RESEARCH ARTICLE

Aberrant Wound Healing in an Epidermal Interleukin-4 Transgenic Mouse Model of Atopic Dermatitis Yan Zhao1, Lei Bao2, Lawrence S. Chan2,3,4, Luisa A. DiPietro1, Lin Chen1* 1 Center for Wound Healing and Tissue Regeneration, College of Dentistry, University of Illinois at Chicago, Chicago, Illinois, United States of America, 2 Departments of Dermatology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America, 3 Departments of Immunology and Microbiology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America, 4 Medicine Service, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois, United States of America * [email protected]

Abstract OPEN ACCESS Citation: Zhao Y, Bao L, Chan LS, DiPietro LA, Chen L (2016) Aberrant Wound Healing in an Epidermal Interleukin-4 Transgenic Mouse Model of Atopic Dermatitis. PLoS ONE 11(1): e0146451. doi:10.1371/ journal.pone.0146451 Editor: Miroslav Blumenberg, NYU Langone Medical Center, UNITED STATES Received: October 30, 2015 Accepted: December 17, 2015 Published: January 11, 2016 Copyright: © 2016 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was supported by NIH Grants R01GM50875 (LAD), College of Dentistry, University of Illinois at Chicago (LC, YZ, and LAD), Dr. Orville J. Stone Endowed Professorship (LSC), and the Albert H. and Mary Jane Slepyan Endowed Fellowship (LB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

Wound healing in a pre-existing Th2-dominated skin milieu was assessed by using an epidermal specific interleukin-4 (IL-4) transgenic (Tg) mouse model, which develops a pruritic inflammatory skin condition resembling human atopic dermatitis. Our results demonstrated that IL-4 Tg mice had delayed wound closure and re-epithelialization even though these mice exhibited higher degrees of epithelial cell proliferation. Wounds in IL-4 Tg mice also showed a marked enhancement in expression of inflammatory cytokines/chemokines, elevated infiltration of inflammatory cells including neutrophils, macrophages, CD3+ lymphocytes, and epidermal dendritic T lymphocytes. In addition, these mice exhibited a significantly higher level of angiogenesis as compared to wild type mice. Furthermore, wounds in IL-4 Tg mice presented with larger amounts of granulation tissue, but had less expression and deposition of collagen. Taken together, an inflamed skin condition induced by IL-4 has a pronounced negative influence on the healing process. Understanding more about the pathogenesis of wound healing in a Th2- dominated environment may help investigators explore new potential therapeutic strategies.

Introduction Wound healing is a well-regulated complicated process that involves interactions among resident and recruited cells such as epithelial cells, fibroblasts, endothelial cells, inflammatory cells, and interactions of those cells with extracellular matrix molecules, growth factors, cytokines, and chemokines. Inflammatory response is a normal part of the healing process following injury that helps eliminate micro-organisms and injured cells. In addition, Inflammation also assists restoring tissue integrity. However, excessive inflammation may be unfavorable to subsequent healing [1–3]. IL-4 is mainly secreted by Th2 cells, mast cells, eosinophils, and basophils. It was first identified as a factor promoting the growth and differentiation of B lymphocytes [4]. IL-4 is actually

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a multifunctional cytokine, which has profound effects on not only hematopoietic cells such as B lymphocyte and monocytes/macrophages [5], but also non-hematopoietic cells, such as fibroblasts, where it stimulates the synthesis of extracellular matrix, especially collagens [6–8]. Furthermore, fibrosis in lung and liver has been reportedly associated with the Th2 immuneresponse, especially the production of cytokines IL-4 and IL-13 [9, 10]. However, there were very limited and conflicting studies investigating the roles of IL-4 in skin wound healing. One study showed that IL-4 was strongly expressed during the early period (days 2–4) of normal wound healing and disappeared after wound closure in a mouse model [11]. This same study reported that topical application of recombinant IL-4 on wounds increased the inflammatory cell infiltrate, but accelerated wound closure while IL-4 anti-sense oligonucleotides significantly inhibited healing [11]. Another study showed that recombinant IL-4 reversed the retardation of wound closure in the bacteria infected acute and chronic wounds accompanied with decreased neutrophil infiltration, but had no effect on closure of acute non-infected wounds [12]. The aim of the present study was to investigate if skin injury in a pre-existing Th2 dominated microenvironment would result in an altered wound healing, such as a chronic wound condition or excessive scar formation. We used a keratin-14 IL-4 epidermal transgenic mouse model [13–22]. This line of mice spontaneously develops a pruritic inflammatory skin condition that resembles human atopic dermatitis, typically demonstrating xerosis, conjunctivitis, chronic skin lesions with T cell, mast cell, macrophage, and eosinophil infiltration, staphylococcus aureus infection and elevation of total serum IgE [13–22]. In the present study, wounds were made on normal looking dorsal skin of IL-4 Tg mice before the onset of skin disease. The results demonstrated that IL-4 Tg mice had significantly impaired wound closure accompanied by drastically elevated levels of inflammation and angiogenesis, as well as decreased deposition of collagen compared to C57BL/6 wild type (WT) mice.

Materials and Methods Animals and wound models The keratin 14 epidermal IL-4 Tg mouse line was initially established in outbred CByB6 mice in 2001 [15]. It was then backcrossed to C57BL/6 mice for more than 10 generations. The majority of mice develop an atopic dermatitis- like skin condition at the age of approximately 13 weeks [22]. In our current experiments, IL4-Tg mice in a C57BL/6 background were wounded when the mice were eight weeks old and had not yet developed skin disease; C57BL/6 wild type (WT) mice were used as controls. Six 3mm full thickness excisional wounds were made on shaved dorsal skin under ketamine (100mg/Kg) and xylazine (5mg/Kg) anesthesia using a biopsy punch (Acuderm, Inc., Ft. Lauderdale, FL). The wounds were photographed at days 0, 1, 3, 5, 7, 10, 14 and 21 after wounding, and wound sizes were determined by software AxioVision (ZEISS, Oberkochen, Germany). Wound tissues were harvested and stored in either RNAlater (Sigma, St. Louis, MO), embedded in OCT compound or fixed in formalin for future processing. IL-4 Tg mice developing dermatitis during the study were removed from the experiment. Wound breaking strength was examined in another set of experiments. A single 2 centimeter incisional wound was made on the shaved dorsal skin and closed with two surgical clips. The clips were removed at day 7 post-wounding; wounds were harvested at days 14 and 21 to assess wound breaking strength. The University of Illinois at Chicago Institutional Animal Care and Use Committee (IACUC) approved this study. Mice were housed at 22 to 24°C on a 12-h:12-h light/dark cycle and received food and water ad libitum. The animals were monitored daily by the animal-care staff and investigators. Any mice displaying signs of infection, severe skin rashes, wasting, and hunching were euthanized. All other mice were sacrificed by lethal CO2 overdose followed by cervical dislocation at the end of the experiment. All animal

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procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health).

Indirect immunofluorescence For immunohistochemical studies, 8μm frozen sections were prepared from wound tissues embedded in OCT compound, air-dried, rehydrated in PBS and fixed in cold acetone for 10 min. Sections were blocked with 10% goat serum for 30 min. Sections were then incubated with either rat anti-mouse Gr-1 (for neutrophil staining, 0.5μg/ml;BD Bioscience, San Jose, CA), rat anti-mouse CD68 (for macrophage staining, 0.5 μg/ml; Abcam, Cambridge, MA), rat anti-mouse CD3 (for T lymphocyte staining, 1.25 μg/ml; SouthernBiotech, Birminham, AL), rat anti-mouse CD31 (for endothelial cell staining, 0.3 μg/ml, BD Biosciences, San Jose, CA), rabbit anti-mouse Ki67 (for proliferating cell staining, 0.5 μg/ml; Abcam), or hamster antimouse dendritic epidermal T cells (DETC) (1.25 μ/ml;, BD Biosciences) for 45 min followed by Alexa fluor 488 or 594 goat anti-rat IgG, or goat anti-rabbit IgG Alexa fluor 594, or goat antihamster IgG Alexa fluor 594 (Invitrogen, Carlsbad, CA). The staining procedures were performed at room temperature. Stained sections were observed using a fluorescence microscope (Axioskop 40, ZEISS, Oberkochen, Germany) and recorded by a digital camera (AxioCam HRc, ZEISS). CD3 and Gr-1 positively stained cells in the wounds and wound margins were counted and the average number per 20x field was calculated. To quantify the density of CD31-stained blood vessels, the area within the wound bed and CD31-positive area were measured using ImageJ [23]. The vessel density was then expressed as the percent CD31 positive area in the wound bed. Since the staining of CD68 was very intense at some time points in the IL4 Tg mice, it was impossible to accurately count the stained cells. Therefore, CD68 was quantified using the same method for quantification of CD31.

Hematoxylin/eosin (HE), Masson’s Trichome, and picrosirious red stainings The rate of re-epithelialization was calculated using the method published previously [24]. Using H&E stained tissue sections, the thickness of epithelium at the wound edge before wound closure or at the center of the wound bed after closure, and the thickness of the wound bed were measured using AxioVision software. Mature and immature collagens were quantified using Image J analysis of the wound bed in picrosirious red stained histologic sections. The percent of mature or immature collagen was calculated as follows: pixels of mature or immature collagen/total pixels of mature and immature collagen x100.

Real time PCR Total RNA was extracted from normal skin and wounds of IL-4 Tg and WT mice using TriZol (Invitrogen). One microgram of each RNA sample was treated with DNAse I, and subjected to reverse transcription using a Retro-script kit (Invitrogen). Relative mRNA expression of IL-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IFN-γ, TNF-α, TGF-β1, FGF-7, FGF-10, EGF, VEGF, CCL-2 (MCP-1), CXCL-1 (MIP-2α or KC), CXCL-2 (MIP-2β), lysyl oxidase (LOX), and NLR family pyrin domain containing 3 (NLRP3) was examined using a StepOne Plus real time PCR system (Applied Biosystems, Carlsbad, CA) that employs SYBR Green PCR mix (Roche, Basel,Switzerland) and gene specific primers. All primer sequences for IL-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IFN-γ, TNF-α, TGF-β1, CCL-2 (MCP-1), and GAPDH were as previously described [25]. The primers for CXCL-1, VEGF-A165, NLRP3, MMP-9, and macrophage 2 (M2) marker YM1/ Arginase 1(Arg1) were as published in [26], [19], [27], [28] and [29] respectively. The primer

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sequences of other molecules are listed in Table 1. Levels of mRNA expression in skin of normal WT mice were used as baseline. GAPDH was used as a house-keeping gene for calibration.

Breaking strength Breaking strength of 2 cm incisional wounds was examined at days 14 and 21 post-wounding using a tensiometer (Mark-10, Copiague, NY) as described previously [26, 30]. Breaking strength was recorded as weight load (pound, lb) at the point of wound breakage. Two strips from each mouse were tested and the average was used as the wound breaking strength for that individual animal.

Cell culture Primary normal human skin epidermal keratinocytes (NHEK, ATCC, Manassas, VA) and human normal dermal fibroblasts (NHDF, PromoCell, Heidelberg Germany) were cultured in 12-well plates to 80–90% confluence and then treated with mitomycin C (10μg/ml, SigmaAldrich, St Louis, MO) for 2 hours to prevent cell proliferation. Scratches were produced by application of 3 horizontal and 3 vertical scratch wounds using 0.2ml pipette tips. After washing, the cells were treated with human recombinant IL-4 (50ng/ml, PeproTech, Rocky Hill, NJ), a cocktail of human recombinant IL-1β, IL-4, IL-6, and TNF-α (50ng/ml, PeproTech), or a positive control, human recombinant EGF (50ng/ml, PeproTech) for 24 hours. The defined areas were photographed at 0 and 24 hours post injury, and the numbers of migrated cells in the wounds were counted. For the proliferation assay, 5x103/well NHEK or NHFB cells were cultured in a 96-well plate. After 24 hours, the cells were treated the same way described above and then incubated for 24 hours. A cell proliferation assay was performed using CellTiter 961 Aqueous Non-Radioactive Cell Proliferation Assay kit (Promega, Madison, WI).

Statistical analyses Results were expressed as means + standard errors (SEM). A multiple t test or t test was performed using GraphPad Prism (GraphPad Software, San Diego, CA). p values less than 0.05 were considered statistically significant.

Results Wound healing is significantly delayed in the epidermis of IL-4 Tg mice IL-4 Tg mice tolerated skin wounding well and all mice survived during the 21 days of study. Three mice developed dermatitis on the dorsal skin in the course of study and were excluded from sample collection. Based on visual observation, wound closure in IL-4 Tg mice was delayed compared to WT mice; this delay was statistically significant at days 5, 7, and 10 (Fig 1A and 1B). Table 1. Primer sequences for real time PCR Forward (5’-3’)

Reverse (5’-3’)

EGF

CCCAGGCAACGTATCAAAGT

CXCL-2

CACTCTCAAGGGCGGTCAA

GGTCATACCCAGGAAAGCAA AGGCACATCAGGTACGATCCA

IGF-1

TCATGTCGTCTTCACACCTCTTCT

CCACACACGAACTGAAGAGCAT

LOX

CAAGGGACATCGGACTTCTTAC

TGGCATCAAGCAGGTCATAG

FGF-7

TTTGGAAAGAGCGACGACTT

GGCAGGATCCGTGTCAGTAT

FGF-10

TCAGTGGAAATCGGAGTTGT

TGCTGCCAGTTAAAAGATGC

Collagen I

GGTATGCTTGATCTGTATCTG

AGTCCAGTTCTTCATTGCATT

Collagen III

AGCACCTGTTTCTCCCTT

CTGGTATGAAAGGACACAGAG

doi:10.1371/journal.pone.0146451.t001

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Fig 1. Wound healing is delayed in the epidermis of IL-4 Tg mice. a) Representative photomicrographs of wounds from days 0 to 21 after injury. Six 3mm full thickness excisional wounds were made on the dorsal skin of IL-4 Tg and WT C57BL/j mice. Bar = 3mm. b) Percent of wound closure. Similar results were obtained in another experiment. c) Photomicrographs of HE stained histologic sections of unwounded skin, days 3, 7, and 21 post-wounding. Bar = 200μm. d) Rate of wound re-epithelialization measured by histomorphometric analysis of tissue sections. e & f) Epithelial thickness and wound/scar thickness respectively, based on HE stained sections. * p