The skin microbiome in pediatric atopic dermatitis and food allergy.

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.

A Malassezia pseudoprotease dominates the secreted hydrolase landscape and is a potential allergen on skin.

Malassezia globosa is abundant and prevalent on sebaceous areas of the human skin. Genome annotation reveals that M. globosa possesses a repertoire of secreted hydrolytic enzymes relevant for lipid and protein metabolism. However, the functional significance of these enzymes is uncertain and presence of these genes in the genome does not always translate to expression at the cutaneous surface. In this study we utilized targeted RNA sequencing from samples isolated directly from the skin to quantify gene expression of M. globosa secreted proteases, lipases, phospholipases and sphingomyelinases. Our findings indicate that the expression of these enzymes is dynamically regulated by the environment in which the fungus resides, as different growth phases of the planktonic culture of M. globosa show distinct expression levels. Furthermore, we observed significant differences in the expression of these enzymes in culture compared to healthy sebaceous skin sites. By examining the in situ gene expression of M. globosa's secreted hydrolases, we identified a predicted aspartyl protease, MGL_3331, which is highly expressed on both healthy and disease-affected dermatological sites. However, molecular modeling and biochemical studies revealed that this protein has a non-canonical active site motif and lacks measurable proteolytic activity. This pseudoprotease MGL_3331 elicits a heightened IgE-reactivity in blood plasma isolated from patients with atopic dermatitis compared to healthy individuals and invokes a pro-inflammatory response in peripheral blood mononuclear cells. Overall, our study highlights the importance of studying fungal proteins expressed in physiologically relevant environments and underscores the notion that secreted inactive enzymes may have important functions in influencing host immunity.

Diphtheria toxin activates ribotoxic stress and NLRP1 inflammasome-driven pyroptosis.

The ZAKα-driven ribotoxic stress response (RSR) is activated by ribosome stalling and/or collisions. Recent work demonstrates that RSR also plays a role in innate immunity by activating the human NLRP1 inflammasome. Here, we report that ZAKα and NLRP1 sense bacterial exotoxins that target ribosome elongation factors. One such toxin, diphtheria toxin (DT), the causative agent for human diphtheria, triggers RSR-dependent inflammasome activation in primary human keratinocytes. This process requires iron-mediated DT production in the bacteria, as well as diphthamide synthesis and ZAKα/p38-driven NLRP1 phosphorylation in host cells. NLRP1 deletion abrogates IL-1ß and IL-18 secretion by DT-intoxicated keratinocytes, while ZAKα deletion or inhibition additionally limits both pyroptotic and inflammasome-independent non-pyroptotic cell death. Consequently, pharmacologic inhibition of ZAKα is more effective than caspase-1 inhibition at protecting the epidermal barrier in a 3D skin model of cutaneous diphtheria. In summary, these findings implicate ZAKα-driven RSR and the NLRP1 inflammasome in antibacterial immunity and might explain certain aspects of diphtheria pathogenesis.

Early initiation of short-term emollient use for the prevention of atopic dermatitis in high-risk infants-The STOP-AD randomised controlled trial.

BACKGROUND: Protecting the skin barrier in early infancy may prevent atopic dermatitis (AD). We investigated if daily emollient use from birth to 2 months reduced AD incidence in high-risk infants at 12 months. METHODS: This was a single-center, two-armed, investigator-blinded, randomized controlled clinical trial (NCT03871998). Term infants identified as high risk for AD (parental history of AD, asthma or allergic rhinitis) were recruited within 4 days of birth and randomised 1:1 to either twice-daily emollient application for the first 8 weeks of life (intervention group), using an emollient specifically formulated for very dry, AD-prone skin, or to standard routine skin care (control group). The primary outcome was cumulative AD incidence at 12 months. AD <6 months was diagnosed based on clinical presence of AD. The UK Working Party Diagnostic Criteria were applied when diagnosing AD between 6 and 12 months. RESULTS: Three hundred twenty-one were randomised (161 intervention and 160 control), with 61 withdrawals (41 intervention, 20 control). The cumulative incidence of AD at 12 months was 32.8% in the intervention group vs. 46.4% in the control group, p = 0.036 [Relative risk (95%CI): 0.707 (0.516, 0.965)]. One infant in the intervention group was withdrawn from the study following development of a rash that had a potential relationship with the emollient. There was no significant difference in the incidence of skin infections between the intervention and control groups during the intervention period (5.0% vs. 5.7%, p > 0.05). CONCLUSIONS: This study has demonstrated that early initiation of daily specialized emollient use until 2 months reduces the incidence of AD in the first year of life in high-risk infants.

Expression of Staphylococcus aureus Virulence Factors in Atopic Dermatitis.

Atopic dermatitis (AD) is a skin inflammatory disease in which the opportunistic pathogen Staphylococcus aureus is prevalent and abundant. S. aureus harbors several secreted virulence factors that have well-studied functions in infection models, but it is unclear whether these extracellular microbial factors are relevant in the context of AD. To address this question, we designed a culture-independent method to detect and quantify S. aureus virulence factors expressed at the skin sites. We utilized RNase-H‒dependent multiplex PCR for preamplification of reverse-transcribed RNA extracted from tape strips of patients with AD sampled at skin sites with differing severity and assessed the expression of a panel of S. aureus virulence factors using qPCR. We observed an increase in viable S. aureus abundance on sites with increased severity of disease, and many virulence factors were expressed at the AD skin sites. Surprisingly, we did not observe any significant upregulation of the virulence factors at the lesional sites compared with those at the nonlesional control. Overall, we utilized a robust assay to directly detect and quantify viable S. aureus and its associated virulence factors at the site of AD skin lesions. This method can be extended to study the expression of skin microbial genes at the sites of various dermatological conditions.

Distinct skin microbiome community structures in congenital ichthyosis.

BACKGROUND: The ichthyoses are rare genetic keratinizing disorders that share the characteristics of an impaired epidermal barrier and increased risk of microbial infections. Although ichthyotic diseases share a T helper (Th) 17 cell immune signature, including increased expression of antimicrobial peptides, the skin microbiota of ichthyoses is virtually unexplored. OBJECTIVES: To analyse the metagenome profile of skin microbiome for major congenital ichthyosis subtypes. METHODS: Body site-matched skin surface samples were collected from the scalp, upper arm and upper buttocks of 16 healthy control participants and 22 adult patients with congenital forms of ichthyosis for whole metagenomics sequencing analysis. RESULTS: Taxonomic profiling showed significant shifts in bacteria and fungi abundance and sporadic viral increases across ichthyosis subtypes. Cutibacterium acnes and Malassezia were significantly reduced across body sites, consistent with skin barrier disruption and depletion of lipids. Microbial richness was reduced, with specific increases in Staphylococcus and Corynebacterium genera, as well as shifts in fungal species, including Malassezia. Malassezia globosa was reduced at all body sites, whereas M. sympodialis was reduced in the ichthyotic upper arm and upper buttocks. Malassezia slooffiae, by contrast, was strikingly increased at all body sites in participants with congenital ichthyosiform erythroderma (CIE) and lamellar ichthyosis (LI). A previously undescribed Trichophyton species was also detected as sporadically colonizing the skin of patients with CIE, LI and epidermolytic ichthyosis subtypes. CONCLUSIONS: The ichthyosis skin microbiome is significantly altered from healthy skin with specific changes predominating among ichthyosis subtypes. Skewing towards the Th17 pathway may represent a response to the altered microbial colonization in ichthyosis. What is already known about this topic? The skin microbiome of congenital ichthyoses is largely unexplored. Microbes play an important role in pathogenesis, as infections are common. The relative abundances of staphylococci and corynebacteria is increased in the cutaneous microbiome of patients with Netherton syndrome, but extension of these abundances to all congenital ichthyoses is unexplored. What does this study add? A common skin microbiome signature was observed across congenital ichthyoses. Distinct microbiome features were associated with ichthyosis subtypes. Changes in microbiome may contribute to T helper 17 cell immune polarization. What is the translational message? These data provide the basis for comparison of the microbiome with lipidomic and transcriptomic alterations in these forms of ichthyosis and consideration of correcting the dysbiosis as a therapeutic intervention.

Shared signatures and divergence in skin microbiomes of children with atopic dermatitis and their caregivers.

BACKGROUND: Atopic dermatitis (AD) is a common chronic skin condition in children (15-20%) that can significantly impair their quality of life. As a result of its relapsing nature and enrichment of Staphylococcus aureus during flares, clinical management can include eradicating S aureus from the skin of children; however, this does not extend to their healthy caregivers, who are potential reservoirs. OBJECTIVE: Our aim was to understand skin microbiome sharing and microbial features in children with AD and their healthy adult caregivers. METHODS: We utilized whole-metagenome profiling at 4 body sites (volar forearm, antecubital fossae, cheeks, and lesions) in combination with sequencing of S aureus isolates to characterize a cohort of children with AD and their healthy caregivers (n = 30 families) compared to matched pairs from control households (n = 30 families). RESULTS: Metagenomic analysis revealed distinct microbiome configurations in the nonlesional skin of AD children and their healthy caregivers versus controls, which were sufficient to accurately predict case-control status (area under the receiver operating characteristic curve > 0.8). These differences were accompanied by significant microbiome similarity between children and their caregivers, indicating that microbiome sharing may play a role in recurrent disease flares. Whole-genome comparisons with high-quality S aureus isolate genomes (n = 55) confirmed significant strain sharing between AD children and their caregivers and AD-specific enrichment of strains expressing enterotoxins Q and K/K2. CONCLUSION: Our results highlight the distinctive skin microbiome features of healthy caregivers for children with AD and support their inclusion in strategies for the treatment of recurrent pediatric AD.

No Association of filaggrin copy number variation and atopic dermatitis risk in White and Black Americans.

Atopic dermatitis (AD) is a chronic, inflammatory skin condition with a multifactorial pathophysiology. The filaggrin gene (FLG) has particularly been implicated given loss of function (LoF) mutations in this gene lead to skin barrier dysfunction and such mutations can increase a patient's likelihood of developing AD. FLG has intragenic copy number variation (CNV), which impacts the total amount of filaggrin produced. Previous research reported a dose-dependent effect such that as amount of FLG increases, risk of AD decreases. To gain a better understanding, we evaluated FLG CNV in a large case-control study of Whites and Blacks with and without AD. The goal of our study was to determine whether FLG CNV has a dose-dependent effect on the risk of developing AD and to determine whether FLG CNV varies by race. The frequencies and odds ratios comparing a given CNV by race or race within those with AD did not significantly vary. It had been thought that FLG CNV might vary by race and represent an important association with AD in Black AD subjects. However, our work suggests that while there are racial differences with respect to CNV, these differences do not appear to explain AD risk.

Skin microbiome of atopic dermatitis.

The skin microbiome is a key component of pathogenesis in atopic dermatitis (AD). The skin of AD patients is characterized by microbial dysbiosis, with a reduction of microbial diversity and overrepresentation of pathogenic Staphylococcus aureus (S. aureus). Recent exciting studies have elucidated an importance of establishing an appropriate immune response to microbes in early life and uncovered the new mechanisms of microbial community dynamics in modulating our skin microbiome. Several microbes are associated with AD pathogenesis, with proposed pathogenic effects from S. aureus and Malassezia. The complex relationships between microbes within the skin microbiome consortia includes various species, such as Staphylococcal, Roseomonas and Cutibacterium strains, that can inhibit S. aureus and are potential probiotics for AD skin. Numerous microbes are now also reported to modulate host response via communication with keratinocytes, specialized immune cells and adipocytes to improve skin health and barrier function. This increased understanding of skin microbiota bioactives has led to new biotherapeutic approaches that target the skin surface microenvironment for AD treatment.

Impact of low-dose calcipotriol ointment on wound healing, pruritus and pain in patients with dystrophic epidermolysis bullosa: A randomized, double-blind, placebo-controlled trial.

BACKGROUND: Wound management is a critical factor when treating patients with the inherited skin fragility disease dystrophic epidermolysis bullosa (DEB). Due to genetic defects in structural proteins, skin and mucous epithelia are prone to blistering and chronic wounding upon minor trauma. Furthermore, these wounds are commonly associated with excessive pruritus and predispose to the development of life-threatening squamous cell carcinomas, underscoring the unmet need for new therapeutic options to improve wound healing in this patient cohort. Vitamin D3 is acknowledged to play an important role in wound healing by modulating different cellular processes that impact epidermal homeostasis and immune responses. In this study, we evaluate the safety and efficacy of low-dose calcipotriol, a vitamin D3 analogue, in promoting wound healing and reducing itch and pain in patients with DEB. METHODS: Eligible DEB patients, aged ≥ 6 years and with a known mutation in the COL7A1 gene, were recruited to a placebo-controlled, randomized, double blind, cross-over phase II monocentric clinical trial. Patients were required to have at least two wounds with a minimum size of 6 cm2 per wound. The primary objective was to evaluate efficacy of daily topical application of a 0.05 µg/g calcipotriol ointment in reducing wound size within a 4-week treatment regimen. Secondary objectives were to assess safety, as well as the impact of treatment on pruritus, pain, and bacterial wound colonization in these patients. RESULTS: Six patients completed the clinical trial and were included into the final analysis. Topical low-dose calcipotriol treatment led to a significant reduction in wound area at day 14 compared to placebo (88.4% vs. 65.5%, P < 0.05). Patients also reported a significant reduction of pruritus with calcipotriol ointment compared to placebo over the entire course of the treatment as shown by itch scores of 3.16 vs 4.83 (P < 0.05) and 1.83 vs 5.52 (P < 0.0001) at days 14 and 28, respectively. Treatment with low-dose calcipotriol did not affect serum calcium levels and improved the species richness of the wound microbiome, albeit with no statistical significance. CONCLUSIONS: Our results show that topical treatment with low-dose calcipotriol can accelerate wound closure and significantly reduces itch, and can be considered a safe and readily-available option to improve local wound care in DEB patients. Trial Registration EudraCT: 2016-001,967-35. Registered 28 June 2016, https://www.clinicaltrialsregister.eu/ctr-search/trial/2016-001967-35/AT.

A cell-based drug discovery assay identifies inhibition of cell stress responses as a new approach to treatment of epidermolysis bullosa simplex.

In the skin fragility disorder epidermolysis bullosa simplex (EBS), mutations in keratin 14 (K14, also known as KRT14) or keratin 5 (K5, also known as KRT5) lead to keratinocyte rupture and skin blistering. Severe forms of EBS are associated with cytoplasmic protein aggregates, with elevated kinase activation of ERK1 and ERK2 (ERK1/2; also known as MAPK3 and MAPK1, respectively), suggesting intrinsic stress caused by misfolded keratin protein. Human keratinocyte EBS reporter cells stably expressing GFP-tagged EBS-mimetic mutant K14 were used to optimize a semi-automated system to quantify the effects of test compounds on keratin aggregates. Screening of a protein kinase inhibitor library identified several candidates that reduced aggregates and impacted on epidermal growth factor receptor (EGFR) signalling. EGF ligand exposure induced keratin aggregates in EBS reporter keratinocytes, which was reversible by EGFR inhibition. EBS keratinocytes treated with a known EGFR inhibitor, afatinib, were driven out of activation and towards quiescence with minimal cell death. Aggregate reduction was accompanied by denser keratin filament networks with enhanced intercellular cohesion and resilience, which when extrapolated to a whole tissue context would predict reduced epidermal fragility in EBS patients. This assay system provides a powerful tool for discovery and development of new pathway intervention therapeutic avenues for EBS.

The Polyamine Regulator AMD1 Upregulates Spermine Levels to Drive Epidermal Differentiation.

Maintaining tissue homeostasis depends on a balance between cell proliferation, differentiation, and apoptosis. Within the epidermis, the levels of the polyamines putrescine, spermidine, and spermine are altered in many different skin conditions, yet their role in epidermal tissue homeostasis is poorly understood. We identify the polyamine regulator, Adenosylmethionine decarboxylase 1 (AMD1), as a crucial regulator of keratinocyte (KC) differentiation. AMD1 protein is upregulated on differentiation and is highly expressed in the suprabasal layers of the human epidermis. During KC differentiation, elevated AMD1 promotes decreased putrescine and increased spermine levels. Knockdown or inhibition of AMD1 results in reduced spermine levels and inhibition of KC differentiation. Supplementing AMD1-knockdown KCs with exogenous spermidine or spermine rescued aberrant differentiation. We show that the polyamine shift is critical for the regulation of key transcription factors and signaling proteins that drive KC differentiation, including KLF4 and ZNF750. These findings show that human KCs use controlled changes in polyamine levels to modulate gene expression to drive cellular behavior changes. Modulation of polyamine levels during epidermal differentiation could impact skin barrier formation or can be used in the treatment of hyperproliferative skin disorders.

High-fat diet induces a predisposition to follicular hyperkeratosis and neutrophilic folliculitis in mice.

BACKGROUND: Neutrophilic folliculitis is an inflammatory condition of hair follicles. In some neutrophilic folliculitis, such as in patients with acne and hidradenitis suppurativa, follicular hyperkeratosis is also observed. Neutrophilic folliculitis is often induced and/or exacerbated by a high-fat diet (HFD). However, the molecular mechanisms by which an HFD affects neutrophilic folliculitis are not fully understood. OBJECTIVE: Our aim was to elucidate how an HFD promotes the development of neutrophilic folliculitis. METHODS: Mice were fed an HFD, and their skin was subjected to histologic, RNA sequencing, and imaging mass spectrometry analyses. To examine the effect of an HFD on neutrophil accumulation around the hair follicles, phorbol 12-myristate 13-acetate (PMA) was used as an irritant to the skin. RESULTS: Histologic analysis revealed follicular hyperkeratosis in the skin of HFD-fed mice. RNA sequencing analysis showed that genes related to keratinization, especially in upper hair follicular keratinocytes, were significantly upregulated in HFD-fed mice. Application of PMA to the skin induced neutrophilic folliculitis in HFD-fed mice but not in mice fed a normal diet. Accumulation of neutrophils in the skin and around hair follicles was dependent on CXCR2 signaling, and CXCL1 (a CXCR2 ligand) was produced mainly by hair follicular keratinocytes. Imaging mass spectrometry analysis revealed an increase in fatty acids in the skin of HFD-fed mice. Application of these fatty acids to the skin induced follicular hyperkeratosis and caused PMA-induced neutrophilic folliculitis even in mice fed a normal diet. CONCLUSION: An HFD can facilitate the development of neutrophilic folliculitis with the induction of hyperkeratosis of hair follicles and increased neutrophil infiltration around the hair follicles via CXCR2 signaling.

Investigating PEGDA and GelMA Microgel Models for Sustained 3D Heterotypic Dermal Papilla and Keratinocyte Co-Cultures.

Hair follicle morphogenesis is heavily dependent on reciprocal, sequential, and epithelial-mesenchymal interaction (EMI) between epidermal stem cells and the specialized cells of the underlying mesenchyme, which aggregate to form the dermal condensate (DC) and will later become the dermal papilla (DP). Similar models were developed with a co-culture of keratinocytes and DP cells. Previous studies have demonstrated that co-culture with keratinocytes maintains the in vivo characteristics of the DP. However, it is often challenging to develop three-dimensional (3D) DP and keratinocyte co-culture models for long term in vitro studies, due to the poor intercellular adherence between keratinocytes. Keratinocytes exhibit exfoliative behavior, and the integrity of the DP and keratinocyte co-cultured spheroids cannot be maintained over prolonged culture. Short durations of culture are unable to sufficiently allow the differentiation and re-programming of the keratinocytes into hair follicular fate by the DP. In this study, we explored a microgel array approach fabricated with two different hydrogel systems. Using poly (ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), we compare their effects on maintaining the integrity of the cultures and their expression of important genes responsible for hair follicle morphogenesis, namely Wnt10A, Wnt10B, and Shh, over prolonged duration. We discovered that low attachment surfaces such as PEGDA result in the exfoliation of keratinocytes and were not suitable for long-term culture. GelMA, on the hand, was able to sustain the integrity of co-cultures and showed higher expression of the morphogens overtime.

Atopic dermatitis microbiomes stratify into ecologic dermotypes enabling microbial virulence and disease severity.

BACKGROUND: Atopic dermatitis (AD) is a common skin disease affecting up to 20% of the global population, with significant clinical heterogeneity and limited information about molecular subtypes and actionable biomarkers. Although alterations in the skin microbiome have been described in subjects with AD during progression to flare state, the prognostic value of baseline microbiome configurations has not been explored. OBJECTIVE: Our aim was to identify microbial signatures on AD skin that are predictive of disease fate. METHODS: Nonlesional skin of patients with AD and healthy control subjects were sampled at 2 time points separated by at least 4 weeks. Using whole metagenome analysis of skin microbiomes of patients with AD and control subjects (n = 49 and 189 samples), we identified distinct microbiome configurations (dermotypes A and B). Blood was collected for immunophenotyping, and skin surface samples were analyzed for correlations with natural moisturizing factors and antimicrobial peptides. RESULTS: Dermotypes were robust and validated across 2 additional cohorts (63 individuals), with strong enrichment of subjects with AD in dermotype B. Dermotype B was characterized by reduced microbial richness, depletion of Cutibacterium acnes, Dermacoccus and Methylobacterium species, individual-specific outlier abundance of Staphylococcus species (eg, S epidermidis, S capitis, S aureus), and enrichment in metabolic pathways (eg, branched chain amino acids and arginine biosynthesis) and virulence genes (eg, ß-toxin, δ-toxin) that defined a pathogenic ecology. Skin surface and circulating host biomarkers exhibited a distinct microbial-associated signature that was further reflected in more severe itching, frequent flares, and increased disease severity in patients harboring the dermotype B microbiome. CONCLUSION: We report distinct clusters of microbial profiles that delineate the role of microbiome configurations in AD heterogeneity, highlight a mechanism for ongoing inflammation, and provide prognostic utility toward microbiome-based disease stratification.