Klk8 is required for microabscess formation in a mouse imiquimod model of psoriasis

Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferative epidermis (acanthosis) with aberrant differentiation (parakeratosis) as well as dendritic cell (DC), T-cell and neutrophil infiltration. Accumulating evidence indicates that the IL-23/T17 pathogenic axis plays a critical role in psoriasis, where IL-17 and IL-22 produced by Th17 and γδ T cells cooperatively stimulate proliferation of keratinocytes and a variety of inflammatory mediators that contribute to the psoriatic phenotype 1, 2. Recent evidence indicates that IL-36 cytokines (IL-36α, IL-36β and IL-36γ) also play an important role in psoriasis 3, 4. These cytokines bind to IL-36 receptor (IL-36R) and activate NF-κB (s1). In addition, mutations in IL-36 receptor antagonist (IL-36RN), an endogenous IL-36R signalling antagonist, have been detected in patients with generalized pustular psoriasis, suggesting a role in neutrophil activation (s2–s4). Kallikrein-related peptidases (KLKs) are a subgroup of 15 secretory chymotrypsin- and trypsin-like serine proteases. KLK5 and KLK7 are expressed in the granular layer of epidermis and contribute to corneocyte desquamation by degrading desmosomal cadherins such as desmoglein-1 and desmocollin-1 (s5). Human KLK8 is also expressed in the granular layer of the epidermis (5, s6). Previous reports indicate that KLK8 is overexpressed in human psoriatic epidermis 6. Moreover, serum KLK8 levels in patients with psoriasis are significantly higher than those in healthy controls, and they are also correlated with disease severity (s7). However, the role of KLK8 in the pathogenesis of psoriasis is still unclear. Recently, topical treatment with imiquimod (IMQ), a ligand for TLR7/8, was shown to induce psoriasis-like skin inflammation in mice 7, 8. In this study, we investigated the role of KLK8 in the IMQ-induced mouse psoriasis model using Klk8 knockout mice. See e-supplement. To investigate possible involvement of Klk8 in the development of IMQ-induced dermatitis, we first examined the expression and localization of Klk8. Quantitative RT-PCR analysis revealed that the expression of Klk8 in the IMQ-treated skin was significantly higher than that in untreated skin (Fig. 1a). Furthermore, while weak Klk8 expression was detected in the upper epidermis of untreated mice, expression was markedly enhanced after the IMQ treatment (Fig. 1b). Klk8 was located on both cytoplasmic and intercellular areas, suggesting its secretion from keratinocytes. These observations indicate that topical application of IMQ stimulates the expression and secretion of Klk8 in the upper epidermis. To further assess the role of Klk8 in a mouse IMQ model, we investigated the development of psoriasis-like dermatitis in Klk8 knockout mice. IMQ-treated skin of wild-type mice showed characteristic neutrophil microabscess formation in the epidermis, which was markedly reduced in Klk8 knockout mice (Fig. 1c, d). In contrast, no statistically significant difference was observed in the number of Ki-67-positive keratinocytes between wild-type and Klk8 knockout mice (Figure S2a), suggesting that Klk8 presence or absence did not affect keratinocyte proliferation in this model. Nor was epidermal thickness affected in Klk8 knockout mice (Figure S2b). Furthermore, consistent with a previous report 8, topical application of IMQ on the ear of wild-type mice resulted in increased ear thickness. Interestingly, the IMQ-induced ear swelling was significantly reduced in Klk8 knockout mice from day 5 post-treatment (Fig. 1e). In histological analysis, this reduced ear swelling was mostly due to the decrease in dermal inflammatory cell infiltration and oedema rather than epidermal acanthosis. Taken together, our data indicate that Klk8 is significantly involved in epidermal microabscess formation but not in keratinocyte proliferation in the IMQ-treated mouse psoriasis model. Because there were far fewer neutrophil microabscesses in Klk8 knockout mice, we investigated the molecular mechanism of the formation of these microabscesses. Recently, the importance of IL-36 in the induction of psoriasiform dermatitis has been established 9. Quantitative RT-PCR analysis results showed that topical IMQ treatment led to increased expression of IL-36α, IL-36β and IL-36γ in the wild-type mouse skin, all of which were significantly reduced in Klk8 knockout mice (Fig. 2a). Immunofluorescence analyses showed that IL-36α was highly expressed in both the cytoplasm and the intercellular space of the upper epidermis of IMQ-treated mice (Fig. 2b). In addition, colocalization of Klk8 and IL-36α was observed in the IMQ-treated mouse epidermis, further suggesting that Klk8 is indeed involved in IL-36 production. In this study, we demonstrated that Klk8 expression is markedly enhanced in the IMQ-treated psoriasiform epidermis. Furthermore, Klk8 was crucial for microabscess formation in the upper epidermis, where Klk8 was significantly involved in IL-36 expression. Munro's microabscesses are among the hallmarks of psoriasis pathology. A recent report showed that skin inflammation including microabscess formation is significantly reduced in IL36R-deficient mice, emphasizing the pathogenic significance of IL-36 at least in the IMQ model 9. Our data suggest that epidermal KLK8 exacerbates human psoriatic lesions through IL-36 expression and microabscess formation in the upper epidermis. Although our data demonstrated that Klk8 promotes inflammatory response in the mouse IMQ model, the precise mechanism is not clear. KLK activation is proceeded by complex proteolytic cascades with sequential activation of multiple KLKs and contributes to protease-activated receptor 2 (PAR2) activation (s8). Several studies have identified PAR2 on keratinocytes to be a major effector of the cutaneous inflammatory response (s9, s10). Further studies are required to determine the mechanism by which Klk8 regulates upper epidermal microabscess formation in the IMQ-induced psoriasis model. Recent understanding of immune molecular system results in new types of drugs targeting inflammatory cytokines, such as TNF-α, IL-23 and IL-17 2 . However, inhibition of these cytokines may increase susceptibility to various infections by general immunosuppression. To avoid this, a pharmacologic approach to keratinocyte-specific molecules would be required. Our data support the idea that the inhibition of KLK8 may be another promising approach for the treatment of psoriasis. We thank Ms. K Nishikura and Ms. Y Nishinome for their technical assistance. SIi, MK, NS, SIg, MH and HT performed the research. SIi analysed the data and wrote the manuscript. YB, SY, HI and AI-Y designed the research study and wrote the manuscript. The authors have no conflict of interests to declare. Appendix S1 Experimental design Appendix S2. References Table S1. Primer sequences used in this study. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.