IL-33 Enhances ACE2 Expression on Epidermal Keratinocytes in Atopic Dermatitis: A Plausible Issue for SARS-CoV-2 Transmission in Inflamed Atopic Skin

Background: Interleukin-33 (IL-33) is an important cytokine in the pathophysiology of atopic dermatitis (AD) and in the progression of COVID-19. Angiotensin converting enzyme 2 (ACE2), the entry receptor for SARS-CoV-2, is expressed in epidermal keratinocytes. Whether IL-33 could regulate the expression of ACE2 mechanistically in keratinocytes warrants investigation. Objective: We questioned whether the ACE2 expression is increased in AD skin. We also questioned whether ACE2 is expressed in keratinocytes; if so, would its expression be enhanced mechanistically by IL-33. Methods: We measured and compared the expression of ACE2 in skin from patients with AD, patients with psoriasis, and healthy controls using immunohistochemistry. Flow cytometry, immunofluorescent exam, and quantitative RT-PCR were used for measuring the ACE2 expression in cultured keratinocytes treated with IL-33 and IL-17. Blocking antibodies were utilized to study the intracellular signaling pathways governing the ACE2 expression using cytokines. Results: The results showed that the ACE2 expression is increased in AD compared with that in healthy skin and psoriasis. In primary epidermal keratinocytes, ACE2 is constitutively expressed. IL-33 induces a time-dependent increase in ACE2 expression in cultured keratinocytes through quantitative PCR, flow cytometry, and immunofluorescent examinations. Furthermore, pretreatment of an ERK inhibitor, but not a STAT3 inhibitor, eliminated the increases in ACE2 by IL-33 in keratinocytes, indicating that IL-33 enhances ACE2 expression through ERK on epidermal keratinocytes. Conclusion: This is the first study to reveal that IL-33 enhances ACE2 expression on keratinocytes via ERK. Although further mechanistic studies are required, the increased ACE2 expression in IL-33 might have a biological implication on the transmission of SARS-CoV-2 in patients with AD.


Introduction
Atopic dermatitis (AD), a common inflammatory skin disease, affects 15-20% of children and 1-3% of adults in industrialized countries [1]. Intensive itching and sleep disturbance in AD cause huge burdens in patients, family, and society. Its pathogenesis includes impaired skin barriers and the aberrant production of T helper-2 (Th2)-associated cytokines [2]. When the epidermal barrier is impaired by exogenous insults or endogenous immune responses, IL-33 is released from epidermal keratinocytes as an alarmin to activate type-2 innate lymphoid cells (ILC2) [3] and basophils to release Th2 cytokines, including IL-4 and IL-13, which reduce the expression of barrier protein filaggrin, forming a viscous cycle to perturb the damaged skin barrier [4]. The IL-33 receptor, known as the membrane-bound stimulation-2 receptor (ST2), is widely expressed in several immune cells, such as ILC2, Th2 cells, regulatory T (Treg) cells, M2 macrophages, and eosinophils, along with epithelial

Skin Samples for Immunohistochemistry
Human skin was obtained from AD through skin biopsy. All of the procedures were performed according to national and institutional ethical guidelines. Here, 5 µm serial tissue sections obtained from the skin of AD patients (age 20-64), patients with psoriasis (age 42-68), and controls (age 8-67) (n = 5, 4, and 9, respectively) were used.

Culture for Primary Keratinocytes
Normal human keratinocytes were obtained from adult foreskins through routine circumcision. The keratinocytes were harvested and cultured as described previously [40]. Briefly, skin specimens were washed with PBS (phosphate buffer saline) (pH 7.2), cut into small pieces, and harvested in Dulbecco's Modified Eagle Medium (DMEM) containing 0.25% trypsin (Gibco, Grand Island, NY, USA) overnight at 4 • C. The epidermal sheet was separated from the dermis using fine-tipped forceps. The epidermal cells were pelleted through centrifugation (500× g, 10 min) and were then dispersed into individual cells by repeated gentle aspiration. The keratinocytes were gently resuspended in 5 mL of keratinocyte in a serum-free medium (Gibco), which contained 25 mg/mL bovine pituitary extract and 5 ng/mL recombinant human epidermal growth factor. Keratinocytes at the third passage were then grown in a keratinocyte in serum-free medium without bovine pituitary extract and recombinant human epidermal growth factor for 24 h before experimentation.

Immunohistochemistry
Tissues were fixed in 10% buffered formalin, dehydrated, and embedded in paraffin at the pathology department. Deparaffinization with xylene was performed in our lab. Subsequently, the sections were washed in PBS (phosphate buffer saline) and incubated with primary antibodies directed against ACE2 (Rabbit, Genetex GTX101395, San Antonio, TX, USA), CD207 (Langerin, Invitrogen 12-2075-82, Waltham, MA, USA), and isotype antibody (Rabbit IgG and Rat IgG2a-PE). The slides were then washed in PBST (phosphate buffer saline with 0.1% Tween 20) and visualization was performed using a fluorescence microscope (Olympus DP80, Shinjuku-ku, Tokyo, Japan) according to the manufacturer's instructions.

Quantitative RT-PCR
The keratinocytes were cultured in a 12-well plate with keratinocyte SFM (serumfree medium). Cytokines were added to the medium with different concentrations and durations. The quantification and purity of the RNA were assessed using A260/A280 absorption (Nanodrop spectrophotometer; Thermo Fisher (Waltham, MA, USA)), and RNA samples with ratios greater than 1.7 were stored at −70 • C for further analysis. Extracted RNA (1 µL) was then subjected to PCR amplification using MPCR kits (Maxim Biotech, San Francisco, CA, USA) according to the manufacturer's instructions. Primer sequences used were designed for human ACE2 FORWARD: CATTGGAGCAAGTGTTGGATCTT and human ACE2 BACKWARD: GAGCTAATGCATGCCATTCTCA. The reactions were carried out under the following conditions: 96 • C for 1 min and 60 • C for 4 min, 30 cycles of 94 • C for 1 min and 60 • C for 3 min, and extension at 70 • C for 10 min.
In order to validate that IL-33 indeed enhances the expression of ACE2 in keratinocytes, we used an immunofluorescent examination to measure the expression of ACE2 in keratinocytes treated with IL-33. Furthermore, we were interested in the mechanism through which IL-33 enhances the expression of ACE2. Therefore, in order to test this hypothesis, inhibitors of ERK (PD98059) or STAT3 (STA21) were added to the keratinocytes treated with IL-33.

Immunofluorescent Exam
Keratinocytes were cultured with keratinocyte SFM (serum-free medium). KCs, attached on 16 mm cover glasses, were cultured in 12-well plates. Cytokines and protein inhibitors were added to the 12-well plate for 24 h. Then, the cells were fixed and holed by 0.1% triton X for 1 h in room air. We stained the keratinocytes with 1:500 rabbit anti-ACE2 (Genetex GTX101395) overnight at 4 • C, followed by Goat anti-rabbit IgG-568 antibody (secondary antibody) binding for 1 h in room air. Immunostained samples were analyzed using the feature of "Analyze Particles" in ImageJ.

ACE2 Expression Was Increased in Atopic Dermatitis Compared with That in Normal Skin
In order to investigate whether the ACE2 receptor was indeed expressed on the skin in vivo, we took skin samples from AD patients and psoriasis patients and performed an immunohistochemical analysis ( Figure 1). The results showed that ACE2 was expressed in the basal and suprabasal epidermis. Of note, the ACE2 expression was significantly increased in AD compared with that in normal skin and psoriasis.

Through Quantitative RT-PCR, ACE2 mRNA Expression Increased in a Time-Dependent Manner under IL-33 Stimulation
We demonstrated the expression of ACE2 in the epidermal keratinocytes in tissue using IHC. We then asked whether ACE2 was expressed in cultured keratinocytes, and if so, whether IL-33 would enhance ACE2 expression. To address this, we performed quantitative RT-PCR to measure the transcriptional expression of ACE2 in keratinocytes treated with IL-33 for 2, 6, and 24 h. The result revealed that the ACE2 mRNA expression is present in keratinocytes ( Figure 2). Under IL-33 stimulation, the ACE2 transcriptional expression was enhanced over time (24 h > 6 h > 2 h). However, there was no dose-dependent effect of IL-33 on the expression of ACE2. In contrast with that of IL-33, the ACE2 expression was not enhanced by IL-17, a cytokine closely relevant to the pathophysiology of psoriasis.

ACE2 Is Constitutively Expressed in the Cultured Keratinocytes -Both IL-33 and IL-17 Enhance the ACE2 Protein Expression in a Time-Dependent Manner
We showed that IL-33 enhanced the ACE2 transcriptional expression in keratinocytes. We then asked whether this would be verified at the translational level. We first used flow cytometry to measure the ACE2 expression in cultured keratinocytes ( Figure 3). The results showed that the treatment of IL-33 or IL-17 at 10 ng/mL both enhanced the expression of ACE2 in cultured keratinocytes in a time-dependent manner. Biomedicines 2022, 10, x FOR PEER REVIEW 5 of 12 Figure 1. The ACE expression is increased in AD skin compared with that in psoriatic skin and healthy skin. We performed immunohistochemistry on the skin from psoriatic, atopic, and healthy patients. The ACE2 receptor (green color) was expressed in the basal and suprabasal epidermis in all three disease conditions. Notably, the ACE2 expression was increased in AD (middle pictures) compared with that in normal skin (left) and psoriasis (right). Green: ACE2; blue: DAPI; red: CD207 (langerin), Langerhans cell. Representative images are shown.

Through Quantitative RT-PCR, ACE2 mRNA Expression Increased in a Time-Dependent Manner under IL-33 Stimulation
We demonstrated the expression of ACE2 in the epidermal keratinocytes in tissue using IHC. We then asked whether ACE2 was expressed in cultured keratinocytes, and if so, whether IL-33 would enhance ACE2 expression. To address this, we performed quantitative RT-PCR to measure the transcriptional expression of ACE2 in keratinocytes treated with IL-33 for 2, 6, and 24 h. The result revealed that the ACE2 mRNA expression is present in keratinocytes ( Figure 2). Under IL-33 stimulation, the ACE2 transcriptional expression was enhanced over time (24 h > 6 h > 2 h). However, there was no dose-dependent effect of IL-33 on the expression of ACE2. In contrast with that of IL-33, the ACE2 expression was not enhanced by IL-17, a cytokine closely relevant to the pathophysiology of psoriasis.  The ACE expression is increased in AD skin compared with that in psoriatic skin and healthy skin. We performed immunohistochemistry on the skin from psoriatic, atopic, and healthy patients. The ACE2 receptor (green color) was expressed in the basal and suprabasal epidermis in all three disease conditions. Notably, the ACE2 expression was increased in AD (middle pictures) compared with that in normal skin (left) and psoriasis (right). Green: ACE2; blue: DAPI; red: CD207 (langerin), Langerhans cell. Representative images are shown. Figure 1. The ACE expression is increased in AD skin compared with that in psoriatic skin and healthy skin. We performed immunohistochemistry on the skin from psoriatic, atopic, and healthy patients. The ACE2 receptor (green color) was expressed in the basal and suprabasal epidermis in all three disease conditions. Notably, the ACE2 expression was increased in AD (middle pictures) compared with that in normal skin (left) and psoriasis (right). Green: ACE2; blue: DAPI; red: CD207 (langerin), Langerhans cell. Representative images are shown.

Through Quantitative RT-PCR, ACE2 mRNA Expression Increased in a Time-Dependent Manner under IL-33 Stimulation
We demonstrated the expression of ACE2 in the epidermal keratinocytes in tissue using IHC. We then asked whether ACE2 was expressed in cultured keratinocytes, and if so, whether IL-33 would enhance ACE2 expression. To address this, we performed quantitative RT-PCR to measure the transcriptional expression of ACE2 in keratinocytes treated with IL-33 for 2, 6, and 24 h. The result revealed that the ACE2 mRNA expression is present in keratinocytes ( Figure 2). Under IL-33 stimulation, the ACE2 transcriptional expression was enhanced over time (24 h > 6 h > 2 h). However, there was no dose-dependent effect of IL-33 on the expression of ACE2. In contrast with that of IL-33, the ACE2 expression was not enhanced by IL-17, a cytokine closely relevant to the pathophysiology of psoriasis.

Enhance the ACE2 Protein Expression in a Time-Dependent Manner
We showed that IL-33 enhanced the ACE2 transcriptional expression in keratinocytes. We then asked whether this would be verified at the translational level. We first used flow cytometry to measure the ACE2 expression in cultured keratinocytes ( Figure  3). The results showed that the treatment of IL-33 or IL-17 at 10 ng/mL both enhanced the expression of ACE2 in cultured keratinocytes in a time-dependent manner. Figure 3. ACE2 is constitutively expressed in the cultured keratinocytes. We performed flow cytometry and immunofluorescence examinations for evaluating the ACE2 expression. Keratinocytes were treated with IL-33 and IL-17 at 10 ng/mL for 2, 6, and 24 h, respectively. The ACE2 protein expression under IL-33 (upper row) or IL-17 stimulation (lower row) was measured using flow cytometry and immunofluorescence examinations (representative data from three repeated experiments). The bar graph represents the quantification of the intensity of the ACE2 expression through immunofluorescence examinations. * indicates p < 0.05 compared with baseline ACE2.

IL-33 Induces the ACE2 Expression, which Is Abrogated by Pretreatment with PD98059
We then used an immunofluorescent examination to measure the expression of ACE2 in keratinocytes treated with IL-33. The results showed that the ACE2 expression increased on the keratinocytes and IL-33 enhanced the expression of ACE2 on keratinocytes. In order to reveal the probable mechanism, inhibitors for ERK (PD98059) or STAT3 (STA21) were added to keratinocytes treated with IL-33. The expression of ACE2 was measured by immunofluorescent examinations and flow cytometry (Figures 4 and 5, respectively). The immunofluorescent examination results showed that IL-33 consistently enhanced the expression of ACE2. Of note, pretreatment of the keratinocytes with PD98059, but not STA21, eliminated the increase in ACE2 expression by IL-33. Through flow cytometry, the data showed that IL-33 induced a modest expression of ACE2, which was abrogated by both PD98059 and STA21. Interestingly, while IL-17 induced a minimally increased expression of ACE2, the pretreatment of STA21 potentiated the Figure 3. ACE2 is constitutively expressed in the cultured keratinocytes. We performed flow cytometry and immunofluorescence examinations for evaluating the ACE2 expression. Keratinocytes were treated with IL-33 and IL-17 at 10 ng/mL for 2, 6, and 24 h, respectively. The ACE2 protein expression under IL-33 (upper row) or IL-17 stimulation (lower row) was measured using flow cytometry and immunofluorescence examinations (representative data from three repeated experiments). The bar graph represents the quantification of the intensity of the ACE2 expression through immunofluorescence examinations. * indicates p < 0.05 compared with baseline ACE2.

IL-33 Induces the ACE2 Expression, Which Is Abrogated by Pretreatment with PD98059
We then used an immunofluorescent examination to measure the expression of ACE2 in keratinocytes treated with IL-33. The results showed that the ACE2 expression increased on the keratinocytes and IL-33 enhanced the expression of ACE2 on keratinocytes. In order to reveal the probable mechanism, inhibitors for ERK (PD98059) or STAT3 (STA21) were added to keratinocytes treated with IL-33. The expression of ACE2 was measured by immunofluorescent examinations and flow cytometry (Figures 4 and 5, respectively). The immunofluorescent examination results showed that IL-33 consistently enhanced the expression of ACE2. Of note, pretreatment of the keratinocytes with PD98059, but not STA21, eliminated the increase in ACE2 expression by IL-33. Through flow cytometry, the data showed that IL-33 induced a modest expression of ACE2, which was abrogated by both PD98059 and STA21. Interestingly, while IL-17 induced a minimally increased expression of ACE2, the pretreatment of STA21 potentiated the expression of ACE2 by IL-17. Taken together, both immunofluorescent examination and flow cytometry data indicated that IL-33 induces the expression of ACE2 through ERK.   With a similar experimental design, we performed flow cytometry to measure ACE2 expression in IL-33treated keratinocytes. Small molecule inhibitors for PD98059 or STA21 (ERK and STAT3, respectively) were pretreated in order to investigate the role of ERK or STAT3 in IL-33-induced ACE2 expression. The data showed that IL-33 induced a modest expression of ACE2, which was abrogated by both PD98059 and STA21. Interestingly, while IL-17 induced a minimally increased expression of ACE2, the pretreatment of STA21 potentiated the expression of ACE2 by IL-17.

Discussion
In this study, we demonstrated that the ACE2 expression was increased in AD skin, but not in psoriatic skin. We also showed that the ACE2 expression in keratinocytes was enhanced in a time-dependent manner by IL-33 at both a transcriptional level and a translational level. In fact, the induction of ACE2 by IL-33 was mediated by ERK.
In our IHC (Figure 1), an increased ACE2 expression was presented in AD, but not in psoriasis. This result was consistent with the results in Xue et al.'s research [38]. In quantitative RT-PCR (Figure 2), we showed the time-dependent expression of ACE2 after IL-33 treatment at a transcriptional level. However, the enhancement of ACE2 by IL-33 was not dose dependent. At a translational level, we utilized flow cytometry to measure the ACE2 protein expression (Figure 3). Under IL-33 stimulation, the ACE2 protein expression increased in a time-dependent manner. To summarize, IL-33 enhanced the ACE2 expression at a transcriptional and translational level. Incidentally, it was paradoxical that IL-17 enhanced the ACE2 expression at a translational level, but not a transcriptional level. It is necessary to conduct more research to reveal the impact of IL-17 on the ACE2 expression.
We demonstrated that IL-33 induced the expression of ACE2 via ERK. In 2016, Ryu et al. showed that IL-33 downregulated the filaggrin expression by inducing signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated protein kinase (ERK) phosphorylation in human keratinocytes [41]. They also demonstrated that IL-33 downregulated the expression of CLDN1, a tight junction protein on keratinocytes, via the phosphorylation of the ERK/STAT3 pathway [42]. Extracellular signal-regulated protein kinase (ERK)1/2 is a mitogen-activated protein kinase (MAPK) family protein with typical cascade signaling characteristics and plays an important role in signal transduction pathways and the function of transcription factors, including activator protein-1, protooncogene c-Fos, and ETS domain-containing protein Elk-1 [43]. In brief, ERK 1/2 has a role Figure 5. IL-33 enhanced ACE2 expression through ERK in keratinocytes by flow cytometry. With a similar experimental design, we performed flow cytometry to measure ACE2 expression in IL-33treated keratinocytes. Small molecule inhibitors for PD98059 or STA21 (ERK and STAT3, respectively) were pretreated in order to investigate the role of ERK or STAT3 in IL-33-induced ACE2 expression. The data showed that IL-33 induced a modest expression of ACE2, which was abrogated by both PD98059 and STA21. Interestingly, while IL-17 induced a minimally increased expression of ACE2, the pretreatment of STA21 potentiated the expression of ACE2 by IL-17.

Discussion
In this study, we demonstrated that the ACE2 expression was increased in AD skin, but not in psoriatic skin. We also showed that the ACE2 expression in keratinocytes was enhanced in a time-dependent manner by IL-33 at both a transcriptional level and a translational level. In fact, the induction of ACE2 by IL-33 was mediated by ERK.
In our IHC (Figure 1), an increased ACE2 expression was presented in AD, but not in psoriasis. This result was consistent with the results in Xue et al.'s research [38]. In quantitative RT-PCR (Figure 2), we showed the time-dependent expression of ACE2 after IL-33 treatment at a transcriptional level. However, the enhancement of ACE2 by IL-33 was not dose dependent. At a translational level, we utilized flow cytometry to measure the ACE2 protein expression (Figure 3). Under IL-33 stimulation, the ACE2 protein expression increased in a time-dependent manner. To summarize, IL-33 enhanced the ACE2 expression at a transcriptional and translational level. Incidentally, it was paradoxical that IL-17 enhanced the ACE2 expression at a translational level, but not a transcriptional level. It is necessary to conduct more research to reveal the impact of IL-17 on the ACE2 expression.
We demonstrated that IL-33 induced the expression of ACE2 via ERK. In 2016, Ryu et al. showed that IL-33 downregulated the filaggrin expression by inducing signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated protein kinase (ERK) phosphorylation in human keratinocytes [41]. They also demonstrated that IL-33 downregulated the expression of CLDN1, a tight junction protein on keratinocytes, via the phosphorylation of the ERK/STAT3 pathway [42]. Extracellular signal-regulated protein kinase (ERK)1/2 is a mitogen-activated protein kinase (MAPK) family protein with typical cascade signaling characteristics and plays an important role in signal transduction pathways and the function of transcription factors, including activator protein-1, protooncogene c-Fos, and ETS domain-containing protein Elk-1 [43]. In brief, ERK 1/2 has a role in delivering extracellular signals to the nucleus, and these signals regulate the cell cycle, cell proliferation, and cell development [44]. Signal transducer and activator of transcription (STAT) is a class of transcription factors that are activated by cytokines, growth factors, and other peptide ligands [45]. In humans, the Stat family consists of seven proteins, including STAT-1, -2, -3, -4, -5A, -5B, and -6 [46]. STAT-3 regulates a variety of functions, including proliferation, cell cycle progression, apoptosis, angiogenesis, and immune evasion [47][48][49]. In AD, the activation of the IL-13/IL-4-JAK-STAT6/STAT3 axis downregulates the expression of filaggrin, loricrin, and involucrin [50]. The dysfunction of these proteins impairs the skin barrier. On the other hand, IL-17 reduces the expression of filaggrin and involucrin via P38/ERK MAPK signaling pathways [51].
Our data showed that the IL-17 expression increased through IL-17 at a protein level, but was not upregulated at an mRNA level. Theoretically, the increased ACE2 protein level on the cell membrane of keratinocytes through IL-17 could result from ACE2 protein trafficking, but not the increased ACE2 expression through the new protein synthesis by transcription and translation. However, the mechanisms for the discrepancy in the transcriptional and translational levels of ACE2 through IL-33 and IL-17 remain unknown. A paper by Dr. Krueger showed that secukinumab, an IL-17 inhibitor, lowers the ACE2 expression in psoriatic skin [52]. Interestingly, in that report, the reduction in ACE2 at a protein level by tissue immunohistochemistry seemed to be more prominent than that at a transcriptional level by real-time tissue PCR.
One of the important limitations of this study was that we only had the pharmacological inhibition, but not RNA interference examination, to demonstrate the regulation of ERK in IL-33-induced ACE2 expression. Additional RNA interference experiments targeting ERK and/or other intracellular signals might provide further evidence. Nevertheless, we found scientific evidence that the ACE2 expression increased in atopic skin and epidermal keratinocytes treated with IL-33.

Conclusions
The ACE2 receptor is expressed not only in pulmonary cells, but also in keratinocytes. The expression of ACE2 is increased in the epidermis in AD. IL-33 increases the ACE2 expression on keratinocytes through ERK. Although SARS-CoV-2 has an affinity to pulmonary cells through ACE2, the expression of ACE2 in epidermal keratinocytes cannot be overlooked. Although further mechanistic studies are required, the increased ACE2 expression in IL-33 might have a biological implication on the transmission of SARS-CoV-2 in patients with AD.