Granzyme A potentiates chemokine production in IL‐17‐stimulated keratinocytes

Plaque psoriasis presents with focal skin inflammation, partially maintained by IL‐17‐mediated interactions between infiltrating epidermal T cells and activated keratinocytes. Here we show that the majority of lesional epidermal CD8 T cells express granzyme A, alone or in combination with IL‐17. To assess proinflammatory properties of granzyme A in psoriasis, primary human keratinocytes were stimulated with granzyme A in the presence or absence of IL‐17. Out of 33 analysed keratinocyte‐derived inflammatory mediators, granzyme A potentiated IL‐17‐induced secretion of CXCL 1, CXCL 12 and CCL 4. Intriguingly, all three chemokines are implicated in psoriasis pathogenesis and are involved in recruitment of T cells, neutrophils and pDCs into inflamed tissues. Our results indicate that granzyme A produced by lesional CD8 T cells specifically increase the chemokine production from inflamed keratinocytes, thereby amplifying a chemotactic inflammatory loop that sustains psoriasis lesions.

Intriguingly, all three chemokines are implicated in psoriasis pathogenesis and are involved in recruitment of T cells, neutrophils and pDCs into inflamed tissues. Our results indicate that granzyme A produced by lesional CD8 T cells specifically increase the chemokine production from inflamed keratinocytes, thereby amplifying a chemotactic inflammatory loop that sustains psoriasis lesions.

| BACKGROUND
Granzymes (Gzms), a family of granule serine proteases that is stored in cytotoxic lymphocytes, have the capacity to induce cellular cytotoxicity in the presence of perforin. [1] In psoriasis, where massive T-cell infiltration occurs in affected skin, [2,3] gene expression of GZMA and GZMB is upregulated in lesional skin alongside with genes related to the Th17/IL-23 axis. [4,5] We have previously reported elevated gene expression of GZMA, GZMB and PRF (perforin) in CD8 T cells sorted from psoriasis lesions as compared to healthy skin. [6] Despite increased frequency of GzmB-and perforinexpressing cells in lesional psoriasis compared to normal skin, atopic (AD) and allergic contact dermatitis (ACD) lesions harbour significantly more granzyme-expressing T cells as compared to psoriasis (s1). [7] Moreover, apoptotic cells are scarce in psoriasis (s3) and keratinocytes from psoriasis lesions show resistance to apoptosis (s2), indicating alternative functionality of Gzms in psoriasis compared to AD and ACD. Non-cytolytic activity of Gzms is reported, and in particular, GzmA induces expression of proinflammatory cytokines IL-1β, IL-6 or IL-8 in primary monocytes, [8] fibroblasts or epithelial cell lines (s4). Interestingly, GzmA-deficient mice display no defects in cellular cytotoxicity but impaired LPS- [8] or bacterial-induced septic shock, [9] further supporting its proinflammatory role (s4).

| QUESTIONS ADDRESSED
We postulated that granzymes act as a proinflammatory mediator in psoriasis. Granzyme and perforin expression in CD8 T cells from psoriasis lesions was assessed and primary keratinocytes were stimulated with GzmA and IL-17 to assess secretion of inflammatory mediators.

| EXPERIMENTAL DESIGN
See Supporting Information.

| Granzyme A potentiates chemokine production in IL-17-stimulated keratinocytes
In psoriasis, CD8 T cells producing IL-17A accumulate in epidermis [6] (s6) in close contact with keratinocytes that respond to IL-17 simulation with production of innate cytokines and chemokines. [3] In the limited number of patients included in this study, no correlation between the frequency of GzmA+ T cells and lesional leucocyte infiltration or disease severity could be determined (data not shown). However, epidermal GzmA-expressing CD8 T cells produced the psoriasisassociated cytokines IFNγ and IL-17A upon stimulation ( Figure 1D,E).
IL-1α secretion was increased by GzmA alone or in combination with IL-17 as previously reported, [9] but this difference did not reach statistical significance.

| CONCLUSIONS
Proinflammatory activity of GzmA [8]  Our results indicate that GzmA produced by lesional CD8 T cells has the capacity to specifically increase chemokine secretion from

ACKNOWLEDGEMENT
We thank all patients who participated in this study, and Lennart Blomqvist, Irène Gallais Sérézal and Emma Wadman for help with sample collection.

CONFLICT OF INTEREST
The authors have declared no conflicting interests.

AUTHOR CONTRIBUTION
SC, EM, KB, DC and BR performed the experiments. SC and LE designed the research study. BR, MS and LE contributed essential reagents or tools. SC, EM and LE analysed the data. SC and LE wrote the manuscript that was read and revised by all authors.

ETHICAL APPROVAL
The study was performed according to the Declaration of Helsinki principles and approved by the Stockholm Regional Committee of Ethics (approval number 2012/50-31/2). Written consent was collected from all donors.

Niacinamide leave-on formulation provides long-lasting protection against bacteria in vivo Abstract
Antimicrobial peptides (AMPs) form a part of the skin's innate immune system. Their primary activity is to provide antimicrobial benefits and hence protect from infections. AMPs that are present on human skin include psoriasin (S100A7), RNase 7, lysozyme, LL-37 and defensins. Niacinamide is a well-known cosmetic ingredient that has been used traditionally for multiple skin benefits.
Recent data indicate that niacinamide treatment can boost AMPs in human gut epithelial cells and in neutrophils. Treatment with niacinamide in mice also provided protection from skin infections by enhancing AMPs. In this article, we find that treatment with niacinamide formulation provides long-lasting protection against bacteria, potentially through the activation of an AMP response.

| BACKGROUND
Skin is an immunologically active organ and harbours immune mediators from the innate and adaptive branches of immunity. Skin innate immunity is critical for cutaneous defense and antimicrobial peptides (AMPs) form a part of the skin's innate immune system. Primary activity of the AMPs is to provide antimicrobial benefits and protect from infections. [1,2] Niacinamide is a well-known cosmetic ingredient that has been used traditionally for skin lightening, anti-ageing and skin barrier building benefits. [3,4] Recent data indicate that niacinamide treatment can boost AMPs in gut epithelial cells and in neutrophils. [5,6] Treatment with niacinamide in mice also provided protection from infections through the boost of AMPs. [6] Based on these studies, we hypothesized that niacinamide can elicit similar responses in human skin resulting in enhanced antimicrobial protection and boosting AMP in skin cells.

| QUESTION ADDRESSED
To evaluate the effect of a niacinamide containing formulation in providing antimicrobial benefits to human skin.

| EXPERIMENTAL DESIGN
Supplementary section Data S1.