Keratinocyte Conjugation by Adhesion Peptide Induces Up-Regulation of Tight Junction Proteins and Immune Regulation Under Inflammatory Conditions

In March 2020 the World Health Organization declares the
COVID-19 disease as a Pandemic [1]. In Mexico there are with
COVID-19 until March 2021, 2, 151, 028 confirmed patients,
440,983 suspected cases and 193,142 deaths..


Introduction
The skin, the body's largest organ, helps to secure the integrity of the host and allows the individual to communicate with the outside world. A network of physical, chemical, and immunologic components ensures a finely tuned balance between protection from harmful pathogens and bidirectional signal exchange [1,2].
Immune responses originating in the skin are executed by cells and molecules belonging to the innate and adaptive immune systems. The innate immune system, which provides immediate host defense, includes physical barriers (e.g., epithelia) and soluble factors such as antimicrobial peptides, chemokines, and cytokines produced by a variety of resident cells (e.g., keratinocytes and fibroblasts) and infiltrating leukocytes (e.g., granulocytes, monocytes/macrophages, and Dendritic Cells (DCs)) [1][2][3]. The most numerous cell within the epithelial compartment is the keratinocyte [1,3]. In addition to its important role in maintaining the physical barrier of the skin, the keratinocyte actively participates in innate immune responses.
When keratinocytes are stimulated by danger signals (e.g., LPS), they release large amounts of cytokines that trigger inflammation,

activate/inhibit T cells, modulate the phenotype and function of
Langerhans cells (skin DCs), and regulate leukocyte trafficking [1][2][3]. An effective immune response in human skin is usually initiated by dendritic Antigen-Presenting Cells (APCs) and is ultimately executed by T lymphocytes or B cell-derived antibodies, or both [1,2].
DCs are professional APCs that play major roles in initiating and regulating adaptive immune responses. They are capable of capturing and processing antigens, migrating into lymphoid organs, stimulating antigen-specific responses by naïve resting T cells, and secreting cytokines to initiate immune responses [2,[4][5][6]. Immature DCs (imDC) regulate skin homeostasis by promoting the development of regulatory T cells, while their mature counterparts (i.e., DCs that have been stimulated by danger signals) induce cyto-destructive reactions [1,[4][5][6]. An important type of immunomodulatory T cell that controls immune responses is the Treg cell, formerly known as the T suppressor cell [7]. Treg cells are induced by immature APCs/DCs and are the key to maintaining tolerance to self-antigens in the periphery [4][5][6]. Tight junction (TJ) proteins expressed by keratinocytes in the epidermal layer play a central role in barrier function. TJ proteins create a barrier by forming cell-cell junctions that connect cells together very tightly, which affects the function of the immune system in epidermal tissues [8]. amino acid sequence of foot protein-1 of mytilus edulis; this protein promotes cell-cell interactions in keratinocytes in just 10 min [9][10][11][12]. This data suggested an effect of Adh-Pep-10-2 on tight junction formation [9][10][11][12]. In addition, AdhPep-10-2 increases expression of TJ proteins. To the best of our knowledge, no study has examined the relationship between increased TJ proteins and DC-mediated immunity; this was the aim of the present study.

Ethics Statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of CHA University (The approved protocol Number: 180136). All surgery was performed under CO 2 gas euthanasia, and all efforts were made to minimize suffering.

Western Blot Analysis
Keratinocytes treated (or not) with AdhPep-10-2 were lysed with pro-prep extraction buffer (iNtRON, Gyeonggi, Korea) containing a 5-protease inhibitor cocktail. The protein concentration was then measured using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein were separated on SDS-PAGE gels and transferred to PVDF membranes (BioRad, CA, USA). The membranes were blocked with 5% skim milk in PBST and then incubated overnight at 4°C with primary antibodies as follows: anti-occludin rabbit mAb (E6B4R, Cell Signaling Technologies, Danvers, MA, USA), anti-claudin-1 rabbit mAb (D3H7C, Cell Signaling Technologies), or anti-GAPDH rabbit mAb (14C10, Cell Signaling Technologies); all diluted 1:1000. The membranes were then washed with PBST and incubated for 2 h at room temperature with an HRP-conjugated secondary antibody (7074, Cell Signaling Technologies); diluted 1:2000. The membrane was then exposed to ECL reagents (Thermo Scientific) and signals were detected using a Luminescent image analyzer (LAS-4000, Fuji Film, Tokyo, Japan).

Statistical Analysis
Data are expressed as the mean ± SD. Statistical comparisons were performed using one way analysis of t tests or variance (ANOVA) followed by the Newman-Keul's test. Differences were considered statistically significant at P < 0.05. Statistical analysis was performed using GraphPad software (GraphPad Prism v5.0; GraphPad Software).

Function of AdhPep-10-2
To confirm the function of AdhPep-10-2 for tight junction protein, we cultured Adh-10-2 peptide with keratinocytes stained with PKH or Cell Vue Burgundy cell labelling reagent for 1 h.
Confocal images revealed that AdhPep-10-2 increased the physical interaction between keratinocytes (Figures 1A & 1B). AdhPep-10-2 significantly increased the Pearson's correlation coefficient (Rr) and percent of co-localization ( Figure 1C). To examine the correlation between increased cell-cell interaction and expression of TJ proteins, we examined expression of TJ proteins by western blotting with antibodies specific for occludin and claudin-1 ( Figure   1D). Cells were exposed to peptide (10 nM) for 18 h. The results showed a marked increase in TJ proteins (occludin and claudin-1) expression by keratinocytes treated with 10 nM of peptide ( Figure   1).

Discussion
Skin is a major entry site for microbial pathogens and allergens; therefore, it is heavily protected by DCs [13,14]. TJ proteins expressed by keratinocytes play a central role in barrier function.
This affects not only epidermal barrier function and regulation by the immune system but is also relevant to skin diseases. The peptides, AdhPep-10-2, for cell conjugation efficiently promotes interaction between keratinocytes and increases expression of TJ proteins. Here, we examined the effect of AdhPep-10-2treated keratinocytes on DCs-mediated immunity. We found that AdhPep-10-2-treated keratinocytes induce anti-inflammatory response by blocking DC maturation and cytokine production. We also found that in the presence of LPS, Pep-Kera-DCs increased the FoxP3 + CD4 + CD25 + Treg population and reduced the IFN-γ +producing T cell population. Therefore, the data suggest that Pep-Kera-DCs induce anti-inflammatory response under conditions of inflammation.
Interestingly, we found that AdhPep-10-2-treated keratinocytes effectively reduced secretion of CXCL1 without affecting that of other pro-inflammatory cytokines. A previous report shows that in the epidermis, CXCL1 plays an important role in recruiting inflammatory cells during wound healing [15,16]. Therefore, we expect that CXCL1 is an important factor for DC-mediated immunity. Moreover, even though CXCL1 is present in the AdhPep-10-2-treated keratinocyte supernatant at levels lower than that in non-treated keratinocyte supernatant, it is not the only component that affects DC maturation and function ( Figure 5). Thus, it is likely that multiple factors act together to inhibit DC maturation. Since DCs express CXCR2, the receptor for CXCL1, CXCL1 could have a direct effect on DC maturation. Moreover, the signaling pathways activated by CXCL1 binding to CXCR2 on DCs are unknown [15,17]. Therefore, future studies will examine different factors and their effects of signaling pathways.
Skin DCs extend dendrites between keratinocytes to develop a dense network [18]. Activated skin DCs gain access to antigen by extending their dendrites through epidermal TJs [19].
AdhPep10-2 increases expression of TJ proteins. We expect that in the skin, AdhPep-10-2 increases expression of TJ proteins by keratinocytes, thereby suppressing penetration by skin DC dendrites and lymphocytes. This has important implications for the pathogenesis of inflammatory skin diseases such as psoriasis and atopic dermatitis and for the healing of skin lesions. In fact, increased expression of CXCL1 has been reported in inflammatory skin diseases such as psoriasis [15,20,21]; indeed, it is suggested to play a role in neutrophil recruitment and epidermal hyperproliferation, which are characteristic of this disease. Furthermore, the number of CD11c + DCs in the dermis of psoriatic lesions is 30fold higher than that in healthy skin [1,15,20]. Therefore, there are several potential avenues for a therapeutic intervention targeting augmented expression of CXCL1 and lymphocyte recruitment.

Conclusion
we showed here that AdhPep-10-2-treated keratinocytes inhibit DC maturation and increase the Treg cell population. These results show the possibility that increased keratinocyte TJ proteins inhibit DC maturation and inflammatory mediator production.
Although the underlying mechanisms still need to be elucidated, we suggest the involvement of CXCL1 which expression is reduced.