Th17 micro-milieu regulates NLRP1-dependent caspase-5 activity in skin autoinflammation

IL-1β is a potent player in cutaneous inflammation and central for the development of a Th17 micro-milieu in autoinflammatory diseases including psoriasis. Its production is controlled at the transcriptional level and by subsequent posttranslational processing via inflammatory caspases. In this study, we detected inflammatory caspase-5 active in epidermal keratinocytes and in psoriatic skin lesions. Further, interferon-γ and interleukin-17A synergistically induced caspase-5 expression in cultured keratinocytes, which was dependent on the antimicrobial peptide psoriasin (S100A7). However, diseases-relevant triggers for caspase-5 activity and IL-1β production remain unknown. Recently, extranuclear DNA has been identified as danger-signals abundant in the psoriatic epidermis. Here, we could demonstrate that cytosolic double-stranded (ds) DNA transfected into keratinocytes triggered the activation of caspase-5 and the release of IL-1β. Further, interleukin-17A promoted caspase-5 function via facilitation of the NLRP1-inflammasome. Anti-inflammatory vitamin D interfered with the IL-1β release and suppressed caspase-5 in keratinocytes and in psoriatic skin lesions. Our data link the disease-intrinsic danger signals psoriasin (S100A7) and dsDNA for NLPR1-dependent caspase-5 activity in psoriasis providing potential therapeutic targets in Th17-mediated skin autoinflammation.


Introduction
Activation of the Th17 pathway has been linked to several autoimmune diseases including the skin. Psoriasis is a autoinflammatory skin disease of unknown origin that affects two percent of the population [1]. In psoriasis, interleukin-1β (IL-1β) is an important player for the PLOS  development of the inflammatory Th17 phenotype [2]. Systemic interference with IL-1β shows beneficial effects in clinical trials with patients suffering from psoriasis, specifically in the inflammatory manifestations of psoriasis [3]. Accordingly, topical interference with IL-1β, such as by anti-psoriatic vitamin D analogues, is of therapeutic interest [4]. In psoriatic lesions, keratinocytes are a major source of active IL-1β [1,5]. Pro-inflammatory mediators like TNFα up-regulate IL-1β through NF-κB signaling on transcriptional level [6]. To be functional, IL-1β precursors need to be activated by proteolytic cleavage and a constitutively enhanced IL-1β maturation alone can cause skin inflammation [7]. IL-1β maturation is mediated by inflammasomes, which can activate inflammatory caspases upon recognition of certain molecular patterns [8]. In psoriasis, caspase-1 is active in epidermal keratinocytes and has been linked to IL-1β production via ASC-dependent inflammasome complexes, such as NLRP3 and AIM2 [5,9]. In comparison, NLRP1 is capable to additionally utilizes inflammatory caspase-5 for IL-1β activation independently of ASC [10][11][12]. So far, pathogen-associated patterns relevant for the defense against invading microbes in infectious diseases are known to activate the NLRP1 inflammasome, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS) [13,14]. Recent studies reveal that NLRP1 gene variants confer susceptibility to non-infectious skin-associated autoinflammatory and autoimmune diseases, including vitiligo, lupus erythematosus, and psoriasis [15][16][17][18]. Accordingly, the enhanced expression of NLRP1and NLRP1-specific caspase-5 have been linked to the pathogenesis of Th17-driven skin diseases [12,[19][20][21]. Therefore, the identification of associated cytokine and intrinsic danger-associated molecular patterns that regulate caspase-5 activity in sterile chronic inflammatory diseases could lead to novel therapeutic approaches that target these patterns.
Intracellular self-DNA is physiologically encapsulated into the nucleus and mitochondria. Under inflammatory conditions, self-DNA can be detected in the cytosol of keratinocytes in sterile autoinflammatory diseases, including psoriasis [9,22]. When cytosolic, self-DNA becomes a trigger, such as to activate the AIM2 inflammasome complex [23]. S100 peptides are another group of danger signals that have been originally discovered as antimicrobial peptides (AMP) [24,25]. Psoriasin (S100A7) is increased in the psoriatic epidermis and exerts diverse immune-stimulating functions in Th17-mediated chronic inflammatory diseases, such as induction of proinflammatory cytokines and leukocyte chemotaxis [26,27]. Here, we investigate regulators and triggers for epidermal NLRP1-dependent caspase-5 activation in a psoriasis-relevant cytokine micro-milieu. This study identifies novel molecular targets for approaches in Th17-mediated diseases using psoriasis as an example.

Patients and skin samples
The study was conducted according to the Declaration of Helsinki Principles. Sample acquisitions were approved by the local ethical committee, Faculty of Medicine, Ludwig-Maximilian University, Munich, Germany. For all the procedures, informed patient's written consent was obtained. Patients suffering from plaque psoriasis did not receive systemic therapy and no topical treatment for at least four weeks before entering the study. 4-mm punch biopsies were taken from a marker psoriatic plaques before treatment with a topical calcipotriol preparation (LEO Pharma, Neu-Isenburg, Germany) containing ointment (0.005%; applied twice daily) and 5 to 7 days after treatment onset. Skin biopsies from untreated lesional skin from psoriasis patients were collected and compared with biopsies from healthy volunteers. The biopsies were directly transferred to 1 ml TRIzol1 (Invitrogen, Karlsruhe, Germany) for RNA extraction or snap frozen in liquid nitrogen for immunofluorescence staining.

Immunofluorescence staining
Immunofluorescence staining was performed on 8 μm serial frozen sections of healthy and psoriatic skin fixed for 5 minutes in acetone at -20˚C. The sections were blocked in 10% normal goat serum, and incubated overnight with anti-caspase-5 (Cell Signaling Technology, Inc., Beverly, MA, USA; 1:50). The sections were then incubated with goat anti-rabbit IgG conjugated with Alexa Fluor 1 647 (Invitrogen, Carlsbad, CA, USA; 1:250), diluted in 10% normal goat serum and incubated for 1 hour at room temperature in a dark humidified chamber. Staining with secondary antibodies only was performed as a negative control. Sections were overlaid with ProLong Gold antifade reagent containing DAPI (Invitrogen, Carlsbad, CA, USA). Fluorescent stained tissues were imaged using a fluorescent microscope Zeiss ImagerZ1 (Zeiss, Jena, Germany) using a 12-bit CCD digital camera PCO PixelFly (PCO, Kelheim, Germany).

Statistical analysis
Data were expressed as pooled means + SEM of three independent experiments conducted in triplicates for each condition. Comparative data were analysed with GraphPad Prism 5.01 software (La Jolla, CA, USA) using Student's t-test and ANOVA. A P-value of less than 0.05 was considered statistically significant.

Inflammatory caspase-5 is induced and activated in psoriatic skin lesions
IL-1β has been identified as an important pro-inflammatory mediator increased in psoriatic skin lesions. To be functional, IL-1β precursors are cleaved into the biologically active form via inflammatory caspases [8]. Data revealed that transcripts of IL-1β, inflammatory caspase-1 and caspase-5 were up-regulated in psoriatic skin lesions (Fig 1A-1C). An epidermal expression and activation of IL-1β and caspase-1 has already been reported in psoriatic skin vs. normal but not for caspase-5 [5,9]. Here, immunofluorescent staining illustrated the increased expression of caspase-5 in psoriasis and showed a prominent distribution throughout the layers of the psoriatic epidermis compared to healthy skin ( Fig 1D). Further, inflammatory procaspase-5 and cleaved caspase-5 could be detected in psoriatic lesions suggesting that caspase-5 activity is regulated by an autoinflammatory micro-milieu in epidermal keratinocytes in psoriasis ( Fig 1E).

Cytosolic dsDNA induces IL-1β release via caspase-5 by IFNγ-primed epidermal keratinocytes
To identify immune-regulatory factors for IL-1β and caspase-5 in the epidermis, keratinocytes were exposed to a psoriasis-relevant cytokines. Data showed that TNFα (2.1-fold) and IL-17A (1.8-fold) induce IL-1β but had no effect on inflammatory caspase-5. In comparison, IFNγ strongly increased caspase-5 expression besides IL-1β in epidermal keratinocytes (Fig 2A and  2B), however keratinocytes stimulated with IFNγ alone did not release IL-1β into the culture supernatant ( Fig 2C). In inflamed psoriatic plaques, free DNA is detectable in the cytosol of keratinocytes [9], and we hypothesized that double-stranded (ds) DNA mediates caspase-5 activation and IL-1β release by keratinocytes. Subsequently, cultured cells primed with IFNγ and transfected with dsDNA [Poly(dA:dT)] led to IL-1β activation compared to single treatment. In this setting, transfection of DNase-treated dsDNA abolished the IL-1β release by keratinocytes confirming dsDNA as a trigger. Beside the activating effect of DNA, we could further show that cytosolic dsDNA induces a pro-IL-1β expression in keratinocytes but not caspase-5 (S1A Fig  and S1B Fig). In addition, IFNγ-primed keratinocytes secreted higher amounts of active caspase-5 subunits (10 KDa bands) in response to cytosolic DNA (-DNase, Fig 2D). When dsDNA was treated with DNase prior transfection, these bands vanished (+DNase) indicating a DNA-dependent activation. To test the functional relevance, the investigated caspases were targeted by siRNA interference (Fig 2E). Data revealed that knock-down of caspase-5 inhibited the DNAdependent IL-1β release by IFNγ-primed keratinocytes (caspase-5 siRNA interference efficacy, S2A Fig). Data suggest regulatory and activating factors in the psoriatic micro-milieu important for epidermal IL-1β production via inflammatory caspases.
The antimicrobial peptide psoriasin (S100A7) mediates caspase-5 and IL-1β release Psoriasin (S100A7) has been discovered as an antimicrobial peptide up-regulated in inflamed psoriatic skin [24,29,30]. IFNγ induces psoriasin release by keratinocytes [24], and we hypothesized that psoriasin is another intrinsic danger-associated molecular pattern that regulates IL-1β production. In IFNγ-primed keratinocytes, targeting psoriasin by siRNA interference suppressed the IL-1β release (0.7-fold) in response to cytosolic DNA (Fig 3A; psoriasin siRNA interference efficacy, S2B Fig). In this setting, psoriasin knock-down induced the expression ofpro-IL-1β but the antimicrobial peptide down-regulated IL-1β activating caspase-5 (0.7-fold) besides caspase-1 (0.8-fold) indicative for the overall suppressed IL-1β production in the presence of IFNγ (Fig 3B). However, the psoriatic epidermis is exposed to a mixed Th1/Th17 cytokine milieu, and IL-17A is a key cytokine in the disease pathogenesis. We could further show that IL-17A enhances the IFNγ-mediated psoriasin expression in keratinocytes suggesting a regulatory role here (Fig 3C). In keratinocytes treated with IFNγ and IL-17A, targeting psoriasin showed a slightly stronger regulatory effect on caspase-5 (0.4-fold) and caspase-1 (0.5-fold), whereas pro-IL-1β expression was not significantly altered ( Fig 3D). Under mixed cytokine conditions, suppression of psoriasin inhibited the overall IL-1β production in response to cytosolic DNA (0.6-fold) (Fig 3E). Data suggest that psoriasin mediates the IFNγ and IL-17A induced regulation of inflammatory caspases and IL-1β production in human epidermal keratinocytes.

IL-17A induces caspase-5 and facilitates NLRP1-mediated IL-1β release
Regarding the relevance of IL-17A in psoriasis, we investigated the caspase-mediated IL-1β activation by epidermal keratinocytes further. In IFNγ-primed keratinocytes, co-stimulation with IL-17A increased the expression of pro-IL-1β (Fig 4A). Under the combined stimulation, IL-17A amplified the IFNγ-mediated expression of caspase-5, whereas caspase-1 was only slightly induced (Fig 4B and 4C; S3A Fig and S3B Fig). Inflammatory caspases are functionally dependent on inflammasome complexes, and IL-17A interfered with the regulation of caspase-1 recipient NLRP3 and dsDNA-sensing AIM2, whereas the expression of NLRP1 which can additionally activate caspase-5, remained unaffected (Fig 4D; S3D Fig and S3E Fig). To analyze the mixed cytokine effect on IL-1β production, cultured keratinocytes were transfected with dsDNA and stimulated with IFNγ and IL-17A (Fig 4E). The combined stimulation induced an IL-1β release similar to treatment with IFNγ, whereas treatment of keratinocytes with IL-17A alone had no enhancing effect when compared to control. The regulatory data suggest that NLRP1-mediated caspase activity contributes to the IFNγ and IL-17A sustained IL-1β release, which was analyzed next. dsDNA-transfected and IFNγ + IL-17A primed keratinocytes secreted cleaved bands of activated caspase-5 (10kDa, 20kDa) and caspase-1 (20 kDa) into the culture supernatant (Co, control siRNA; Fig 4F). Under this condition, co-transfection of keratinocytes with siRNA targeting NLRP1 vanished these bands (N1, NLRP1siRNA; Fig 4F, NLRP1 siRNA interference efficacy; Figs 3C and S3) as quantified below. Further, knock-down of either NLRP1, associated caspase-5 or caspase-1 led to a significant decrease in IL-1β release by IFNγ and IL-17A-treated keratinocytes when transfected with dsDNA (0.7-fold, 0.7-fold, 0.3-fold, respectively) ( Fig 4G; caspase-1 siRNA interference efficacy, S2C Fig). Data suggest that the mixed pro-inflammatory micro-milieu in Th1/Th17-mediated psoriasis provides a relevant setting for NLRP1-dependent inflammasome activation. Under these conditions, the dsDNA-sensing NLRP1 can contribute to IL-1β release besides other inflammasomes active in epidermal keratinocytes.

Vitamin D interferes with IL-1β release by epidermal keratinocytes and suppresses caspase-5 in psoriatic skin lesions
Based on data above, topical interference with caspase-5 mediated IL-1β production in lesional psoriatic skin, such as by anti-inflammatory anti-psoriatic vitamin D analogues, might be of therapeutic interest. To test this in a mixed psoriatic micro-milieu, IFNγ and IL-17A primed keratinocytes were transfected with dsDNA, and the IL-1β release was measured dependent on hormonally active vitamin D (calcitriol, 1,25 D 3 ) (Fig 5A). In this environment, co-treatment with 1,25 D 3 reduced the IL-1β release by keratinocytes dose-dependently (up to 0.7-fold). Data further showed that increasing concentrations of 1,25 D 3 interfered with the cytokineinduced production of caspase-5 (Fig 5B), which is regulated on transcriptional level (0.5-fold) (Fig 5C). However, the expression of caspase-1, NLRP1, or IL-1β were not affected by 1,25 D 3 in keratinocytes (data not shown). Vitamin D analogues are standard therapeutics for chronic inflammatory diseases, such as psoriasis [31]. Based on our data, we hypothesized that calcipotriol suppresses caspase-5 expression in the epidermis of psoriatic plaques. Corresponding skin sections of psoriasis showed a reduced epidermal thickening in response to calcipotriol which is accompanied by an attenuated caspase-5 staining in the epidermis (Fig 5D). This was further supported by immunoblot analysis from corresponding skin biopsies showing reduced caspase-5 levels in psoriasis by calcipotriol (0.8-fold) (Fig 5E and 5F). The calcipotriol-mediated suppression of caspase-5 mRNA levels in the psoriatic tissue suggests a transcriptional regulation, which was also detected in 1,25 D 3 -treated keratinocytes (Fig 5C and 5G). Similar to caspase-5, associated NLRP1 was also suppressed, IL-1β-levels reduced (0.6-fold, p = 0.16), whereas caspase-1 levels remained unaffected (Fig 5H, data not shown). Together, data suggest that targeting caspase-5 may contribute to the anti-inflammatory effects of vitamin D that could be relevant for treatment of Th17-mediated autoinflammatory diseases with caspase-5 overexpression, such as psoriasis or lupus erythematosus (S4 Fig, S5 Fig), or others with proposed NLRP1 inflammasome activity [20,21].

Discussion
Here, we observed elevated epidermal expression and increased caspase-5 activation in psoriasis. In epidermal keratinocytes, the intrinsic danger signals cytosolic DNA and the antimicrobial peptide psoriasin (S100A7) were identified as disease-relevant triggers for caspase-5 activity and IL-1β production in a Th1/Th17 environment. Patients suffering from psoriasis have lower vitamin D levels [32], and we observed that application of vitamin D suppresses caspase-5 in keratinocytes in psoriasis. Current studies link IL-1β production and Th17-mediated effects in autoimmune diseases, including psoriasis, and these observations could contribute to the developing understanding of their pathogenesis and therapeutic interference [2,33,34].
Inflammatory caspase-5 and associated IL-1β production have been found predominantly in highly immunogenic tissues with close contact to pathogens, such as in leukocytes and in some epithelial cell types [35][36][37][38]. There are controversial studies about the expression of caspase-5 in resident skin keratinocytes [12,39]. Here, we demonstrated that caspase-5 is constitutively expressed in the epidermis of normal skin and functionally active in cultured epidermal keratinocytes.
In the skin, keratinocytes are a major source of IL-1β but the mechanisms of IL-1β production via caspase-5 in a psoriatic Th1/Th17-environment remain unknown [5]. So far, caspase-1 has been identified active and linked to IL-1β maturation in psoriasis [5,12]. This study showed that caspase-5 is induced in the psoriatic epidermis and activated in psoriatic skin lesions. A scattered single cell stain in the psoriatic dermis might be indicative for caspase-5 expressing immune cells, such as macrophages, which could contribute to the caspase-5 levels detected in lesional psoriasis, also after vitamin D treatment (not shown) [36,40,41].
Increasing evidence connects the NLRP1 inflammasome to the pathogenesis of skin-associated autoimmune diseases, such as vitiligo, lupus erythematosus, pemphigus vulgaris, and psoriasis [15,17,20,45]. Compared to other inflammasome complexes, such as NLPR3 and AIM2 expressed in keratinocytes, NLRP1 can activate caspase-5 besides inflammasome ubiquitous caspase-1 [8,11]. The suppression of caspase-1 recipient NLRP3 and AIM2 by the investigated Th1/Th17 setting in keratinocytes suggests that the steady IL-1β activity is co-sustained by unaffected NLRP1 and induced caspase-5 and caspase-1activity. Thus, besides other inflammasomes active in keratinocytes, NLRP1 is able to contribute to the IL-1β production, particularly in the presence of IL-17A.So far, certain microbial components have been identified to activate the NLRP1 inflammasome, such as muramyl dipeptide (MDP), anthrax lethal factor (LF) from Bacillus anthracis, and LPS [13,14,36]. Recently, free cytosolic DNA has been found in psoriatic keratinocytes, which can activate caspase-1 via the DNA-sensing inflammasome AIM2 [9,23]. Here, we could show a DNA-dependent activation of IL-1β in keratinocytes, which is mediated by the NLRP1 and associated caspase-5 and caspase-1. In comparison to previous studies, we were able to detect NLRP1-dependent caspase-5 and caspase-1 cleavage in keratinocytes, which is consistent with reports in other cell types [8,11,46]. Our observations on an enhanced caspase-5 regulation in the presence of IL-17A and NLRP1 inflammasome activity in keratinocytes suggests a functional contribution for epidermal IL-1β production in psoriasis and likely other diseases, where Th17 may also contribute, such as lupus erythematosus. The pleiotropic functions of IL-1β produced by epidermal keratinocytes exert local inflammatory effects in the skin, such as release of cytokines and Th17-immunocyte chemotaxins [47]. Skin-attracted and activated immune cells in turn amplify the local inflammation by Th17 cytokine release [48] and subsequent epidermal IL-1β induction as proposed here. This suggests that targeting of local IL-1β production in the skin contributes to the reduction of the local inflammatory phenotype, such as directly through regulation of inflammasomes in the epidermis or through interference with the adjacent Th1/Th17 micro-milieu.
Vitamin D therapeutic effects have been continuously evaluated for numerous chronic inflammatory diseases and cancers [49,50]. Topical vitamin D analogues are a hallmark in the treatment of psoriasis by affecting skin inflammation through various mechanisms, including the local reduction of Th17 immunocytes [51]. Previous studies underscore the importance of IL-1β regulation in keratinocytes and psoriatic skin by calcipotriol [4,52]. Here, our data indicate that vitamin D further acts as a direct suppressor of caspase-5 and IL-1β production in analyzed by ELISA, n = 4. B, Keratinocytes stimulated with IFNγ, IL-17A and vitamin D-dependent caspase-5 levels were detected by immunoblotting, n = 3. C, Vitamin D-dependent regulation of caspase-5 in keratinocytes stimulated with IFNγ, IL-17A and analyzed by RTqPCR and normalized to β-actin. Data represent mean + SEM *, p < 0.05 determined by Student's t test, n = 9. D, Representative immunofluorescent staining of caspase-5 in psoriatic skin was reduced after calcipotriol treatment, scale bar = 50 μm. Skin sections of three psoriatic patients were examined. E, F, Representative caspase-5 immunoblotting of caspase-5 levels in skin lysates from psoriatic patients reduced after calcipotriol treatment, protein levels were quantified by densitometry versus β-actin. Data represent mean + SEM, *, p < 0.05 determined by Student's t test. Skin lysates of three psoriatic patients were examined. G, H, Regulation of caspase-5 and IL-1β levels in skin lysates from psoriatic patients after calcipotriol treatment as analyzed by RTqPCR and normalized to PBGD. Data represent mean + SEM *, p < 0.05; **, p < 0.01 determined by Student's t test, n = 3. https://doi.org/10.1371/journal.pone.0175153.g005 Th17 regulates caspase-5 activity in autoinflammation keratinocytes suggesting a novel anti-inflammatory mechanism relevant in psoriasis and other Th17-driven inflammatory skin diseases.
Together, this study identified disease-intrinsic regulators, triggers and therapeutic mechanisms of caspase-5 dependent epidermal IL-1β production in psoriasis. These observations may provide insides into current therapies and suggest NLRP1and associated caspase-5 as novel targets for Th17-mediated autoimmune diseases. The autoinflammatory Th1/Th17 milieu in psoriasis contains IFNγ and IL-17A, which induces psoriasin (S100A7)-dependent inflammatory caspases-5 over caspase-1 in keratinocytes. Free dsDNA present in the cytosol of psoriatic keratinocytes activates caspase-5 and caspase-1 dependent on NLPR1 and leads to a subsequent IL-1β release. Topical vitamin D/ calcipotriol treatment suppresses caspase-5 and NLRP1 regulation in psoriasis and interferes with IL-1β release by epithelial keratinocytes. (PDF) S5 Fig. Differential regulation of IL-1β, caspase-1 and caspase-5 in chronic inflammatory skin diseases. Expression levels of IL-1β, caspase-1, caspase-5 in healthy skin compared to tissues from patients with atopic dermatitis, lupus erythematosus and lichen planus analyzed by RTqPCR and normalized to PBGD. Data represent mean + SEM, Ã , p < 0.05 determined by Student's t test. Skin lysates of five patients were examined for each inflammatory skin disease. (PDF)