Menopause is accompanied by skin changes, including complaints of itch, redness, and dry skin, linked to epidermal thinning and reduced sebaceous gland activity. These changes can cause microbiome dysbiosis, leading to dermatoses. Malassezia, a ubiquitous, lipid-dependent, and dominant skin fungus, is associated with multiple skin and systemic diseases. However, the mechanism for its pathogenicity in the context of host physiological changes, such as menopause, remains unexplored. We investigated the skin metagenomes of 345 Asian women spanning pre-, peri-, and post-menopause, revealing menopausal stage-specific differences in prevalence and abundance of Malassezia species. In keratinocyte co-culture, Malassezia elicited cytotoxicity beyond a critical fungal threshold, invaded keratinocytes, and induced inflammation. Transcriptomic profiling of keratinocytes exposed to toxic fungal loads revealed a gene expression profile characteristic of the hyperproliferative and undifferentiated phenotype present in many inflammatory dermatological diseases. Our findings reveal a mechanistic link between menopause-driven changes in skin physiology and Malassezia-mediated keratinocyte dysfunction.

Environmental toxins that cause irritant dermatitis remain poorly understood as activators of innate immune pathways. Here, we identify rove beetle (Paederus) and blister beetle (Meloidae) toxins as previously unrecognized triggers of the human NLRP1 inflammasome in keratinocytes. Rove beetles, likely through the ribosome inhibitor pederin, activate NLRP1 via translational stalling and the ZAKα-dependent ribotoxic stress response. In contrast, the phosphatase inhibitor cantharidin from blister beetles induces NLRP1 through TAK1-driven hyperphosphorylation of its linker region, independent of ZAKα. In their hyperactivated states, ZAKα and TAK1 share overlapping phosphosites on the NLRP1 disordered linker, including a common essential ′TZ motif′. In addition, we show that TAK1 and ZAKα are jointly responsible for NLRP1 linker phosphorylation and activation caused by dsRNA and CHIKV infection. These findings reveal medically relevant insect toxins as activators of NLRP1, and uncover parallel MAP3 kinase pathways as converging upstream activating signals for the human NLRP1 inflammasome. ### Competing Interest Statement The authors have declared no competing interest. National Medical Research Council, MOH-001499, NMRC OF-LCG: MOH001635 Ministry of Education - Singapore, RT23/23, MOE-T2EP30222-0008 Skin Research Institute of Singapore, H22J1a0040

Abstract Introduction The skin forms a mechanical barrier against invading pathogens and environmental allergens. As such, genetic deficiencies in crucial barrier proteins such as filaggrin could result in skin barrier dysfunction. Clinically, filaggrin deficiency is correlated with predisposition to ichthyosis vulgaris (IV), increasing severity to atopic dermatitis (AD) and susceptibility to cutaneous infections. However, immunological mechanisms of how filaggrin-deficiency drives these skin disorders and augment the interactions with microbes remain unclear. Methods Using a mouse model with specific deficiency for filaggrin, we topically administered the ears with MC903 and colonized with Staphylococcus aureus to interrogate the effects on atopic dermatitis and pathobiontic bacterial colonization. Results We first show that filaggrin-deficiency results in comparable trans-epidermal water loss (TEWL) at baseline. However, filaggrin deficient (Flg-/-) mice exhibited increased inflammation and barrier dysfunction upon a biophysical challenge such as tape-stripping. Post atopic induction with calcipotriol (MC903) challenge, elevated tissue swelling and TEWL were also observed in Flg-/- animals relative to wildtype (WT) mice. The proinflammatory cytokine- IL18 was also significantly upregulated in Flg-/- mice compared to WT. Importantly, prophylactic inhibition of IL-18 significantly reduced skin swelling and restored barrier function in filaggrin-deficient mice to WT levels, underscoring its potential as a therapeutic target for inhibition during AD. Moreover, IL-18 expression was elevated in the keratinocytes in our mouse model of AD and in human AD patients. Colonization of filaggrin-deficient mice with S.aureus also resulted in the elevation of inflammatory swelling compared to WT controls. Conclusion We have thus described an animal model of skin barrier dysfunction with filaggrin-deficient mice to study the initiation events of disease and identified IL-18 as an immunopathogenic factor during atopic dermatitis. Funding Source Skin Research Institute of Singapore Joint Research Grant (SRIS_JRG_2010) Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

While skin microbiome studies have increasingly highlighted its importance in health and disease, our understanding of inter-individual heterogeneity in structure and function remains limited, impacting the ability to develop microbiome-based stratification and therapeutics. Powered by comprehensive skin microbiome characterization in a multi-ethnic population-based cohort (>3,550 shotgun metagenomes across 18 sampling sites), we established significant undescribed inter-individual heterogeneity and the extensive prevalence of distinct microbial configurations (17 species-resolution dermotypes) in seven out of nine body sites. Combining functional in silico and in vitro studies revealed insights into how these dermotypes assemble as a function of niche-dependent microbial interactions (e.g. hypoxia-dependent inhibition of S. hominis by S. epidermidis / M. luteus ) and metabolic resource utilization (e.g. differential galactose and histidine metabolism). Integration of demographic, skin physiological, and behavioral data further identified >30 significant associations with host attributes. Cross-site analysis revealed remarkable coordination across disparate skin regions (predictive AUC-ROC>0.8) and bilateral consistency (Pearson π>0.95), emphasizing the role of specific microbial and host factors in shaping dermotypes. Finally, we provide multiple lines of evidence that dermotype states impact the risk for skin discomfort (e.g. irritation, itch) and diseases (e.g. eczema), that when combined with our highly accurate dermotype classifiers (AUC-ROC>0.98), provide a new paradigm for understanding skin microbiome function and stratifying patients in the context of skin and other diseases. ### Competing Interest Statement The authors have declared no competing interest. Shotgun metagenomic sequencing data is available from the European Nucleotide Archive (ENA, ) under project accession number PRJEB87291. Demographic data and skin physiological measurements can be found in Supplementary File 18 . Large datasets and metabolomics data are available on Figshare at . The authors of this study do not own the rights to the HELIOS dataset, and this dataset is under controlled access to ensure good data governance, responsible data use, and that the dataset is only used for the intended research purposes in compliance with HELIOS study cohort IRB and ethics approval. Access to individual level data for study participants should be submitted to the HELIOS Data Access Committee (email: helios_science{at}ntu.edu.sg).

Sebaceous free fatty acids are metabolized by multiple skin microbes into bioactive lipid mediators termed oxylipins. This study investigated correlations between skin oxylipins and microbes on the superficial skin of pre-pubescent children (N = 36) and adults (N = 100), including pre- (N = 25) and post-menopausal females (N = 25). Lipidomics and metagenomics revealed that Malassezia restricta positively correlated with the oxylipin 9,10-DiHOME on adult skin and negatively correlated with its precursor, 9,10-EpOME, on pre-pubescent skin. Co-culturing Malassezia with keratinocytes demonstrated a link between 9,10-DiHOME and pro-inflammatory cytokines IL-1β and IL-6 production. We also observed strong correlations between other skin oxylipins and microbial taxa, highlighting life stage differences in sebum production and microbial community composition. Our findings imply a complex host-microbe communication system mediated by lipid metabolism occurring on human skin, warranting further research into its role in skin health and disease and paving the way towards novel therapeutic targets and treatments.

Abstract In 2020–2021, a “mysterious illness” struck Senegalese fishermen, causing severe acute dermatitis in over one thousand individuals following exposure through drift-net fishing activity. Here, by performing deep analysis of the environmental samples we reveal the presence of the marine dinoflagellate Vulcanodinium rugosum and its associated cyclic imine toxins. Specifically, we show that the toxin PortimineA, strongly enriched in environmental samples, impedes ribosome function in human keratinocytes, which subsequently activates the stress kinases ZAKα and P38 and promotes the nucleation of the human NLRP1 inflammasome, leading to the release of IL-1β/IL-18 pro-inflammatory cytokines and cell death. Furthermore, cell-based models highlight that naturally occurring mutations in the P38-targeted sites of human NLRP1 are unable to respond to PortimineA exposure. Finally, the development and use of human organotypic skins and zebrafish models of PortimineA exposure demonstrate that the ZAKα-NLRP1 axis drives skin necrosis and inflammation. Our results exemplify the threats to human health caused by emerging environmental toxins and identify ZAKα and NRLP1 as important pharmacological targets to mitigate PortimineA toxicity.

In vitro 3D full-thickness reconstituted human skin has high physiological relevance due to the presence of differentiation features often lacking in 2D cell cultures. Here, we present a protocol to reconstruct a 3D skin model using human fibroblasts, keratinocytes, and rat-tail collagen I. We describe steps for cell expansion, the casting of cellular and acellular layers, seeding keratinocytes, the air lifting of culture, and incubation. We also demonstrate the use of chitosan to prevent tissue contraction in 12-well inserts. For complete details on the use and execution of this protocol, please refer to Robinson et al.1,2.

Background Post-menopausal women undergo significant dermatological changes, including thinning skin and reduced sebaceous gland activity, alongside increased incidence of dermatological diseases and hair loss. These changes reshape the skin’s ecological niche, influencing the skin mycobiome composition and behavior. Malassezia , a lipid-dependent human pathobiont and dominant fungal resident of skin, has been implicated in several dermatological disorders. We hypothesize that shifts in Malassezia populations contribute to post-menopausal skin disorders through altered host–microbe interactions. Results Shotgun metagenomics of facial and scalp skin from 345 Asian women were stratified by menopausal stage (pre- [N=171], peri- [N=36], and post-menopausal [N=138]) and revealed the presence of seven out of the seventeen recognized Malassezia species: M. globosa , M. restricta , M. arunalokei , M. furfur , M. dermatis , M. japonica , and M. sympodialis . Detection frequencies of several species varied markedly across menopausal groups. Notably, M. globosa was detected 20% more frequently on the scalp of post- versus pre-menopausal women. Reduced sebum concentration on post-menopausal women’s skin correlated with increased M. globosa abundance. In vitro co-culture of keratinocytes with Malassezia spp. showed cells tolerated fungal loads up to 104.5 CFU/cm², but severe cytotoxicity was observed at ≥105.5 CFU/cm². M. globosa elicited the highest cytotoxicity towards keratinocytes. All Malassezia spp. tested invaded keratinocytes and triggered strong pro-inflammatory responses. Notably, IL-1α, IL-1β, IL-6, IL-8, IL-21, TNF-α, GM-CSF, G-CSF, and MMP1 were significantly overproduced. Transcriptomics of keratinocytes exposed to toxic fungal loads revealed a gene expression profile characteristic of hyperproliferative and undifferentiated cells, alongside elevated expression of NLRP3 , a key inflammasome sensor involved in pyroptosis. Conclusions Menopause is associated with distinct shifts in Malassezia spp. prevalence and abundance. Reduced skin lipids and thickness may increase fungal burden relative to host cells, promoting inflammation and barrier dysfunction. Malassezia ’s ability to invade keratinocytes suggests a mechanism for immune evasion and induction of chronic inflammation. Furthermore, keratinocytes exposed to high fungal loads exhibited a transcriptomic profile indicative of hyperproliferation and impaired differentiation, resembling patterns observed in psoriasis, seborrheic dermatitis, and other inflammatory skin conditions. Our co-culture model provides mechanistic insight into Malassezia -driven skin inflammation and offers a platform to develop targeted therapies for post-menopausal skin disorders. ### Competing Interest Statement The authors have declared no competing interest.

Metatranscriptomics methods for the skin are hampered by low microbial biomass, contamination with host cells and low RNA stability. In this study, we developed a robust, clinically tractable skin metatranscriptomics workflow that provides high technical reproducibility of profiles, uniform coverage across gene bodies and strong enrichment of microbial mRNAs. Paired application of this protocol with metagenomics to five skin sites in a cohort of 27 healthy adults identifies a notable divergence between transcriptomic and genomic abundances. Specifically, Staphylococcus species and the fungi Malassezia had an outsized contribution to metatranscriptomes at most sites, despite their modest representation in metagenomes. Species-level analysis shows signatures of microbial adaptation to their niches. Gene-level analysis identifies diverse antimicrobial genes transcribed by skin commensals in situ, including several uncharacterized bacteriocins. Correlation of microbial gene expression with organismal abundances uncovers more than 20 genes that putatively mediate interactions between microbes. This work highlights how skin metatranscriptomics identifies active species and microbial functions in situ. Skin metagenomic and metatranscriptomic analysis shows divergence between microbial abundance and activity.

Solar UVB light causes damage to the outermost layer of skin. This insult induces rapid local responses, such as dermal inflammation, keratinocyte cell death, and epidermal thickening, all of which have traditionally been associated with DNA damage response signaling. Another stress response that is activated by UVB light is the ribotoxic stress response (RSR), which depends on the ribosome-associated mitogen-activated protein 3 kinases (MAP3K) ZAKα and culminates in p38 and JNK activation. Using ZAK knockout mice, we here show that it is the RSR that is responsible for the early manifestation of UVB-induced skin inflammation and keratinocyte death and subsequent proliferation in vivo. We also show that the RSR controls both p38-mediated pyroptotic and JNK-mediated apoptotic programmed cell death of human keratinocytes in vitro. In sum, our work highlights that skin cells rely on a cytoplasmic and ribosomal stress signal rather than a nuclear and DNA-templated signal for rapid inflammatory responses to UV exposure.

The skin microbiome is an extensive community of bacteria, fungi, mites, viruses and archaea colonizing the skin. Fluctuations in the composition of the skin microbiome have been observed in atopic dermatitis (AD) and food allergy (FA), particularly in early life, established disease, and associated with therapeutics. However, AD is a multifactorial disease characterized by skin barrier aberrations modulated by genetics, immunology, and environmental influences, thus the skin microbiome is not the sole feature of this disease. Future research should focus on mechanistic understanding of how early-life skin microbial shifts may influence AD and FA onset, to guide potential early intervention strategies or as microbial biomarkers to identify high-risk infants who may benefit from possible microbiome-based biotherapeutic strategies. Harnessing skin microbes as AD biotherapeutics is an emerging field, but more work is needed to investigate whether this approach can lead to sustained clinical responses.

Strategies for prevention of atopic dermatitis (AD) are likely most effective with the help of skin biomarkers to identify high-risk infants in the pre-disease stage. This study aimed to identify early skin microbiome and metabolome signature predictive of infantile AD at 6 and 18 months of life.

Palmoplantar pustulosis is a chronic, recurrent skin disorder characterized by sterile pustules and erythematous scaling on the palms and soles. In our study, dupilumab treatment showed efficacy and safety in PPP patients, potentially resulting from PPP patients' serum Th2 signatures and the similarities between PPP and atopic dermatitis based on serum Olink proteomics.image

It is clear that significant breakthroughs have been achieved in the field of artificial intelligence (AI) in dermatology. This year, 2023, has already witnessed notable milestones, including the introduction of AI and Image Analysis as a distinct abstract category for posters and oral presentations at a major international investigative dermatology conference. In addition, the inaugural AI in Dermatology session was held as a Satellite Symposium at the International Societies for Investigative Dermatology (ISID).

Loss-of-function (LoF) variants in the FLG gene are causative for ichthyosis vulgaris (IV) and the major genetic risk factor for atopic dermatitis (AD) (Barker et al, 2007; Weidinger et al, 2006). Owing to its extremely repetitive nature and sequence similarity of intragenic repeats, the FLG gene is technically challenging to genetically analyze and therefore determine the contribution of LoF variants to disease status.

Genital Herpes Simplex Virus-2 (HSV-2) infection is a highly prevalent and chronic sexually transmitted disease without any cures or FDA-approved vaccines. Symptomatic recurrences occur episodically to trigger genital lesions, compromising the barrier function of the vaginal epithelium and perivaginal skin and tripling the risk of secondary human immunodeficiency virus (HIV)-1 acquisition. Understanding how the immune system interacts with HSV-2 would thus enable us to develop better therapeutics against novel inflammatory and pathological targets. We used a female mouse infection model to define immunopathogenic pathways and revealed that sustained type I interferon (IFN) signalling is a driver of pathogenic neutrophil responses, identifying IL-18 as a novel component of ulcerative disease during genital HSV-2 infection. We further show that IL-18 drives natural killer (NK) cells to produce the serine protease granzyme B. Similar to autoimmune blistering skin diseases, accumulation of extracellular granzyme B in the vagina coincided with epithelial ulceration. Remarkably, genetic loss of granzyme B or therapeutic inhibition by a specific protease inhibitor was sufficient to reduce genital disease and restore epithelial integrity without altering viral control. Moreover, both IL-18 and granzyme B were markedly elevated in human herpetic ulcers compared to non-herpetic ulcers, suggesting these host mechanisms may be engaged in HSV-infected patients. Collectively, our study reveals that IL-18 induced by sustained type-I IFN signalling in neutrophils drive NK cells to promote granzyme B accumulation in the vagina, which in turn drives the destruction of vaginal epithelium during HSV-2 infection. Granzyme B is identified as a novel therapeutic target that can augment the treatment of genital herpes by restoring the barrier function of the epithelium. Our study also calls into question the immunopathogenic potential of these different players in the destruction of the epithelial and skin barrier during other disease conditions.

Immune dysregulation and a defective epidermal barrier drives the pathogenesis of atopic dermatitis (AD). The IL-1 signaling network plays a critical role in innate immunity, affecting skin barrier function and impacting on the pathogenesis of AD. We identified the upregulation of IL-1R2, an understudied IL-1 signaling pathway decoy receptor, in basal and differentiating keratinocytes with in vitro 3D skin models mimicking AD. IL-1R2 has previously been show to be upregulated in immune cells in AD patients however the relevance and function in the epidermis has not been reported before. To further investigate IL-1R2 in epidermal cells we stimulated skin explants with AD related cytokines (IL-4, IL-13, IL17A, IL-22 and TNF). qPCR and RNAScope indicated that gene expression of IL-1R2 increased when stimulated with IL-4. RNAScope of IL-4 stimulated explants also showed that IL-1R2 expression was specifically increased in the basal and differentiating keratinocytes within the stratum granulosum compared to control explants. Combined RNAScope-Immuno Histochemistry also showed an upregulation of IL-1R2 in epidermal Langerhans cells, indicating additional cellular contribution to IL-1R2 decoy activity within the epidermis. Additionally, we identified that IL-1R2 expression was increased in AD patients' lesional epidermis compared to non-lesional epidermis and also observed increased expression in keratinocytes from publicly available scRNAseq AD datasets . These findings in patient samples corroborated our results from the IL-4 treated 3D keratinocyte and explant skin models. These results show that IL-4 stimulates IL-1R2 expression in epidermal keratinocytes that could account for the upregulation observed in patient lesional skin. The upregulation of IL-1R2 in AD would reduce IL-1 family signaling as a pathomechanism to control inflammation in lesional AD skin.