Effects of pioglitazone on the differentiation and inflammation in vitiligo keratinocytes

Abstract

Vitiligo is an acquired hypopigmentary disorder characterized by progressive melanocyte loss due to the cytotoxic action of CD8+ cells. Although the triggering events specifically leading to the onset and spread of the disease have yet to be defined, accumulating data demonstrate the occurrence of defects in hypopigmented and normally pigmented skin.1 Degeneration of keratinocytes and deposits of extracellular granular material extended into normal-appearing epidermis have been described in early reports.2 Acanthosis of the non-lesional skin has also been reported.3 We identified alterations in keratinocyte differentiation and skin barrier lipid composition associated with upregulation of inflammatory mediators in pigmented vitiligo epidermis, suggesting that skin barrier defects may constitute upstream players involved in the activation of autoimmune responses causing melanocyte death.4 Due to the multifaceted aetiopathogenesis of the disease, an effective/preventive therapeutic approach remains to be identified. PPARγ is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily. In the skin, PPARγ inhibits keratinocyte proliferation, improves differentiation and permeability barrier homeostasis while reducing inflammation.5, 6 Pioglitazone is a PPARγ agonist belonging to the synthetic thiazolidinediones that has been proposed as an effective treatment for chronic inflammatory skin diseases.7 Here, we investigated the potential effects of pioglitazone in ameliorating keratinocyte differentiation and skin barrier assembly and reducing inflammation of vitiligo epidermis. We employed primary keratinocytes collected from non-lesional skin of patients with vitiligo cultured in high-calcium medium to promote the differentiation programme8 and treated with pioglitazone. Morphological evaluation showed the induction of differentiation-related traits including the presence of clustered and compact keratinocytes following pioglitazone treatment (Figure 1a). To form an efficient skin barrier, keratinocytes differentiate, progressively express early (keratin1/10) and late (involucrin/filaggrin) differentiation markers and stratify up to form the terminally differentiated corneocytes embedded in a lipid matrix in the stratum corneum.9 Pioglitazone-treated VHKs showed a significantly higher involucrin mRNA (IVL) and a trend of increased filaggrin (FLG) expression in comparison to untreated cells. Parallel evaluation of the transcripts for the enzymes of the ceramide (CER)-associated pathway involved in the formation of the extracellular lipid matrix also revealed the induction of the very long-chain fatty acids protein 4 (ELOVL4), patatin-like phospholipase domain containing 1 (PNPLA1), arachidonate 12-lipoxygenase, 12R type (ALOX12B) and transglutaminase 1 (TGM1) genes and a tendency to increase for ceramide synthase 3 (CERS3) in cells exposed to pioglitazone (Figure 1b). This induction was also confirmed at the protein level (Figure 1c,d). Quantitative analysis of the fluorescence signal demonstrated that pioglitazone-treated keratinocytes showed a significant increase in the number of positive cells for involucrin (mean ± SD, 22.2 ± 3.8 vs. 7.2 ± 1.5 of untreated cells, p < 0.01), filaggrin (mean ± SD, 9.9 ± 2.7 vs. 3.8 ± 2.4, p < 0.05) and TGM1 (mean ± SD, 51.9 ± 13.9 vs. 27.4 ± 5.3, p < 0.05). Likewise, the fluorescence intensity for ELOVL4 detected in VHKs upon pioglitazone treatment was significantly higher (2.3-fold, p < 0.01). The differentiation programme promoted in response to pioglitazone was accompanied by the downregulation of CXCL10 at mRNA level (0.18 ± 0.01 vs. 0.40 ± 0.12, p < 0.01) and chemokine secretion (Figure 1e), indicating that pioglitazone is effective in inhibiting the production of one of the main inflammatory mediators released by keratinocytes and involved in the activity of the disease.10 These findings demonstrate the ability of pioglitazone to improve the defective differentiation/barrier constitution and reduce the production of inflammatory messengers in vitiligo keratinocytes (Figure 2). As a result, pioglitazone may preserve and reinforce the skin barrier homeostasis in patients with vitiligo, thus possibly counteracting environmental stressor effects and the subsequent triggering of inflammation. Employing agents able to act on the skin barrier composition and function may allow to approach vitiligo from a previously unexplored perspective to treat and/or slow down the disease and its spreading. This research was funded by the Italian Ministry of Health (RC). E.B., D.K. and M.P. are named as inventors on a patent for selective activators of peroxisome proliferator-activated receptors (PPARs) for the treatment of vitiligo. The other authors have no conflicts of interests to declare. The study was approved by the Medical Ethical Committee of the San Gallicano Dermatological Institute and it was conducted in agreement with the Declaration of Helsinki principle guidelines. Participants gave their written informed consent. All data needed to evaluate the conclusions are present in the paper.

Topics & Concepts