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

Abstract

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.

Development and characterization of a human Th17‐driven ex vivo skin inflammation model

“…[17,18] The model described by Smith SH et al 17 is reported to be essential for a sustained Th17 differentiation. [14] Moreover, the use of animal-derived substances, such as 64% bovine collagen solution and 2% fetal bovine serum added to the tissue culture wells and to the culture medium, respectively, creates potential variability in the model described by Smith SH et al [17] compared with the model described by Garret SM et al [18] and to the InflammaSkin® model, which are both developed in 100% serumand animal-free settings. The model described by Garret SM et al [18] is based on an ex vivo culture of defatted but not dermatomed skin from healthy individuals, stimulated with different differentiation cocktails, but all containing anti-CD3 and anti-CD28 antibodies, IL-1β and IL-23 and thus has more similarity to the InflammaSkin® model.…”

“…However, in their model, skin biopsies are cultured in an air-liquid interphase where the dermis is submerged directly in the culture medium, creating an artificial wet setting for the dermal compartment that may induce spongiosis due to continuous absorption of liquid. [18] Consequently, the feasibility to culture skin tissue in this condition is limited to a short period (48 hours), as described by Garret SM et al [18] Furthermore, culture conditions used in their setting (3-5 biopsies of 3 mm diameter per well in a semi-submerged condition) does not allow topical application of formulations, limiting F I G U R E 4 Pharmacological response of the InflammaSkin® model to prophylactic treatment with BDP gel and PDE4 inhibitor cream. InflammaSkin® models were cultured for 7 d either untreated or topically treated with placebo gel, Betamethasone dipropionate gel (BDP gel), placebo cream or PDE4 inhibitor cream (PDE4 inh.…”

“…Other skin models using in situ T‐cell activation and Th17 polarization have been recently published and share some similarities to the InflammaSkin® model. [ 17,18 ] The model described by Smith SH et al 17 employs skin samples defatted and dermatomed to 750 µm, which could imply that some cellular architecture and dermal immune cells may not been fully represented. Importantly, the Th17 polarization cocktail used in this model does not contain IL‐23, which is reported to be essential for a sustained Th17 differentiation.…”

“…[ 14 ] Moreover, the use of animal‐derived substances, such as 64% bovine collagen solution and 2% fetal bovine serum added to the tissue culture wells and to the culture medium, respectively, creates potential variability in the model described by Smith SH et al [ 17 ] compared with the model described by Garret SM et al [ 18 ] and to the InflammaSkin® model, which are both developed in 100% serum‐ and animal‐free settings. The model described by Garret SM et al [ 18 ] is based on an ex vivo culture of defatted but not dermatomed skin from healthy individuals, stimulated with different differentiation cocktails, but all containing anti‐CD3 and anti‐CD28 antibodies, IL‐1β and IL‐23 and thus has more similarity to the InflammaSkin® model. However, in their model, skin biopsies are cultured in an air‐liquid interphase where the dermis is submerged directly in the culture medium, creating an artificial wet setting for the dermal compartment that may induce spongiosis due to continuous absorption of liquid.…”

Insulin-like growth factor (IGF)-II- mediated fibrosis in pathogenic lung conditions

“…Primers used include ACTA2 (Hs00426835_g1), B2M (Hs00187842_m1), Collagen 1A1 (Hs00164004_m1), Fibronectin 1 (Hs00365052_m1), GAPDH (H202758991_g1), IGF1R (Hs00609566_m1), IGF2R (Hs00974474_m1), IR (Hs00961560_m1), MMP3 (Hs00968305_m1), TGFβ1 (Hs00998133_m1), TGFβ2 (Hs00234244_m1), TGFβ3 (Hs01086000_m1), TIMP1 (Hs00171558_m1), and TIMP4 (Hs00162784_m1). Gene expression was determined by the delta-delta Ct method [22].…”