-67ccd0624c57","added_by":"auto","created_at":"2025-04-16 09:27:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3001782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe impact of cytokines on the proliferation and apoptosis of canine primary epidermal organoids (cPEOs).\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea. Increased Ki67 immunopositivity was perceivable in treatments involving Th2 cytokines; b. Objectively, Ki67 immunopositive pixels per organoid area pixels were calculated after pixels were captured \u0026nbsp;Qby imageJ. Relative Ki67 immunopositive pixels compared to control were displayed (c). The data were analysed using Student’s t-test and the statistical significance in denoted as * p \u0026lt; 0.05 and ** p \u0026lt; 0.01 compared to the control. c. TNF-α, with or without Th2 cytokines, slightly increased Caspase 3-positive signals. The representative photos in Panels a and c were taken at various magnifications to highlight the significant differences between each treatment with scale bars in the bottom-right corners.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6308044/v1/ab15f88d4edf343ceaf0ac44.png"},{"id":84450922,"identity":"ac608f7a-b0eb-4e8d-8e66-83ec62bbad27","added_by":"auto","created_at":"2025-06-12 06:47:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17841746,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6308044/v1/1ce045c4-b5b8-4031-a6b5-216de3298f89.pdf"},{"id":80712634,"identity":"f1b39ef8-81ca-42d3-961b-060ce0901a01","added_by":"auto","created_at":"2025-04-16 09:19:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":97296,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6308044/v1/5857bcde5284f8f949c28f16.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eTNF- α and TH2 Cytokines Induced Canine Atopic Dermatitis–like Morphologic and Molecular Characteristics in the Canine Epidermal Organoid Culture System\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCanine Atopic Dermatitis (CAD) is a genetically predisposed allergic skin disease characterized by inflammation and itching, primarily triggered by IgE antibodies directed against environmental allergens \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Similar to Atopic Dermatitis (AD) in humans, the pathogenesis of CAD is believed to encompass intricate interactions between genetic factors and environmental influences that alter both the immune response and skin barrier function \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Also, in most cases, skin barrier dysfunction and abnormal immune responses are interlinked aspects of this disease, exacerbating each other. This complexity impedes the identification of the primary event that initiates the cascade, thereby hindering the understanding of the critical events in the pathogenesis. A carefully controlled \u003cem\u003ein vitro\u003c/em\u003e model, free of complex interactions, is required to study the key events leading to clinical CAD.\u003c/p\u003e \u003cp\u003eRecently, there has been a growing number of research publications utilizing the organoid system to investigate diseases of veterinary importance \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Organoids are \u003cem\u003ein vitro\u003c/em\u003e structures resembling organs, which faithfully recapitulate various characteristics of a specific organ, and have the capability of prolonged expansion of these mini-organs within chemically inducible systems \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. These organoids can be expanded without major chromosomal aberrations or loss of differentiation potential, proving them to be an excellent model in disease investigation. Simple epidermal organoids mimicking epidermis were generated from canine keratinocytes and this epidermal organoid also expressed key marker proteins consistent with normal canine skin \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe pathogenesis of atopic dermatitis in both humans and dogs has been strongly suggested to be associated with T-helper 2-polarized (Th2) immune responses triggered by various cytokines \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. A dysregulation of immune responses in CAD manifested heightened levels of Th2, Th17 and regulatory T (Treg) cells \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Canine atopic dermatitis manifests both acute and chronic phases \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. During the acute phase, atopic skin overexpresses Th2 cytokines. IL-4 and its homologous cytokine IL-13 play crucial roles as primary drivers of the Th2 immune response, while in chronic lesions, there is a more complex immune profile characterized by the presence of Th2, Th1, and Treg cells \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In this chronic stage, pro-inflammatory cytokines such as TNF-α are recognized to play a pivotal role in the development of itch sensation and inflammation. The aim of current study was to tease out the specific roles of various cytokines that play in the development of morphologic and molecular changes occurring in CAD skin, using a well-controlled model, without secondary infections. Focus was placed on the epidermal differentiation and barrier proteins. Specifically, we generated canine primary epidermal organoids (cPEOs) by utilizing epidermal stem cells sourced directly from the canine epidermis, with the culture medium initially supplemented with recombinant canine IL-4, IL-13 (Th2 cytokines) and TNF-a (pro-inflammatory cytokine) singly or a combination during the 8-day culture. This model enables assessment of the controlled induction of the effects of various cytokines on the formation of the epidermal barrier.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. Canine Primary Epidermal Organoids (cPEOs) Recapitulate the Epidermis\u003c/h2\u003e \u003cp\u003eCanine primary epidermal organoids were generated from keratinocytes isolated from normal canine skin which are grown in Matrigel supplemented with various growth factors. The multicellular spherical structures were easily visible by phase contrast microscopy by 3 days of culture. The organoids achieved maximum growth by 8 days in culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In this model, the basal cells are present at the periphery of the organoid and as the cells grow inward, they display differentiation and keratinization at the centre of the organoid. In order to determine the structure and protein expression pattern of cPEOs and to investigate the similarities cPEOs with normal canine skin, Hematoxylin and Eosin (H\u0026amp;E) staining and immunohistochemistry (IHC) were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe histologic structure and expression of proteins in various layers of the organoids and normal canine skin suggest that the \u003cem\u003ein vitro\u003c/em\u003e cPEOs closely mimic normal canine skin. The organoids appear as round, densely cellular structures. The outermost cell layer exhibits a basaloid appearance, akin to the stratum basale, while the central cells are larger with enlarged nuclei, reminiscent of the more differentiated layers of the epidermis, such as the stratum spinosum and stratum granulosum. In certain organoids, structures resembling keratohyalin granules of the stratum granulosum were visible, and some organoids displayed a cornified core, consistent with keratinised stratified squamous epithelium observed in a healthy epidermis. The cPEOs also displayed a PAS-positive membrane at the periphery, suggesting the presence of a basement membrane, further resembling normal canine epidermis.\u003c/p\u003e \u003cp\u003eIn normal epidermis, keratinocytes in the basal layer express Keratin 5 (K5), while Keratin 10 (K10) is expressed in the suprabasal layer. Similarly, in cPEOs, K5 expression was unevenly distributed, with stronger signals at the periphery, while K10 was primarily found in the central areas. In cPEOs, the expression of Ki67 and p63 indicated proliferating cells in the outermost cell layer, which is consistent with seen in the basal layer of normal canine skin. Additionally, structural proteins Filaggrin (FLG), Involucrin (IVL), and Loricrin (LOR), which are essential components of cornified envelope in stratum corneum, were present in the innermost layers of the organoid. This pattern aligns with a differentiation process from the outer to the inner layers, similar to the formation of a 'basal-out' epidermis in healthy skin.\u003c/p\u003e \u003cp\u003eCornification is the final stage in stratum corneum formation, representing the endpoint of epidermal differentiation and cell death \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, apoptosis, highlighted by caspase-3, was absent in the superficial stratum corneum of the skin and the center of cPEOs, instead showing scattered, randomly distributed signals.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2. Cytokines Lead to Spongiosis and Detachment of Desmosomal Contacts of cPEOs\u003c/h3\u003e\n\u003cp\u003eOur next experiments were aimed at developing an \u003cem\u003ein vitro\u003c/em\u003e model for canine atopic dermatitis. Th2 cytokines and TNF-α are cytokines known to be involved in canine atopic dermatitis \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In order to mimic the histologic features of atopic skin, we have grown organoids in the presence of IL-4, IL-13, TNF-α either individually or in combination as described in the methods section. On day 8, the organoids were collected and processed for histologic evaluation. The representative phase contrast images of the organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) revealed that the impact of cytokine treatments on the size of the organoids was not significantly perceivable. However, organoid size was found to be strongly influenced by organoid density (data not shown). Debris surrounding the organoids in the TNF-α group was more readily observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistologic examination of cPEOs revealed that after exposure to TNF-α and/or Th2 cytokines, they exhibited increased intercellular spaces between the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), a characteristic feature known as spongiosis, commonly seen in atopic skin. The induced spongiosis was confirmed at transmission electron microscopy (TEM), which displayed widening of intercellular spaces between keratinocytes of cPEOs with cytokine treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Detachment of desmosomal contacts was observed in spongiotic regions because of enlarged intercellular spaces (denoted by double arrow in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). In contrast, desmosomal contacts remained intact in areas without perceivable spongiosis when stimulated with TNF-α and/or Th2 cytokines. Lengths of intercellular spaces were measured in the spongiotic areas in the layer equivalent to stratum spinosum for each treatment group. Comparing the control group, significant enlargement in the lengths of intercellular spaces was observed in the induced spongiotic areas in the treatment group by statistical analysis. The results were also consistent with the observations made with H\u0026amp;E staining. The most significant effect was observed with the combination of IL-4, IL-13, and TNF-α. Additionally, the combined treatment with IL-4 and IL-13 showed larger intercellular spaces compared to individual treatments, highlighting their synergistic effect when used together. Furthermore, TNF-α also exacerbates the outcomes of Th2 immune responses. However, the mechanism triggered by TNF-α may differ from that of IL-4 and IL-13, as indicated by the increased presence of apoptotic structures among keratinocytes treated with TNF-α observed by TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e3. Cytokines Decreased the Epidermal Suprabasal Differentiation\u003c/h3\u003e\n\u003cp\u003eHistologic analysis of cPEOs showed that after exposures to cytokine stimulations, especially Th2 cytokines, cPEOs exhibited decreased production of keratohyalin granules (hypogranulosis) and reduced formation of cornified cores, indicative of reduced differentiation, and potential alterations in the balance between proliferation and terminal differentiation.\u003c/p\u003e \u003cp\u003eTo investigate for the effect on keratinocyte differentiation at the molecular level, the expression of a panel of genes, which are differentially expressed in the various layers of the epidermis was analysed at the mRNA level using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The fold gene expression levels of each gene of interest, relative to the control, are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared to the control group of cPEOs, the transcriptional expression levels of K10, FLG, IVL, and LOR were significantly diminished following exposure to IL-4, IL-13, and TNF-α individually or in combination. Following TNF-α treatments, the mean fold gene expressions for FLG, IVL, and LOR showed a relatively smaller decrease while for K10, there was a marked reduction in gene expression. This finding potentially indicates different mechanisms between Th2 cytokines and TNF-α affecting suprabasal differentiations. Transcriptional changes of K10, FLG, IVL, and LOR were validated by evaluating protein expression by IHC. K10 showed immunolabelling in the suprabasal layers towards the centre of the cPEOs. As cornified envelope proteins, FLG, IVL, and LOR were strongly expressed in the layer equivalent to the stratum corneum. The expression levels of these markers appeared to decrease in intensity and area following each cytokine treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared to the control group of cPEOs, the transcriptional expression levels of K5, CLD1, and OCD were not significantly changed or showed any trends after IL-4, IL-13 and/or TNF-α treatments. This suggests that these cytokines might not directly influence basal layer markers or impair the tight junction composition.\u003c/p\u003e\n\u003ch3\u003e4. The Effect of Cytokines on Proliferation and Apoptosis\u003c/h3\u003e\n\u003cp\u003eIn the present study, an increase in Ki67 relative expression compared to the control was observed following exposure to Th2 cytokines, either individually or in combination with IL-4 and IL-13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). This can also be significantly detected when measuring immunopositive pixels per organoid area (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). With TNF-α treatment, either alone or in combination with Th2 cytokines, changes in the Ki67 relative expressions were less noticeable or varied inconsistently across different animals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eApoptosis signals, highlighted by Caspase 3 through IHC, showed that TNF-α appeared to increase apoptosis, as positive signals were more easily observed in each organoid structure in the groups involving TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). This observation is consistent with the findings from phase contrast and TEM images of TNF-α group (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have successfully developed a canine epidermal organoid system to mimic the morphological changes observed in the canine skin. Using this model, we demonstrated the effect of Th2 cytokines and TNF-α on epidermal barrier function and epidermal differentiation. As the concentrations of IL-4, IL-13, and TNF-α in canine atopic skin tissue remain unknown, the concentration in our study was set according to published data on atopic research on human 3D skin models \u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. We selected relatively high concentrations of cytokines, aiming to induce discernible changes effectively. The treatment period was determined based on the day-gradient experiments (4-, 6-, and 8-day treatments, data not shown). The 8-day treatment revealed the most significant spongiotic changes in cPEOs, closely mimicking the atopic skin morphology.\u003c/p\u003e \u003cp\u003eCanine atopic dermatitis manifests as a vicious cycle of an allergic immune response leading to an altered skin biome and permeability, increasing exposure to multiple antigens. \u003cem\u003eIn vivo\u003c/em\u003e, such alterations, which signify impairment of the skin barrier, become responsible for activation of the immune system because they promote penetration of pathogens or allergens, thereby creating the vicious circle for the development of atopic dermatitis. Our results demonstrated the roles of Th2 cytokines (IL-4 and IL-13) and the proinflammatory cytokine (TNF-α) in the morphological alterations such as tissue spongiosis, hypogranulosis, and hyperproliferation. Epidermal barrier damage, which are featured by reduced differentiations identified by altered mRNA and protein expression levels of suprabasal markers (including cornified envelope proteins), represent one of the hallmarks of canine atopic dermatitis\u0026ndash;like characteristics in our study.\u003c/p\u003e \u003cp\u003eSpongiosis that describes the presence of intercellular oedema is a characteristic histopathologic appearance in diseases of the skin, such as atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, certain types of psoriasis and pemphigus. Spongiosis in the epidermis are characterized by impairment or loss of cohesion between keratinocyte and the influx of fluid \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In our investigation, we found that IL-4, IL-13, and TNF-α each induce spongiosis independently. The mechanism of spongiosis remains unclear. A study revealed that IL-4, IL-13, and IFN-γ are capable of inducing epidermal spongiosis accompanied by heightened hyaluronic acid (HA) accumulation and reduced E-cadherin expression \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. This indicates a potential osmotic flow of water into the intercellular compartment of the epidermis due to hyaluronan accumulation and weakened adherens junctions. Apoptosis may also contribute to the formation of spongiosis, particularly evident when exposed to TNF-α, as evidenced by the frequent presence of apoptotic structures in our study. TNF-induced apoptosis as an extrinsic apoptotic pathway via TNF-R1 may explain the spongiosis caused by apoptosis-driven cell separation and subsequent fluid accumulation in the epidermis \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These findings suggests that exact mechanisms leading to spongiosis can vary or be multifactorial depending on the underlying disease.\u003c/p\u003e \u003cp\u003eFurthermore, TNF-α has a myriad of pro-inflammatory effects on the skin, different from atopy-associated cytokines, and which is a major mediator in the pathogenesis of many other inflammatory disorders, e.g. rheumatoid arthritis, Crohn\u0026rsquo;s disease, and psoriasis in humans. In the chronic phase of CAD, TNF-α also plays a role involved in the general inflammatory reaction. TNF-α is reported to have a mild inhibitory effect on keratinocyte proliferation \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, which was not identified in our study, probably the effect depends on the concentration of TNF-α used in various experiments.\u003c/p\u003e \u003cp\u003eEpidermal differentiation and turnover are a complex process \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Keratinocytes originate from proliferative cells in the basal layer of the epidermis and mature into flattened, dead cells in the outermost layer. Keratinocytes in the basal layer express K5 and in the suprabasal layer express K10. Keratohyalin granules, composed of keratin and keratin-binding proteins like FLG or IVL or LOR, form in the stratum granulosum. The stratum granulosum cell layer is sealed by functional tight junctions in the epidermis. Proteins within tight junctions, such as CLD1 and OCD, are associated with CAD, showing decreased expression and abnormal distribution patterns in atopic dogs \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, in our study, the transcriptional expressions of CLD1 and OCD did not change significantly following exposure to Th2 cytokines and/or TNF-α. This observation is consistent with the findings by using a 3D model using normal human skin biopsies \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The mechanisms behind the reduced expression of tight junction molecules in the atopic epidermis have not been fully elucidated, which was found associated to other cytokines and \u003cem\u003eStaphylococcus\u003c/em\u003e species-dominant microbiome dysbiosis \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The stratum corneum, most crucial for the epidermal barrier, consists of terminally differentiated keratinocytes (\u0026lsquo;corneocytes\u0026rsquo;) embedded in an extracellular lipid-rich matrix, known as the \"bricks and mortar\" structure \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. That the critical role of the stratum corneum works as air-fluid barrier in the skin barrier is increasingly recognized as the primary focus in AD, and also in CAD, too. Cornified envelope, assembled by terminally differentiated keratinocytes, comprises proteins like FLG, IVL, LOR, periplakin, envoplakin, and the small proline-rich protein family (SPRRs) \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. It was reported reduced expressions of CE proteins occur in both lesional and non-lesional skin of atopic dogs \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Similarly in our research, Th2 cytokines and TNF-α decrease the expression of CE proteins (FLG, IVL, and LOR). Th2 cytokines more significantly decreased the expression of cornified envelope (CE) proteins (FLG, IVL, and LOR) with increased proliferation compared to TNF-α, suggesting distinct underlying influenced pathway. In contrast, markers of basal cells (K5) and tight junction proteins (CLD1 and OCD) in stratum granulosum did not exhibit significant changes or trends following exposure to Th2 cytokines or TNF-α. These findings indicate that after the induction of Th2 cytokines and TNF-α, keratinocytes specifically modify the suprabasal structural components and/or the barrier function, particularly in the stratum corneum, which is consistent with the finding in patients with AD \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our study, we did not observe any significant change in the size of organoids following the administration of Th2 cytokines. This finding did not correspond with epidermal hyperplasia and hyperkeratosis in atopic skin \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The inconsistency may be partially due to the fact that the 8-day culture period of the organoid model is likely insufficient to recapitulate the real epidermal turnover, which typically occurs over around 30 days in native human skin \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The simplicity of this epidermal model, consisting of only keratinocyte clusters, also limits its ability to fully mimic the function and structure of real epidermis. Furthermore, the current epidermal organoid model is a basal-out model, and does not form cornified layers facing the surrounding environment directly. Thus, barrier function tests such as transepidermal water loss and permeability assays are hard to achieve in this model system. In CAD, less dense lipid matrix is also a prominent feature in the damaged skin barrier, leading to a fragile and dry epidermis, which was not investigated in the present study. Although IL-4 and IL-13 are not major factors in inducing apoptosis nor directly involved in the apoptotic pathway, a study by Kamsteeg et al. (2011) found that IL-4 and IL-13 triggered DNA fragmentation and cell death in keratinocytes using a human skin equivalent model. In our study, after performing IHC staining for caspase-3, no perceivable differences were observed between the Th2 cytokine groups and the control. We observed only a few apoptotic cells in our cPEOs, carrying low sensitivity. However, in TNF-α group, an increased number of positive cells were identified. These findings suggest that increased apoptosis of keratinocytes in CAD is primarily driven by factors other than IL-4 and IL-13. These limitations and uncertainties are anticipated to be addressed in the future research.\u003c/p\u003e \u003cp\u003ePathophysiological concepts underlying the development of CAD are multifactorial and skin barrier defects in CAD result from the dysregulation of multiple pathways. Nevertheless, our epidermal organoid system derived from normal skin samples offers a robust \u003cem\u003ein vitro\u003c/em\u003e model to tease out roles of each cytokine in the disease progression. To our knowledge, this is the first study to investigate the effects of cytokines using epidermal organoids, even within the broader field of human medicine. Moreover, as in humans, CAD is a group of heterogeneous diseases caused by genetic variants that can differ among individuals, leading to unpredictable responses to therapy \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. For example, loss-of-function mutations in FLG, the gene encoding profilaggrin and filaggrin, is a significant genetic predisposing factor for AD [33] but implicated in only some atopic dogs [8]. Given the unclear genetic background of each CAD case, we assume the canine epidermal organoid system derived from patient skin samples preserves the genetic information and has the great potential to be a platform for testing therapeutic options tailored to each individual dog. However, obtaining atopic skin samples is challenging because CAD diagnosis is typically made by excluding other diseases, without necessarily requiring a biopsy. Further research is planned to artificially create a model with FLG knockout mimicking clinical CAD cases and also to test the feasibility of epidermal organoids as a validated tool to screen CAD treatment options.\u003c/p\u003e \u003cp\u003eIn conclusion, it is suggested that the present model system can be used to study CAD, recognising the critical role of the stratum corneum plays as the maintenance of the air-fluid barrier and which appears to be a primary defect in the development of CAD. Th2 cytokines and TNF-α elicit a CAD-like phenotype including epidermal spongiosis, and reduced epidermal differentiation especially in stratum corneum, Th2 cytokines increased epidermal proliferation and TNF-α appeared to induce cellular apoptosis. It is anticipated cytokine-supplemented cPEOs could serve as an excellent candidate for the testing of prospective therapies for CAD.\u003c/p\u003e "},{"header":"Materials \u0026 Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eIsolation and culture of canine primary keratinocyte\u003c/h2\u003e \u003cp\u003eSkin samples with no visible abnormalities were obtained from freshly scavenged tissues that were submitted for biopsy. Tissue scavenging was approved by the Animal Ethics Committee (AEC) of the University of Melbourne (Ethics ID #22006). All methods were performed in accordance with the University of Melbourne\u0026rsquo;s policies on the use of animals for research, in compliance with the Australian Code for the Care and Use of Animals for Scientific Purposes. A representative skin sample was processed for histology to ensure that there were no other significant pathological changes. Keratinocytes were isolated by the two-step digestion. Isolated keratinocytes were collected and immediately flash frozen.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eOrganoid culture and cytokine treatment\u003c/h3\u003e\n\u003cp\u003eOrganoids were cultured as previously described with some modifications \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Keratinocytes within soluble Matrigel were plated on the surface of previously layered Matrigel in the organoid culture medium with/without cytokines. Organoid media formulations were as indicated in the Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online. The cytokine concentrations and combinations for six groups are A) Control; B) 30 ng/mL IL-4; C) 30 ng/mL IL-13; D) 5 ng/mL TNF-α; E) 30 ng/mL IL-4, 30 ng/mL IL-13; F) 30 ng/mL IL-4, 30 ng/mL IL-13 and 5 ng/mL TNF-α. The day of seeding cells was defined as Day 0. Fresh culture medium with cytokines was replaced every 3 days until Day 8. On Day 7, phase contrast images were captured by an inverted microscope with camera (CKX-53 and DP23; Olympus).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin and Eosin (HE) Staining and Immunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eOrganoids were directly fixed in 4% paraformaldehyde (PFA) at room temperature for 2 h after removing the medium. Fixed samples were then embedded in a solid agarose pad followed by paraffin. Paraffin-embedded samples were cut at 3 \u0026micro;m and slides were stained with H\u0026amp;E and PAS after deparaffinization. As routine IHC methods, tissue sections were deparaffinized and rehydrated in xylene and a graded series of ethanol solutions, and then microwaved for antigen retrieval. After endogenous peroxidase block and incubation with antibodies, the slides were incubated with secondary antibody REAL Envision Detection System Peroxidase/DAB+, mouse/rabbit (Dako) according to the manufacturer\u0026rsquo;s instructions. All antibodies used are listed in Supplementary Table S2 online. Negative controls included incubation with non-immune goat serum instead of primary antibody. Dog skin was used as the positive control. Images were obtained using a light microscope with camera (BX-41 and DP74; Olympus).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRNA Extraction and Real-Time RT-qPCR\u003c/h2\u003e \u003cp\u003eOrganoid samples were lysed by TRIzol reagent (Invitrogen) and RNA was extracted following the manufacturer\u0026rsquo;s instructions. The RNA purity and integrity were measured using NanoPhotometer N120 (IMPLEN). A260/280 were in the desired range (1.80\u0026ndash;2.20) for all tested samples. The extracted RNA was reverse transcribed using GoScript\u0026trade; Reverse Transcriptase Mix, Oligo(dT) (Promega) and used for first-strand complementary DNA (cDNA) synthesis as per manufacturer\u0026rsquo;s instructions. Quantitative real-time PCR for the selected gene and calculations of cycle threshold (Ct) values were performed using Rotor-Gene Q (Qiagen) with a system using GoTaq\u0026reg; Flexi DNA polymerase (Promega, USA) according to the manufacturer\u0026rsquo;s protocol. The primers used are listed in Supplementary Table S3 online. All primers were validated by generating standard curves with R2 value \u0026gt;99%. As a stably expressed gene in canine skin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is selected as the reference gene. The mRNA expression levels was normalized to GAPDH expression based on the ΔCt method and calculated based on fold gene change (2^-(∆∆Ct)) \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The experiment was performed in duplication while differences between two Ct values in the same repetition should be less than 0.5.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eUltrastructural Study: Transmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eOrganoid samples for transmission electron microscopy were fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer according to the standard procedures and embedded in epon resin. Ultrathin sections (70\u0026ndash;85 nm) were cut, stained with uranyl acetate and lead citrate, and observed with a FEI Talos L120C cryo electron microscope (Thermo Fisher Scientific) at 60 kV and a CCD (1024 x 1024 pixel) camera was used for image recording.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was all conducted by GraphPad Prism version 10.2.0. For the analysis of intercellular spaces measured under TEM, five representative photos (11000\u0026times;) for each group were captured to measure the lengths of intercellular spaces. A two-way ANOVA was performed, followed by Tukey's multiple comparisons test to compare each treatment group with the control. For gene expression analysis using fold gene change (2^-(∆∆Ct)), an unpaired Student\u0026rsquo;s t-test was performed to compare each treatment group with the control. For the IHC results for Ki67, five organoids with the most advanced differentiation and similar size were selected from each group. The labelling index was determined by calculating the average percentage of Ki67 immunopositive pixels relative to the total area pixels of the organoid, with pixel data obtained using ImageJ. Relative Ki67 immunopositive pixels were statistically analyzed using an unpaired Student\u0026rsquo;s t-test (two tailed) to compare each treatment group with the control.\u003c/p\u003e \u003cp\u003eA P-value of \u0026le;\u0026thinsp;0.05 was considered statistically significant. Data from at least three independent experiments are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eEthics Approval Statement\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue scavenging was approved by the Animal Ethics Committee (AEC) of the University of Melbourne (Ethics ID #22006) before the investigation. However, AEC approval for activities involving scavenged tissue is not required because no animals were harmed or killed for the purpose of this study. All experiments were conducted in accordance with the University of Melbourne\u0026rsquo;s policies on the use of animals for research, in compliance with the Australian Code for the Care and Use of Animals for Scientific Purposes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAvailability of Data and Materials\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eCompeting Interest Statement\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by the China Scholarship Council (CSC) \u0026ndash; University of Melbourne Ph.D. Scholarship organized by CSC (File No. 202008320396) and University of Melbourne.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAuthors\u0026apos; Contributions\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBC, RFS, and SRG conceptualized and designed the study. BC was responsible for data acquisition, analysis, and interpretation. BC, OSO, and MB contributed to the investigation and methodology. BC prepared the initial draft of the manuscript. All authors reviewed, revised, and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAcknowledgements\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful for support from the Asia-Pacific Centre for Animal Health group (Melbourne Veterinary School, Faculty of Science, the University of Melbourne) and Ian Holmes Imaging Centre (Bio21, the University of Melbourne). We thank Dr. Dijina Swaroop (Department of Medicine, Monash University) for sharing scientific expertise.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eDeclaration of generative AI in scientific writing\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this manuscript, generative AI tools (ChatGPT by OpenAI and Grammarly) were only used to improve readability and language. All scientific concepts, conclusions, and interpretations were developed by the authors.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHalliwell, R. Revised nomenclature for veterinary allergy. \u003cem\u003eVet. Immunol. 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Sci.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 220\u0026ndash;223. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0923-1811(95)00407-J\u003c/span\u003e\u003cspan address=\"10.1016/0923-1811(95)00407-J\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1995).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"canine atopic dermatitis, canine primary epidermal organoids, Th2 cytokines, skin barrier, molecular signature","lastPublishedDoi":"10.21203/rs.3.rs-6308044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6308044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAtopic Dermatitis (AD) is a chronic inflammatory and pruritic skin disease that affects both human and veterinary patients. Canine atopic dermatitis is prevalent in 27% of the canine population. This frequently encountered disease leads to discomfort and reduced quality of life in affected animals. Our methodology utilizes a well-controlled model system of the canine primary epidermal organoids (cPEOs) derived from normal canine keratinocytes and exhibits morphological characteristics and key marker proteins consistent with normal canine skin. We investigated the direct impact of specific immune mediators, namely IL-4, IL-13 (Th2 cytokines), and TNF-α (a pro-inflammatory cytokine), both individually and in combination, on skin barrier components using this model system. The results demonstrated that cytokines induce CAD-like morphological and molecular characteristics in the canine epidermal organoid system, including epidermal spongiosis and reduced suprabasal epidermal differentiation. Th2 cytokines increased epidermal proliferation and TNF-α appeared to induce cellular apoptosis. These findings indicate that the canine epidermal organoid system holds promise as a valuable tool for understanding the pathogenesis of AD in both humans and veterinary patients and can become a potential platform for assessing individual treatment options or screening drug candidates for canine atopic cases.\u003c/p\u003e","manuscriptTitle":"TNF- α and TH2 Cytokines Induced Canine Atopic Dermatitis–like Morphologic and Molecular Characteristics in the Canine Epidermal Organoid Culture System","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 09:18:58","doi":"10.21203/rs.3.rs-6308044/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"755a0e9c-bc78-467c-b3d0-0153bb31ec2c","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47154273,"name":"Biological sciences/Cell biology"},{"id":47154274,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-06-12T06:39:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-16 09:18:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6308044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6308044","identity":"rs-6308044","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}