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Published in final edited form as: Exp Mol Pathol. 2020 May 21;115:104470. doi: 10.1016/j.yexmp.2020.104470

Skin remodeling and wound healing in the Gottingen minipig following exposure to sulfur mustard

Jeffrey D Laskin 1, Gabriella Wahler 2, Claire R Croutch 3, Patrick J Sinko 4, Debra L Laskin 2, Diane E Heck 5, Laurie B Joseph 2,*
PMCID: PMC7374066  NIHMSID: NIHMS1598432  PMID: 32445752

Abstract

Sulfur mustard (SM), a dermal vesicant that has been used in chemical warfare, causes inflammation, edema and epidermal erosions depending on the dose and time following exposure. Herein, a minipig model was used to characterize wound healing following dermal exposure to SM. Saturated SM vapor caps were placed on the dorsal flanks of 3-month-old male Gottingen minipigs for 30 min. After 48 hr the control and SM wounded sites were debrided daily for 7 days with wet to wet saline gauze soaks. Animals were then euthanized, and full thickness skin biopsies prepared for histology and immunohistochemistry. Control skin contained a well differentiated epidermis with a prominent stratum corneum. A well-developed eschar covered the skin of SM treated animals, however, the epidermis beneath the eschar displayed significant wound healing with a hyperplastic epidermis. Stratum corneum shedding and a multilayered basal epithelium consisting of cuboidal and columnar cells were also evident in the neoepidermis. Nuclear expression of proliferating cell nuclear antigen (PCNA) was contiguous in cells along the basal epidermal layer of control and SM exposed skin; SM caused a significant increase in PCNA expression in basal and suprabasal cells. SM exposure was also associated with marked changes in expression of markers of wound healing including increases in keratin 10, keratin 17 and loricrin and decreases in E-cadherin. Trichrome staining of control skin showed a well-developed collagen network with no delineation between the papillary and reticular dermis. Conversely, a major delineation was observed in SM-exposed skin including a web-like papillary dermis composed of filamentous extracellular matrix, and compact collagen fibrils in the lower reticular dermis. Although the dermis below the wound site was disrupted, there was substantive epidermal regeneration following SM-induced injury. Further studies analyzing the wound healing process in minipig skin will be important to provide a model to evaluate potential vesicant countermeasures.

Keywords: sulfur mustard, vesicants, wound healing, Gottingen minipig, keratin 10, keratin 17, loricrin, E-cadherin, PCNA

Sulfur mustard (SM, bis 2-chloroethyl sulfide) is a skin vesicant that has been used in chemical warfare (Etemad et al., 2019; Jiang and Maibach, 2018; Lowenstein, 2011; Wattana and Bey, 2009). The severity of SM-induced dermal injury is influenced by the dose and time of exposure, as well as the location on the skin (Graham and Schoneboom, 2013; Shakaijian et al., 2010). Typical cutaneous toxicity in humans resulting from SM exposure involves inflammation of delayed onset (Steinritz et al., 2019). This is followed by the formation of fluid filled vesicles which can coalesce to form pendulous blisters; rupture of these blisters results in the formation of a necrotic layer and an eschar (Kehe and Thiermann, 2009; Salamati and Razavi, 2015). Wound healing following SM induced injury is prolonged and remodeling of the tissue can result in depigmentation and scar formation (Poursaleh et al., 2012; Rice, 2003).

Mechanisms underlying SM-induced cutaneous injury are not well understood (Wolfe et al., 2019). As a bifunctional alkylating agent, mustards modify many targets in tissues including DNA, proteins, amino acids and antioxidants such as glutathione and thioredoxin (Laskin et al., 2010; Naghii, 2002; Pal et al., 2009). SM induced degenerative changes in basal epithelial cells involving detachment from the basement membrane zone, along with infiltration and activation of inflammatory macrophages and neutrophils, are thought to underlie blister formation (Joseph et al., 2016; Poursaleh et al., 2012; Vavra et al., 2004).

A number of animal models including pigs, mice, rabbits and guinea pigs have been used to investigate the cytotoxic actions of SM on the skin and consequent wound healing responses (Barillo et al., 2019; Dachir et al., 2012; Joseph et al., 2014). Pig skin is of particular interest as there is a high degree of similarity to human skin in the wound healing response (Meyer, 1978; Seaton et al., 2015; Turner et al., 2015). SM-induced injury in pig skin has been characterized both in vivo and in vitro; the effects of potential therapeutics and barrier creams have also been assessed (Chilcott et al., 2007; Dachir et al., 2017; Kadar et al., 2003; Matar et al., 2019). epidermal cell death and subepidermal microblisters are evident which are associated with significant stromal and vascular changes typical of inflammatory responses including edema and inflammatory cell infiltration (Brown and Rice, 1997; Lindsay and Rice, 1995; Smith et al., 1996). In the present studies we characterized the wound healing response in pig skin following SM exposure using the Gottingen minipig. A better understanding of mechanisms underlying SM-induced wound healing are key to identifying potential targets for the development of effective countermeasures.

Materials and Methods

Animals and Treatments.

All animal experiments and SM treatments were performed at MRIGlobal, Kansas City, MO in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility following MRIGlobal Institutional Animal Care and Use Committee (IACUC) approval, as previously described (Barillo et al., 2019). Animals received humane care in compliance with the institution’s guidelines, as outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Three-month-old male Gottingen minipigs (Marshall Farms, North Rose, NY) were exposed to SM using a saturated vapor cap. Neat liquid SM (10 μ,L) was applied to filter paper in the top of the vapor cap which were then placed onto a glass surface for at least 5 min to allow for the formation of a vapor. Control caps did not contain SM. Caps were then placed on the dorsal skin of anesthetized minipigs and removed after 30 min. After an additional 48 hr, minipigs were anesthetized and skin debrided using wet-to-wet gauze soaks; debridement continued daily for 7 days. After each debridement, exposed sites were covered with a saline gauze dressing (0.9% NaCl, sterile; Hospira, Inc., Lake Forest, IL) which was changed daily. At the end of the skin at wound sites taken, immediately fixed in 10% formalin, and then embedded in paraffin.

Histology and Immunostaining.

Tissue sections (6 μm) were prepared and stained with hematoxylin and eosin (H&E) or Gomori’s trichrome containing methyl (aniline) blue for analysis of collagen I/III (Histopathology Core Facility, Rutgers University, Piscataway, NJ). For immunohistochemistry, tissue sections were deparaffinized, and blocked at room temperature with 1% goat or 1% horse serum for 2 hr and then incubated at room temperature for 30 min, or overnight at 4°C with primary rabbit affinity purified polyclonal antibodies to loricrin (1:400, Abcam, Cambridge, MA), keratin 10 or keratin 17 (1:200, Covance, Princeton, NJ), or with monoclonal antibodies to E-cadherin (1:300, Cell Signaling, Danvers, MA), or PCNA (1:300, Millipore, Burlington, MA) and mouse IgG (ProSci Inc., Poway, CA), or rabbit IgG (ProSci Inc.) as controls. After washing, tissue sections were incubated for 30 min with biotinylated goat anti-rabbit- or goat anti-mouse-secondary antibodies (Vector Labs, Burlingame, CA). Antibody binding was visualized using a DAB Peroxidase Substrate Kit (Vector Labs). Images of tissue sections were acquired at high resolution using an Olympus VS120 Virtual Microscopy System and analyzed using OlyVIA version 2.9 software (Center Valley, PA).

Results

SM-induced structural changes in pig skin during wound healing.

In initial studies we analyzed morphological changes in pig skin during the wound healing process following SM administration. Control dorsal skin sections showed a well-developed laminated stratum corneum overlying a differentiated epidermis (Figs. 1 and 2). The epidermis contained a well-defined basal layer with keratinocytes displaying prominent nuclei. Overlying the basal layer was a 4–6 cell layer thick spiny layer and a distinct 3–4 cell layer thick granular layer (stratum capillaries were scattered throughout the dermis. The dermis was homogeneous with no demarcation between the upper papillary dermis and the lower reticular dermis.

Figure 1. Structural changes in pig skin following exposure to SM.

Figure 1.

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Histological sections, prepared from control (CTL) pig skin and pig skin 9 days post SM, were stained with H&E. Panel A, Sections showing interfollicular epidermis and dermis; Panel B, Sections showing hair follicles embedded in epidermis and dermis. B, basal layer; D, dermis; E, epidermis; Es, eschar; G, granular layer; HF, hair follicle; ORS, outer root sheath; S, spiny layer; SC, stratum corneum; Asterisk, papillary dermis. Panels on the left, scale bar = 100 μm; panels on the right, scale bar = 50 μm.

Figure 2. Trichrome staining of pig skin following exposure to SM.

Figure 2.

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Histological sections, prepared from control (CTL) pig skin and pig skin 9 days post SM, were stained with Gomori’s trichrome containing hematoxylin which stains nuclei dark blue/black, eosin which stains keratin and cytoplasm red, and aniline blue which stains collagen I/III royal blue. Panel A, Sections showing interfollicular epidermis and dermis; Panel B, Sections showing hair follicles embedded in epidermis and dermis. B, basal layer; D, dermis; E, epidermis; Es, eschar; HF, hair follicle; ORS, outer root sheath; SC, stratum corneum; Asterisk, papillary dermis. Panels on the left, scale bar = 100 μm; panels on the right, scale bar = 50 μm.

Nine days following SM administration, extensive wound healing was noted, as evidenced by the presence of a hyperplastic neoepidermis (Figs. 1 and 2). This was associated with a significant increase in full skin thickness and epidermal thickness (Fig. 3). Increases in full skin thickness was due to both increases in dermal and epidermal thickness. The basal cell layer was 3–4 cells thick and comprised of disorganized cuboidal and columnar cells. The epidermis also contained a hyperplastic spiny layer with flattened keratinocytes. A thickened stratum lucidum and a stratum granulosum containing 5–7 cell layers were also evident. Numerous birds-eye nuclei were present in basal and suprabasal keratinocytes. The epidermis of SM treated skin was also covered by an eschar directly above the stratum lucidum; despite extensive wound healing, there was no contiguous stratum corneum.

Figure 3. Effect of SM on epidermal thickness in pig skin.

Figure 3.

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H&E stained sections of control (CTL) pig skin and pig skin 9 days post SM exposure were assessed for epidermal thickness (left panel) and full skin thickness (right panel). Note that both epidermal thickness and full skin thickness increased following treatment of skin with SM. Data are presented as mean + S.E. (n = 3). *Significant from control p < 0.05.

Following SM exposure, the interfollicular papillary dermis appeared disorganized and edematous near the basement membrane; inflammatory cells including macrophages and neutrophils were also present at the dermal/epidermal junction. In SM treated dermis, but not control skin, a marked delineation in the papillary and reticular dermis was evident (Fig. 2). Stratum corneum shedding, basal cell karyolysis and elongated rete ridges composed of multilayered columnar epithelial cells were also apparent in the neoepidermis during wound healing following SM administration (Figs. 1 and 2 and not shown). Conversely, the dermis surrounding the hair follicles was homogeneous in both control and SM treated tissue.

Epidermal proliferation, differentiation and wound healing.

PCNA, a marker of cellular proliferation, was present within the nuclei of basal cells in the epidermis and the outer root sheath of hair follicles in control skin. A marked increase in the number of PCNA expressing expressed higher levels of PCNA when compared to basal cells from control skin (Fig. 4). In both control and SM-treated skin, scattered PCNA expressing cells were evident in flattened suprabasal cells. PCNA expression in hair follicles was similar in control and SM treated skin (not shown). These data indicate that epidermal hyperplasia in the neoepidermis following SM treatment is due to extensive proliferation of cells in the basal layer. This may account, in part, for the appearance of disorganized cuboidal and columnar cells in the basal layer, as well as aberrant differentiation of the suprabasal cells in the neoepidermis during wound healing.

Figure 4. Effects of SM on PCNA expression in pig skin.

Figure 4.

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Histological sections, prepared from control (CTL) pig skin (upper panels) and pig skin 9 days post SM (lower panels), were visualized with an antibody to proliferating cell nuclear antigen (PCNA). B, basal layer; HF, hair follicle; Black/white star, flattened suprabasal nuclei. Panels on the left, scale bar = 100 μm; panels on the right, scale bar = 50 μm.

Keratins are intermediate filaments that are structural elements in keratinocytes and are also important in regulating keratinocyte proliferation, differentiation and would healing activity (Moll et al., 2008). We found that basal cells from control and SM-treated skin expressed no or very low levels of keratin 10 (Fig. 5). In contrast, suprabasal cells from control and SM-treated skin expressed high levels of keratin 10; significantly more keratin 10 was expressed in suprabasal cells from SM-treated skin when compared to control skin. Keratin 10 was not expressed in the stratum corneum (Fig. 5). Keratin 10 is known to be activated in suprabasal cells and is important in regulating keratinocyte terminal differentiation. Increased levels of keratin 10 were noted in keratinocytes in the inner root sheath of hair follicles following SM treatment (Fig. 5).

Figure 5. Effects of SM on keratin-10 expression in pig skin.

Figure 5.

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Histological sections, prepared from control (CTL) pig skin and pig skin 9 days post SM, were visualized with an antibody to keratin-10. Panel A, Sections showing interfollicular epidermis and dermis; Panel B, Sections showing hair follicles embedded in epidermis and dermis. B, basal layer; D, dermis; E, epidermis; HF, hair follicle; IRS, inner root sheath; ORS, outer root sheath; S, spiny layer; SC, stratum corneum. Panels on the left, scale bar = 100 μm; panels on the right, scale bar = 50 μm.

Keratin 17, which is largely present in epidermal appendages, but not interfollicular keratinocytes, was expressed at low constitutive levels in the epidermis of control skin (Kirfel et al., 2003) (Fig. 6). SM treatment caused a marked increase in keratin 17 expression in both basal and suprabasal keratinocytes in the neoepidermis. Significantly greater amounts of keratin 17 were also expressed in basal keratinocytes relative to suprabasal keratinocytes. Increased expression of keratin 17 in the neoepidermis of SM-treated skin is important in maintaining keratinocytes in a proliferative state during the wound healing process (McGowan and suprabasal cells in the hyperplastic epidermis. The outer root sheath of hair follicles in control and SM treated skin also expressed generally similar amounts of keratin 17 where it functions in structural support (Fig. 6).

Figure 6. Effects of SM on keratin-17 expression in pig skin.

Figure 6.

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Histological sections, prepared from control (CTL) pig skin and pig skin 9 days post SM, were stained with an antibody to keratin-17. Panel A, Sections showing interfollicular epidermis and dermis; Panel B, Sections showing hair follicles embedded in epidermis and dermis. D, dermis; E, epidermis; B, basal layer; HF, hair follicle; IRS, inner root sheath; ORS, outer root sheath; S, spiny layer; SC, stratum corneum. Panels on the left, scale bar = 100 μm; panels on the right, scale bar = 50 μm.

We next examined two proteins important in developing the structural integrity of the epidermis, loricrin and E-cadherin. Loricrin, a glycine-serine-cysteine-rich insoluble protein, is critical for the formation of keratinocyte cornified envelopes (Candi et al., 1995). In control skin, loricrin was expressed in the granular layer in a tight band of cells just beneath the stratum corneum (Fig. 7). In the neoepidermis of SM-treated skin, loricrin was expressed in 2–3 cell layers just below the eschar. Increased expression of loricrin in the neoepidermis likely reflects an increase in formation of well differentiated keratinocytes required for wound healing. Loricrin was also expressed in differentiating cells in the infundibulum of the hair follicle keratinocytes in the outer root sheath of SM-treated skin. (Fig. 7).

Figure 7. Effects of SM on loricrin expression in pig skin.

Figure 7.

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Histological sections, prepared from control (CTL) pig skin and pig skin 9 days post SM, were stained with an antibody to showing hair follicles embedded in epidermis and dermis. G, granular layer; HF, hair follicle; I, infundibulum. Panels on the left, scale bar = 100 μm; panels on the right, scale bar = 50 μm.

E-cadherin is an adhesion protein essential for the formation and maintenance of epithelial adherens junctions (Young et al., 2003). In control skin, E-cadherin was expressed at junctions between basal cells and in differentiating suprabasal cells of the epidermis (Hynes, 1992) (Fig. 8). Expression of E-cadherin was significantly reduced in the epidermis after SM treatment; in these tissues, it was largely confined to epithelial adherens junctions of suprabasal cells below the granular layer (Fig. 8). Generally similar amounts of E-cadherin were expressed in adherens junctions of outer root sheath cells in hair follicles of control and SM-treated skin. Expression of E-cadherin in the outer root sheath is likely necessary to maintain structural integrity of hair follicles in damaged skin following treatment with SM, as well as in control skin during the hair growth cycle (Fig. 8).

Figure 8. Effects of SM on E-cadherin expression in pig skin.

Figure 8.

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Histological sections, prepared from control (CTL) pig skin and pig skin 9 days post SM, were stained with antibody to E- cadherin. Panel A, Sections showing interfollicular epidermis and dermis; Panel B, Sections showing hair follicles embedded in epidermis and dermis. Es, eschar; HF, hair follicle; IRS, inner root sheath; ORS, outer root sheath; Star, suprabasal cells. Panels on the left, scale bar = 100 μm; panels on the right, scale bar = 50 μm.

Discussion

The repair of skin wounds following chemical burns is key to restoration of normal tissue structure and function. In the skin, wound healing is a dynamic process involving not only the development of a well differentiated neoepidermis that provides barrier functions, but also restoration of the dermis with limited scarring. The present studies demonstrate significant impairment in the wound healing process in the skin of a Gottingen mini pig following exposure to SM. This may contribute to chronic disorders in wound healing observed in victims exposed to SM, including epidermal proliferative disorders, scar formation and cancer (Firooz et al., 2011; Ghanei and Harandi, 2007; Rowell et al., 2009).

Although a neoepidermis developed over the SM-injured site in the pig skin, indicating initiation of wound healing, significant edema and epidermal hyperplasia remained; formation of a stratum corneum was also incomplete. The presence of edema 9 days after SM exposure, along with macrophages and neutrophils at the dermal/epidermal junction demonstrate that inflammation is prolonged. Epidermal hyperplasia following SM exposure was characterized by increased numbers of cells in the basal layer, granular layer, and stratum lucidium. Additionally, basal cells appeared disorganized with a mixture of cuboidal and columnar cells indicating aberrant regulation of proliferation. Components of the basement membrane zone in the skin are known to regulate epidermal cell growth and differentiation; it is likely that SM disrupts basal cell-matrix interactions. In this regard, earlier studies have shown that SM targets basal cell adhesion complexes known to regulate cell-cell and cell-matrix interactions including α3βi- and α6β4-integrins and extra cellular matrix components such as laminin 332, fibronectin and heparan sulfate proteoglycan (Chang et al., 2018; Werrlein and Madren-Whalley, 2003; Zhang et al., 1995) disrupted in SM treated skin, despite significant regeneration of the epidermis. Thus, wound healing in areas between the papillary and reticular dermis was incomplete; only sporadic collagen deposition was evident. In contrast, dermis of control skin contained a dense well- developed collagen network. Fibroblasts are known to be essential for collagen biosynthesis; SM may target these cells resulting in impaired collagen production during the wound healing process. This is supported by previous reports of SM-induced cytotoxicity in fibroblasts derived from mouse and human skin (Black et al., 2011; Long et al., 2016). SM has also been reported to alter expression of proteases such as matrix metalloproteinase (MMP)-2- and MMP-9 in guinea pig and mouse skin (Dachir et al., 2012; Mouret et al., 2015). It is possible that upregulation of collagenases in the dermis following SM treatment impairs the turnover of collagens important in wound repair.

In the epidermis and dermal appendages, keratins function as important structural proteins; they also control dermal cell growth and differentiation, as well as motility and migration during wound healing and tissue regeneration (Cheng and Eriksson, 2017; Patel et al., 2006). Keratin 10 is a suprabasal specific intermediate filament expressed in the epidermis (Fischer et al., 2016; Walter, 2001). In the minipig model, we found that keratin 10 was primarily expressed in differentiating suprabasal keratinocytes. Following SM administration, a marked increase in expression of keratin 10 was noted in the hyperplastic epidermis; this is consistent with its role in regenerating the mechanical integrity of the skin following regrowth of the epidermis, as well as in restoring skin hydration and permeability barrier function (Baba et al., 2005; Jensen et al., 2000). Keratin 10 was also found in keratinocytes within the inner root sheath following SM exposure where it likely functions to strengthen the hair follicle during wound repair (Coulombe et al., 2004; Koch and Roop, 2004). This suggests that increased expression of keratin 10 in the neoepidermis of SM-treated skin plays a role in restoring a In accord with previous observations in domestic pigs and humans, keratin 10 was also expressed in minipigs in the inner root sheath of hair follicles (Stark et al., 1990; Wollina et al., 1992). Increases in expression of keratin 10 in the hair follicle following SM treatment may be important in suppressing hair growth in the interfollicular epidermis as the skin regenerates during wound repair (Wollina et al., 1992).

Keratin 17 has been reported to be expressed in dermal appendages including hair follicles and sebaceous glands, but not in epidermis (Jin and Wang, 2014; Yang et al., 2019). Consistent with this report, we found only very low constitutive levels of keratin 17 in the epidermis of pig skin. However, expression of this keratin was markedly increased during wound healing after SM most prominently in basal cells (Ekanayake-Mudiyanselage et al., 1998; Mazzalupo et al., 2003). Earlier studies showed that keratin 17 expression is upregulated in human epidermis following injury induced by punch biopsies, in proliferative disorders including psoriasis and cancer, and after viral infections (Markey et al., 1992; Patel et al., 2006). Keratin 17 is known to be regulated by proinflammatory cytokines and growth factors and is important in controlling keratinocyte growth during wound repair and remodeling; it is presumably involved in these functions in pig skin following SM-induced injury (Depianto et al., 2010; Gibbs et al., 2000; Mazzalupo et al., 2003). We also found that keratin 17 was expressed in the outer root sheath of pig skin hair follicles. These data are in line with earlier studies showing that keratin 17 is expressed in the outer root sheath of hair follicles in mouse and human skin (Bianchi et al., 2005; Panteleyev et al., 1997). SM had no effect on expression of keratin 17 in hair follicles. Keratin is important for cell viability in the hair bulb, as well as for structural and mechanical integrity of the hair shaft (Tong and Coulombe, 2006; Wang et al., 2003). The fact that SM did not alter hair follicle keratin 17 expression suggests that hair follicles in pig skin are resistant to in pig skin following SM administration.

Loricrin is an abundant cornified envelop protein present in differentiating keratinocytes; it is important in the development of the skin barrier (Candi et al., 1995; Nemes and Steinert, 1999). During this process, loricrin cross-links with other components of the cornified cell envelop via the formation of isodipeptidic bonds, a process catalyzed by the action of epidermal transglutaminases (Hitomi, 2005; Inada et al., 2000; Steinert and Marekov, 1995). Our findings that loricrin is expressed in the upper granular layer of minipig epidermis are in accord with its barrier function in these animals. Similar expression patterns of loricrin have been described in the epidermis of a number of other mammalian species (Candi et al., 2005; Hohl et al., 1993; Jorgensen et al., 2018; Streubel et al., 2018). Expression of loricrin was noted in the granular layer of the hyperplastic epidermis following SM exposure. Increased loricrin expression was also observed in keratinocytes in the infundibulum of the hair follicle in SM treated skin. These data indicate increased formation of cornified envelopes in the hair follicle, possibly due to tissue remodeling in response to SM-induced injury. Loricrin expression in the hair follicle also presumably functions to maintain the structural integrity of the hair follicles post-SM exposure (Akiyama et al., 2002).

E-cadherin is a key component of adherens junctions, a protein complex important in intercellular adhesion in epidermis and hair follicles (Young et al., 2003). It contributes to the ability of the epidermis to withstand mechanical stress; it is also known to regulate keratinocyte proliferation, migration and differentiation, as well as wound healing (Andl et al., 2003; Kuwahara et al., 2001; Suffoletto et al., 2018). In normal pig skin, we found that E- cadherin was expressed at adherens junctions of cells in the basal and suprabasal differentiating layers of the epidermis (Muranaka et al., 2018). Reduced amounts of E-cadherin were expressed within the basal layer during wound repair following treatment with SM, where it was largely localized integrity, decreases in expression of this protein may be necessary to allow cells to proliferate and migrate as well as remodel the tissue during wound healing. Earlier studies have shown that decreases in E-cadherin can lead to progressive hyperproliferation of mouse and human keratinocytes (Chung et al., 2005; Deng et al., 2014; Jamora et al., 2005). Decreases in E- cadherin are important for increased growth and differentiation of keratinocytes during wound healing and providing sufficient numbers of keratinocytes for wound closure (Oda et al., 2017). In this regard, decreases in E-cadherin have been reported in migrating mouse and equine keratinocytes during wound repair (Jorgensen et al., 2019; Kuwahara et al., 2001).

Of note was our observation that E-cadherin was expressed at the adherens junctions in the outer root sheath of hair follicles in both control and SM-treated pig skin. These findings are in accord with earlier studies reporting localization of this protein at adherens junctions in human and mouse hair follicles and support the critical structural role of E-cadherin in the hair follicle (Tinkle et al., 2004). Increased levels of E-cadherin were noted in outer root sheath cells adjacent to the hair cuticle, presumably because of its importance in maintaining mechanical strength against the continuing growth of the hair shaft (Alibardi and Bernd, 2013).

In summary, our results demonstrate several novel aspects of the wound healing process in minipig skin 9 days following exposure to SM. Although a stratified epidermis formed over the skin wound, differentiation was incomplete. Thus, reduced amounts of E-cadherin, keratin 10 and keratin 17 were observed. There was also significant epidermal hyperplasia, and the basal cell layer was disorganized and multilayered with increased numbers of proliferating cells relative to control skin. The dermis below the wound was disrupted and a major delineation was observed between the upper papillary dermis and the lower reticular dermis which contained compact collagen fibrils. These observations are important as they suggest potential mechanistic targets for the development of countermeasures against dermal vesicants. These include agents factor as well as agents that stimulate collagen production such as fibroblast growth factor and cholesterol ester which may enhance papillary dermal repair and/or regrowth of the neoepidermis. Success in this endeavor will depend not only on a better understanding of the skin remodeling process following exposure to these agents, but also on establishing methods to improve the wound healing process.

Acknowledgements

This work was supported by the NIH under grants AR055073, ES005022 and T32ES007148.

Footnotes

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Disclosure of interest

The authors have no conflict of interest for the subject matter of this paper.

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