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-1β 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.

Partially Hydrolysed Whey-Based Infant Formula Improves Skin Barrier Function

Specific partially hydrolysed whey-based infant formulas (pHF-W) have been shown to decrease the risk of atopic dermatitis (AD) in infants. Historically, AD has been associated primarily with milk allergy; however, defective skin barrier function can be a primary cause of AD. We aimed to ascertain whether oral supplementation with pHF-W can improve skin barrier function. The effect of pHF-W was assessed on transepidermal water loss (TEWL) and antibody productions in mice epicutaneously exposed to Aspergillus fumigatus. Human primary keratinocytes were stimulated in vitro, and the expression of genes related to skin barrier function was measured. Supplementation with pHF-W in neonatal mice led to a significant decrease in TEWL and total IgE, but not in allergen-specific antibody levels. The whey hydrolysate was sufficient to decrease both TEWL and total IgE. Aquaporin-3 gene expression, linked with skin hydration, was modulated in the skin of mice and human primary keratinocytes following protein hydrolysate exposure. Skin barrier improvement may be an additional mechanism by which pHF-W may potentially reduce the risk of AD development in infants. Further human studies are warranted to confirm the clinical efficacy of these observations.

Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery

In recent years, lipid nanoparticles (LNPs) have gained considerable attention in numerous research fields ranging from gene therapy to cancer immunotherapy and DNA vaccination. While some RNA-encapsulating LNP formulations passed clinical trials, DNA-loaded LNPs have been only marginally explored so far. To fulfil this gap, herein we investigated the effect of several factors influencing the microfluidic formulation and transfection behavior of DNA-loaded LNPs such as PEGylation, total flow rate (TFR), concentration and particle density at the cell surface. We show that PEGylation and post-synthesis sample concentration facilitated formulation of homogeneous and small size LNPs with high transfection efficiency and minor, if any, cytotoxicity on human Embryonic Kidney293 (HEK-293), spontaneously immortalized human keratinocytes (HaCaT), immortalized keratinocytes (N/TERT) generated from the transduction of human primary keratinocytes, and epidermoid cervical cancer (CaSki) cell lines. On the other side, increasing TFR had a detrimental effect both on the physicochemical properties and transfection properties of LNPs. Lastly, the effect of particle concentration at the cell surface on the transfection efficiency (TE) and cell viability was largely dependent on the cell line, suggesting that its case-by-case optimization would be necessary. Overall, we demonstrate that fine tuning formulation and microfluidic parameters is a vital step for the generation of highly efficient DNA-loaded LNPs.

Interferon-Lambda 1 Inhibits Staphylococcus aureus Colonization in Human Primary Keratinocytes

Atopic dermatitis (AD) is a common inflammatory skin disease. Staphylococcus aureus (S. aureus) colonization in skin lesions occurs in approximately 70% of AD patients. It has been found that IFN-λ1 can inhibit the colonization of S. aureus in normal human nasal mucosa. IFN-λ1 can increase IL-28RA in infected human keratinocytes. In this study, we found that IFN-λ1 can increase mRNA expression of FLG and antimicrobial peptides (AMPs) and inhibit TSLP mRNA expression in infected human keratinocytes. IFN-λ1 can increase intracellular ROS level, decrease STAT1 phosphorylation, and inhibit the colonization of S. aureus in human primary keratinocytes. These effects were attenuated by knocking-down IL-28R and NADPH oxidase inhibitor, suggesting that this function was mediated by JAK-STAT1 signaling pathway. These results suggest that IFN-λ1 might have an inhibitory effect on S. aureus colonization in AD lesions. Our findings might have potential value in the treatment for AD.

Plasma exosomal miR-375-3p regulates mitochondria-dependent keratinocyte apoptosis by targeting XIAP in severe drug-induced skin reactions

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe drug-induced cutaneous reactions characterized by keratinocyte apoptosis. Exosomes are nanometer-sized membranous vesicles in body fluids. They contain functional proteins, mRNAs, and miRNAs, which induce immune dysfunction and influence disease progression. However, their roles and mechanisms in SJS/TEN remain unknown. Our results demonstrate that exosomes isolated from the plasma of patients with SJS/TEN were 30 to 200 nm in diameter and expressed CD9, CD63, CD81, and TSG101 exosome marker proteins. miR-375-3p was markedly up-regulated in 35 patients with SJS/TEN and correlated with clinical severity. Plasma exosomes were internalized by human primary keratinocytes and promoted keratinocyte apoptosis in vitro. Furthermore, miR-375-3p overexpression promoted intrinsic (mitochondria-dependent) apoptosis of human primary keratinocytes via down-regulation of the X-linked inhibitor of apoptosis protein (XIAP), a key apoptosis regulator in primary human keratinocytes. In sum, our study indicates that the circulating exosomal miR-375-3p enters keratinocytes, down-regulates XIAP, and induces keratinocyte apoptosis in patients with SJS/TEN.

In vitro protective effects of Paeonia mascula subsp. hellenica callus extract on human keratinocytes

Natural ingredients have been used to improve the state of health in humans. The genus Paeonia has been studied only limited yet it’s reported to have many activities such as antioxidant and anti-inflammatory. To this context, here we focused on an endemic Paeonia species in Attica. This study aims to present the development of the Paeonia mascula subsp. hellenica callus extract and its pleiotropic bioactivity on human primary keratinocytes exploring its potential application as an active agent in skin-related products. This extract showed a high scavenging activity with high phenolic content and an interesting metabolic profile. At a molecular level, the study on the transcript accumulation of genes revealed that this extract exhibits in vitro skin-related protection properties by mediating mitochondrial energy, cell proliferation, immune and inflammatory response and positively regulates genes involved in epidermal and in stratum corneum function. Besides, the extract is proven not skin irritant on reconstructed human skin model. These findings indicate that the specific P. mascula subsp. hellenica extract possesses significant in vitro protection activity on human epidermis and provides new insights into its beneficial role in skin confirming that the advent of biotechnology contribution the past few decades.