The mechanisms of inflammation in acne are currently subject of intense investigation. This study focused on the activation of adaptive and innate immunity in clinically early visible inflamed acne lesions and was performed in two independent patient populations. Biopsies were collected from lesional and non-lesional skin of acne patients. Using Affymetrix Genechips, we observed significant elevation of the signature cytokines of the Th17 lineage in acne lesions compared to non-lesional skin. The increased expression of IL-17 was confirmed at the RNA and also protein level with real-time PCR (RT-PCR) and Luminex technology. Cytokines involved in Th17 lineage differentiation (IL-1β, IL-6, TGF-β, IL23p19) were remarkably induced at the RNA level. In addition, proinflammatory cytokines and chemokines (TNF-α, IL-8, CSF2 and CCL20), Th1 markers (IL12p40, CXCR3, T-bet, IFN-γ), T regulatory cell markers (Foxp3, IL-10, TGF-β) and IL-17 related antimicrobial peptides (S100A7, S100A9, lipocalin, hBD2, hBD3, hCAP18) were induced. Importantly, immunohistochemistry revealed significantly increased numbers of IL-17A positive T cells and CD83 dendritic cells in the acne lesions. In summary our results demonstrate the presence of IL-17A positive T cells and the activation of Th17-related cytokines in acne lesions, indicating that the Th17 pathway is activated and may play a pivotal role in the disease process, possibly offering new targets of therapy.

Vitiligo affects 1% of the worldwide population. Halting disease progression and repigmenting the lesional skin represent the two faces of therapeutic challenge in vitiligo. We performed transcriptome analysis on lesional, perilesional, and non-depigmented skin from vitiligo patients and on matched skin from healthy subjects. We found a significant increase in CXCL10 in non-depigmented and perilesional vitiligo skin compared with levels in healthy control skin; however, neither CXCL10 nor other immune factors were deregulated in depigmented vitiligo skin. Interestingly, the WNT pathway, which is involved in melanocyte differentiation, was altered specifically in vitiligo skin. We demonstrated that oxidative stress decreases WNT expression/activation in keratinocytes and melanocytes. We developed an ex vivo skin model and confirmed the decrease activation of the WNT pathway in human skin subjected to oxidative stress. Finally, using pharmacological agents that activate the WNT pathway, we treated ex vivo depigmented skin from vitiligo patients and successfully induced differentiation of resident stem cells into pre-melanocytes. Our results shed light on the previously unrecognized role of decreased WNT activation in the prevention of melanocyte differentiation in depigmented vitiligo skin. Furthermore, these results support further clinical exploration of WNT agonists to repigment vitiligo lesions.

Proteases play a pivotal role in epidermal differentiation and desquamation. Separation of a total protein extract from human reconstructed epidermis by two-dimensional gel electrophoresis and subsequent peptide analysis of a specific protein spot identified a new protein exhibiting similarities with the retroviral aspartic protease family. Cloning of the corresponding full-length cDNA revealed an open reading frame encoding for a new protease of 343 amino acids, containing a putative aspartic protease catalytic domain. We named this protein Skin ASpartic Protease (SASPase). RT-PCR and northern blot analysis of various human tissues revealed that SASPase was specifically expressed within the epidermis. Immunohistochemical analysis showed a particularly intense expression restricted to the granular layers, whereas in diseased skin, its expression was changed. Western blot analysis, using a monoclonal antibody, revealed the expression of two forms of the enzyme: a 28 kDa putative proform and the active 14 kDa form. Recombinant truncated SASPase (SASP28) was generated from a prokaryotic expression system in Escherichia coli as a fusion protein with GST. SASP28 degraded insulin and to a lesser extent casein with a pH optimum of 5. As seen for retroviral proteases, an auto-activation processing was evidenced, generating a 14 kDa protein (SASP14). Site-directed mutagenesis inhibited auto-activation of the enzyme. Indinavir, a potent HIV protease inhibitor used in AIDS therapy, had a significant inhibitory effect on rSASPase auto-activation, which could explain its side effects on skin.

After separating by two-dimensional gel electrophoresis an extract of total proteins from human stratum corneum, two spots were extracted and analyzed for their peptide sequence. The resulting internal protein sequences provided evidence for the identification of a new calcium-binding protein. Cloning of the corresponding full-length cDNA was achieved by reverse transcriptase-polymerase chain reaction using two keratinocyte libraries, one from proliferating cultured keratinocytes and one from differentiated keratinocytes of reconstructed human epidermis. The cDNA had an open reading frame encoding a new calcium-binding protein of 146 amino acids, a member of the calmodulin family. We named this new protein calmodulin-like skin protein (CLSP), since reverse transcriptase-polymerase chain reaction studies of CLSP expression in 10 different human tissues revealed that this protein was particularly abundant in the epidermis where its expression is directly related to keratinocyte differentiation. Expression of the cloned cDNA in Escherichia coli yielded a recombinant protein which allowed its further characterization. rCLSP is able to bind calcium, and similarly to calmodulin, exposes thereafter hydrophobic parts which most likely interact with target proteins. Epidermal proteins retained by CaM affinity column are quantitatively and qualitatively distinct from those of the rCLSP column. Sequencing of a rCLSP affinity purified protein revealed 100% identity with transglutaminase 3, a key enzyme in terminal differentiation, indicating an important role of CLSP in this process.

Desquamation is described as a protease-dependent phenomenon where serine proteases with a basic pH optimum play a key role. Recently proteases with an acidic pH optimum were identified in the stratumcorneum and associated with desquamation, e.g., cathepsin D and the stratum corneum thiol protease. The purpose of this study was to investigate if human stratum corneum contains proteases different from the above, exhibiting similar properties. After gel filtration, we identified four distinct proteolytic activities in a human stratum corneum extract, a cathepsin-E-like activity (80 kDa), a cathepsin-D activity (40 kDa), a yet unknown cathepsin-L-like form (28 kDa) exhibiting the highest caseinolytic activity, and a chymotrypsin-like protein (24 kDa) containing the acidic activity of the well described stratum corneum chymotryptic enzyme. We named the new 28 kDa protease stratum corneum cathepsin-L-like enzyme. Characterization of stratum corneum cathepsin-L-like enzyme provided clear evidence that this new protease, despite its membership to the cathepsin-L-like family, is distinct from cathepsin L and from the recently described stratum corneum thiol protease. Its ability to hydrolyze corneodesmosin, a marker of corneocyte cohesion, was in favor of a role of stratum corneum cathepsin-L-like enzyme in the desquamation process. A more detailed analysis did not allow us to identify stratum corneum cathepsin-L-like enzyme at the molecular level but revealed that stratum corneum thiol protease is identical with the recently described cathepsin L2 protease. Reverse transcription polymerase chain reaction studies and the use of a specific antibody revealed that, in contrast to earlier reports, expression of stratum corneum thiol protease in human epidermis is not related to keratinocyte differentiation. Our results indicate that the stratum corneum thiol protease is probably expressed as a pro-enzyme in the lower layers of the epidermis and in part activated by a yet unidentified mechanism in the upper layers during keratinocyte differentiation.