The NLRP1 inflammasome in skin diseases.

Healthy human skin is constantly exposed to sterile and microbial agents. The skin immune system plays an important role in immune surveillance between tolerance and immune activation. This is mainly mediated by neutrophils, macrophages and most importantly lymphocytes. Keratinocytes, which form the outer skin barrier (epidermis) are also critical for cutaneous homeostasis. Being a non-professional immune cell, recognition of danger signals in keratinocytes is mediated by innate immune receptors (pattern recognition receptors, PRR). While Toll-like receptors are located on the cell membrane or the endosomes, nucleotide-binding domain and leucine-rich repeat containing gene family receptors (NLR) are intracellular PRRs. Some of these, once activated, trigger the formation of inflammasomes. Inflammasomes are multiprotein complexes and serve as platforms that mediate the release of innate cytokines after successful recognition, thereby attracting immune cells. Moreover, they mediate the pro-inflammatory cell death pyroptosis. Best characterized is the NLRP3 inflammasome. The function of inflammasomes differs significantly between different cell types (keratinocytes versus immune cells) and between different species (human versus mouse). In recent years, great progress has been made in deciphering the activation mechanisms. Dysregulation of inflammasomes can lead to diseases with varying degrees of severity. Here we focus on the structure, function, and associated pathologies of the NLRP1 inflammasome, which is the most relevant inflammasome in keratinocytes.

Transcriptional adaptation of staphylococci during colonization of the authentic human environment: An overview of transcriptomic changes and their relationship to physiological conditions.

Staphylococci are commensals of human skin and mucous membranes, but some species can also cause serious infections. Host niches during both colonization and infection differ greatly and are characterized by specific environmental conditions (pH, temperature, oxygen, nutrient availability, and microbiota) that can affect gene expression and virulence of microbes. To successfully occupy extremely different habitats at different anatomical sites, Staphylococci are equipped with a variety of regulatory elements that allow specific adaptation to the changing environments. Not surprisingly, gene expression in vivo can be significantly different from the expression pattern observed in vitro. Niche specific stimuli that influence the bacterial ability to either cause infection or maintain colonization are only partially understood. Here, we describe habitat specific conditions and discuss the available literature analyzing staphylococcal gene expression, focusing on Staphylococcus aureus and S. epidermidis during colonization of the nose and skin.

The Staphylococcus epidermidis Transcriptional Profile During Carriage.

The virulence factors of the opportunistic human pathogen Staphylococcus epidermidis have been a main subject of research. In contrast, limited information is available on the mechanisms that allow the bacterium to accommodate to the conditions during carriage, a prerequisite for pathogenicity. Here, we tested the hypothesis that the adaptation of S. epidermidis at different anatomical sites is reflected by differential gene regulation. We used qPCR to profile S. epidermidis gene expression in vivo in nose and skin swabs of 11 healthy individuals. Despite some heterogeneity between individuals, significant site-specific differences were detected. For example, expression of the S. epidermidis regulator sarA was found similarly in the nose and on the skin of all individuals. Also, genes encoding colonization and immune evasion factors (sdrG, capC, and dltA), as well as the sphingomyelinase encoding gene sph, were expressed at both anatomical sites. In contrast, expression of the global regulator agr was almost inactive in the nose but readily present on the skin. A similar site-specific expression profile was also identified for the putative chitinase-encoding SE0760. In contrast, expression of the autolysine-encoding gene sceD and the wall teichoic acid (WTA) biosynthesis gene tagB were more pronounced in the nose as compared to the skin. In summary, our analysis identifies site-specific gene expression patterns of S. epidermidis during colonization. In addition, the observed expression signature was significantly different from growth in vitro. Interestingly, the strong transcription of sphingomyelinase together with the low expression of genes encoding the tricarboxylic acid cycle (TCA) suggests very good nutrient supply in both anatomical niches, even on the skin where one might have suspected a rather lower nutrient supply compared to the nose.

ASC Speck Formation after Inflammasome Activation in Primary Human Keratinocytes.

Chronic UV irradiation results in many changes in the skin, including hyperplasia, changes in dermal structures, and alteration of pigmentation. Exposure to UVB leads to cutaneous damage, which results in inflammation characterized by increased NF-κB activation and the induction of inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin- (IL-) 1, or IL-8. IL-1 secretion is the result of inflammasome activation which is besides apoptosis, a result of acute UVB treatment. Inflammasomes are cytosolic protein complexes whose formation results in the activation of proinflammatory caspase-1. Key substrates of caspase-1 are IL-1ß and IL-18, and the cytosolic protein gasdermin D (GSDMD), which is involved in inflammatory cell death. Here, we demonstrate that UVB-induced inflammasome activation leads to the formation of ASC specks. Our findings show that UVB provokes ASC speck formation in human primary keratinocytes prior to cell death, and that specks are, opposed to the perinuclear cytosolic localization in myeloid cells, formed in the nucleus. Additionally, we showed by RNAi that NLRP1 and not NLRP3 is the major inflammasome responsible for UVB sensing in primary human keratinocytes. Formation of ASC specks indicates inflammasome assembly and activation as their formation in hPKs depends on the presence of NLRP1 and partially on NLRP3. Nuclear ASC specks are not specific for NLRP1/NLRP3 inflammasome activation, as the activation of the AIM2 inflammasome by cytosolic DNA results in ASC specks too. These nuclear ASC specks putatively link cell death to inflammasome activation, possibly by binding of IFI16 (gamma-interferon-inducible protein) to ASC. ASC can interact upon UVB sensing via IFI16 with p53, linking cell death to ASC speck formation.

Adaptation of Staphylococcus aureus to the Human Skin Environment Identified Using an ex vivo Tissue Model.

The healthy human epidermis provides physical protection and is impenetrable for pathogenic microbes. Nevertheless, commensal and pathogen bacteria such as Staphylococcus aureus are able to colonize the skin surface, which may subsequently lead to infection. To identify and characterize regulatory elements facilitating adaptation of S. aureus to the human skin environment we used ex vivo tissue explants and quantified S. aureus gene transcription during co-culture. This analysis provided evidence for a significant downregulation of the global virulence regulator agr upon initial contact with skin, regardless of the growth phase of S. aureus prior to co-culture. In contrast, the alternative sigma factor B (sigB) and the antimicrobial peptide-sensing system (graRS) were expressed during early colonization. Consistently, sigB target genes such as the clumping factor A (clfA) and fibrinogen and fibronectin binding protein A (fnbA) were strongly upregulated upon skin contact. At later timepoints of the adhesion process, wall teichoic acid (WTA) synthesis was induced. Besides the expression of adhesive molecules, transcription of molecules involved in immune evasion were increased during late colonization (staphylococcal complement inhibitor and staphylokinase). Similar to nasal colonization, enzymes involved in cell wall metabolism (sceD and atlA) were highly transcribed. Finally, we detected a strong expression of proteases from all three catalytic classes during the entire colonization process. Taken together, we here present an ex vivo skin colonization model that allows the detailed characterization of the bacterial adaptation to the skin environment.

NLRP1 Is the Key Inflammasome in Primary Human Keratinocytes.

The epidermis is the primary area of contact between the body and the environment, and it distinguishes between harmful exposures and those that should be tolerated. Discrimination between insults, and in particular the recognition of danger signals such as UVB, is mediated by innate immune receptors. Inflammasomes are one major innate mechanism that activate inflammatory caspases. In human keratinocytes, the importance of inflammasomes and the sensing of UVB and other danger signals are a matter of debate. Fenini et al. now provide evidence that the NLRP1 (rather than the NLRP3) inflammasome plays a key role in UVB sensing and subsequent IL-1ß and -18 secretion by human keratinocytes.

Differential induction of ATF3 and HO-1 in myeloid cells and keratinocytes via Dimethylfumarate or Cyclosporine A.

BACKGROUND: Chronic inflammatory skin diseases are characterized by controlled proliferation of keratinocytes. Here, activating transcription factor 3 (ATF3) might play a fundamental role. In these inflammatory diseases, proliferation is controlled and only rarely leads to cancer development which can be supported by an inflammatory microenvironment. ATF3 is a dual function protein as it suppresses pro-inflammatory IL-6 and IL-8, but also acts as a pro-oncogenic factor by the suppression of p53. We therefore analyzed ATF3 expression comparing myeloid cells with keratinocytes. OBJECTIVE: To dissect the bi-modal role of ATF3 we pharmacologically induced ATF3 and analyzed its influence on cytokine expression and secretion in a cell type specific manner. METHODS: Since inflammatory skin diseases can be treated systemically with Cyclosporin A or Dimethylfumarate we stimulated myeloid cells and primary human keratinocytes with these drugs and analyzed gene expression by quantitative real-time PCR. Cytokine secretion was measured by ELISA. RESULTS: In the present study, we could show that ATF3 is induced in PBMCs by DMF and weakly by Ebselen, while CsA is the most prominent inducer of ATF3 in keratinocytes without enhancing HO-1 transcription. Further we could show that induction of stress by LPS treatment elevates IL-1ß and IL-6 and weakly ATF3 transcription in PBMCs. While transcription of both cytokines is elevated, LPS treatment mediates IL-6 secretion with only little IL-1ß secretion. Treatment with DMF dampens LPS-induced transcription. CONCLUSIONS: Taken together, our results shed light into the different carcinogenic potential of CsA and DMF, which both target ATF3. Collectively our data demonstrate that CsA strongly induces pro-carcinogenic ATF3 in keratinocytes, whereas ATF3 induction by DMF in myeloid cells acts anti-inflammatory.

Human commensals producing a novel antibiotic impair pathogen colonization.

The vast majority of systemic bacterial infections are caused by facultative, often antibiotic-resistant, pathogens colonizing human body surfaces. Nasal carriage of Staphylococcus aureus predisposes to invasive infection, but the mechanisms that permit or interfere with pathogen colonization are largely unknown. Whereas soil microbes are known to compete by production of antibiotics, such processes have rarely been reported for human microbiota. We show that nasal Staphylococcus lugdunensis strains produce lugdunin, a novel thiazolidine-containing cyclic peptide antibiotic that prohibits colonization by S. aureus, and a rare example of a non-ribosomally synthesized bioactive compound from human-associated bacteria. Lugdunin is bactericidal against major pathogens, effective in animal models, and not prone to causing development of resistance in S. aureus. Notably, human nasal colonization by S. lugdunensis was associated with a significantly reduced S. aureus carriage rate, suggesting that lugdunin or lugdunin-producing commensal bacteria could be valuable for preventing staphylococcal infections. Moreover, human microbiota should be considered as a source for new antibiotics.

The νSaα Specific Lipoprotein Like Cluster (lpl) of S. aureus USA300 Contributes to Immune Stimulation and Invasion in Human Cells.

All Staphylococcus aureus genomes contain a genomic island, which is termed νSaα and characterized by two clusters of tandem repeat sequences, i.e. the exotoxin (set) and 'lipoprotein-like' genes (lpl). Based on their structural similarities the νSaα islands have been classified as type I to IV. The genomes of highly pathogenic and particularly epidemic S. aureus strains (USA300, N315, Mu50, NCTC8325, Newman, COL, JH1 or JH9) belonging to the clonal complexes CC5 and CC8 bear a type I νSaα island. Since the contribution of the lpl gene cluster encoded in the νSaα island to virulence is unclear to date, we deleted the entire lpl gene cluster in S. aureus USA300. The results showed that the mutant was deficient in the stimulation of pro-inflammatory cytokines in human monocytes, macrophages and keratinocytes. Purified lipoprotein Lpl1 was further shown to elicit a TLR2-dependent response. Furthermore, heterologous expression of the USA300 lpl cluster in other S. aureus strains enhanced their immune stimulatory activity. Most importantly, the lpl cluster contributed to invasion of S. aureus into human keratinocytes and mouse skin and the non-invasive S. carnosus expressing the lpl gene cluster became invasive. Additionally, in a murine kidney abscess model the bacterial burden in the kidneys was higher in wild type than in mutant mice. In this infection model the lpl cluster, thus, contributes to virulence. The present report is one of the first studies addressing the role of the νSaα encoded lpl gene cluster in staphylococcal virulence. The finding that the lpl gene cluster contributes to internalization into non-professional antigen presenting cells such as keratinocytes highlights the lpl as a new cell surface component that triggers host cell invasion by S. aureus. Increased invasion in murine skin and an increased bacterial burden in a murine kidney abscess model suggest that the lpl gene cluster serves as an important virulence factor.

The secrets of dermcidin action.

Antimicrobial peptides (AMPs) are important effector molecules of the innate immune defense of diverse species. The majority of known AMPs are cationic therefore facilitating the initial binding of the positively charged peptides to the negatively charged bacterial membrane. Dermcidin (DCD) is constitutively expressed in eccrine sweat glands, secreted into sweat and transported to the epidermal surface where it is proteolytically processed giving rise to several truncated DCD peptides. Its processed forms such as the anionic 48mer DCD-1L and the 47mer DCD-1 possess antimicrobial activity against numerous bacteria including Staphylococcus aureus. Here, the latest knowledge regarding the mode of action of the anionic DCD-1(L) and the functional consequences of their interaction with bacterial membranes is reviewed. There is evidence that the interaction of DCD-1(L) with negatively charged bacterial phospholipids leads to Zn(2+) dependent formation of oligomeric complexes in the bacterial membrane, which subsequently leads to ion channel formation resulting in membrane depolarization and bacterial cell death.

Quantitative proteomics of the human skin secretome reveal a reduction in immune defense mediators in ectodermal dysplasia patients.

In healthy human skin host defense molecules such as antimicrobial peptides (AMPs) contribute to skin immune homeostasis. In patients with the congenital disease ectodermal dysplasia (ED) skin integrity is disturbed and as a result patients have recurrent skin infections. The disease is characterized by developmental abnormalities of ectodermal derivatives and absent or reduced sweating. We hypothesized that ED patients have a reduced skin immune defense because of the reduced ability to sweat. Therefore, we performed a label-free quantitative proteome analysis of wash solution of human skin from ED patients or healthy individuals. A clear-cut difference between both cohorts could be observed in cellular processes related to immunity and host defense. In line with the extensive underrepresentation of proteins of the immune system, dermcidin, a sweat-derived AMP, was reduced in its abundance in the skin secretome of ED patients. In contrast, proteins involved in metabolic/catabolic and biosynthetic processes were enriched in the skin secretome of ED patients. In summary, our proteome profiling provides insights into the actual situation of healthy versus diseased skin. The systematic reduction in immune system and defense-related proteins may contribute to the high susceptibility of ED patients to skin infections and altered skin colonization.

Activation of the alternative sigma factor SigB of Staphylococcus aureus following internalization by epithelial cells - an in vivo proteomics perspective.

Staphylococcus aureus is a versatile pathogen that can be a commensal but also cause a wide range of different infections. This broad disease spectrum is a reflection of the complex regulation of a large collection of virulence factors that together with metabolic fitness allow adaptation to different niches. The alternative sigma factor SigB is one of the global regulators mediating this adaptation. However, even if SigB contributes to expression of many virulence factors its importance for successful infection greatly varies with the strain and the infection setting analyzed. We have recently established a proteomics workflow that combines high efficiency cell sorting with sensitive mass spectrometry and allows monitoring of global proteome adaptations with roughly one million bacterial cells. Thus, we can now approach the adaptation of pathogens to the intracellular milieu. In the current study this proteomics workflow was used in conjunction with qRT-PCR and confocal fluorescence microscopy to comparatively analyze the adaptation of the S. aureus wild type strain HG001 and its isogenic sigB mutant to the intracellular milieu of human S9 bronchial epithelial cells. The study revealed fast and transient activation of SigB following internalization by human host cells and the requirement of SigB for intracellular growth. Loss of SigB triggered proteome changes reflecting the different residual growth rates of wild type and sigB mutant, respectively, the resistance to methicillin, adaptation to oxidative stress and protein quality control mechanisms.

The alternative sigma factor B modulates virulence gene expression in a murine Staphylococcus aureus infection model but does not influence kidney gene expression pattern of the host.

Infections caused by Staphylococcus aureus are associated with significant morbidity and mortality and are an increasing threat not only in hospital settings. The expression of the staphylococcal virulence factor repertoire is known to be affected by the alternative sigma factor B (SigB). However, its impact during infection still is a matter of debate. Kidney tissues of controls or mice infected with S. aureus HG001 or its isogenic sigB mutant were analyzed by transcriptome profiling to monitor the host response, and additionally expression of selected S. aureus genes was monitored by RT-qPCR. Direct transcript analysis by RT-qPCR revealed significant SigB activity in all mice infected with the wild-type strain, but not in its isogenic sigB mutant (p<0.0001). Despite a clear-cut difference in the SigB-dependent transcription pattern of virulence genes (clfA, aur, and hla), the host reaction to infection (either wild type or sigB mutant) was almost identical. Despite its significant activity in vivo, loss of SigB did neither have an effect on the outcome of infection nor on murine kidney gene expression pattern. Thus, these data support the role of SigB as virulence modulator rather than being a virulence determinant by itself.

Time-resolved quantitative proteome profiling of host-pathogen interactions: the response of Staphylococcus aureus RN1HG to internalisation by human airway epithelial cells.

Staphylococcus aureus is a versatile gram-positive pathogen that gains increasing importance due to the rapid spreading of resistances. Functional genomics technologies can provide new insights into the adaptational network of this bacterium and its response to environmental challenges. While functional genomics technologies, including proteomics, have been extensively used to study these phenomena in shake flask cultures, studies of bacteria from in vivo settings lack behind. Particularly for proteomics studies, the major bottleneck is the lack of sufficient proteomic coverage for low numbers of cells. In this study, we introduce a workflow that combines a pulse-chase stable isotope labelling by amino acids in cell culture approach with high capacity cell sorting, on-membrane digestion, and high-sensitivity MS to detect and quantitatively monitor several hundred S. aureus proteins from a few million internalised bacteria. This workflow has been used in a proof-of-principle experiment to reveal changes in levels of proteins with a function in protection against oxidative damage and adaptation of cell wall synthesis in strain RN1HG upon internalisation by S9 human bronchial epithelial cells.

Regulatory adaptation of Staphylococcus aureus during nasal colonization of humans.

The nasopharynx is the main ecological niche of the human pathogen Staphylococcus aureus. Although colonization of the nares is asymptomatic, nasal carriage is a known risk factor for endogenous staphylococcal infection. We quantified S. aureus mRNA levels in nose swabs of persistent carriers to gain insight into the regulatory adaptation of the bacterium to the nasal environment. We could elucidate a general response of the pathogen to the surrounding milieu independent of the strain background or the human host. Colonizing bacteria preferentially express molecules necessary for tissue adherence or immune-evasion whereas toxins are down regulated. From the analysis of regulatory loci we found evidence for a predominate role of the essential two-component system WalKR of S. aureus. The results suggest that during persistent colonization the bacteria are metabolically active with a high cell surface turnover. The increased understanding of bacterial factors that maintain the colonization state can open new therapeutic options to control nasal carriage and subsequent infections.

Temporal expression of adhesion factors and activity of global regulators during establishment of Staphylococcus aureus nasal colonization.

The human pathogen Staphylococcus aureus successfully colonizes its primary reservoir, the nasal cavity, most likely by regulatory adaptation to the nose environment. Cotton rats represent an excellent model for the study of bacterial gene expression in the initial phases of colonization. To gain insight into the expression profile necessary for the establishment of colonization, we performed direct transcript analysis by quantitative real-time reverse-transcription polymerase chain reaction on cotton rat noses removed from euthanized animals on days 1, 4, or 10 after instillation of 2 human S. aureus nose isolates. Global virulence regulators (agr, sae) were not active in this early phase, but the essential 2-component regulatory system WalKR seems to play an important role. Accordingly, an elevated expression of walKR target genes (sak, sceD) could be detected. In agreement with previous studies that demonstrated the essential role played by wall teichoic acid (WTA) polymers in nasal colonization, we detected a strongly increased expression of WTA-biosynthetic genes. The expression profile switched to production of the adhesive proteins ClfB and IsdA at later stages of the colonization process. These data underscore the temporal differences in the roles of WTA and surface proteins in nasal colonization, and they provide the first evidence for a regulation of WTA biosynthesis in vivo.

Transcription analysis of the extracellular adherence protein from Staphylococcus aureus in authentic human infection and in vitro.

BACKGROUND: Wound infections caused by Staphylococcus aureus are associated with significant morbidity and mortality. The staphylococcal extracellular adherence protein (Eap) has been shown to delay wound healing by interfering with host defense and angiogenesis, yet the expression of Eap in the human wound is required to exert these functions. METHODS: A protocol was developed to determine eap transcription levels in vivo (human wounds) relative to those in vitro. In parallel, isolates derived from positive blood cultures were analyzed for eap transcription. RESULTS: Transcription of eap was found in vivo as well as in vitro for all isolates, with eap transcription in vivo being significantly elevated relative to that in the in vitro cultures. In vivo, isolates from deep wounds yielded higher transcription than did those from superficial wounds, whereas in vitro transcription levels for blood culture isolates exceeded those for wound isolates. CONCLUSION: This is the first comprehensive transcription analysis of S. aureus eap in authentic human wounds, and our findings support the hypothesis that Eap contributes to the development of chronic infections by interfering with wound-healing mechanisms. These findings open the door to a novel approach for exploring the complex in vivo interactions between bacteria and the host in such settings.