naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation.

Neutrophil extracellular traps (NETs) are a key antimicrobial feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs). NETs counteract microbes but are also linked to inflammation in atherosclerosis, arthritis, or psoriasis by unknown mechanisms. Here, we report that NET-associated RNA (naRNA) stimulates further NET formation in naive PMNs via a unique TLR8-NLRP3 inflammasome-dependent pathway. Keratinocytes respond to naRNA with expression of psoriasis-related genes (e.g., IL17, IL36) via atypical NOD2-RIPK signaling. In vivo, naRNA drives temporary skin inflammation, which is drastically ameliorated by genetic ablation of RNA sensing. Unexpectedly, the naRNA-LL37 'composite damage-associated molecular pattern (DAMP)' is pre-stored in resting neutrophil granules, defining sterile NETs as inflammatory webs that amplify neutrophil activation. However, the activity of the naRNA-LL37 DAMP is transient and hence supposedly self-limiting under physiological conditions. Collectively, upon dysregulated NET release like in psoriasis, naRNA sensing may represent both a potential cause of disease and a new intervention target.

PARP1 expression predicts PARP inhibitor sensitivity and correlates with metastatic potential and overall survival in melanoma.

Metastatic melanoma is still a difficult-to-treat cancer type owing to its frequent resistance mechanisms to targeted and immunotherapy. Therefore, we aimed to unravel novel therapeutic strategies for melanoma patients. Preclinical and clinical studies show that melanoma patients may benefit from a treatment with poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). In this study, we focus on PARP1 as a potential biomarker to predict the response of melanoma cells to PARPi therapy. We found that melanoma cells with high basal PARP1 expression exhibit significantly increased cell death after PARPi treatment owing to higher PARP1 trapping compared with melanoma cells with low PARP1 expression. In addition, we could demonstrate that PARP1 expression levels are low in nonmalignant skin cells, and metastatic melanomas show considerably higher PARP1 levels compared with primary melanomas. Most strikingly, we found that high PARP1 levels correlate with worse overall survival of late stage metastasized melanoma patients. In conclusion, we show that PARP1 might act as a biomarker to predict the response to PARPi therapy, and that in particular the late stage metastasized melanoma patients are especially sensitive to PARPi therapy owing to elevated PARP1 expression. Our data suggest that the PARPi cytotoxicity primarily will affect the high PARP1 expressing melanoma cells, rather than the low PARP1 expressing nonmalignant skin cells resulting in only low side effects.

Targeting Gαi2 in neutrophils protects from myocardial ischemia reperfusion injury.

Neutrophils are not only involved in immune defense against infection but also contribute to the exacerbation of tissue damage after ischemia and reperfusion. We have previously shown that genetic ablation of regulatory Gαi proteins in mice has both protective and deleterious effects on myocardial ischemia reperfusion injury (mIRI), depending on which isoform is deleted. To deepen and analyze these findings in more detail the contribution of Gαi2 proteins in resident cardiac vs circulating blood cells for mIRI was first studied in bone marrow chimeras. In fact, the absence of Gαi2 in all blood cells reduced the extent of mIRI (22,9% infarct size of area at risk (AAR) Gnai2-/- → wt vs 44.0% wt → wt; p < 0.001) whereas the absence of Gαi2 in non-hematopoietic cells increased the infarct damage (66.5% wt → Gnai2-/- vs 44.0% wt → wt; p < 0.001). Previously we have reported the impact of platelet Gαi2 for mIRI. Here, we show that infarct size was substantially reduced when Gαi2 signaling was either genetically ablated in neutrophils/macrophages using LysM-driven Cre recombinase (AAR: 17.9% Gnai2fl/fl LysM-Cre+/tg vs 42.0% Gnai2fl/fl; p < 0.01) or selectively blocked with specific antibodies directed against Gαi2 (AAR: 19.0% (anti-Gαi2) vs 49.0% (IgG); p < 0.001). In addition, the number of platelet-neutrophil complexes (PNCs) in the infarcted area were reduced in both, genetically modified (PNCs: 18 (Gnai2fl/fl; LysM-Cre+/tg) vs 31 (Gnai2fl/fl); p < 0.001) and in anti-Gαi2 antibody-treated (PNCs: 9 (anti-Gαi2) vs 33 (IgG); p < 0.001) mice. Of note, significant infarct-limiting effects were achieved with a single anti-Gαi2 antibody challenge immediately prior to vessel reperfusion without affecting bleeding time, heart rate or cellular distribution of neutrophils. Finally, anti-Gαi2 antibody treatment also inhibited transendothelial migration of human neutrophils (25,885 (IgG) vs 13,225 (anti-Gαi2) neutrophils; p < 0.001), collectively suggesting that a therapeutic concept of functional Gαi2 inhibition during thrombolysis and reperfusion in patients with myocardial infarction should be further considered.

Dormancy of cutaneous melanoma.

Many cancer-related deaths including melanoma result from metastases that develop months or years after the initial cancer therapy. Even the most effective drugs and immune therapies rarely eradicate all tumor cells. Instead, they strongly reduce cancer burden, permitting dormant cancer cells to persist in niches, where they establish a cellular homeostasis with their host without causing clinical symptoms. Dormant cancers respond poorly to most drugs and therapies since they do not proliferate and hide in niches. It therefore remains a major challenge to develop novel therapies for dormant cancers. In this review we focus on the mechanisms regulating the initiation of cutaneous melanoma dormancy as well as those which are involved in reawakening of dormant cutaneous melanoma cells. In recent years the role of neutrophils and niche components in reawakening of melanoma cells came into focus and indicate possible future therapeutic applications. Sophisticated in vitro and in vivo melanoma dormancy models are needed to make progress in this field and are discussed.

Enhanced expression of p21 promotes sensitivity of melanoma cells towards targeted therapies.

Metastatic melanoma patients benefit from the approved targeted BRAF inhibitor (BRAFi) therapy. Despite the great progress in the therapeutic approach to combat metastatic melanoma, fast emerging drug resistance in patients limits its long-term efficacy. In this study, we aimed to unravel the role of the p53 target gene CDKN1A/p21 in the response of melanoma cells towards BRAFi. We show that p53 activation increases BRAFi sensitivity in a synergistic manner exclusively in cells with a high expression of CDKN1A/p21. In a similar way, high expression of p21 was associated with a better response towards the mouse double minute 2 inhibitor (MDM2i) compared to those with low p21 expression. Indeed, p21 knockdown decreased the sensitivity towards both targeted therapies. The results indicate that the sensitivity of melanoma cells towards targeted therapies (BRAFi and MDM2i) is dependent on the p21 protein level in the cells. In addition to that, we found that p53 negatively regulates p73 expression; however, p73 seems not to have an influence on p53 expression. These findings offer new potential strategies for the treatment improvement of melanoma patients with high basal p21 levels with BRAFi by increasing treatment efficacy using combination therapies with p53 activating substances, which are able to further increase p21 expression levels. Furthermore, the data suggest that the expression and induction level of p21 could be used as a predictive biomarker in melanoma patients to forecast the outcome of a treatment with p53 activating substances and BRAFi. All in all, this manuscript shows the distinct role of p53 family members and its impact on melanoma therapy. In future, individualized treatment regimens based on p21 basal and induction levels could help melanoma patients with limited treatment options.

Bacterial membrane vesicles shape Staphylococcus aureus skin colonization and induction of innate immune responses.

Staphylococcus aureus colonization is abundant on the skin of atopic dermatitis (AD) patients where it contributes to skin inflammation. S. aureus produces virulence factors that distinguish it from commensal skin bacteria such as S. epidermidis and S. lugdunensis. However, it has remained unclear, which of these virulence factors have the strongest impact on AD. Membrane vesicles (MVs) are released by pathogenic bacteria and might play an essential role in the long-distance delivery of bacterial effectors such as virulence factors. We show that MVs are also released by skin commensals in a similar quantity and membrane lipid amount as those from pathogenic S. aureus. Interestingly, MVs from skin commensals can protect against S. aureus skin colonization by conditioning human skin for enhanced defence. In contrast, MVs released by S. aureus are able to induce CXCL8 and TNF-α in primary human keratinocytes, recruit neutrophils and induce neutrophil extracellular traps, which enhance S. aureus skin colonization. CXCL8 induction is TLR2- and NFkB-dependent and the induction level correlates with the membrane lipid and protein A content of the MVs. Interestingly, MVs of S. aureus strains from the lesional skin of AD patients show an enhanced membrane lipid and protein A content compared to the strains from the non-lesional sites and have an enhanced proinflammatory potential. Our data underline the complex interplay in host- and bacterial derived factors in S. aureus skin colonization and the important role of bacterial derived MVs and their membrane lipid and protein A content in skin inflammatory disorders.

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.

Beneficial effects of coagulase-negative Staphylococci on Staphylococcus aureus skin colonization.

Our skin is constantly exposed to a large number of pathogens while at the same time undergoing selective colonization by commensal microorganisms such as coagulase-negative Staphylococci. Staphylococcus aureus, however, is a facultative pathogen that is usually absent from healthy skin but frequently colonizes the inflamed skin of atopic dermatitis patients, where it further promotes inflammation. Enhanced S. aureus skin colonization was shown to correlate with a loss of microbiome diversity indicating a role for skin commensals to shape pathogen colonization. Together, keratinocytes and immune cells in the skin need to discriminate commensals from pathogens and orchestrate subsequent immune reactions in response to colonizing microbes. However, the mechanisms how individual commensals cooperate with keratinocytes and the immune system of the skin to prevent pathogen colonization are barely understood. In this review, we discuss the current knowledge on the functional effects of coagulase-negative staphylococci, the most frequently isolated skin commensals, on S. aureus skin colonization.

Staphylococcus aureus Lpl protein triggers human host cell invasion via activation of Hsp90 receptor.

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein-like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat-inducible and appears to play a major role in Lpl1 interaction. Pre-incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1-Hsp90 interaction induces F-actin formation, thus, triggering an endocytosis-like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl-Hsp90 interaction.

Quantitative Proteomics Links the Intermediate Filament Nestin to Resistance to Targeted BRAF Inhibition in Melanoma Cells.

Targeted inhibition of mutated kinases using selective MAP kinase inhibitors in malignant melanoma often results in temporary improvement of clinical symptoms followed by rapid development of resistance. To gain insights in molecular processes that govern resistance, we performed SILAC-based quantitative proteomics profiling of vemurafenib-resistant and -sensitive melanoma cells. Among downregulated proteins in vemurafenib-resistant cell lines we detected multiple proteins involved in cytoskeletal organization and signaling, including the intermediate filament nestin, which was one of the most downregulated proteins. Previous studies showed that nestin is expressed in various types of solid tumors and its abundance correlates with malignant phenotype of transformed cells. However, the role of nestin in cancer cells regarding acquired resistance is still poorly understood. We performed CRISPR/Cas9 knockout of the nestin gene (NES) in vemurafenib-sensitive cells and showed that loss of nestin leads to increased cellular proliferation and colony formation upon treatment with BRAFV600E and MEK inhibitors. Moreover, nestin depletion led to increased invasiveness and metalloproteinase activity like the phenotype of melanoma cells with acquired resistance to the BRAF inhibitor. Finally, phosphoproteome analysis revealed that nestin depletion influenced signaling through integrin and PI3K/AKT/mTOR pathways and led to increased focal adhesion kinase abundance and phosphorylation. Taken together, our results reveal that nestin is associated with acquired vemurafenib resistance in melanoma cells.

Staphylococcal (phospho)lipases promote biofilm formation and host cell invasion.

Most Staphylococcus aureus strains secrete two lipases SAL1 and SAL2 encoded by gehA and gehB. These two lipases differ with respect to their substrate specificity. Staphylococcus hyicus secretes another lipase, SHL, which is in contrast to S. aureus lipases Ca2+-dependent and has a broad-spectrum lipase and phospholipase activity. The aim of this study was to investigate the role of staphylococcal (phospho) lipases in virulence. For this we constructed a gehA-gehB double deletion mutant in S. aureus USA300 and expressed SHL in agr-positive (accessory gene regulator) and agr-negative S. aureus strains. The lipases themselves have no hemolytic or cytotoxic activity. However, in agr-negative strains SHL-expression caused an upregulation of hemolytic activity. We further show that SHL-expression significantly enhanced biofilm formation probably due to an increase of extracellular DNA release. SHL-expression also increased host cell invasion 4-6-fold. Trioleate, a main triacylglycerol component of mammalian skin, induced lipase production. Finally, in the mouse sepsis and skin colonization models the lipase producing and mutant strain showed no significant difference compared to the WT strain. In conclusion, we show that staphylococcal lipases promote biofilm formation and host cell invasion and thereby contribute to S. aureus virulence.

Entry, Intracellular Survival, and Multinucleated-Giant-Cell-Forming Activity of Burkholderia pseudomallei in Human Primary Phagocytic and Nonphagocytic Cells.

The human pathogen Burkholderia pseudomallei and the related species Burkholderia thailandensis are facultative intracellular bacteria characterized by the ability to escape into the cytosol of the host cell and to stimulate the formation of multinucleated giant cells (MNGCs). MNGC formation is induced via an unknown mechanism by bacterial type VI secretion system 5 (T6SS-5), which is an essential virulence factor in both species. Despite the vital role of the intracellular life cycle in the pathogenesis of the bacteria, the range of host cell types permissive for initiation and completion of the intracellular cycle is poorly defined. In the present study, we used several different types of human primary cells to evaluate bacterial entry, intracellular survival, and MNGC formation. We report the capacity of B. pseudomallei to enter, efficiently replicate in, and mediate MNGC formation of vein endothelial and bronchial epithelial cells, indicating that the T6SS-5 is important in the host-pathogen interaction in these cells. Furthermore, we show that B. pseudomallei invades fibroblasts and keratinocytes and survives inside these cells as well as in monocyte-derived macrophages and neutrophils for at least 17 h postinfection; however, MNGC formation is not induced in these cells. In contrast, infection of mixed neutrophils and RAW264.7 macrophages with B. thailandensis stimulated the formation of heterotypic MNGCs in a T6SS-5-dependent manner. In summary, the ability of the bacteria to enter and survive as well as induce MNGC formation in certain host cells may contribute to the pathogenesis observed in B. pseudomallei infection.

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.

Excreted Cytoplasmic Proteins Contribute to Pathogenicity in Staphylococcus aureus.

Excretion of cytoplasmic proteins in pro- and eukaryotes, also referred to as "nonclassical protein export," is a well-known phenomenon. However, comparatively little is known about the role of the excreted proteins in relation to pathogenicity. Here, the impact of two excreted glycolytic enzymes, aldolase (FbaA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), on pathogenicity was investigated in Staphylococcus aureus Both enzymes bound to certain host matrix proteins and enhanced adherence of the bacterial cells to host cells but caused a decrease in host cell invasion. FbaA and GAPDH also bound to the cell surfaces of staphylococcal cells by interaction with the major autolysin, Atl, that is involved in host cell internalization. Surprisingly, FbaA showed high cytotoxicity to both MonoMac 6 (MM6) and HaCaT cells, while GAPDH was cytotoxic only for MM6 cells. Finally, the contribution of external FbaA and GAPDH to S. aureus pathogenicity was confirmed in an insect infection model.

Casein kinase 1α has a non-redundant and dominant role within the CK1 family in melanoma progression.

BACKGROUND: We previously identified CK1α as a novel tumor suppressor in melanoma and reported that the loss of CK1α leads to increased proliferation and invasive growth of melanoma cells by strong activation of the Wnt/ß-catenin signaling pathway. METHODS: In this study we analyzed expression and the functional effects of the dominantly expressed CK1- isoforms α, δ and ε in melanoma cells by quantitative real-time PCR, western blot and immunohistochemistry. We down-regulated CK1 kinase activity with isoform specific siRNAs and small molecule inhibitors. Vice versa we overexpressed the CK1 isoforms α, δ and ε using viral vectors and tested the biological effects on melanoma cell proliferation, migration and invasion. RESULTS: We show that protein expression of all three CK1-isoforms is downregulated in metastatic melanoma cells compared to benign melanocytic cells. Furthermore, the CK1δ and ε isoforms are able to negatively regulate expression of each other, whereas CK1α expression is independently regulated in melanoma cells. Inhibition of the expression and activity of CK1δ or CK1ε by specific inhibitors or siRNAs had no significant effect on the growth and survival of metastatic melanoma cells. Moreover, the over-expression of CK1δ or CK1ε in melanoma cells failed to induce cell death and cell cycle arrest although p53 signaling was activated. This is in contrast to the effects of CK1α where up-regulated expression induces cell death and apoptosis in metastatic melanoma cells. CONCLUSION: These data indicate that CK1α has a dominant and non-redundant function in melanoma cells and that the CK1δ and ε isoforms are not substantially involved in melanoma progression.

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.

Biological functions of casein kinase 1 isoforms and putative roles in tumorigenesis.

Isoforms of the casein kinase 1 (CK1) family have been shown to phosphorylate key regulatory molecules involved in cell cycle, transcription and translation, the structure of the cytoskeleton, cell-cell adhesion and receptor-coupled signal transduction. They regulate key signaling pathways known to be critically involved in tumor progression. Recent results point to an altered expression or activity of different CK1 isoforms in tumor cells. This review summarizes the expression and biological function of CK1 family members in normal and malignant cells and the evidence obtained so far about their role in tumorigenesis.

Crosstalk between keratinocytes and neutrophils shapes skin immunity against S. aureus infection.

Introduction: Staphylococcus aureus (S. aureus) infection of the skin leads to a rapid initial innate immune response with keratinocytes in the epidermis as the initial sensors. Polymorphonuclear neutrophils (PMNs) are the first innate immune cells to infiltrate infection sites where they provide an effective first-line of defense. Previous work of our group showed that in inflamed skin a crosstalk between PMNs and keratinocytes results in enhanced S. aureus skin colonization. Methods: In this work, we used an in vitro co-culture model to studied the crosstalk between primary human keratinocytes (PHKs) and PMNs in a sterile environment and upon S. aureus infection. We investigated the influence of PHKs on PMN activation by analyzing PMN lifespan, expression of degranulation markers and induction of proinflammatory cytokines. Furthermore, we analyzed the influence of PMNs on the inflammatory response of PHKs. Finally, we investigated the influence of the skin microbiome on PMN-mediated skin inflammation. Results: We show that co-culture of PMNs with PHKs induces activation and degranulation of PMNs and significantly enhances their lifespan compared to PMN cultivation alone by an IL-8 mediated mechanism and, furthermore, primes PMNs for enhanced activity after S. aureus infection. The prolonged incubation with PMNs also induces inflammatory responses in PHKs which are further exacerbated in the presence of S. aureus and induces further PMN recruitment thus fueling skin inflammation. Interestingly, infection of PHKs with the skin commensal S. epidermidis reduces the inflammatory effects of PMNs in the skin and exhibits an anti-inflammatory effect. Discussion: Our data indicate that skin infiltrating PMNs and PHKs influence each other in such a way to enhance skin inflammation and that commensal bacteria are able to reduce the inflammatory effect.

Histological and functional characterization of 3D human skin models mimicking the inflammatory skin diseases psoriasis and atopic dermatitis.

Three-dimensional (3D) human skin equivalents have emerged as valuable tools in skin research, replacing animal experimentation and precluding the need for patient biopsies. In this study, we advanced 3D skin equivalents to model the inflammatory skin diseases atopic dermatitis and psoriasis by cytokine stimulation, and were successful in integrating TH1 T cells into skin models to develop an immunocompetent 3D psoriasis model. We performed in-depth histological and functional characterization of 3D skin equivalents and validated them in terms of tissue architecture, pathological changes, expression of antimicrobial peptides and Staphylococcus aureus colonization using 3D reconstruction by multiphoton microscopy and phenotyping by highly multiplexed 'co-detection by indexing' (CODEX) microscopy. We show that our skin equivalents have a structural architecture with a well-developed dermis and epidermis, thus resembling human skin. In addition, the skin models of atopic dermatitis and psoriasis show several phenotypic features of inflammatory skin disease, including disturbed epidermal differentiation and alterations in the expression of epidermal barrier genes and antimicrobial peptides, and can be reliably used to test novel treatment strategies. Therefore, these 3D equivalents will be a valuable tool in experimental dermatological research.