Skin-Homing Exacerbates Atopic Dermatitis-like Symptoms by Participating in Th2 Responses in BALB/c Mice

Skin-Homing Exacerbates Atopic Dermatitis-like Symptoms by Participating in Th2 Responses in BALB/c Mice | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 12 February 2025 V1 Latest version Share on Skin-Homing Exacerbates Atopic Dermatitis-like Symptoms by Participating in Th2 Responses in BALB/c Mice Authors : Huimin Yuan 0000-0001-9005-4739 , Aorou Li , Yanru Yu , Jingwei Sun , Xinyue Li , Huimin Guo , Jing Feng , Shujing Zhang , Yang Tang , and Fengjie Zheng [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173935869.91835425/v1 289 views 136 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: The ”internal and external crosstalk” between skin barrier dysfunction and immune inflammatory response is the main pathogenesis of atopic dermatitis (AD), with the Th2 immune inflammatory response being the main link. Migration of circulating lymphocytes to inflammatory sites is essential for the immune response. Although AD is characterized by lymphocyte infiltration into the dermis, its skin-homing effect remains poorly defined. Methods: In this study, we induced an AD mouse model using DNFB to observe the skin barrier function changes and examine the Th2 immune inflammatory response. Additionally, we analyzed the homing of Th2 cells from peripheral blood to the skin and the gene and protein expression of key homing molecules. Furthermore, we explored the relationship between skin-homing and AD. Results: In the AD mouse model, skin barrier damage was observed, along with the skin-homing of Th2 lymphocytes and related immune inflammatory responses. Correlation analysis showed a significant positive correlation between the levels of homing cells and homing molecule proteins in peripheral blood and the protein levels of skin Th2 immune cells and transcription factors. Additionally, there was a significant linear relationship between the gene levels of homing molecules and AD symptoms. Conclusions: This study found that AD mice mainly exhibited skin barrier damage and Th2 cell immune responses, along with the phenomenon of skin-homing of Th2 lymphocytes, which can exacerbate AD symptoms. By intervening in the skin-homing of Th2 lymphocytes, AD symptoms can be effectively improved, and recurrences could be prevented. 1 INTRODUCTION Atopic dermatitis (AD) is a common chronic, recurrent inflammatory skin disease characterized by polymorphic skin lesions, lichenification, dry skin, and severe itching. Various neuropsychiatric disorders and allergic reactions can accompany it. Globally, the prevalence of AD among adults reaches 7% to 10%, and among children, it can be as high as 25%, showing a trend of increasing year by year [1] . Its long duration, easy recurrence, and difficulty in curing severely affect the quality of life of patients and their families. The etiology and pathogenesis of AD are complex and closely related to factors such as genetics, environment, skin barrier damage, and immune dysregulation [2] . The important pathological mechanism of ”internal-external crosstalk” between skin barrier dysfunction and immune imbalance has become an industry consensus for the occurrence and development of AD [3] . The ”external” factor represents skin barrier dysfunction caused by genetic factors or acquired environmental factors, while the ”internal” factor refers to systemic allergic inflammation resulting from immune dysregulation in the body [4] . The interaction between skin barrier dysfunction and the immune inflammatory disorder affects the process of occurrence, development, and prognosis in AD. In recent years, there has been a general consensus in the academic community that immune and inflammatory responses play a more critical role in AD as a systemic inflammatory disease. Studies have indicated that the imbalance of Th1/Th2 immune responses serves as the foundation and key link in the pathogenesis of AD, leading to abnormal cytokine expression and elevated inflammation levels in patients [5-6] . As research progresses, both clinical and experimental studies have confirmed that Th2-type inflammatory responses are activated in both acute and chronic phases of AD [7-8] making it a crucial mechanism in the development of the disease [9-10] . The activation of Th2 cells promotes the expression and release of cytokines such as interleukin-4 (IL-4), IL-10, and IL-13. IL-4 can continuously activates Th2 cells while inhibiting Th1 cell activation and the production of interferon-gamma (IFN-γ). Cytokines like IL-4 and IL-13 can suppress the expression of skin barrier structural proteins such as filaggrin and loricrin, thereby exacerbating skin barrier dysfunction [11] . The impaired skin barrier function, in turn, activates Th2 immune responses by triggering factors like thymic stromal lymphopoietin, intensifying inflammatory reactions, forming an ”internal-external crosstalk” [12] , leading to recurrent and persistent AD. Circulating lymphocytes in the blood selectively traverse capillary high endothelial venules (HEV), migrating directionally to settle in sites of skin inflammation. This phenomenon is known as lymphocyte homing to the skin or skin-homing. Studies have shown that lymphocytes homing to the skin play a crucial role in the pathogenesis and progression of various skin diseases, including allergic and inflammatory skin conditions [13] . The molecular basis of lymphocyte homing lies in the interaction between homing receptors on the lymphocyte surface and adhesion molecules, known as vascular addressins, on the surface of vascular endothelial cells. Cutaneous lymphocyte-associated antigen (CLA) is a key receptor for lymphocyte homing on T-cells with skin tropism, and is considered a biomarker of skin immune activation [14] . CLA + T-cells exhibit preferential responsiveness to the skin microenvironment and can migrate to inflamed skin. After selective binding to the vascular adhesion molecule, the endothelial leukocyte adhesion molecule-1 (E-selectin), expressed on endothelial cells of HEV, these cells mediate lymphocyte adhesion and rolling, providing favorable conditions for T-cell adhesion [14] . Subsequently, another important receptor for skin lymphocyte homing, CC chemokine receptor 4 (CCR4), binds to its ligand, thymus, and activation-regulated chemokine (TARC/CCL17), acting as a ”navigator” for T-cells [15] . This induces the activation of lymphocyte integrin, lymphocyte function-associated antigen 1 (LFA1), which then tightly adheres to intercellular adhesion molecule-1 (ICAM1) on endothelial cells. This process mediates the migration of lymphocytes to extravascular sites of skin inflammation, where they exert their immune function, completing the process of lymphocyte skin homing [16] . Increasing evidence suggests that lymphocyte skin homing plays a crucial role in the pathogenesis of AD [17-18] . However, whether skin-homing exacerbates the Th2 immune inflammatory response and skin barrier dysfunction in AD by regulating the migration and colonization of Th2 lymphocytes to sites of skin inflammation remains to be further investigated and elucidated. Therefore, this study intends to explore the mechanism and influence of lymphocyte skin-homing on AD on the basis of reproducing important AD features such as skin barrier dysfunction and Th2 immune inflammatory response by replicating the AD mouse model. 2 MATERIALS AND METHODS A complete description of all methods used in this study is supplied in Data S1. 2.1 Ethics Statement Experiments in this study were performed strictly according to the principles and procedures outlined by Animal Ethics Committee of the Beijing University of Chinese Medicine (BUCM-4-2022083101-3087, Beijing, China). All efforts were made to minimize suffering and ensure the highest ethical and humane standards according to the 3R principles [19] . 2.2 Animals The specific pathogen-free male BALB/c mice aged 6-8 weeks were purchased from Beijing Vital River Laboratory Animal Technology (SCXK [Beijing] 2016-0006, Beijing, China). The mice were housed in individually ventilated cages at the Animal Facility of Beijing University of Chinese Medicine, where they were acclimatized to a controlled environment with a temperature of 22-25℃, relative humidity of 33%-40%, and a 12-h light/dark cycle. They had free access to food and water and were able to breathe normally. 2.3 DNFB-induced Model of AD Induction of an AD-like skin phenotype by Dinitrofluorobenzene (DNFB) was based on Myoung-schook Yoou, et al [20] . In short, DNFB (No. M24218031, Macklin, Shanghai, China) was repeatedly applied to mouse skin to produce an AD-like mouse model (200ul of 1% DNFB stimulation for two weeks and 0.2% DNFB stimulation for 3 weeks, twice a week). We have successfully replicated AD-like mouse models according to this method in previous research [21-22] 。 2.4 Skin Barrier All details of observation and evaluation of skin barrier function are outlined in Data S1. 2.5 Cell Preparation and Flow cytometry Detailed methods for isolation of cells from skin tissue and peripheral blood, cell staining for flow cytometry are described in Data S1. 2.6 ELISA, Western Blot and RT-qPCR Total IgE in serum, the protein expression of GATA3, CLA and CCR4 in skin, and mRNA relative expression of skin-homing are outlined in Data S1. 2.7 Immunohistochemistry and Multiplex immunofluorescence staining All details of immunohistochemistry and multiplex immunofluorescence staining are presented in Data S1. 2.8 Quantification and statistical analysis All data were presented as means ± SD. An unpaired two-tailed student’s t-test with Welch’s correction was used to determine the Statistical significance of the two groups. Pearson correlation and linear regression analysis were used to assess the relationships between the skin-homing and AD symptom presentations. All statistical analyses were performed in Prism version 9.0 (GraphPad, California, SD, United States). A P -value of less than 0.05 was considered statistically significant. Significance was indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 3 RESULTS 3.1 Skin barrier function A growing number of studies indicate that the disruption of skin barrier function, as a significant contributor to AD pathogenesis, not only leads to skin water loss resulting in dryness and itching but also increases the sensitivity of lesion sites to external chemicals or pathogens, promoting inflammatory responses and causing chronic, persistent skin inflammation [29-30] . This, in turn, exacerbates the impairment of skin barrier function, forming a vicious cycle. In this experiment, we induced AD-like lesions in BALB/c mice using hapten DNFB to replicate an AD mouse model. This model exhibits the characteristic skin lesions of chronic or chronically relapsing dermatitis, such as scaling, lichenification, and dry skin [21] . Additionally, AD mice show pronounced itching and scratching behaviors, along with anxiety- and depression-like emotional disturbances [28] , which better reflect the clinical manifestations of AD (Figure 1A-1B). Specifically, the AD mice model exhibited significantly increased skin lesion scores, scratching frequency, skin moisture, oil content, and inflammatory status (average P < 0.001, Figure 1C-1G). Histological changes using Masson staining revealed that the lesioned skin of AD mice showed markedly thickened, red-stained stratum corneum, primarily due to hyperplasia of the spinous cell layer. The dermis also shows visibly thinner blue-stained collagen fibers (Figure 1H). Suggesting an impairment of the skin barrier function. Figure 1. Skin barrier function evaluation of mice in each group Notes: (A) Back skin and its microscopic manifestations. (B-G) Comparison of weight, number of scratches, and skin score, moisture, oil content, and inflammatory status (n = 8). (H) Morphological observation of skin by Masson staining. ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. the CONT group. CONT, normal mice; AD, DNFB-treated mice. 3.2 Th2 immune inflammatory response In this study, an AD animal model was established through repeated skin stimulation with the hapten DNFB, and the immune response-related investigations were conducted under the premise of confirming significant skin barrier dysfunction in this AD model. In this experiment, flow cytometry detected a significant decrease in the proportion of CD3 + CD4 + Th cells among lymphocytes in the skin tissue of AD mice ( P = 0.022), a notable increase in the proportion of CD3 + CD8 + CTL cells ( P = 0.002), and there was a marked increase in the proportion of IL-4 + CD4 + CD3 + Th2 cells among lymphocytes ( P = 0.037), suggesting an increase of Th2 cells in the skin tissue of AD mice (Figure 2A-2F). And the GATA-binding protein 3 (GATA-3) is expressed exclusively in Th2 cells and is a Th2-specific transcription factor [34] . It can promote the synthesis of Th2 cytokines in differentiating or already differentiated Th0 cells [35] . In this experiment, the protein expression levels of GATA-3 in the skin of AD mice were significantly increased ( P = 0.037, Figure 2H-2I). Meanwhile, previous studies found that the serum levels of Th2 cell-related factor IL-4 were significantly increased, and the relative mRNA expression of IL-4 and IL-10 were also significantly upregulated in skin tissues [22] . Further toluidine blue staining showed massive infiltration of mast cells in the skin tissue of AD mice (P < 0.001, Figure 2J-2K). Again, it further suggesting that the activation of Th2 inflammatory response is a key mechanism leading to the development of AD [9-10] . Figure 2. Th2 immune inflammatory response of mice in each group Notes: (A) Skin IL-4 + Th2 cell related flow gating. (B-F) comparison of CD3 + T cell%, CD4 + CD3 + Th cell%, CD8 + CD3 + CTL cell%, CD4 + CD3 + Th cell% /CD8 + CD3 + CTL cell%, IL-4 + CD4 + Th2 cell%, respectively (n = 4). (G) Serum IgE levels (n = 8). (H-I) Western blot pattern of GATA-3 protein (β-Actin was the internal reference), comparison of the relative expression of GATA-3 protein in the skin of mice in each group (n = 4). (J-K) Histomorphology observation of skin mast cell staining and comparison of mast cell count (n = 8). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. the CONT group. CONT, normal mice; AD, DNFB-treated mice. 3.3 CLA + and CCR4 + (CD4 + Th2) in peripheral blood CLA, expressed by skin-homing T cells, is a post-translational modification of P-selectin glycoprotein ligand-1 (PSGL-1) catalyzed by fucosyltransferase VII (FucT-VII). IL-12 is the best-known mediator that induces FucT-VII expression, thereby conferring skin-homing ability to Th1 and Th2 cells [38] . AD is predominantly characterized by a Th2 immune response; therefore, this study examined the Th2 cell-related skin-homing phenomenon of peripheral blood cells. In this experiment, the proportion of CD3 + CD4 + Th cells among lymphocytes in the peripheral blood of AD mice was significantly reduced ( P = 0.011), while the proportion of CD3 + CD8 + CTL cells among lymphocytes was significantly increased ( P = 0.038), and the significantly increased ratio of CD3 + CD4 + Th cells to CD3 + CD8 + CTL cells indicates a severe immune inflammatory response in these AD mice ( P = 0.002), suggesting a severe immune-inflammatory response in the AD mice (Figure 3A-3E). Simultaneously, the proportions of both CLA + Th2 cells and CCR4 + Th2 cells among Th cells in the peripheral blood of AD mice were increased significantly ( P = 0.031 and P = 0.025), indicating the presence of skin-homing of circulating Th2 lymphocytes in these AD mice (Figure 3A, 3F-3G). Figure 3. Expression of CLA + , CCR + (CD4 + Th2) cells in peripheral blood of mice in each group Notes: (A) Peripheral blood CLA + , CCR + (CD4 + Th2) cell related flow gating. (B-H) are CD3 + T cell%, CD4 + CD3 + Th cell%, CD8 + CD3 + CTL cell%, CD4 + CD3 + Th cell% /CD8 + CD3 + CTL cell%, CLA + CD4 + Th2, respectively Comparison of cell%, CCR4 + CD4 + Th2 Cells %, CCR4 + CLA + CD4 + Th2 Cells % (n = 4). * P < 0.05, ** P < 0.01 vs. the CONT group. CONT, normal mice; AD, DNFB-treated mice. 3.4 Expression of skin homing molecules in skin The process by which lymphocytes selectively migrate from the bloodstream to the skin is mediated by the interaction between skin-homing receptors on lymphocyte surfaces and adhesion molecules on endothelial cell surfaces, and is further regulated by multiple factors, including the tissue microenvironment, cytokines, and superantigens. Lymphocytes that home to the skin play a crucial role in the development and progression of various skin diseases, including allergic and inflammatory skin conditions [39] . In this experiment, the mRNA expression levels of CLA ( P = 0.001), E-selectin ( P < 0.001), CCR4 ( P < 0.0001), CCL17 ( P < 0.0001), LFA1 ( P = 0.010), and ICAM1 ( P = 0.002), which are key molecules for lymphocyte homing to the skin, were significantly upregulated in AD mice skin (Figure 4A-4F). Additionally, the protein expression levels of CLA and CCR4 were also notably increased ( P = 0.039 and P = 0.008, Figure 4G-4I). There was a marked increase in the deposition of E-selectin and CCL17 positive proteins in the dermal tissue (average P < 0.0001, Figure 4J-4K). Furthermore, fluorescent labeling of LFA1 and ICAM1 was visibly increased in the skin tissue of AD mice, and there was a significant increase in the fluorescent adhesion binding labeling of LFA1 and ICAM1 (Figure 4L). Therefore, it can be concluded that there is a widespread overexpression of key factors for lymphocyte homing to the skin in AD. Figure 4. Expression of molecules of skin homing in each group of mice skin Notes: (A-F) shows the mRNA relative expression levels of CLA, E-selectin, CCR4, CCL17, LFA1, and ICAM1 in each group of mice (n = 8), respectively. (G-I) Western blot strip diagram of CLA and CCR4 protein (β-Actin was the internal reference), expression of CLA and CCR4 protein in the skin of mice in each group (n = 4). (J-K) shows the localization and expression of E-selectin and CCL17 in skin (n=6), respectively. (L) Adhesion localization of LFA1 and ICAM1. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. the CONT group. CONT, normal mice; AD, DNFB-treated mice. 3.5 Correlation analysis between skin-homing molecules and AD symptoms Studies have found that in AD patients of different age groups, the expression of CLA + Th2 cells is significantly higher compared to normal individuals. Additionally, the expressions of serum TARC/CCL17, skin CCR4 and E-selectin are specifically increased in AD patients and correlate with disease severity [40-41] . And when the expression of CCL17 and CCR4 is decreased, both dermatitis symptoms and type 2 inflammatory factors levels improve in AD mice [42] . In this study, we observed a positive linear correlation between key skin-homing cells in the peripheral blood of AD mice, including CLA + Th2, CCR4 + Th2, CLA + CCR4 + Th2, and skin cells of IL-4 + Th2. Furthermore, there was a positive linear correlation between the protein levels of key homing molecules CLA and CCR4, and the protein level of the Th2-specific transcription factor GATA3 in skin tissue. These findings suggest that lymphocyte skin homing can exacerbate the infiltration of Th2 lymphocytes and related immune inflammatory responses in the local skin tissue of AD mice (Figures 5A-5B). Further analysis revealed significant positive or negative linear correlations between the mRNA levels of key skin-homing molecules (CLA, E-selectin, CCL17, CCR4) and AD evaluation indicators (such as Score, number of scratching, body weight, moisture, oil content, and inflammatory status) (Figures 5C-5F). Additionally, there was a significant positive correlation between LFA1 and ICAM1 mRNA levels and lesion Scores, moisture, oil content, and inflammatory status (Figures 5G-5H). These results suggest that intervening in the lymphocyte skin-homing process could influence skin Th2 immune responses or directly improve AD symptoms, providing valuable insights for the treatment of AD. Figure 5. Correlation analysis between key molecules of lymphocyte skin homing and AD symptoms Notes: (A) Correlation analysis of peripheral blood CLA + Th2 cells%, CCR4 + Th2 Cells%, CLA + CCR4 + Th2 cells% and skin IL-4 + Th2 cells%. (B) Correlation analysis between expression levels of skin-homing key molecules CLA, E-selectin and GATA-3 protein. (C-H) The correlation between the mRNA expression levels of CLA, E-selectin, CCR4, CCL17, LFA1, ICAM1 and skin Score, number of scratching, body weight, moisture, oil content, and inflammatory status in AD mouse models. 4 DISCUSSION AD is a common chronic inflammatory skin disease characterized by polymorphic skin lesions, dry skin, and recurrent itching. It is has high incidence and recurrence rate, is difficul to cure, and often presents with various psychological and economic burdens during disease progression, making it one of the research hotspots and difficulties in the field of dermatology in recent years. In this study, an AD animal model was established by repeatedly stimulating the skin with the hapten DNFB. Based on the premise that this AD model exhibits significant skin barrier dysfunction, it was found that there is a phenomenon of skin-homing of Th2 lymphocytes in this AD mouse model. This can exacerbate the infiltration of Th2 lymphocytes in the skin and related immune and inflammatory responses, thereby aggravating AD symptoms and leading to recurrent episodes. Research has confirmed that individuals with elevated levels of Th2 cytokines in cord blood cells have a significantly increased risk of developing AD in the future. The elevated IgE levels in the peripheral blood of AD patients are positively correlated with excessive activation of Th2 cells. Among these cytokines, IL-4, IL-5, and IL-10 are recognized as the main Th2 cytokines involved in the pathogenesis of AD [43] . The differentiation of Th0 cells into Th2 cells requires the induction of interleukin IL-4, which is produced by eosinophils, mast cells, or already differentiated Th2 cells. Consistently, we found that the proportion of IL-4 + Th2 cells in the AD mice skin significantly increased as well as GATA-3 protein, and a large infiltration of mast cells in the skin, resulting the aggravation of pigment and inflammation. Combined with the previous study results, we can clarify that the Th2 immune inflammatory response in this AD mice is mainly Th2 cell activation. However, it should be noted that in this experiment, the serum IgE levels in the AD group of mice did not show a significant increasing trend. Some studies have shown that although IgE is one of the main diagnostic indicators of AD and is used as a criterion for AD classification, as well as an important measure for the condition of AD patients and the success of AD animal model establishment [5, 49] , the clinical diversity of AD has increased with the growing number of cases and the progress of clinical research. Some scholars have found that the serum IgE levels of approximately 20% to 40% of AD patients have no significant correlation with the occurrence and prognosis of the disease. This suggests that under immune regulation, IgE levels may exhibit phenomena that contradict traditional beliefs, and diagnosis should be based on a combination of serum inflammatory factors and specific clinical manifestations rather than solely on IgE levels [50-51] . Increasing evidence suggests that lymphocyte skin-homing plays a significant role in the pathogenesis of AD [52] . In this experiment, we observed a significant decrease in the proportion of CD4 + CD3 + Th cells among lymphocytes, a marked increase in the proportion of CD8 + CD3 + CTL cells, and a notable elevation in the ratio of CD4 + CD3 + Th cells to CD8 + CD3 + CTL cells in the peripheral blood of AD mice. Additionally, there was a pronounced increase in the proportion of CLA + CD4 + Th2 cells and CCR4 + CD4 + Th2 cells among lymphocytes, accompanied by an upward trend in the proportion of CCR4 + CLA + CD4 + Th2 cells. Simultaneously, the mRNA relative expression levels of key molecules related to skin-homing, such as CLA, CCR4, E-selectin, and CCL17, were significantly increased in the skin of AD mice. Additionally, the protein expression levels of CLA and CCR4, as well as the positive localization of E-selectin and CCL17 proteins, were notably elevated in the skin tissue. Furthermore, multiple immunofluorescence staining revealed a marked increase in the adhesive localization expression markers of LFA1 and ICAM1. These findings suggest the presence of homing of Th2 lymphocytes from peripheral blood to the skin lesions in this AD mice. Th2 lymphocytes in the blood selectively migrate to the site of skin inflammation, realizing homing and producing cytokines, which further affects the skin barrier, leading to repeated episodes of skin inflammation in AD. Future work will be needed to address in AD mice if skin tissue, like blood, is overexpressed within the homing of Th2 lymphocytes. Recent studies have reported a high degree of overlap between T cells in AD-affected skin and circulating T cells marked by CLA + and specific antigens [53] . In AD patients of different age groups, CLA + Th2 cell expression is significantly increased compared to healthy individuals. Both serum TARC/CCL17 and skin CCR4 and E-selectin expressions are specifically increased in AD patients and correlate with disease severity [40-41] , and the decreased expressions of CCL17 and CCR4 lead to improved dermatitis symptoms and reduced levels of Th2 inflammatory factors in AD mice [54] . In this experiment, our results indicated that the skin-homing of lymphocytes in peripheral blood can participate in and exacerbate the infiltration of Th2 cells and related immune inflammatory responses in the skin tissue of AD mice. Studies have indicated that Efalizumab can improve AD clinical symptoms by blocking the interaction of homing adhesion molecules [55] . During treatment, patients develop secondary CLA + T cell proliferation, and disease exacerbation occurs after treatment interruption. This suggests normal T cell recirculation/turnover between skin and blood, where tissue-resident memory T cells can migrate from the skin back into the blood [56] , exhibiting CLA + Th2 characteristics, along with increased expressions of GATA-3 and IL-13 [57] . Further correlation analysis revealed that, apart from body weight and scratching frequency, the major molecules involved in skin-homing exhibited significant or notable linear relationships with almost all other AD barrier function indicators. This might be attributed to the fact that body weight and scratching frequency are not directly involved in barrier function. Collectively, these findings suggest that lymphocyte skin-homing mediates the migration and infiltration of Th2 cells into the skin, thereby exacerbating the ”internal-external crosstalk” response. Effective regulation of lymphocyte skin-homing could be a key to maintaining stable therapeutic efficacy in AD. Although previous studies have captured the homing phenomenon in zebrafish using dynamic tracking methods [58] , the process of lymphocyte skin-homing mediating the migration and infiltration of Th2 cells into the skin cannot be replicated by the expression of related molecules in peripheral blood and skin tissue. This requires finding a more suitable and intuitive AD model for dynamic observation of the entire process in vivo. As skin homing is a multistep process that requires the interaction of additional molecules such as VLA-4, CCR10, CCL22, and VCAM-1 to mediate, as well as the high expression of CLA + natural killer (NK) cells playing a role (the frequency of CLA + CD56 bright and CLA + CD56 dim NK cell populations increases in the peripheral blood of patients with severe AD [59] ), further detailed studies are needed to reveal the impact of the interactions between these molecules and cells on AD. In addition, the occurrence of AD is closely related to innate immune cells such as granulocytes, macrophages, innate-like T cells, and innate lymphocytes, as well as the human microbiota (including skin microbiota) [60] . Lymphocyte skin homing in AD also involves an increase in various immune cells, changes in the expression of skin-homing markers, alterations in the composition of the microbiota, and disruption of skin barrier function [61] . These factors can both contribute to the onset and exacerbation of AD symptoms and are important reasons for inducing Th2 lymphocyte homing to the skin in AD, thereby triggering recurrent episodes of AD. The intricate relationships among these factors require further in-depth study. In summary, lymphocyte homing exhibits dynamism and complexity. AD is generally characterized by the overexpression of key factors involved in lymphocyte skin homing. This mediates the migration and infiltration of Th2 cells into the skin, exacerbating the ”internal-external crosstalk” and intensifying AD symptoms. As a result, AD often recurs and is difficult to cure. Effective regulation of Th2 lymphocyte in skin-homing may represent an important direction for maintaining stable efficacy in the treatment of AD. Author Contributions Yuan and R. Li performed experiments, analysed data and drafted the manuscript; Y. Yu, J. Sun, X. Li and H. Guo performed experiments and analysed data; J. Feng and S. Zhang directed and manipulated the staining of the histopathological sections, acquired and analysed data; F. Zheng and Y. Tang designed and supervised the study, edited the manuscript and acquired funding. All authors contributed to data discussion and review of the manuscript. Acknowledgements The authors would like to thank the scientific Research and Experimental Center of the College of School of Traditional Chinese Medicine of Beijing University of Chinese Medicine for providing equipment services and experimental operation sites, and thank the staff of the Experimental Animal Center of Beijing University of Traditional Chinese Medicine for caring the mice. Conflicts of Interest The authors have declared no conflict of interest. Data Availability Statement The data that supports the findings of this study are available in the Supporting Information of this article. ORCID Huimin Yuan https://orcid.org/0000-0001-9005-4739 Fengjie Zheng https://orcid.org/0000-0003-0450-388X Yang Tang https://orcid.org/0009-0007-3923-4997 References 1. M. R. Laughter, M. B. C. Maymone, S. Mashayekhi, et al., “The Global Burden of Atopic Dermatitis: Lessons From the Global Burden of Disease Study 1990-2017,” The British journal of dermatology 184, no. 2 (2021): 304-309. 2. 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Ständer, “Atopic Dermatitis,” The New England journal of medicine 384, no. 12 (2021): 1136-1143. 61. J. Clowry, D. J. Dempsey, T. J. Claxton, et al., “Distinct T cell Signatures are Associated with Staphylococcus Aureus Skin Infection in Pediatric Atopic Dermatitis,” JCI insight 9, no. 9 (2024): e178789. Supplementary Material File (figure-1.tif) Download 14.07 MB File (figure-2.tif) Download 18.24 MB File (figure-3.tif) Download 21.80 MB File (figure-4.tif) Download 29.04 MB File (figure-5.tif) Download 18.81 MB Information & Authors Information Version history V1 Version 1 12 February 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords animal models atopic dermatitis basic immunology basic mechanisms flow cytometry lymphocytes signal transduction t cells Authors Affiliations Huimin Yuan 0000-0001-9005-4739 Beijing University of Chinese Medicine School of Traditional Chinese Medicine View all articles by this author Aorou Li Beijing University of Chinese Medicine School of Traditional Chinese Medicine View all articles by this author Yanru Yu Beijing University of Chinese Medicine School of Traditional Chinese Medicine View all articles by this author Jingwei Sun Beijing University of Chinese Medicine School of Traditional Chinese Medicine View all articles by this author Xinyue Li Beijing University of Chinese Medicine School of Traditional Chinese Medicine View all articles by this author Huimin Guo Beijing University of Chinese Medicine School of Traditional Chinese Medicine View all articles by this author Jing Feng Beijing University of Chinese Medicine School of Traditional Chinese Medicine View all articles by this author Shujing Zhang Beijing University of Chinese Medicine School of Traditional Chinese Medicine View all articles by this author Yang Tang Beijing University of Chinese Medicine School of Traditional Chinese Medicine View all articles by this author Fengjie Zheng [email protected] Beijing University of Chinese Medicine School of Traditional Chinese Medicine View all articles by this author Metrics & Citations Metrics Article Usage 289 views 136 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Huimin Yuan, Aorou Li, Yanru Yu, et al. Skin-Homing Exacerbates Atopic Dermatitis-like Symptoms by Participating in Th2 Responses in BALB/c Mice. Authorea . 12 February 2025. DOI: https://doi.org/10.22541/au.173935869.91835425/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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