Mouse Skin Cells Research Articles

Atmospheric pressure plasma jet-mouse skin interaction: Mitigation of damages by liquid interface and gas flow control

The possible benefits of an atmospheric pressure plasma jet skin treatment have been tested in vivo on mouse skin. Many studies have been conducted in vitro on mouse skin cells, but only a few in vivo where, due to the complexity of the biological system, plasma can cause severe damages. For this reason, we investigated how kHz plasma generated in a jet that is known to inflict skin damage interacts with mouse skin and explored how we can reduce the skin damage. First, the focus was on exploring plasma effects on skin damage formation with different plasma gases and jet inclinations. The results pointed to the perpendicular orientation of a He plasma jet as the most promising condition with the least skin damage. Then, the skin damage caused by a He plasma jet was explored, focusing on damage mitigation with different liquid interfaces applied to the treatment site, adding N2 to the gas mixture, or alternating the gas flow dynamics by elongating the jet's glass orifice with a funnel. All these mitigations proved highly efficient, but the utmost benefits for skin damage reduction were connected to skin temperature reduction, the reduction in reactive oxygen species (ROS), and the increase in reactive nitrogen species (RNS).

Galangin Reverses H2O2-Induced Dermal Fibroblast Senescence via SIRT1-PGC-1α/Nrf2 Signaling.

UV radiation and H2O2 are the primary factors that cause skin aging. Both trigger oxidative stress and cellular aging. It has been reported that deacetylase silent information regulator 1 (SIRT1), a longevity gene, enhances activation of NF-E2-related factor-2 (Nrf2), as well as its downstream key antioxidant gene hemeoxygenase-1 (HO-1), to protect cells against oxidative damage by deacetylating the transcription coactivator PPARγ coactivator-1α (PGC-1α). Galangin, a flavonoid, possesses anti-oxidative and anti-inflammatory potential. In the present study, we applied Ultraviolet B/H2O2-induced human dermal fibroblast damage as an in vitro model and UVB-induced photoaging of C57BL/6J nude mice as an in vivo model to investigate the underlying dermo-protective mechanisms of galangin. Our results indicated that galangin treatment attenuates H2O2/UVB-induced cell viability reduction, dermal aging, and SIRT1/PGC-1α/Nrf2 signaling activation. Furthermore, galangin treatment enhanced Nrf2 activation and nuclear accumulation, in addition to inhibiting Nrf2 degradation. Interestingly, upregulation of antioxidant response element luciferase activity following galangin treatment indicated the transcriptional activation of Nrf2. However, knockdown of SIRT1, PGC-1α, or Nrf2 by siRNA reversed the antioxidant and anti-aging effects of galangin. In vivo evidence further showed that galangin treatment, at doses of 12 and 24 mg/kg on the dorsal skin cells of nude mice resulted in considerably reduced UVB-induced epidermal hyperplasia and skin senescence, and promoted SIRT1/PGC-1α/Nrf2 signaling. Furthermore, enhanced nuclear localization of Nrf2 was observed in galangin-treated mice following UVB irradiation. In conclusion, our data indicated that galangin exerts anti-photoaging and antioxidant effects by promoting SIRT1/PGC-1α/Nrf2 signaling. Therefore, galangin is a potentially promising agent for cosmetic skin care products against UV-induced skin aging.

Localization of IL-1α Responsive Dermal Populations in an Ex Vivo Human Model of Wound Healing

While playing a critical role in skin wound healing, the inflammatory phase of this process is poorly understood. To gain a better understanding of the inflammatory phase of wound healing, we developed an ex vivo skin culture model of skin injury-induced inflammation. Previous work in our laboratory showed ex vivo culture of human skin induces an interleukin 1 alpha (IL-1α)-dependent response characterized by increased transcript and protein levels for the inflammatory cytokines/chemokines, IL-6, CXCL1, and CSF3. However, the cellular sources of these factors in ex vivo cultured human skin have not been determined. Prior work with ex vivo cultured mouse skin and single cell RNA sequencing suggested fibroblasts and endothelial cells were potential cellular sources for these inflammatory mediators. The current studies used spatial transcriptomics analysis of ex vivo cultured human skin to localize the IL-1α target cell populations/skin tissue regions that produce IL-6, CXCL1 and CSF3. The Visium Gene Expression Solution platform (10x Genomics Inc.) was used to generate spatial transcriptomics data from skin specimens preserved immediately after biopsy or after skin culture for 24 hours. Loupe Browser version 5.1.0 (10x Genomics Inc) was used for data analysis to identify and characterize cell populations/regions expressing IL6, CXCL1, and CSF3 and associated differentially expressed genes (including cell type-specific transcripts). Notably, these IL-1α-induced transcripts were localized to the parent dermis region cluster. Analysis of subclusters in the dermal region showed differential expression of these inflammatory transcripts in regions enriched with either or both fibroblast and endothelial cell specific-type markers. Potential novel markers of this inflammatory response, like SOD2, were identified and warrant future investigation. Subsequent studies in identifying the targets of IL-1α in skin inflammation is called for, as they may lead to better understanding of this processes in wound healing and better clinical outcomes.

Protective effect of oligosaccharides isolated from Panax ginseng C. A. Meyer against UVB-induced skin barrier damage in BALB/c hairless mice and human keratinocytes

Ethnopharmacological relevanceSkin barrier dysfunction can lead to water and electrolyte loss, triggering homeostatic imbalances that can trigger atopic dermatitis and anaphylaxis. Panax ginseng C.A. Meyer is a traditional Chinese medicinal herb with known therapeutic benefits for the treatment of skin diseases, including photodamage repair effects and reduction of pigmentation. However, few reports exist that describe effectiveness of ginseng active components for repair of skin barrier damage. Materials and methodsGinseng oligosaccharide extract (GSO) was prepared from P. ginseng via water extraction followed by ethanol precipitation and resin and gel purification. GSO composition and structural characteristics were determined using LC-MS, HPLC, FT-IR, and NMR. To evaluate GSO as a skin barrier repair-promoting treatment, skin of UVB-irradiated BALB/c hairless mice was treated with or without GSO then skin samples were evaluated for epidermal thickness, transepidermal water loss (TEWL), and stratum corneum water content. In addition, UVB-exposed skin samples and HaCaT cells were analyzed to assess GSO treatment effects on levels of epidermal cornified envelope (CE) protein and other skin barrier proteins, such as filaggrin (FLG), involucrin (IVL), and aquaporin-3 (AQP3). Meanwhile, GSO treatment was also evaluated for effects on UVB-irradiated hairless mouse skin and HaCaT cells based on levels of serine protease inhibitor Kazal type-5 (SPINK5), trypsin-like kallikrein-related peptidase 5 (KLK5), chymotrypsin-like KLK7, and desmoglein 1 (DSG1). These proteins are associated with UVB-induced skin barrier damage manifesting as dryness and desquamation. ResultsGSO was shown to consist of oligosaccharides comprised of seven distinct types of monosaccharides with molecular weights of approximately 1 kDa that were covalently linked together via β-glycosidic bonds. In vivo, GSO applied to dorsal skin of BALB/c hairless mice attenuated UVB-induced epidermal thickening and moisture loss. Furthermore, GSO ameliorated UVB-induced reductions of levels of FLG, IVL, and AQP3 proteins. Additionally, GSO treatment led to increased DSG1 protein levels due to decreased expression of KLK7. In vitro, GSO treatment of UVB-irradiated HaCaT cells led to increases of FLG, IVL, and AQP3 mRNA levels and corresponding proteins, while mRNA levels of desquamation-related proteins SPINK5, KLK5, KLK7, and DSG1 and associated protein levels were restored to normal levels. ConclusionA P. ginseng oligosaccharide preparation repaired UVB-induced skin barrier damage by alleviating skin dryness and desquamation symptoms, highlighting its potential as a natural cosmetic additive that can promote skin barrier repair after UVB exposure.

Dysregulation of epidermal barrier in mouse skin wounds induced by sulfur mustard

Sulfur mustard (2,2′-dichlorodiethyl sulfide, SM), is a potent chemical vesicant. Skin exposure to SM results in blistering, persistent epidermal hyperplasia, severe tissue damage, and prolonged wound repairs. SM, a bifunctional alkylating agent, causes DNA damage in mouse skin cells; however, the mechanistic action of SM in tissue injuries is still unclear. Studies showed mouse skin following SM exposure activates several stress signaling pathways including oxidative stress, and induces strong inflammatory responses. Skin structured with multilayers of keratinocytes (KCs), serves as a protection barrier against toxic chemicals. Keratins (Ks), the major structural intermediate filaments proteins in KCs, may regulate skin wound healing. In normal skin epidermis, KCs express K5 in basal cells and K10 in the differentiated suprabasal layers. Upon skin injury, KCs, activated by inflammation, express K6 and K17 to promote KCs migration for wound repair. Dysregulation of activated epidermal KCs may result in barrier breach and lead to uncontrolled inflammation and skin diseases. In addition, the cornified envelope (CE) precursor molecules, the small proline-rich (Sprr) proteins, present to protect skin from reactive oxidative species (ROS) damage. The present study was focused on SM induced alteration of skin barrier alarmin molecules in mouse ear skin. Time course studies (1-7 days) of mouse ear skin exposing to a single dose (0.08 mg) of SM showed characteristic histopathology changes over time. Significant changes of skin barrier alamin molecules (K10, K6, K17, and Sprr1B) gene expression were observed in SM injured skin post exposure. Immunohistochemistry results showed the K6 and K17 staining was restricted to the skin appendages in the naïve control, whereas samples exposed to SM had increased expression over time in the suprabasal epithelium. The activated KCs highly expressed K6, K17, and Sprr1B at the wound edge. In contrast, reduced expression of the differentiation marker, K10, indicated incompletely differentiated epithelium, suggesting weakened barrier protection in skin damaged by SM. Multiplex immunofluorescent studies showed heterogeneous coexpression of K5/ K6/K10 and K5/K10/Sprr1B in the hyperplasia behind the leading edge, suggesting hyperactivated KCs in the SM skin wounds. Overall, K10, K6, K17, and Sprr1B have value as skin wound and repair markers of SM induced injury. Understanding the molecular mechanism of action of vesicant-induced skin injury and repair involved in the dysregulation of epidermal barrier may help to identify new targets of medical countermeasures to chemical vesicant skin injury.

585 UBE2N is essential for normal epidermal homeostasis and controlling inflammation

UBE2N, also named Ubc13, is a K63-Ub-specific E2 conjugase. UBE2N plays an essential role in immunity and embryonic tissue development and represents a potential therapeutic target for cancer and immunological disorders. However, its function in adult skin homeostasis is unknown. Here, we demonstrate that topically induced deletion of UBE2N in mouse skin cells resulted in apparent skin abnormalities, including hair growth defects, skin blistering, and subsequent thickening that resembled psoriatic plaques.

Disruption of DNA polymerase ζ engages an innate immune response

In mammalian cells, specialized DNA polymerase ζ (pol ζ) contributes to genomic stability during normal DNA replication. Disruption of the catalytic subunit Rev3l is toxic and results in constitutive chromosome damage, including micronuclei. As manifestations of this genomic stress are unknown, we examined the transcriptome of pol ζ-defective cells by RNA sequencing (RNA-seq). Expression of 1,117 transcripts is altered by ≥4-fold in Rev3l-disrupted cells, with a pattern consistent with an induction of an innate immune response. Increased expression of interferon-stimulated genes at the mRNA and protein levels in pol ζ-defective cells is driven by the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-signaling partner stimulator of interferon genes (STING) pathway. Expression of key interferon-stimulated chemokines is elevated in basal epithelial mouse skin cells with a disruption of Rev3l. These results indicate that the disruption of pol ζ may simultaneously increase sensitivity to genotoxins and potentially engage parts of the innate immune response, which could add an additional benefit to targeting pol ζ in cancer therapies.

Analysing Mouse Skin Cell Behaviour under a Non-Thermal kHz Plasma Jet

Plasma jets are extensively used in biomedical applications, particularly for exploring cell viability behaviour. However, many experimental parameters influence the results, including jet characteristics, secondary liquid chemistry and protocols used, slowing research progress. A specific interest of the presented research was skin cell behaviour under a non-thermal kHz plasma jet—a so-called cold plasma jet—as a topical skin treatment. Our research was focused on in vitro mouse skin cell direct plasma treatment with argon as an operating gas. The research was complemented with detailed gas-phase diagnostics and liquid-phase chemical analysis of the plasma and plasma-treated medium, respectively. The obtained results showed that direct plasma jet treatment was very destructive, leading to low cell viability. Even with short treatment times (from 35 s to 60 s), apoptosis was observed for most L929 murine fibroblasts under approximately the same conditions. This behaviour was attributed to plasma species generated from direct treatment and the types of cell lines used. Importantly, the research exposed important points that should be taken under consideration for all further research in this field: the urgent need to upgrade and standardise existing plasma treatment protocols of cell lines; to monitor gas and liquid chemistries and to standardise plasma discharge parameters.

Distinct Distribution of RTN1A in Immune Cells in Mouse Skin and Lymphoid Organs.

The endoplasmic reticulum-associated protein reticulon 1A (RTN1A) is primarily expressed in neuronal tissues but was recently identified also specifically in cells of the dendritic cell (DC) lineage, including epidermal Langerhans cells (LCs) and dermal DCs in human skin. In this study, we found that in mice major histocompatibility complex class II (MHCII)+CD207+ LCs but not dermal DCs express RTN1A. Further, RTN1A expression was identified in CD45+MHCII+CD207+ cells of the lymph node and spleen but not in the thymus. Of note, RTN1A was expressed in CD207low LCs in adult skin as well as emigrated LCs and DCs in lymph nodes and marginally in CD207hi cells. Ontogeny studies revealed that RTN1A expression occurred before the appearance of the LC markers MHCII and CD207 in LC precursors, while dermal DC and T cell precursors remained negative during skin development. Analogous to the expression of RTN1A in neural tissue, we identified expression of RTN1A in skin nerves. Immunostaining revealed co-localization of RTN1A with nerve neurofilaments only in fetal but not in newborn or adult dermis. Our findings suggest that RTN1A might be involved in the LC differentiation process given its early expression in LC precursors and stable expression onward. Further analysis of the RTN1A expression pattern will enable the elucidation of the functional roles of RTN1A in both the immune and the nervous system of the skin.

Stem Cells from Human Exfoliated Deciduous teeth Promote Hair Regeneration in Mouse

Stem cells in different types may interact with each other to maintain homeostasis or growth and the interactions are complicated and extensive. There is increasing evidence that mesenchymal-epithelial interactions in early morphogenesis stages of both tooth and hair follicles show many similarities. In order to explore whether stem cells from one tissue could interact with cells from another tissue, a series of experiments were carried out. Here we successfully extracted and identified stem cells from human exfoliated deciduous teeth (SHED) of 8–12 years old kids, and then found that SHED could promote hair regeneration in a mouse model. In vitro, SHED shortened the hair regeneration cycle and promoted the proliferation and aggregation of dermal cells. In vivo, when SHED and skin cells of C57 mice were subcutaneously co-transplanted to nude mice, more hair was formed than skin cells without SHED. To further explore the molecular mechanism, epidermal and dermal cells were freshly extracted and co-cultured with SHED. Then several signaling molecules in hair follicle regeneration were detected and we found that the expression of Sonic Hedgehog (Shh) and Glioma-associated oncogene 1 (Gli1) was up-regulated. It seems that SHED may boost the prosperity of hairs by increase Shh/Gli1 pathway, which brings new perspectives in tissue engineering and damaged tissue repairing.

Inter-α-Trypsin Inhibitor Heavy Chain 5 (ITIH5) Is a Natural Stabilizer of Hyaluronan That Modulates Biological Processes in the Skin

Introduction: Hyaluronan (HA) is a major component of the skin that exerts a variety of biological functions. Inter-α-trypsin inhibitor heavy chain (ITIH) proteins comprise a family of hyaladherins of which ITIH5 has recently been described in skin, where it plays a functional role in skin morphology and inflammatory skin diseases including allergic contact dermatitis (ACD). Objective: The current study focused on the ITIH5-HA interaction and its potential clinical and functional impact in extracellular matrix (ECM) stabilization. Methods: Studying the molecular effects of ITIH5 in skin, we established skin models comprising murine skin cells of Itih5 knockout mice and corresponding wild-type controls. In addition, human dermal fibroblasts with an ITIH5 knockdown as well as a murine recombinant Itih5 protein were established to examine the interaction between ITIH5 and HA using in vitro adhesion and HA degradation assays. To understand more precisely the role of ITIH5 in inflammatory skin diseases such as ACD, we generated ITIH5 knockout cells of the KeratinoSens® cell line. Results: Using murine skin models, ITIH5 knockdown fibroblasts, and a reactive oxygen species (ROS)-mediated HA degradation assay, we proved that ITIH5 binds to HA, thereby acting as a stabilizer of HA. Moreover, microarray profiling revealed the impact of ITIH5 on biological processes such as skin development and ECM homeostasis. Performing the in vitro KeratinoSens skin sensitization assay, we detected that ITIH5 decreases the sensitizing potential of moderate and strong contact sensitizers. Conclusion: Taken together, our experiments revealed that ITIH5 forms complexes with HA, thereby on the one hand stabilizing HA and facilitating the formation of ECM structures and on the other hand modulating inflammatory responses.

Abstract 5840: IRE1a is required for ROS homeostasis and PERK protein stability in response to UVB irradiation

Abstract IRE1α is required for ROS homeostasis and PERK protein stability in response to UVB irradiation UV induced stress is one of the primary driving factors of human skin cancer. UV irradiation can activate Endoplasmic Reticulum (ER) stress and the Unfolded Protein Response (UPR). Inositol-requiring enzyme 1a (IRE1α) is one of the key players in the UPR. Previous studies have shown that UVB irradiation can activate IRE1α in human and mouse skin cells, but the function of IRE1α in the UV response has not yet been characterized. Here we show that IRE1α expression is transiently downregulated after UVB irradiation and that in the absence of IRE1α the stability and activation of PKR-like endoplasmic reticulum kinase (PERK) is compromised. Basal levels of PERK are reduced in the absecence of IRE1α. Following UV irradiation, loss of IRE1α not only causes rapid degradation of PERK protein but also suppresses phosphorylation of the PERK target, Nuclear factor erythroid-2-related factor 2 (NRF2), and NRF2 dependent antioxidant gene expression. Similarly, PERK protein is rapidly degraded following thapsigargin treatment in the absence of IRE1α, suggesting a general regulation of PERK stability by IRE1α. Additionally, keratinocytes with either knockout or knockdown of IRE1α have increased intracellular ROS and cytosolic calcium. Overexpression of both wildtype and RNase defective human IRE1α can rescue the increased ROS levels. Additionally, the increased ROS is also suppressed by treatment with an inhibitor of the ER localized Inositol triphosphate receptor (InsP3R) which regulates calcium efflux from the ER. Because PERK regulates cytosolic calcium through inhibiting InsP3R, our result suggests that IRE1α is critical for suppressing intracellular ROS through stabilization of PERK. We conclude that crosstalk between IRE1α and PERK is essential for the basal and UVB- induced oxidative stress response, and this may have important implications for UV related damage and skin cancer development. Citation Format: Jeongin Son, Stephen Worrel, Adam Glick. IRE1a is required for ROS homeostasis and PERK protein stability in response to UVB irradiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5840.

Bucillamine Inhibits UVB-Induced MAPK Activation and Apoptosis in Human HaCaT Keratinocytes and SKH-1 Hairless Mouse Skin.

Ultraviolet B (UVB) radiation is known as a culprit in skin carcinogenesis. We have previously reported that bucillamine (N-[2-mercapto-2-methylpropionyl]-L-cysteine), a cysteine derivative with antioxidant and anti-inflammatory capacity, protects against UVB-induced p53 activation and inflammatory responses in mouse skin. Since MAPK signaling pathways regulate p53 expression and activation, here we determined bucillamine effect on UVB-mediated MAPK activation in vitro using human skin keratinocyte cell line HaCaT and in vivo using SKH-1 hairless mouse skin. A single low dose of UVB (30mJcm-2 ) resulted in increased JNK/MAPK phosphorylation and caspase-3 cleavage in HaCaT cells. However, JNK activation and casaspe-3 cleavage were inhibited by pretreatment of HaCaT cells with physiological doses of bucillamine (25 and 100µm). Consistent with these results, bucillamine pretreatment in mice (20mgkg-1 ) inhibited JNK/MAPK and ERK/MAPK activation in skin epidermal cells at 6-12 and 24h, respectively, after UVB exposure. Moreover, bucillamine attenuated UVB-induced Ki-67-positive cells and cleaved caspase-3-positive cells in mouse skin. These findings demonstrate that bucillamine inhibits UVB-induced MAPK signaling, cell proliferation and apoptosis. Together with our previous report, we provide evidence that bucillamine has a photoprotective effect against UV exposure.

Conjugated linoleic acid attenuates 2,4-dinitrofluorobenzene-induced atopic dermatitis in mice through dual inhibition of COX-2/5-LOX and TLR4/NF-κB signaling

Conjugated linoleic acid (CLA), commonly found in beef, lamb and dairy products, has been reported to exhibit anti-inflammatory and antipruritus effects and to inhibit the release of chemical mediators such as histamine and eicosanoid in laboratory rodents. The chief objective of the study is to assess the efficacy of CLA on atopic dermatitis (AD) in mice and to explore possible mechanisms with CLA treatments. To develop a new therapy for AD, the anti-AD potential of CLA was investigated by inducing AD-like skin lesions in mice using 2,4-dinitrofluorobenzene. We evaluated dermatitis severity; histopathological changes; serum levels of T helper (Th) cytokines (interferon-γ, interleukin-4); changes in protein expression by western blotting and immunohistochemistry staining for cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), toll like receptor 4 (TLR-4), myeloid differentiation factor 88 (MyD88), nuclear factor-κB (NF-κB) and tumor necrosis factor α (TNF-α); and production of the proinflammatory lipid mediators, such as prostaglandin E2 and leukotriene B4, in the skin lesions. Treatment with CLA ameliorated the development of AD-like clinical symptoms and effectively inhibited epidermal hyperplasia and infiltration of mast cells and CD4+ T cells in the AD mouse skin. Total serum immunoglobulin E levels and the expression levels of Th1/Th2 cytokines and lipid mediators in dorsal skin were dramatically suppressed by CLA. Furthermore, CLA down-regulated the expressions of COX-2, 5-LOX, TLR4, MyD88, NF-κB and TNF-α. Taken together, our findings demonstrate the potential usefulness of CLA as an anti-inflammatory dietary supplement or drug for the prevention and management of AD skin diseases by modulating the COX-2/5-LOX and TLR4/MyD88/NF-κB signaling pathways.

Dihydroartemisinin ameliorates psoriatic skin inflammation and its relapse by diminishing CD8+ T-cell memory in wild-type and humanized mice.

Conventional immunosuppressants cause side effects and do not prevent the recurrence of autoimmune diseases. Moreover, they may not inhibit autoimmunity mediated by pathogenic memory T-cells. Dihydroartemisinin (DHA) has been shown to regulate autoimmunity. However, it remains unknown whether DHA impacts psoriasis and its recurrence. The objective of this study was to determine therapeutic effects of DHA on psoriasis and its relapse as well as its underlying mechanisms.Methods: We established animal models of imiquimod (IMQ)-induced psoriasis-like wild-type mice and humanized NSG mice receiving lesional human skin from patients with psoriasis. Many immunoassays, including immunohistochemistry, flow cytometry, quantitative RT-PCR and Western blotting, were performed.Results: We found that DHA not only ameliorated acute skin lesion of psoriatic mice, but also alleviated its recurrence by diminishing CD8+ central memory T (TCM) and CD8+ resident memory T (TRM) cells. It attenuated epidermal pathology and T-cell infiltration in the skin of IMQ-induced psoriatic mice while suppressing expression of IL-15, IL-17 and other proinflammatory cytokines in the skin. Surprisingly, DHA reduced the frequency and number of CD8+, but not CD4+, subset of CD44highCD62Lhigh TCM in psoriatic mice, whereas methotrexate (MTX) lowered CD4+, but not CD8+, TCM frequency and number. Indeed, DHA, but not MTX, downregulated eomesodermin (EOMES) and BCL-6 expression in CD8+ T-cells. Furthermore, DHA, but not MTX, reduced the presence of CD8+CLA+, CD8+CD69+ or CD8+CD103+ TRM cells in mouse skin. Interestingly, treatment with DHA, but not MTX, during the first onset of psoriasis largely prevented psoriasis relapse induced by low doses of IMQ two weeks later. Administration of recombinant IL-15 or CD8+, but not CD4+, TCM cells resulted in complete recurrence of psoriasis in mice previously treated with DHA. Finally, we demonstrated that DHA alleviated psoriatic human skin lesions in humanized NSG mice grafted with lesional skin from psoriatic patients while reducing human CD8+ TCM and CD103+ TRM cells in humanized mice.Conclusion: We have provided the first evidence that DHA is advantageous over MTX in preventing psoriasis relapse by reducing memory CD8+ T-cells.

Search of itch-producing substance(s) released from skin cells of atopic dermatitis model mice

Search of itch-producing substance(s) released from skin cells of atopic dermatitis model mice

APCDD1 as a Co-receptor Positively Regulates Wnt5a/c-Jun Non-Canonical Signaling Pathway

Adenomatosis polyposis down-regulated 1 (APCDD1) is a transmembrane glycoprotein that negatively regulates Wnt/β-catenin canonical signaling by binding with Wnt ligands and receptors. We analyzed the role of APCDD1 in the Wnt5a/c-Jun non-canonical signaling pathway and demonstrated that APCDD1 can interact in vitro with Wnt5a, a classical ligand, and Ror2, a receptor of non-canonical Wnt signaling. Furthermore, we verified the binding of APCDD1 and Ror2 in primary cells of mouse skin. Moreover, APCDD1 seems to form a complex with Ror2 and Vangl2 in the cell, and complex formation can be improved by adding Wnt5a. In the presence of Wnt5a and Ror2, APCDD1 can induce the phosphorylation of c-Jun, a transcription factor of Wnt5a non-canonical signaling, and its phosphorylation level is a readout of Wnt5a signaling. Wound-healing assay shows that APCDD1 accelerates polarized cell migration during Wnt5a-induced wound closure. Therefore, it is very likely that APCDD1 regulates Wnt5a/c-Jun non-canonical signaling as co-receptor binding with both Wnt5a and Ror2.

DNA-Damaging Effect of Different Wavelength (206 and 257 nm) Femtosecond Laser Pulses.

Objective: The purpose of this study was to investigate the possible cytotoxic and genotoxic impact of new-generation 206 nm femtosecond solid-state laser irradiation on murine skin cells in vitro, and to compare the cell and DNA damage caused by different wavelength (206 vs. 257 nm) femtosecond laser pulses. Background data: The first attempts to evaluate the possible genotoxic impact of ultrashort laser pulses on the murine bone marrow cells in vitro revealed the unlooked-for DNA-damaging effect. However, the impact of far-ultraviolet (UV) radiation on genetic material of internal and external organs' cells may differ due to differences in size, structure, and biochemical composition of the cells. Methods: Mouse skin cells were exposed to different doses of 206 and 257 nm wavelength femtosecond laser, and 254 nm UV lamp irradiation. Comet assay in two versions-the standard alkaline and the enzyme-linked-was used for the evaluation of DNA damage. Results: The irradiation determined by different parameters demonstrated intensity-dependent genotoxic impact. The pyrimidine dimers made up the greater part of DNA photodamage, but with rising exposure dose the increase of relative amount of more energy-consuming primary damage-DNA strand breaks-was detected. Conclusions: The 206 nm femtosecond laser irradiation was much more cytotoxic but caused less primary DNA damage than the same pulse duration longer wavelength (257 nm) laser irradiation. DNA-damaging effect of 206 nm femtosecond laser pulses with extremely low penetration force may highly depend on the size, structure, and biochemical composition of the cells of organ or tissue targets.

Single-Cell Transcriptomics of Regulatory T Cells Reveals Trajectories of Tissue Adaptation

SummaryNon-lymphoid tissues (NLTs) harbor a pool of adaptive immune cells with largely unexplored phenotype and development. We used single-cell RNA-seq to characterize 35,000 CD4+ regulatory (Treg) and memory (Tmem) T cells in mouse skin and colon, their respective draining lymph nodes (LNs) and spleen. In these tissues, we identified Treg cell subpopulations with distinct degrees of NLT phenotype. Subpopulation pseudotime ordering and gene kinetics were consistent in recruitment to skin and colon, yet the initial NLT-priming in LNs and the final stages of NLT functional adaptation reflected tissue-specific differences. Predicted kinetics were recapitulated using an in vivo melanoma-induction model, validating key regulators and receptors. Finally, we profiled human blood and NLT Treg and Tmem cells, and identified cross-mammalian conserved tissue signatures. In summary, we describe the relationship between Treg cell heterogeneity and recruitment to NLTs through the combined use of computational prediction and in vivo validation.

Brahma deficiency in keratinocytes promotes UV carcinogenesis by accelerating the escape from cell cycle arrest and the formation of DNA photolesions

BackgroundUltraviolet radiation (UVR) is the principal cause of keratinocyte skin cancers. Previous work found that levels of the chromatin remodelling protein, Brahma (Brm), are diminished during the progression from actinic keratoses to cutaneous squamous cell carcinomas in humans, and its loss in UV-irradiated mouse skin causes epidermal hyperplasia and increased tumour incidence. MethodsThe skins of mice and mouse and human keratinocytes deficient in Brm were exposed to UVR and evaluated for cell cycle progression and DNA damage response. ObjectiveTo identify the mechanisms by which loss of Brm contributes to UVR-induced skin carcinogenesis. ResultsIn both mouse keratinocytes and HaCaT cells, Brm deficiency led to an increased cell population growth following UVR exposure compared to cells with normal levels of Brm. Cell cycle analysis using a novel assay showed that Brm-deficient keratinocytes entered cell cycle arrest normally, but escaped from cell cycle arrest faster, enabling them to begin proliferating earlier. In mouse keratinocytes, Brm primarily affected accumulation in G0/G1-phase, whereas in HaCaT cells, which lack normal p53, accumulation in G2/M-phase was affected. Brm-deficient keratinocytes in mouse skin and human cell cultures also had higher levels of UVR-induced cyclobutane pyrimidine dimer photolesions. These effects occurred without any compensatory increase in DNA repair or cell death to remove photolesions or the cells that harbor them from the keratinocyte population. ConclusionThe loss of Brm in keratinocytes exposed to UVR enables them to resume proliferation while harboring DNA photolesions, leading to an increased fixation of mutations and, consequently, increased carcinogenesis.