Maren Simanski

Cytosolic DNA triggers inflammasome activation in keratinocytes in psoriatic lesions

In psoriasis, cytosolic DNA in keratinocytes triggers maturation of the cytokine IL-1β via the AIM2 inflammasome. Taking AIM at Psoriasis Everyday life is filled with dangers—from road construction and wet floors to biohazards and radiation. Warning signs, whether the ubiquitous ‘!’ or the classic skull and crossbones, signal impending peril, giving us the chance to change our course of action to avoid the hazards. Cells in the body also respond to danger signals, such as cytosolic DNA, which warns of microbial presence, activating inflammasomes that ramp up the body’s inflammatory response to damage. Now, Dombrowski et al. implicate cytosolic DNA in the inflammation associated with psoriasis, an autoimmune skin disease, and suggest how vitamin D may be able to counteract this response. Psoriasis is a chronic inflammatory disease that commonly causes red, itchy plaques on the skin. Dombrowski et al. inspected both psoriatic and healthy human skin and found higher amounts of cytosolic DNA and AIM2—a DNA sensor associated with inflammasomes—in psoriatic keratinocytes, the predominant cell type in skin. Cytosolic DNA activated AIM2 inflammasomes in keratinocytes, which resulted in the production of proinflammatory cytokines. Importantly, the authors found that the cathelicidin peptide LL-37, which has antimicrobial function, also served as an anti-inflammatory agent by blocking activation of the DNA-sensing inflammasomes. Vitamin D controls cathelicidin production in human skin, and both topical vitamin D treatment and ultraviolet (UV) B light, which up-regulates vitamin D, are currently used to treat psoriasis. The new data from Dombrowski et al. not only provide a mechanistic explanation for the success of these therapies but also suggest that cathelicidin may constitute a new target that specifically affects the inflammasome. For psoriasis, the danger signal itself may be the hazard, and cathelicidin could be a means of defense. The proinflammatory cytokine interleukin-1β (IL-1β) plays a central role in the pathogenesis and the course of inflammatory skin diseases, including psoriasis. Posttranscriptional activation of IL-1β is mediated by inflammasomes; however, the mechanisms triggering IL-1β processing remain unknown. Recently, cytosolic DNA has been identified as a danger signal that activates inflammasomes containing the DNA sensor AIM2. In this study, we detected abundant cytosolic DNA and increased AIM2 expression in keratinocytes in psoriatic lesions but not in healthy skin. In cultured keratinocytes, interferon-γ induced AIM2, and cytosolic DNA triggered the release of IL-1β via the AIM2 inflammasome. Moreover, the antimicrobial cathelicidin peptide LL-37, which can interact with DNA in psoriatic skin, neutralized cytosolic DNA in keratinocytes and blocked AIM2 inflammasome activation. Together, these data suggest that cytosolic DNA is an important disease-associated molecular pattern that can trigger AIM2 inflammasome and IL-1β activation in psoriasis. Furthermore, cathelicidin LL-37 interfered with DNA-sensing inflammasomes, which thereby suggests an anti-inflammatory function for this peptide. Thus, our data reveal a link between the AIM2 inflammasome, cathelicidin LL-37, and autoinflammation in psoriasis, providing new potential targets for the treatment of this chronic skin disease.

RNase 7 Contributes to the Cutaneous Defense against Enterococcus faecium

Background Human skin is able to mount a fast response against invading microorganisms by the release of antimicrobial proteins such as the ribonuclease RNase 7. Because RNase 7 exhibits high activity against Enterococcus faecium the aim of this study was to further explore the role of RNase 7 in the cutaneous innate defense system against E. faecium. Methodology/Principal Findings Absolute quantification using real-time PCR and ELISA revealed that primary keratinocytes expressed high levels of RNase 7. Immunohistochemistry showed RNase 7 expression in all epidermal layers of the skin with an intensification in the upper more differentiated layers. Furthermore, RNase 7 was secreted by keratinocytes in vitro and in vivo in a site-dependent way. RNase 7 was still active against E. faecium at low pH (5.5) or high NaCl (150 mM) concentration and the bactericidal activity of RNase 7 against E. faecium required no ribonuclease activity as shown by recombinant RNase 7 lacking enzymatic activity. To further explore the role of RNase 7 in cutaneous defense against E. faecium, we investigated whether RNase 7 contributes to the E. faecium killing activity of skin extracts derived from stratum corneum. Treatment of the skin extract with an RNase 7 specific antibody, which neutralizes the antimicrobial activity of RNase 7, diminished its E. faecium killing activity. Conclusions/Significance Our data indicate that RNase 7 contributes to the E. faecium-killing activity of skin extracts and suggest an important role for RNase 7 in the protection of human skin against E. faecium colonization.

The Pattern Recognition Receptor NOD2 Mediates Staphylococcus aureus-Induced IL-17C Expression in Keratinocytes

IL-17C is an important epithelial cell-derived cytokine activating innate immunity by the induction of antimicrobial peptides and cytokines. Here, we investigated the role of the cytosolic pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) for the Staphylococcus aureus-mediated induction of IL-17C. Activation of NOD2 in HEK293 cells overexpressing NOD2 induced the IL-17C promoter, an activity that was significantly reduced in cells overexpressing the Crohns disease-associated NOD2 mutation 3020insC (1007fs) or the Crohns disease- and atopic dermatitis-associated NOD2-R702W variant. The first NF-κB-binding site in the IL-17C promoter was critical for NOD2-mediated IL-17C induction. Infection of human primary keratinocytes with S. aureus induced NOD2 and IL-17C gene expression. Overexpression of NOD2 in keratinocytes augmented S. aureus-mediated IL-17C gene expression as compared with NOD2-R702W overexpression. S. aureus-induced IL-17C expression was diminished in NOD2 small interfering RNA (siRNA)-treated keratinocytes. Moreover, significantly less S. aureus bacteria survived in keratinocytes overexpressing NOD2 but not in cells overexpressing the NOD2-R702W variant. Finally, S. aureus showed an increased survival in keratinocytes treated with NOD2 or IL-17C siRNA. In summary, our study provides evidence that S. aureus activates NOD2 in keratinocytes, resulting in an increased expression of IL-17C, a mechanism that may be dysregulated in atopic dermatitis.

Antimicrobial RNases in Cutaneous Defense

Antimicrobial proteins (AMP) are small endogenous proteins which are capable of rapidly inactivating microorganisms at low micro- and nanomolar concentrations. Their significance in host defense is reflected by their wide distribution in nature. Several AMP have been isolated from human skin, and there is increasing evidence that AMP may play an important role in cutaneous defense. One important human AMP class comprises several antimicrobial members of the RNase A superfamily. Of these, two members, RNase 7 and RNase 5, have been implicated in cutaneous defense. This review gives an overview about our current knowledge on the potential role of RNase 7 and RNase 5 in protecting human skin from infection.

IL-17A and IFN-γ Synergistically Induce RNase 7 Expression via STAT3 in Primary Keratinocytes

Human keratinocytes produce several antimicrobial peptides and proteins (AMP) which contribute to the protection of human skin against infection. RNase 7 is a major AMP involved in cutaneous defense with a high expression in keratinocytes and a broad spectrum of antimicrobial activity. The cytokine IL-17A has been recently identified as a potent inducer of several AMP in keratinocytes. Since the role of IL-17A to induce RNase 7 expression is unknown we analyzed IL-17A alone and in combination with other cytokines to induce RNase 7 expression in keratinocytes. Whereas IL-17A alone only weakly induced RNase 7 expression, the synergistic combination of IL-17A and IFN-γ (IL-17A/IFN-γ) was identified as a potent inducer of RNase 7 expression. This combination was more effective in inducing RNase 7 than the combination of IL-17A/TNF-α, a combination previously identified as a strong inducer of psoriasis-related immune response genes including several AMP. IFN-γ and IL-17A both have been reported to activate the transcription factor STAT3 (Signal transducer and activator of transcription 3). Therefore we investigated the influence of STAT3 on the IL-17A/IFN-γ -mediated RNase 7 induction. The use of a STAT3 inhibitor as well as siRNA-mediated downregulation of STAT3 resulted in a diminished IL-17A/IFN-γ -mediated RNase 7 induction in keratinocytes indicating that STAT3 is involved in this process. Similarly as seen with RNase 7, treatment of keratinocytes with IL-17A/IFN-γ revealed also a synergistic induction of gene expression of the AMP human beta-defensin (hBD)-2 and -3 as well as the S100 protein psoriasin (S100A7) indicating that the combination of IL-17A/IFN-γ is a potent inducer of various AMP classes in general. This was also reflected by an increase of the Staphylococcus aureus-killing activity of IL-17A/IFN-γ -treated keratinocytes.

Infection of keratinocytes with Trichophytum rubrum induces epidermal growth factor-dependent RNase 7 and human beta-defensin-3 expression.

Human keratinocytes are able to express various antimicrobial peptides (AMP) to protect the skin from exaggerated microbial colonization and infection. Recently, in vitro growth-inhibiting activity of the skin-derived AMP psoriasin, RNase 7 and human beta-defensin (hBD)-2 against dermatophytes such as Trichophyton (T.) rubrum have been reported. To evaluate whether keratinocytes are able to respond to T. rubrum infection by an induced expression of AMP we exposed primary keratinocytes to living conidia of T. rubrum. This led to conidia germination and mycelial growth which was paralleled by a strong gene induction of the skin-derived AMP RNase 7 and hBD-3. Gene expression of the AMP psoriasin (S100A7) and hBD-2 were only slightly induced. The T. rubrum-mediated RNase 7 gene induction was accompanied by increased secretion of RNase 7. Parallel treatment of the keratinocytes with T. rubrum and the cytokine combination IL-17A/IFN-γ resulted in synergistic induction of RNase 7 and hBD-3 expression. Since patients receiving therapy by inhibition of the epidermal growth factor receptor (EGFR) more often suffer from dermatophytoses we investigated whether EGFR may be involved in the T. rubrum-mediated RNase 7 and hBD-3 induction. Primary keratinocytes incubated with an EGFR blocking antibody as well as with the EGFR antagonist AG1478 showed a significantly diminished RNase 7 and hBD-3 induction upon exposure of the keratinocytes to T. rubrum indicating that EGFR is involved in the T. rubrum-mediated induction of RNase 7 and hBD-3. The growth of T. rubrum in vitro was inhibited by hBD-3 in a dose-dependent manner suggesting that hBD-3 may contribute to cutaneous innate defense against T. rubrum. Taken together our data indicate that keratinocytes are able to initiate a fast defense response towards T. rubrum by the increased expression of AMP active against T. rubrum. A dysregulation of AMP may contribute to chronic and recurring dermatophytoses.

Paraoxonase 2 Acts as a Quorum Sensing–Quenching Factor in Human Keratinocytes

TO THE EDITOR Many pathogenic bacteria are characterized by their ability to express virulence factors such as proteases, toxins, and biofilms. The expression of several virulence factors is controlled by quorum sensing (QS), a bacterial cell-to-cell signaling system. QS is mediated by the production of small signal molecules termed autoinducers, which can activate or repress gene expression upon reaching a certain minimal threshold concentration (Antunes et al., 2010). In many Gram-negative pathogens including Pseudomonas aeruginosa, N-acyl homoserine lactones (AHLs) are used as autoinducers to regulate the expression of virulence factors (Galloway et al., 2011). AHLs can be degraded by lactonases, a process termed quorum quenching (Zhang and Dong, 2004). Production of AHLdegrading enzymes appears to be an effective strategy to interfere with bacterial cell communication and to hamper the expression of virulence factors. Paraoxonase (PON)1–3 are three human lactonases originally described as enzymes that are capable of hydrolyzing organophosphates (Precourt et al., 2011). Although much research interest has been focused on the antioxidative and anti-inflammatory properties of PON1–3 (Precourt et al., 2011), the lactonase activity of these enzymes led to the hypothesis that they may also have a role in attenuation of bacterial virulence through interfering with QS (Camps et al., 2011). In line with this hypothesis, it has been shown that murine tracheal epithelial cells express PON1–3, and loss of PON2 in murine PON2-deficient tracheal epithelia cultures enhanced P. aeruginosa QS (Stoltz et al., 2007). Overexpression of PON2 increased AHL inactivation in human airway epithelial cell lysates (Stoltz et al., 2007), and PON2 small interfering RNA (siRNA)–treated hepatoma and endothelial cell line lysates showed a decreased capacity to hydrolyse N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL), one of the major AHLs produced by P. aeruginosa (Teiber et al., 2008). In another study, it was shown that PON1-overexpressing transgenic Drosophila was protected from P. aeruginosa lethality and that protection was dependent on the activity of the PON1 lactonase (Stoltz et al., 2008). Together, these studies suggest that PONs have an important role in the quenching of bacterial QS. As nothing is known about the capacity of human skin to interfere with QS, we sought to investigate whether PONs are functionally expressed in human keratinocytes. First, we analyzed gene expression of PON1–3 in human primary keratinocytes using real-time PCR. Plasmids containing inserts with the PON1–3 PCR products served as standards for absolute quantification of PON transcripts. As shown in Figure 1, we detected high levels of PON2 gene expression in primary keratinocytes, whereas PON3 was less expressed and PON1 was below the level of detection (Figure 1a). This is in line with a recent report in which a PON2 but not a PON1 band in gelbased PCR of skin extract was detected (Mackness et al., 2010). In addition, we detected no induction of PON2 gene expression in keratinocytes stimulated with various cytokines, growth factors, and P. aeruginosa (Figure 1b and c). Protein expression of PON2 was analyzed by immunohistochemistry with PON2 antibodies using paraffin sections of normal and psoriatic skin. Strongest PON2 immunoreactivity was detected in the outermost epidermal layers of normal and psoriatic skin (Supplementary Figure S1 online). We detected no obvious differences in the staining intensities between normal and psoriatic skin. In line with these data, a real-time PCR analysis revealed no significant differences in PON2 gene expression between three lesional and non-lesional psoriasis samples (not shown). It has been shown that of the three PON enzymes PON2 exhibits the highest activity toward the important AHL 3OC12-HSL (Teiber et al., 2008). To assess whether 3OC12-HSL is able to induce PON2 expression, we stimulated keratinocytes with different concentrations of 3OC12-HSL for 3 and 16 hours. 3OC12-HSL was not able to induce PON2 gene expression in keratinocytes (Figure 1d). The high expression level of PON2 in keratinocytes together with its strong lactonase activity suggests that PON2 may have a role in cutaneous defence against bacterial pathogens through interference with bacterial QS by signal molecule inactivation. To verify the functional relevance of PON2 expression in keratinocytes, we incubated primary keratinocytes with 20 mM 3OC12-HSL. Following 3 hours of incubation, the medium was removed and analyzed for the presence of 3OC12-HSL by the use of the biosensor strain Pseudomonas putida F117 (pASC8), which expresses green fluorescent protein (GFP) in the presence of AHLs with acyl side chains ranging from C8 to C12 (Steidle et al., 2001). A 50-ml LETTERS TO THE EDITOR

Staphylococcus aureus subverts cutaneous defense by d‐alanylation of teichoic acids

The Gram‐positive bacterium Staphylococcus aureus is a frequent skin colonizer that often causes severe skin infections. It has been reported that neutralizing the negatively charged bacterial surface through the incorporation of d‐alanine in its teichoic acids confers reduced susceptibility of S. aureus towards cationic antimicrobial peptides (AMPs). Using a S. aureus strain deficient in d‐alanylated teichoic acids (dltA mutant), we demonstrate that d‐alanylation of its surface reduces the susceptibility of S. aureus to skin‐derived AMPs such as RNase 7 and human beta‐defensins. This is accompanied by a higher killing activity of skin extracts towards the S. aureus dltA mutant as well as towards clinical isolates expressing lower levels of dltA. We conclude that modulation of cell envelope d‐alanylation may help S. aureus to persist on human skin through evasion of cutaneous innate defense provided by cationic skin‐derived AMPs.

Human skin engages different epidermal layers to provide distinct innate defense mechanisms

Keratinocytes are able to sense bacteria or bacterial products leading to a rapid defense reaction by the increased expression of antimicrobial peptides and cytokines. Recent data by Percoco and collaborators published in Experimental Dermatology indicate that bacteria colonizing the skin surface induce a differential spatial expression pattern of antimicrobial peptides and cytokines. Using laser capture microdissection followed by real‐time PCR as well as immunohistochemistry and transmission electron microscopy, the authors provide evidence that antimicrobial peptides such as human beta‐defensin (hBD)‐2 and ‐3 were more strongly induced in the uppermost epidermal layers, whereas the main induction of cytokines such as IL‐1beta and IL‐6 occurred in the lower parts of the epidermis.

The role of RNase 7 in innate cutaneous defense against Pseudomonas aeruginosa.

The ribonuclease RNase 7 is a major skin‐derived human antimicrobial protein expressed in keratinocytes. Here we show that the gram‐negative pathogen Pseudomonas aeruginosa secretes factor(s) that induced RNase 7 gene and protein expression in human primary keratinocytes. The metalloprotease inhibitor marimastat, the epidermal growth factor receptor (EGFR) inhibitor AG1478 and the EGFR blocking antibody cetuximab significantly attenuated this induction, indicating an important role of the EGFR for the P. aeruginosa‐mediated RNase 7 induction. In line with this, siRNA‐mediated downregulation of ADAM17, a metalloprotease known to proteolytically mediate the release of soluble EGFR ligands, decreased the P. aeruginosa‐mediated RNase 7 induction in keratinocytes. The impact of the EGFR was also demonstrated in a human 3D skin equivalent where blockade of the EGFR diminished induction of RNase 7 by P. aeruginosa. Blockade of Toll‐like receptor 5 (TLR5), a pattern recognition receptor (PRR) known to be activated by P. aeruginosa, only moderately reduced the P. aeruginosa‐mediated RNase 7 induction in keratinocytes. The functional relevance of RNase 7 to participate in cutaneous defense against P. aeruginosa was demonstrated by antibodies that neutralized the antimicrobial activity of RNase 7. These antibodies significantly inhibited the capacity of human stratum corneum skin extracts to control growth of P. aeruginosa. Taken together, our results indicate that P. aeruginosa induces the expression of RNase 7 in keratinocytes in an EGFR‐dependent manner. Enhanced release of RNase 7 contributes to control cutaneous growth of P. aeruginosa.