Combined Application of Dinitrofluorobenzene and Ovalbumin Induced AD-Like Dermatitis With Upregulated Helper T Cell Cytokines and Prolonged Th2 Response

doi:10.21203/rs.3.rs-1974685/v1

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Combined Application of Dinitrofluorobenzene and Ovalbumin Induced AD-Like Dermatitis With Upregulated Helper T Cell Cytokines and Prolonged Th2 Response

https://doi.org/10.21203/rs.3.rs-1974685/v1

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Backgroud: Progression of acute-to-chronic atopic dermatitis is accompanied by multiple helper T cell cytokine responses, but the mechanisms and relative importance of these changes remains unclear. There is no animal model for atopic dermatitis that recapitulates these cytokine responses.

Objective: We sought to build a novel mouse model for Atopic Dermatitis(AD) that recapitulates these helper T cell responses and some dynamic changes of cytokine responses in the progression of AD.

Methods: Female BALB/c mice were subjected to the application of Dinitrofluorobenzene(DNFB) and Ovalbumin(OVA) to induce AD-like dermatitis. Skin lesions and serum were collected from mice of acute and chronic phase to detect the changes of cytokine responses and other features of AD.

Results: Combined application of DNFB and OVA successfully induced AD-like dermatitis and histological changes as well as epidermal barrier dysfunction. In acute phase of AD-like dermatitis, Th2-associated cytokines were mainly increased in serum and skin lesions. In chronic phase of AD-like dermatitis,Th2-associated cytokines were still highly expressed, while Th1- and Th17-associated cytokines were also gradually increased. Compared with acute phase, the JAK-STAT signaling pathway was highly expressed in chronic phase of AD-like dermatitis.

Conclusion: The combined application of DNFB and OVA could be used to build a new mouse model for atopic dermatitis. This mouse model recapitulates the helper T cell responses and some dynamic changes of cytokine responses in the progression of acute-to-chronic in human AD. JAK-STAT signaling pathway plays a pivotal role in the chronicity of AD.

Atopic Dermatitis

Dinitrofluorobenzene

Ovalbumin

BALB/c Mice

Atopic dermatitis (AD) is one of the most common skin diseases that predominantly manifests as chronic inflammation, affecting up to 10%-20% of children and adults in developed countries[1, 2], and the prevalence is still increasing. Many patients with AD also suffer from allergic rhinitis and asthma along with intense itching and skin infection[3]. The hallmark clinical sign of AD is eczematous lesions, which may present acutely or chronically, it is unclear whether chronic and acute phases of AD are driven by different immune pathways or just represent a different extent of inflammatory response. AD is a result of skin barrier disruption and imbalanced innate and adaptive immune systems[4]. Data from human and animal studies suggest that AD is a Th2-dominant inflammatory skin disease at the acute stage followed by Th1 and Th17 involvement at the chronic stage[5, 6]. Activated Th2 cells release IL4 and IL13, inducing IgE class switching in B cells and secretion of antigen-specific IgE through the JAK-STAT signalling pathway, which may leads to the chronicity of AD[7].

Over the last several decades, many kinds of animal disease models have enabled us to deepen our understanding of the pathogenesis of AD[8]. Epidermal barrier dysfunction, skin-laden immune cells, and keratinocytes all play critical roles in the progression of AD and psoriasis. Thus, AD animal models are frequently built by epicutaneous application of exogenous allergens[9, 10]. Human and animal skin exhibit many species-specific differences, such as tissue architecture, frequency of hair cycling,immune responses and percutaneous penetration[11]. To date, there is no animal model for AD that comprehensively recapitulates all aspects of human AD characteristics. Actually, most existing mouse models for AD, such as MC903 mouse model, dermatophagoides farinae mouse model, only induce AD-like dermatitis and Th2-associated cytokine response[12]. An ideal animal model for AD would recapitulate as many features of human AD as possible, including keratinocyte hyperproliferation, thickening and altered differentiation of the epidermis, increased T-cell infiltration, elevated Th2/Th1 cytokine expression and serum IgE levels and disrupted skin barrier[13]. Given little is known about the transition of AD from acute to chronic stages, therefore, further investigation of mechanisms behind this transition requires a new mouse model.

Exposure to DNFB was found to induce contact dermatitis in mice[14]. When the shaved back skin of mice is exposed to DNFB, cytotoxic T lymphocytes are recruited to the skin and result in keratinocyte apoptosis and further skin barrier dysfunction[15]. OVA is a chicken protein allergen mainly found in egg white that is commonly used to sensitize immune reactions[16]. Prolonged exposure to OVA induces the development of an AD-like dermatitis characterized by OVA-specific IgE and total serum IgE, as well as increased expression of Th2 cytokines and IFN-γ. However, the induction period of AD-like dermatitis using OVA is time-consuming, and the skin lesions are usually mild. Therefore, we designed this study to evaluate the potential of the combined application of DNFB and OVA to build a new AD mouse model that recapitulates the dynamic changes of helper T cell responses in the progression of acute-to-chronic atopic dermatitis. In this study, female BALB/C mice were subjected to the application of DNFB three times a week and OVA twice a week, and the same protocol was repeated for 4 weeks. The mice in the experimental group developed an impaired skin barrier and typical cutaneous manifestations of AD, and they exhibited classic pathological features, as evidenced by prominent infiltration of inflammatory cells and epidermal thickening. The expression of Th2-associated cytokines (IL4,IL13,TSLP) was elevated in the acute stage, while in the chronic stage, the expression of Th2-associated cytokines were still at high level, meanwhile, the expression of Th1- and Th17-associated cytokines also got prominently elevated. In addition, high JAK-STAT signaling pathway expression was also observed in the chronic stage of this mouse model.

Combined Application of DNFB and OVA Induced AD-like Dermatitis

Figure 2A shows the dynamic changes of skin lesions in each group. With prolonged induction time,the skin lesions of mice in the DNFB group tended to be stabilized, which predominantly presented with dryness, erythema and scaling. However, the skin lesions of mice in the DNFB + OVA group were consistently exacerbated with increasing exposure time, as evidenced by skin thickening, erythema area enlargement and severe oedema and exudation. To compare the morphological changes of skin lesions between different groups, skin lesion scoring was performed and recorded every four days (Fig. 2B). After approximately two weeks, mice in the DNFB group exhibited dryness, scaling, and poorly defined erythema, but neither oedema nor exudation was observed. However, in the DNFB + OVA group, apart from erythema, scaling and dryness, mild oedema and exudation were also observed. At the end point of this experiment, scratching behaviour estimation was performed, and the results indicated that the scratching scores of mice in the DNFB + OVA group were prominently higher than those in the other groups (Fig. 2C). As illustrated in Fig. 2D, the serum total IgE levels of the DNFB + OVA group were 3.89-fold (P<0.0001) and 1.17-fold (P<0.01) higher than those of the DNFB group and OVA group, respectively. As shown in Fig. 2E, the serum OVA-specific IgE levels of the DNFB + OVA group were remarkably higher than those of the DNFB and OVA groups, respectively. Figure 2F shows the comparison of serum IgE levels between acute phase and chronic phase within the DNFB + OVA group. Overall, compared to other groups, the skin lesions of mice in the DNFB + OVA group were the most severe, whereas no significant skin lesions were observed in the OVA and control groups during the entire induction period.

Combined Application of DNFB and OVA induced AD-like Histological Changes

To probe the histological changes among these groups, sections were stained with H&E and toluidine blue to examine the epidermal thickness and the infiltration of mast cells, respectively. Compared to the other groups, both the epidermal thickness and dermal thickness of the mice in the DNFB + OVA group were significantly higher, accompanied by simultaneous alleviation of spongiosis (Fig. 3A-C). Mast cell infiltration was analysed in Toluidine Blue stained sections using Iviewer software, and mast cell counting was performed in five randomly selected fields from each section. As illustrated in Fig. 3D, the infiltration of mast cells in the DNFB + OVA group was markedly higher than that in any other group. Figure 3E shows the comparison of hitological changes between acute phase and chronic phase within the DNFB + OVA group, clearly, the inflammatory cell infiltration and skin thickening in chronic lesions were both more severe than those in acute phase.

Combined Application of DNFB and OVA Aggravates Epidermal Barrier Dysfunction

As Fig. 4A illustrates, our data suggest that the TEWL values of the DNFB + OVA group were 1.48-fold and 2.97-fold than those of the DNFB group and OVA group, respectively (P<0.001). In addition, our data confirm that the application of DNFB + OVA upregulated filaggrin expression in the skin tissue of mice(Fig. 4B). To explore the mechanism behind this finding, we performed immunohistochemistry to investigate the protein expression level of filaggrin. The results(Fig. 4C) showed that the protein expression levels of mice in the DNFB + OVA group were not higher than thosein the DNFB group but were much higher than those in the control group and OVA group. Figure 4D shows the comparison of epidermal barrier related indicators between acute phase and chronic phase within DNFB + OVA group. d1: the FILAGGRIN positive staining area in the epidermis of chronic skin lesions was less than that of acute phase. d2: The mRNA expression in chronic lesions was higher than that in acute lesions. d3: The TEWL values in chronic lesions was much higher than that in acute lesions. Given that the mice in the DNFB + OVA group showed the most severe skin barrier dysfunction and high expression of filaggrin, this result is intriguing and yet difficult to comprehend.

The AD-LIKE Dermatitis is Dominated by Th2 Immune Response

Atopic dermatitis is usually considered a T helper 2 (Th2) cell-mediated disease. Besides, as a master regulator of Th2-driven inflammation, TSLP promote the early diffrentiation of distinct Th2 cell populations. Activated Th2 cells release IL4 and IL13, which together aggravate the inflammatory cascade. As shown in Fig. 5A, our data showed that the expression level of TSLP in the DNFB + OVA group was 1.92-fold higher than that in the control group (P<0.001). In addition, DNFB + OVA also enhanced the expression of IL4 and IL13 both in serum and skin lesion tissues(Fig. 5B-E), albeit the enhancement of IL4 was more subtle. Figure 5F: Within DNFB + OVA group, the expression of Th2 related cytokines in chronic phase was also higher than that in the acute phase, which further confirms that Th2 immune response dominates the progression of atopic dermatitis.

Expression of Helper T cell-associated Cytokines is Upregulated in Chronic Stage of AD-Like Dermatitis

Recently, upregulation of Th1- and Th17-mediated responses has been found to be present in chronic skin lesions[17]. Given the fact that chronic AD is strongly associated with the enhanced expression of Th1 and Th17 related cytokines, we also examined these cytokines in the skin tissues and serum using PCR or ELISA. Figure 6A-F illustrates the comparison of helper T cell-associated cytokine expression between different groups at the end of the experiment. Figure 6G shows the comparison of helper T cell-associated cytokines expression in the acute and chronic phase within the DNFB + OVA group. Namely, a-c: The expression of Th1 cell -cytokines, viz., IL2, IFN-γ was elevated (1.22-fold, P<0.001; 1.38-fold, P<0.001; 1.31-fold, P<0.001, respectively) in serum or skin tissue of DNFB + OVA(chronic) compared to DNFB + OVA (acute). d: The expression of IL17A was upregulated() in the skin tissue of DNFB + OVA(chronic) compared to that of DNFB + OVA(acute). e: The expression of IL19 was also upregulated(2.67-fold, P<0.01) in the skin tissue of DNFB + OVA(chronic) compared to that of DNFB + OVA(acute). f: The expression of IGFL1 (a newly found cytokine that is closely related to the activation of IL17) was increased (1.73-fold, P<0.001) in skin tissue of DNFB + OVA (chronic) compared to that of DNFB + OVA (acute).

Up-Regulation of the JAK-STAT Pathway in Chronic Lesion

It is well known that activated Th2 cells secrete IL4 and IL13, and these cytokines promote IgE class switching in B cells and the production of IgE via the JAK-STAT signaling pathway, which may leads to the chronicity of AD. Thus, we quantified the expression levels of JAK-STAT signaling pathways. As Fig. 7A-C shows, our data indicate that the application of DNFB + OVA increased JAK1 (2.89-fold, P<0.001),STAT3 (3.47-fold, P<0.001), and STAT6 (1.23-fold, P<0.05) compared to the control group. Figure 7D: Within DNFB + OVA group, the comparison of JAK-STAT pathway expression between acute phase and chronic phase, the expression of JAK1 and STAT3 in chronic phase was also higher than that in the acute phase.

Atopic dermatitis is one of the most common skin diseases and poses a significant socioeconomic and public health burden[18]. AD affects people of all ages and ethnicities and has a severe physical and mental impact on patients and families. The pathogenesis is highly complicated and involves strong T-cell-driven inflammation, epidermal barrier dysfunction, and genetic predisposition[19]. Currently, in the field of AD research, mouse models are the most commonly used model system due to a multitude of favourable properties, such as ease of raising, rapid breeding and inbred capability. However, because of mice and human exhibit many species-specific differences in immune responses, skin tissue architecture and percutaneous drug penetration[20], there is no mouse model that recapitulates the dynamic changes of cytokine response in the progression of AD.

In this study, female BALB/C mice were subjected to the application of DNFB and OVA for 4 consecutive weeks. The mice in the DNFB + OVA group developed an impaired skin barrier and typical cutaneous manifestations of AD, including erythema, oedema, scaling, high frequency of scratchings, and they also exhibited classic pathological features, as evidenced by prominent infiltration of inflammatory cells and epidermal thickening. At the second week, mice in the DNFB group exhibited dryness, scaling, and poorly defined erythema, but neither oedema nor exudation was observed. However, in the DNFB + OVA group, apart from erythema, scaling and dryness, mild oedema and exudation were also observed. With prolonged induction time,the skin lesions of mice in the DNFB + OVA group tended to be stabilized and chronic, which predominantly presented with dryness, erythema and scaling. In terms of cytokine response, at the acute stage, the immune response was dominated by Th2-promoting and Th2 cytokines, namely, TSLP, IL13 and IL4, while after the AD-like dermatitis progressed to its chronicity, other cytokine responses, such as Th1-or Th17-related cytokines, IGFL1 and IL19 also got remarkably elevated.

The immune responses of atopic dermatitis are a complex interplay between CD4 + T-cell activation and differentiation, the Th1/Th17 immune pathway, and mainly type 2 skewed immune dysregulation[21]. The type-2 immune response is dominant in the acute phase of AD, mainly manifested as increased levels of IL4 and IL13, as well as increased serum IgE concentrations[22]. As a Th2-related cytokine, TSLP is involved greatly in the pathogenesis of AD. First, after its release in keratinocytes, TSLP activates tissue-resident DCs that subsequently migrate into the local lymph node, where they prime naïve CD4 + T cells into Th2 cells[23]. TSLP further triggers the secretion of proinflammatory cytokines such as IL4, IL13 and TNFα from CD4 + T cells and DCs, which further activate B cells and mast cells[24]. It is reported that at the chronic phase of atopic dermatitis, activation of Th1- and Th17-mediated responses has been observed[25], but the relative importance of these pathways in atopic dermatitis is currently unclear. Among all the mouse models for atopic dermatitis, our model firstly recapitulates some dynamic changes of Th-related cytokines in the progression of AD from acute-to-chronic.

Our results also showed that JAK-STAT pathway expression was significantly up-regulated in chronic lesions. Janus kinases(JAKs) are a family of cytoplasmic tyrosine kinases(TYKs) comprising JAK1,JAK2,JAK3,TYK2. The JAK-STAT pathway plays a central role in the maturation of B-lymphocyte cells[26]. In vitro studies indicate that inhibitors targeting JAK1 decrease the number,activation,and function of T cells,B cells,DCs,and mast cells[27]. Given these cells are greatly involved in the progression of AD, among all the JAKs, JAK1 plays a more important role in the pathogenesis of AD. STATs family comprising STAT1,STAT2,STAT3,STAT5A/B, and STAT6. It is well known that STAT6 is involved in B cell differentiation, IgE class switching, and MHC class Ⅱ production, therefore STAT6 is associated with IgE production and progression of allergic diseases[28]. In terms of STAT3, it is vital for Th17 lymphocyte differentiation, and STAT3 is also involved in mediating the inflammation process and the changes of natural skin barriers, as well as increased TEWL by stimulating the expression of IFN-γ,IL31,and IL22[29, 30]. Our result confirm that the expression of JAK-STAT pathway was remarkably increased in both acute and chronic phases, and the increase was more significant in chronic phase. Taken together, we speculate that the high expression of JAK-STAT pathway may promote the chronicity of AD.

Incresed IL19 expression was usually observed in inflammatory skin, such as atopic dermatitis and aging[31]. The Th17-related cytokines IL17A and IL22 act synergistically to enhance IL19 expression in cultured keratinocytes in vitro[32], since Th17 related cytokines is a marker of chronic AD, IL19 may also be an indicator for chronicity of AD. IGFL1 has been reported as a candidate target gene for AD, it may be related to the abnormal activation of immune response in AD patients, especially the activation of Th17 immunity, which is one of the key subtypes of T cell pathway[33]. Since IL19 and IGF1 were associated with Th17 and our results also showed that both the expression of IL19 and IGFL1 were up-regulated in chronic lesions, we speculated that IL19 and IGFL1 might be new indicators of AD chronicity.

Our findings suggest that the combined application of DNFB + OVA successfully induced AD-like skin lesions and histological findings in BALB/c mice with a rather short induction period. This model also partially recapitulates the dynamic changes of cytokine response in the acute-to-chronic progression of AD.

Drugs and Chemicals

Ovalbumin was purchased from Sigma‒Aldrich (Saint Louis, Missouri, 63103, USA). Dinitrofluorobenzene was purchased from Macklin (Shanghai, 201203, China). Antibodies used for immunohistochemistry were purchased from Abcepta (San Diego, California, 92121, USA). Other required chemicals were purchased from Beyotime (Shanghai, 201611, China). The concentration of DNFB was 0.15%, and a 3:1 mixture of acetone and olive oil was used as the solvent. The0.15% ovalbumin solution was prepared with normal saline. All the drugs and chemicals used in this study were of analytical grade.

Animals

Six-week-old female BALB/c mice weighing 20–25 g were procured from the Experimental Animal Center of the Army Medical University (Third Military Medical University, Chongqing, 400038, China). All the animals were housed in polypropylene cages and were given unlimited access to sterilized fodder and water. All experimental procedures were approved by the Laboratory Animal Welfare and Ethics Committee of Army Medical University (AMUWEC20223374). Animals were acclimatized to feeding conditions for 1 week before experimentation. At specific time point,mice were sacrificed by cervical dislocation.

Experimental Design

Mice were divided into four groups as listed below.

i. Control (NC) group (n = 4): Mice were given solvent or saline, and no hapten, allergen or drugs were given to them.

ii: Dinitrofluorobenzene (DNFB)-only group (n = 4): Dinitrofluorobenzene (100 µl each time) and saline were given to the mice as detailed in Fig. 1.

iii. Ovalbumin (OVA)-only group (n = 4): Ovalbumin (100 µl each time) and solvent were given to the mice as per the details given in Fig. 1.

iv. Dinitrofluorobenzene + Ovalbumin (D + O) group (n = 8): DNFB and OVA were administered to the mice three times a week and two times a week, respectively. Both drugs were administered at doses of 100 µl each time. See Fig. 1 for detailed information. Four of the mice were euthanized at day 14 by cervical dislocation, and the other four mice were euthanized in the same way at day 28.

Sensitization Protocol

After acclimation to the laboratory conditions, the back skin hair of all the mice was shaved with an electric razor, and then the bare skin was treated with hair removal cream. The application of hair removal cream was repeated every three days to keep the skin clean. One hundred microlitres of DNFB or solvent was uniformly smeared onto the shaved back of the mice three times a week. One hundred microlitres of ovalbumin or saline was given twice a week, once for application to the shaved back skin, and once for intraperitoneal injection. Animals were sacrificed 24 h after the last exposure to reagents for procurement of blood samples and skin tissue. All reagents were softly and evenly applied to the mice. Cages were sanitized and replenished with chow and water weekly.

Skin Lesions Evaluation

There are five main items to be scored: erythema, scaling, dryness/scarring, oedema, and exudation. Each item has four grades, namely, 0 (none), 1 (mild), 2 (moderate), or 3 (severe). The total dermatitis score was calculated as the sum of these individual scores. The score of each item was assessed by two independent observers. Skin lesion evaluation was performed every four days, and eight evaluations were performed at the end of the experiment.

Scratching Behaviour Estimation

In addition to skin lesion evaluation, the scratching behaviour estimation was also conducted at the end point of the experiment. The scoring criteria for scratching referred to a previous study[20]. Briefly, estimation was performed at one minute intervals by an observer who was unaware of the treatment status. Estimation was performed for five consecutive minutes, and the scoring criteria were as follows: 1 if the scratching time duration was less than 1 second, 2 if the duration was less than 2 seconds, and 3 if the duration was more than 2 seconds. The scratching behaviour estimation was performed at 24 hours after the last exposure to reagents.

Histological Examination

Skin specimens were fixed in 10% neutral formalin and embedded in paraffin. Slides of 5-µm-thick sections were prepared and stained with H&E to examine the epidermal thickness and inflammatory cell infiltration. Toluidine blue staining was performed to examine the infiltration of mast cells. Slides were then digitally scanned and analysed using Iviewer Analysis Software (UNIC Technologies, Beijing, China). Mast cell counts and epidermal/dermal thickness measurements were performed in five randomly selected fields from each sample.

Transepidermal Water Loss Assay

Transepidermal water loss (TEWL) measurements were conducted with a Cutometer Dual MPA580 (Courage + Khazaka electronic Gmbh, Köln, Germany) to examine skin barrier dysfunction. Each mouse was measured 10 times in a randomly selected area.

RNA Isolation and Quantitative Real-Time PCR

Total RNA was isolated from skin tissues using TRIzol™ reagents according to the manufacturer’s instructions. cDNA synthesis was performed using the PrimeScript™ RT Reagent Kit (TaKaRa, Japan) according to the manufacturer’s instructions. The expression values of each target gene were normalized to the expression values of β-actin. Real-time PCR was performed on a CFX Connect™ Real-Time System (BIO-RAD, CA, USA) with TB Green Premix Ex Taq™ II (TaKaRa, Japan). The amplification programs involved 40 cycles of preincubation for 2 min at 95°C and then amplification cycles consisting of 5 s at 95°C and 30 s at 60°C. The analysed genes and primer sequences of these genes are given in Table 1.

Enzyme-linked Immunosorbent Assay

The next day after the final exposure to reagents, blood was taken from the eyeballs of mice, and then the serum was separated. Frozen serum samples were used to measure the serum levels of IL2, IL4, IL13, IFN-γ, TARC, total IgE and OVA-specific IgE. ELISA was performed with commercial ELISA kits (JM-02339M1, JM-03061M1, JM-02981M1, JM-02448M1, JM-02456M1, J2291-A; Jingmei Biology Technology, Jiangsu, China) according to the manufacturer’s instructions.

Immunohistochemistry

Immunohistochemical staining was performed for FILAGGRIN. Primary and secondary antibodies used were as follows: FILAGGRIN (Host: Rabbit; Reactivity: Human, Mouse, Rat; Abcepta, San Diego, CA, 92121, USA). Goat Anti-Rabbit IgG (H&L) HRP (Source: Goat; Immunogen: Rabbit IgG; Conjugate: HRP; Abcepta, San Diego, CA, 92121, USA). Tissues were fixed in 10% formalin at 4°C overnight and then embedded in paraffin. Then, 5-µm-thick sections were cut and routinely dewaxed and hydrated. Endogenous peroxidase activity and endogenous avidin binding activity were blocked. Sections were incubated with primary antibodies for 12 hours at 4°C overnight, and then the slides were rinsed three times in PBS for five minutes each wash. Sections were incubated with secondary antibodies for 30 minutes at 37°C. Slides were counterstained with haematoxylin. Positively stained cells were counted from five random fields (magnification ⋅400) in each section.

Statistical Analysis

Data are expressed as the mean ± standard error of the mean or standard deviation. Statistical analysis was performed by Student’s t test or one-way analysis of variance (ANOVA) using GraphPad Prism software (GraphPad Software, Inc. La Jolla, CA, USA). Statistical significance was defined as P<0.05. Significance levels of data were denoted as *P < 0.05, **P < 0.01, and *** P < 0.001, ****P<0.0001.

AD：Atopic Dermatitis；DNFB：Dinitrofluorobenzene；OVA：Ovalbumin；

Ethics Approval

This animal study and experiments were assessed and approved by Laboratory Animal Welfare and Ethics Committee of Third Military Medical University. All experimental procedures involving animals were performed in strict accordance with the guidelines and requirements of the Laboratory Animal Werlfare and Ethics Committee of the Third Military Medical University. Animal Ethical Statement Number: AMUWEC20223374.

This study is reported in accordance with ARRIVE guidelines.

Data Availability Statement

The data and materials that support the findings of this study are available from the corresponding author upon reasonable request.

Competing Interests

The authors declare that they have no competing interests.

Consent to publication

Not Applicable.

Funding

This research work was supported by the National Natural Science Foundation of China (No. 82073442).

Author Contributions

PJ and YW designed the experiments; Experiments were performed by PJ,YW,LZ,and LL; Analysis of the data was done by PJ and YW; YW and ZS reviewed the paper; ZS directed the study. The manuscript was prepared by PJ and all authors approved the final manuscript.

Acknowledgement

We thank professor Bing Ni(Director of pathophysiology department of Army Medical University) for kindly sharing the laboratory equipments.

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Table 1 is available in the Supplementary Files section

No competing interests reported.

Table1.jpg
Table 1.Primer Sequeces.

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Combined Application of Dinitrofluorobenzene and Ovalbumin Induced AD-Like Dermatitis With Upregulated Helper T Cell Cytokines and Prolonged Th2 Response

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Version 1

Abstract

Figures

Background

Results

Combined Application of DNFB and OVA Induced AD-like Dermatitis

Combined Application of DNFB and OVA induced AD-like Histological Changes

Combined Application of DNFB and OVA Aggravates Epidermal Barrier Dysfunction

The AD-LIKE Dermatitis is Dominated by Th2 Immune Response

Expression of Helper T cell-associated Cytokines is Upregulated in Chronic Stage of AD-Like Dermatitis

Up-Regulation of the JAK-STAT Pathway in Chronic Lesion

Discussion

Conclusion

Methods

Drugs and Chemicals

Animals

Experimental Design

Sensitization Protocol

Skin Lesions Evaluation

Scratching Behaviour Estimation

Histological Examination

Transepidermal Water Loss Assay

RNA Isolation and Quantitative Real-Time PCR

Enzyme-linked Immunosorbent Assay

Immunohistochemistry

Statistical Analysis

Abbreviations

Declarations

References

Table

Additional Declarations

Supplementary Files

Status:

Version 1