Induction of a Th17 Phenotype in Human Skin—A Mimic of Dermal Inflammatory Diseases

Th17 cells are a subset of effector T helper cells that produce interleukin (IL)-17A, IL-17F, IL-22, and IL-26, which can promote tissue inflammation and contribute to the pathogenesis of rheumatic, fibrosing, and other diseases. Research into these diseases is often limited by a lack of an animal model that closely mimics human disease and the paucity of patient clinical tissues. Therefore, the development of relevant experimental models is crucial. Three media formulations of Th17-skewing cocktail (CT) were evaluated for the ability to induce a Th17 signature in an ex vivo human skin model: CT9 contained αCD3, αCD28, IL-23, IL-1β, IFNγ, IL-4, IL-6, IL-21, and TGFβ; CT8 lacked IL-1β; and CT4 only contained αCD3, αCD28, IL-23, and IL-1β. Healthy donor skin was defatted, distributed as 3 mm punch biopsies, and incubated with one of the cocktail formulations or vehicle for 48 h. All of the cocktail formulations independently significantly stimulated the expression of each gene examined. CT4 induced IL-17A expression 1024-fold, significantly higher than CT9 and CT8. IL-17F was robustly stimulated by CT4 (1557-fold), CT9 (622-fold), and CT8 (111-fold), with significant differences between the CT groups. All of the formulations significantly induced IL-22 (16–42-fold). CT9 stimulated the highest IL-26 response (41-fold), which was significantly higher than CT4 and CT8. IL-10 was stimulated significantly higher with CT8 (10-fold) than CT4 or CT9. The secretion of IL-17A was significantly elevated with all cocktail formulations. Robust IL-17A/IL-17F cytokine induction was preferentially mediated by CT4, which suggested that its components are the minimal constituents necessary for the full induction of these genes in this human skin explant model, while the downstream cytokines were preferentially upregulated by CT4 (IL-22), CT9 (IL-26), or CT8 (IL-10). In summary, our findings suggest that the induction of a Th17 phenotype in human skin is feasible and can be used as a model for rheumatic and fibrosing diseases where Th17 skewing is observed.

IL-17A and IL-17F belong to a family of IL-17 cytokines containing six members (IL-17A-IL-17F). The effects of IL-17A are situational; in murine colitis models, IL-17A functions in a tissue-protective

Procedure
Ex vivo human skin model. Acquire human skin from healthy donors undergoing elective surgery following the rules of the Declaration of Helsinki and in accordance with guidelines set forth by the Institutional Review Board of the Medical University of South Carolina. This study is designated as non-human subject research, as the skin tissues are remnants from plastic surgery obtained without identifiers. Table 1 elaborates on the anatomical sources of donor skin.

1.
Prior to treating skin, ensure that all reagent stocks have been prepared and treatment components have been resuspended in the vehicle recommended by the manufacturer (most require PBS). The suggested stock concentrations should range between 500×-1000× whenever possible.
1.1. Aliquot into small or single-use volumes to minimize freeze-thaw cycles and store at −80 • C until the day of the assay.

2.
Defat and clean skin with ethanol and PBS, as previously described [21].

2.1.
Briefly, place skin dermal-side down atop a layer of aluminum foil on the working surface in a biological safety cabinet and clean blood/debris off of dermis using 70% ethanol.

2.2.
Separate the dermal and adipose layers using scissors and razor blade/scalpel.

2.4.
Add serum-free DMEM with 2× antibiotic/antimycotic to cover the bottom of the dish, leaving the top of the skin exposed to air.

3.
Using a 3 mm punch biopsy, punch and distribute 3-5 biopsies from cleaned, defatted skin into each well of six-well tissue culture plate to be used for the experiment ( Figure 1A-D).

3.1.
Place each biopsy with the dermal side contacting the plastic and the epidermal side facing up ( Figure 1D).

3.2.
Time for Completion: Time is dependent on the size of the experiment. Allot~20 min per 6-well plate.

3.3.
Allow skin punches to adhere approximately 15 min. before adding treatment media.

4.
While waiting for punches to adhere, prepare treatments as follows: Prepare cocktail and vehicle master mixes per Table 2

4.2.
Whether treating a single well or more than one well with the same formulation, it is recommended to make a master mix, combining all of the components that are required in a conical tube first.

4.3.
Mix the tubes containing the cocktail component formulations, and then distribute treatments into well(s) containing the skin punches in a dropwise fashion to avoid dislodging the skin punches from the plastic ( Figure 1E).

4.3.1.
For six-well tissue culture plates, a volume of 1 mL-2 mL is recommended, depending on the height of the skin punch. 4.3.2. Add sufficient media to cover the dermal layer of skin, leaving the epidermal layer exposed to air.
4.3.2.1. When viewed from the side of the well, the dermal layer should be completely submerged in media, leaving the topmost epidermal portion exposed to the air above the surface of the media.
Harvest skin punches and supernatants into Eppendorf tubes and freeze at −80 • C until analysis ( Figure 1F-G).

6.1.
Label three sets of Eppendorf tubes, one set to store skin punches and two sets for supernatants. 6.2.
Using a P1000 pipette, transfer media from each treatment well into a corresponding pre-labeled Eppendorf tube ( Figure 1F).
6.2.1. Centrifuge at 5000 RPM for 5 min to pellet cellular debris and dead cells. 6.2.2. Transfer liquid portion (supernatant) to clean pre-labeled Eppendorf tube on ice. Discard the tube containing pellet.

6.3.
Transfer skin punches to corresponding Eppendorf tube on ice ( Figure 1G).
6.3.1. Skin punches from the same treatment well can be frozen into one Eppendorf tube. Stagger the punches along the inside wall of the tube to ease future removal once frozen.
Time for completion: Time is dependent on the size of the experiment. Allot~20 min per 6-well plate.

7.
RNA isolation, reverse transcription, and quantitative real-time polymerase chain reaction.

7.1.
Homogenize one skin punch per treatment in 1 mL TRIzol TM using a Bead Ruptor 24 tissue homogenizer for 30 s at a speed of 6.95, followed by two minutes on dry ice, repeated for a total of 4-5 cycles.

7.5.
In a 10 µL final reaction volume including best-coverage TaqMan ® Gene Expression primers, include 2 µL cDNA and plate in replicate for real-time PCR. 7.5.1. The baseline expression of target genes tends to be undetectable after 55 cycles in vehicle-treated samples when starting from 1 µg of RNA and 1 µL of cDNA. Using greater quantities of starting material specified in this protocol helps to ensure a calculable Ct value for vehicles, which aids in quantifiable data analysis. 7.5.2. Time for Completion: 3 h.

7.6.
Determine gene expression using the delta delta Ct method [24], normalizing data to the housekeeping gene peptidylprolyl isomerase B (PPIB).

8.1.
Measure the secreted IL-17A in supernatants diluted 1:1 using a commercially available sandwich ELISA kit with a dynamic detection range between 1.6 pg/mL-100 pg/mL per manufacturer's instructions.

Expected Results
Three Th17-skewing cocktail formulations were scrutinized for their ability to stimulate skinresident cells into producing Th17-related cytokines. Cocktail components were identified from a search of the literature and formulations were designed in order to determine the minimal components that are necessary to elicit maximal gene expression of Th17-related cytokines in this ex vivo model. The graphs in Figures 2 and 3 represent mean values +/− standard errors of the mean. Statistical significance of p < 0.05 utilizing analysis of variance with Tukey's multiple comparison test with significance is denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001. The following descriptions represent the expected results and they include data obtained over time and across multiple anatomical skin locations. The lack of significant stimulation of gene or protein expression could indicate failure of stimulation, most likely either due to a non-responsive donor tissue or to expired component(s) in the cocktail formulations.

Regulation of Gene Expression by Different Cocktail Formulations
IL-17A is significantly induced by CT4 (1024-fold), CT8 (552-fold), and CT9 (270-fold) as compared to their respective vehicle controls, with CT4 induction of IL-17A approximately twice that of CT8 and 3.8-times that of CT9 (Figure 2A).
IL-22 expression follows the same trend as IL-17A with respect to Th17-skewing cocktail stimulation ( Figure 2C), though with a more modest induction ranging from 16-fold (CT9) to 42-fold (CT4). CT8 induction of IL-22 is similar to that of CT9 (~20-fold), and there is a significant difference between CT4 and the other two groups.
Gene expression levels of IL-10 are more modest in comparison to the other genes examined ( Figure 2E). IL-10 also exhibits a unique stimulation profile in that CT8 elicits the highest expression level of the gene (10-fold), followed by CT9 (4-fold), and a very modest yet significant effect with CT4 (1.4-fold).   Table 2 in serum-free DMEM +1% antibiotic/antimycotic.

Expected Results
Three Th17-skewing cocktail formulations were scrutinized for their ability to stimulate skin-resident cells into producing Th17-related cytokines. Cocktail components were identified from a search of the literature and formulations were designed in order to determine the minimal components that are necessary to elicit maximal gene expression of Th17-related cytokines in this ex vivo model. The graphs in Figures 2 and 3 represent mean values +/− standard errors of the mean. Statistical significance of p < 0.05 utilizing analysis of variance with Tukey's multiple comparison test with significance is denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001. The following descriptions represent the expected results and they include data obtained over time and across multiple anatomical skin locations. The lack of significant stimulation of gene or protein expression could indicate failure of stimulation, most likely either due to a non-responsive donor tissue or to expired component(s) in the cocktail formulations. and, IL-10 (E) with n = 6-19 unique donors. Samples were tested in technical replicates of 2-3 and averages used to determine fold change of gene expression relative to appropriate vehicle using the delta delta Ct method and graphed in histograms as mean ± standard error of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001 using ordinary one-way ANOVA with Tukey's multiple comparison test.

Regulation of protein levels of IL-17A by different cocktail formulations
The IL-17A protein levels are measured as a proof of concept in matching 48 h-treated supernatants by ELISA to assess whether the induction of gene expression correlates with the increased production of the corresponding protein ( Figure 3). The absolute amount of IL-17A protein released by cocktail stimulation is significantly higher than by the respective vehicle in all three formulations; however, there is not a statistically significant difference between the cocktail groups. On average, CT4 led to the secretion of 84 pg/mL IL-17A, CT8 causes 30 pg/mL IL-17A to be secreted, unique donors. Samples were tested in technical replicates of 2-3 and averages used to determine fold change of gene expression relative to appropriate vehicle using the delta delta Ct method and graphed in histograms as mean ± standard error of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001 using ordinary one-way ANOVA with Tukey's multiple comparison test. and CT9 causes an intermediate amount of IL-17A to be secreted (49 pg/mL) into media conditioned by the tissue cores following 48 h stimulation.

Interpretation/Discussion:
Various stimuli have been reported to induce Th17 cell development and promote Th17-related cytokine release. IL-23 has consistently been found to be crucial for in vivo Th17 cell development, but is itself (without other cytokines) insufficient for Th17 stimulation; likewise, IL-6 alone cannot elicit an increase in IL-17A expression in naïve CD4+ T cells in vitro [25,26]. Further research has determined that Th17 cell differentiation can occur through the combined effects of the cytokines IL-1β, IL-6, and IL-23 in the absence [25] or presence [10] of TGFβ. Other groups have determined, through murine studies, that naïve T cells can be differentiated into Th17 cells through only IL-6 and TGFβ or with the addition of IL-23 [1,27]. Another study on the contribution of TGFβ and IL-6 in Th17 cell formation led a different group to determine that these factors are critical to Th17 lineage commitment in mice, but that IL-23 and IL-1β or IL-1β and IL-6 are necessary for Th17 development from naïve T cells in humans [20,25]. While some studies have shown that the initial TGFβ/IL-1β signal mediating Th17 differentiation can also cause IL-10 induction [28], others have shown that IL-1β can inhibit IL-10 production by purified Th17 cell populations in vitro and in vivo [29]. Alternatively, IL-10 production could indicate a shift from Th17 cells to regulatory T cells (Tregs) [28]. Differences in components deemed to be necessary and sufficient to elicit the secretion of Th17 cytokines can be explained by gathering results from multiple sources across species, from purified systems utilizing naïve CD4+ T cells to whole body animal models. Thus, Th17 development seems to require a combination of cytokines and factors, regardless of the in vitro or in vivo model system. Our ex vivo skin model bridges the gap between in vitro naïve T cell cultures and in vivo murine experiments, and extends relevance of the findings to human tissues and disease. We have shown herein that Th17 cytokines can be induced in an ex vivo skin model using three different combinations of cocktails, containing a variety of cytokines that are known to be essential for Th17 cell development.
Each of the Th17-skewing cocktail formulations induced the gene expression of IL-17A, IL-17F, IL-22, IL-26, and IL-10, as well as the secretion of IL-17A. Though there was no clear cocktail formulation that resulted in the highest induction of all genes tested, CT4 stimulated the highest responses in three out of the five genes, as well as the highest secretion of IL-17A. CT9 clearly induced IL-26 over that of the other formulations, while CT8 produced the greatest response in IL-10 stimulation (which may be due to exclusion of IL-1β from CT8 or may indicate more of a shift towards Protein expression of IL-17A measured from 48 h supernatants in duplicate using a commercially available ELISA kit is graphed as a histogram showing mean ± standard error of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001 using ordinary one-way ANOVA with Tukey's multiple comparison test.

Regulation of Gene Expression by Different Cocktail Formulations
IL-17A is significantly induced by CT4 (1024-fold), CT8 (552-fold), and CT9 (270-fold) as compared to their respective vehicle controls, with CT4 induction of IL-17A approximately twice that of CT8 and 3.8-times that of CT9 (Figure 2A).
IL-22 expression follows the same trend as IL-17A with respect to Th17-skewing cocktail stimulation ( Figure 2C), though with a more modest induction ranging from 16-fold (CT9) to 42-fold (CT4). CT8 induction of IL-22 is similar to that of CT9 (~20-fold), and there is a significant difference between CT4 and the other two groups.
Gene expression levels of IL-10 are more modest in comparison to the other genes examined ( Figure 2E). IL-10 also exhibits a unique stimulation profile in that CT8 elicits the highest expression level of the gene (10-fold), followed by CT9 (4-fold), and a very modest yet significant effect with CT4 (1.4-fold).

Regulation of Protein Levels of IL-17A by Different Cocktail Formulations
The IL-17A protein levels are measured as a proof of concept in matching 48 h-treated supernatants by ELISA to assess whether the induction of gene expression correlates with the increased production of the corresponding protein ( Figure 3). The absolute amount of IL-17A protein released by cocktail stimulation is significantly higher than by the respective vehicle in all three formulations; however, there is not a statistically significant difference between the cocktail groups. On average, CT4 led to the secretion of 84 pg/mL IL-17A, CT8 causes 30 pg/mL IL-17A to be secreted, and CT9 causes an intermediate amount of IL-17A to be secreted (49 pg/mL) into media conditioned by the tissue cores following 48 h stimulation.

Interpretation/Discussion:
Various stimuli have been reported to induce Th17 cell development and promote Th17-related cytokine release. IL-23 has consistently been found to be crucial for in vivo Th17 cell development, but is itself (without other cytokines) insufficient for Th17 stimulation; likewise, IL-6 alone cannot elicit an increase in IL-17A expression in naïve CD4+ T cells in vitro [25,26]. Further research has determined that Th17 cell differentiation can occur through the combined effects of the cytokines IL-1β, IL-6, and IL-23 in the absence [25] or presence [10] of TGFβ. Other groups have determined, through murine studies, that naïve T cells can be differentiated into Th17 cells through only IL-6 and TGFβ or with the addition of IL-23 [1,27]. Another study on the contribution of TGFβ and IL-6 in Th17 cell formation led a different group to determine that these factors are critical to Th17 lineage commitment in mice, but that IL-23 and IL-1β or IL-1β and IL-6 are necessary for Th17 development from naïve T cells in humans [20,25]. While some studies have shown that the initial TGFβ/IL-1β signal mediating Th17 differentiation can also cause IL-10 induction [28], others have shown that IL-1β can inhibit IL-10 production by purified Th17 cell populations in vitro and in vivo [29]. Alternatively, IL-10 production could indicate a shift from Th17 cells to regulatory T cells (Tregs) [28]. Differences in components deemed to be necessary and sufficient to elicit the secretion of Th17 cytokines can be explained by gathering results from multiple sources across species, from purified systems utilizing naïve CD4+ T cells to whole body animal models. Thus, Th17 development seems to require a combination of cytokines and factors, regardless of the in vitro or in vivo model system. Our ex vivo skin model bridges the gap between in vitro naïve T cell cultures and in vivo murine experiments, and extends relevance of the findings to human tissues and disease. We have shown herein that Th17 cytokines can be induced in an ex vivo skin model using three different combinations of cocktails, containing a variety of cytokines that are known to be essential for Th17 cell development.
Each of the Th17-skewing cocktail formulations induced the gene expression of IL-17A, IL-17F, IL-22, IL-26, and IL-10, as well as the secretion of IL-17A. Though there was no clear cocktail formulation that resulted in the highest induction of all genes tested, CT4 stimulated the highest responses in three out of the five genes, as well as the highest secretion of IL-17A. CT9 clearly induced IL-26 over that of the other formulations, while CT8 produced the greatest response in IL-10 stimulation (which may be due to exclusion of IL-1β from CT8 or may indicate more of a shift towards Treg formation). Though the differences between the CT groups in IL-17A secretion were non-significant, the pattern of the absolute IL-17A secreted matched that of IL-17F gene expression (CT4 > CT9 > CT8), rather than that of IL-17A (CT4 > CT8 > CT9). This apparent mismatch in patterning can be explained by several factors. Gene expression data represents the transcription of IL-17A, whereas the protein measurement reflects its secretion, and therefore additional time may be necessary to see optimal protein secretion. Moreover, IL-17A RNA and protein discordance could be due to several other factors at the post-transcriptional and/or post-translational level(s), including RNA-mediated decay, translation inhibition, translation into non-functional protein, protein degradation, and epigenetic modification [30][31][32]. IL-17A and IL-17F are located immediately adjacent to each other (3' end to 3' end) on chromosome 6p12.2 and due to their neighboring proximity, may be co-regulated [33,34]. Additionally, IL-17A and IL-17F, while being able to form individual homodimers, can heterodimerize with each other to bind target receptors [11,35]. The ELISA assay does not distinguish between IL-17A homodimers and IL-17A/IL-17F heterodimers, thus the results may have been influenced by the presence of IL-17F in the supernatants and they ultimately represent both populations of IL-17A homodimers and IL-17A/IL-17F heterodimers.
Other models utilizing different Th17-skewing cytokines have been previously described, including the extensive use of purified naïve T cell cultures [1,3,10,20,28]. Furthermore, investigators have generated Th17 cytokine-expressing dendritic cells or Langerhans' cells from peripheral blood mononuclear cells through treatment with GM-CSF and IL-4 (±TGFβ), followed by a proinflammatory cytokine cocktail of IL-1β, IL-6, PGE2, and TNFα for eight days [18], a cocktail of IL-4, GM-CSF, TNFα, and PGE2, or a cocktail of IL-13, GM-CSF, IFNγ, and a TLR2/4 agonist from Klebsiella pneumoniae [19]. The limitations for these models include the fact that they are in vitro models representing responses primarily from one cell type and methodology that includes incubation with non-human sera. A more complex model, deemed the skin resident immune cell activation assay (sRICA), in which skin is microtomed to a depth of 750 µm, includes incubation with bovine collagen, bovine serum, and cornification medium, followed by a Th17-skewing cocktail of CD3, CD28, IL-1β, IFNγ, IL-4, IL-6, IL-21, and TGFβ, similar to our CT9, but lacking IL-23 [36]. Our system addresses some of the limitations of the sRICA assay, including the incorporation of different combinations of Th17-skewing cytokines, cleaner experimental system without additional bovine collagen or serum added, and a more comprehensive system representing a larger portion of reactive skin depth, including the entirety of both the epidermal and dermal layers. When considering that the normal epidermal thickness is approximately 100 µm according to a recent systematic and comprehensive review of skin microanatomy [37], the sRICA system likely incorporates all of the epidermis and some portion of the underlying dermal layer. Since the thickness of the epidermis can be affected by skin pigmentation, smoking, vascularization, and sex [38], only a portion of the dermal contribution of fibroblasts, macrophages, other resident cells, and matrix components is captured in the sRICA assay. Furthermore, our system does not include a microtoming step that truncates the dermis, rather it incorporates a larger dermal contribution overall and allows for maintaining the skin in an air-liquid interphase with the epidermis exposed to the air, providing a comprehensive skin experimental system that more closely mimics that of living systems and that can easily translate into clinical applications of drug discovery. Therefore, our ex vivo Th17-skewing cocktail methodology has direct relevance to human dermal disease research that incorporates Th17 cytokine involvement, including but not limited to SSc, psoriasis, neutrophilic dermatoses, and SLE [5]. As Th17 cytokines have been identified to promote the disruption of the respiratory epithelial barrier leading to mucosal leakage [39], this same methodology could be potentially applied to an ex vivo lung explant model to study lung diseases with Th17 cytokine involvement.
In summary, using any of the three Th17-skewing cocktail formulations resulted in the upregulation and secretion of Th17 cytokines. Since each of the cocktail formulations uniquely performed when considering the gene expression output, they can be selectively utilized to study various diseases manifesting with unique gene expression profiles. Using the human skin in organ culture provides a model for human diseases where Th17 cells and their cytokines are implicated in disease pathogenesis, and it is a valuable tool for assessing therapies that target Th17 cells/Th17 cytokines.

Conflicts of Interest:
One of the authors is an employee of Bristol-Myers Squibb, who partly funded this work. The other funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.