Keratinocyte-derived defensins activate neutrophil-specific receptors Mrgpra2a/b to prevent skin dysbiosis and bacterial infection.

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that the epithelial-cell-derived antimicrobial peptides defensins activated orphan G-protein-coupled receptors (GPCRs) Mrgpra2a/b on neutrophils. This signaling axis was required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster in keratinocytes or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Defensins and Mrgpra2 were critical for combating S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggered neutrophil release of IL-1ß and CXCL2 which are vital for proper amplification and propagation of the antibacterial immune response. This study demonstrated the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.

Staphylococcus aureus proteases trigger eosinophil-mediated skin inflammation.

Staphylococcus aureus skin colonization and eosinophil infiltration are associated with many inflammatory skin disorders, including atopic dermatitis, bullous pemphigoid, Netherton's syndrome, and prurigo nodularis. However, whether there is a relationship between S. aureus and eosinophils and how this interaction influences skin inflammation is largely undefined. We show in a preclinical mouse model that S. aureus epicutaneous exposure induced eosinophil-recruiting chemokines and eosinophil infiltration into the skin. Remarkably, we found that eosinophils had a comparable contribution to the skin inflammation as T cells, in a manner dependent on eosinophil-derived IL-17A and IL-17F production. Importantly, IL-36R signaling induced CCL7-mediated eosinophil recruitment to the inflamed skin. Last, S. aureus proteases induced IL-36α expression in keratinocytes, which promoted infiltration of IL-17-producing eosinophils. Collectively, we uncovered a mechanism for S. aureus proteases to trigger eosinophil-mediated skin inflammation, which has implications in the pathogenesis of inflammatory skin diseases.

Clinical and molecular change induced by repeated low-dose visible light exposure in both light-skinned and dark-skinned individuals.

BACKGROUND: Visible light (VL) is known to induce pigmentation in dark-skinned individuals and immediate erythema in light-skinned individuals. However, the effects of accumulated low-dose VL exposure across skin types are not well established. METHODS: Thirty-one healthy subjects with light (Fitzpatrick skin types [FST] I-II, n = 13) and dark (FST V-VI, n = 18) skin types were enrolled. Subjects' buttocks were exposed daily to VL, wavelength 400-700 nm, with a dose of 120 J/cm2 at 50 mW/cm2 , for four consecutive days. Microarray using Affymetrix GeneChip (49,395 genes) was performed followed by qRT-PCR on skin samples. RESULTS: Repeated low-dose VL irradiation induced immediate pigment darkening and delayed tanning in dark-skinned individuals while no discernable pigmentation and erythema were observed in light-skinned individuals. Top ten upregulated genes by repeated VL exposure in microarray included melanogenic genes such as tyrosinase (TYR), tyrosinase-related protein-1 (TYRP1), dopachrome tautomerase (DCT), premelanosome protein (PMEL), melan-A (MLANA), and solute carrier family 24, member 5 (SLC24A5) and genes involved in inflammation/matrix remodeling/cell signaling including chemokine (C-C motif) ligand 18 (CCL18), BCL2-related protein A1 (BCL2A1), and cartilage oligomeric matrix protein (COMP). In qRT-PCR CCL18 was upregulated in light skin with a greater extent (mean fold change ± SD; 4.03 ± 3.28, p = .04) than in dark-skinned individuals (1.91 ± 1.32, p = .07) while TYR was not significantly upregulated in both skin types. CONCLUSION: This study highlights the genes upregulated by cumulative VL exposure involved in pigmentation, immune response, oxidation/reduction, and matrix remodeling across skin types providing relevant information on daily solar exposure.

Through the lens of hair follicle neogenesis, a new focus on mechanisms of skin regeneration after wounding.

Wound-induced hair follicle neogenesis (WIHN) is a phenomenon that occurs in adult mammalian skin, where fully functional hair follicles are regenerated in the center of large full-thickness excisional wounds. Although originally discovered over 50 years ago in mice and rabbits, within the last decade it has received renewed interest, as the molecular mechanism has begun to be defined. This de novo regeneration of hair follicles largely recapitulates embryonic hair development, requiring canonical Wnt signaling in the epidermis, however, important differences between the two are beginning to come to light. TLR3 mediated double stranded RNA sensing is critical for the regeneration, activating retinoic acid signaling following wounding. Inflammatory cells, including Fgf9-producing γ-δ T cells and macrophages, are also emerging as important mediators of WIHN. Additionally, while dispensable in embryonic hair follicle development, Shh signaling plays a major role in WIHN and may be able to redirect cells fated to scarring wounds into a regenerative phenotype. The cellular basis of WIHN is also becoming clearer, with increasing evidence suggesting an incredible level of cellular plasticity. Multiple stem cell populations, along with lineage switching of differentiated cells all contribute towards the regeneration present in WIHN. Further study of WIHN will uncover key steps in mammalian development and regeneration, potentially leading to new clinical treatments for hair-related disorders or fibrotic scarring.

Gene expression profiling suggests severe, extensive central centrifugal cicatricial alopecia may be both clinically and biologically distinct from limited disease subtypes.

The natural history of central centrifugal cicatricial alopecia (CCCA) is widely variable. Some patients experience rapid progression to extensive, end-stage disease while others never approach extensive involvement over decades, suggesting heterogeneity in CCCA disease phenotype. To better characterize clinically severe disease in CCCA, tissue samples were obtained from the peripheral, hair-bearing lesional scalp of women with clinically focal, limited and extensive CCCA disease involvement. A microarray analysis was conducted to identify differential expression of genes previously identified to be preferentially expressed in the lesional scalp vs. non-lesional scalp of CCCA patients. Clinically extensive, severe CCCA was characterized by increased expression of MMP9, SFRP4 and MSR1 when directly compared with focal and limited disease. These biomarkers correspond to dysregulated pathways of fibrosis, Wnt signalling and macrophage-mediated inflammatory processes respectively. These findings hold significance for both possible targets for future study of prognostic markers of disease severity and new potential therapeutic targets. In summary, this study suggests clinically extensive, severe CCCA may have a differential gene expression pattern in the lesional scalp of affected patients, in addition to its clinical distinction.

Neutrophil extracellular traps impair regeneration.

Fibrosis is a major health burden across diseases and organs. To remedy this, we study wound-induced hair follicle neogenesis (WIHN) as a model of non-fibrotic healing that recapitulates embryogenesis for de novo hair follicle morphogenesis after wounding. We previously demonstrated that TLR3 promotes WIHN through binding wound-associated dsRNA, the source of which is still unclear. Here, we find that multiple distinct contexts of high WIHN all show a strong neutrophil signature. Given the correlation between neutrophil infiltration and endogenous dsRNA release, we hypothesized that neutrophil extracellular traps (NETs) likely release nuclear spliceosomal U1 dsRNA and modulate WIHN. However, rather than enhance regeneration, we find mature neutrophils inhibit WIHN such that mice with mature neutrophil depletion exhibit higher WIHN. Similarly, Pad4 null mice, which are defective in NET production, show augmented WIHN. Finally, using single-cell RNA sequencing, we identify a dramatic increase in mature and activated neutrophils in the wound beds of low regenerating Tlr3-/- mice. Taken together, these results demonstrate that although mature neutrophils are stimulated by a common pro-regenerative cue, their presence and NETs hinder regeneration.

Diverse cellular players orchestrate regeneration after wounding.

Fibrosis is one of the largest sources of human morbidity. The skin is a complex organ where interplay between diverse cell types and signalling pathways is essential both in homeostasis and wound repair, which can result in fibrosis or regeneration. This makes skin a useful model to study fibrosis and regeneration. While fibrosis often occurs postinjury, both clinical and laboratory observations suggest skin regeneration, complete with reconstituted cell diversity and de novo hair follicles, is possible. Extensive research performed in pursuit of skin regeneration has elucidated the key players, both cellular and molecular. Interestingly, some cells known for their homeostatic function are not implicated in regeneration or wound-induced hair neogenesis (WIHN), suggesting regeneration harnesses separate functional pathways from embryogenesis or other non-homeostatic mechanisms. For example, classic bulge cells, noted for their role in normally cycling hair follicles, do not finally contribute to long-lived cells in the regenerated tissue. During healing, multiple populations of cells, among them specific epithelial lineages, mesenchymal cells, and immune cells promote regenerative outcomes in the wounded skin. Ultimately, targeting specific populations of cells will be essential in manipulating a postwound environment to favour regeneration in lieu of fibrosis.

Injury, dysbiosis, and filaggrin deficiency drive skin inflammation through keratinocyte IL-1α release.

BACKGROUND: Atopic dermatitis (AD) is associated with epidermal barrier defects, dysbiosis, and skin injury caused by scratching. In particular, the barrier-defective epidermis in patients with AD with loss-of-function filaggrin mutations has increased IL-1α and IL-1ß levels, but the mechanisms by which IL-1α, IL-1ß, or both are induced and whether they contribute to the aberrant skin inflammation in patients with AD is unknown. OBJECTIVE: We sought to determine the mechanisms through which skin injury, dysbiosis, and increased epidermal IL-1α and IL-1ß levels contribute to development of skin inflammation in a mouse model of injury-induced skin inflammation in filaggrin-deficient mice without the matted mutation (ft/ft mice). METHODS: Skin injury of wild-type, ft/ft, and myeloid differentiation primary response gene-88-deficient ft/ft mice was performed, and ensuing skin inflammation was evaluated by using digital photography, histologic analysis, and flow cytometry. IL-1α and IL-1ß protein expression was measured by means of ELISA and visualized by using immunofluorescence and immunoelectron microscopy. Composition of the skin microbiome was determined by using 16S rDNA sequencing. RESULTS: Skin injury of ft/ft mice induced chronic skin inflammation involving dysbiosis-driven intracellular IL-1α release from keratinocytes. IL-1α was necessary and sufficient for skin inflammation in vivo and secreted from keratinocytes by various stimuli in vitro. Topical antibiotics or cohousing of ft/ft mice with unaffected wild-type mice to alter or intermix skin microbiota, respectively, resolved the skin inflammation and restored keratinocyte intracellular IL-1α localization. CONCLUSIONS: Taken together, skin injury, dysbiosis, and filaggrin deficiency triggered keratinocyte intracellular IL-1α release that was sufficient to drive chronic skin inflammation, which has implications for AD pathogenesis and potential therapeutic targets.

Hypothesis: Wound-induced TLR3 activation stimulates endogenous retinoic acid synthesis and signalling during regeneration.

Although the mechanism is unclear, it has been shown that genetically normal adult mice with a large wound form de novo morphogenesis of hair follicles in wound-induced hair neogenesis (WIHN)(1). We focused on how tissues recognize damage signals and identified that double-stranded RNA (dsRNA)-mediated toll-like receptor 3 (TLR3) activation stimulates WIHN. Here, we propose a hypothesis that TLR3 stimulates retinoic acid synthesis and signalling to allow for regeneration, suggesting that common clinical methods of facial rejuvenation in human subjects through damage (such as lasers or dermabrasion), and the use of topical retinoids reflect the same biologic pathway.