Effect of Ultraviolet Irradiation on Mast Cell-Deficient W/Wv Mice

The effect of UV irradiation on the skin was investigated in (WB-W/+) × (C57BL/6J-Wv/+)F1-W/Wv mice, which are genetically deficient in tissue mast cells. Their congenic littermates (+/+) and normal albino mice (ICR or Balb/c) were used as controls. Mice were irradiated with 500 mJ/cm2 of UVB and the increment of ear thickness was measured before and 6, 12, and 24 h after irradiation was significantly smaller than that in +/+ and ICR mice. In contrast, the number of sunburn cells formed 24 h after UVB irradiation (200 or 500 mJ/cm2) was similar in W/Wv, +/+ and ICR mice. On the other hand, when mice were treated with 8-methoxy-psoralen (0.5%) plus UVA irradiation (4 J/cm2) (topical PUVA), ears of W/Wv and BALB/c mice, which were both white in color, were thickened similarly 72 h after treatment, but less swelling was observed in +/+ mice, which were black in skin color. The amount of prostaglandin D2 (PGD2) in ears, determined by radioimmuno-assay specific for PGD2, was elevated 3-fold in +/+ and ICR mice at 3 h after irradiation with 500 mJ/cmv of UVB in comparison with basal level without irradiation. However, such elevation was not observed in W/Wv mice. These results suggest that mast cells play an important role in UVB-induced inflammation, and PGs from mast cells are responsible at lest in part for the development of this reaction. However, neither mast cells nor PGs contribute to the sunburn cell formation and ear swelling response by PUVA treatment

An optimized protocol to identify keratinocyte subpopulations in vitro by single-cell RNA sequencing analysis

Here, we describe a protocol for single-cell isolation from the primary culture of normal human epidermal keratinocytes derived from neonatal foreskin. The cell culture conditions have been optimized for inducing expression of keratinocyte differentiation markers. Cells are cultured in the absence or presence of a bioactive lipid lysophosphatidic acid (LPA). Single cells are isolated by Fluidigm C1 system. This is followed by cDNA library preparation using Takara SMART-Seq v4 Ultra and Illumina Nextera XT kit for RNA sequencing

Critical role of TNF-alpha-TNFR1 signaling in intracranial aneurysm formation

BACKGROUND: Intracranial aneurysm (IA) is a socially important disease due to its high incidence in the general public and the severity of resultant subarachnoid hemorrhage that follows rupture. Despite the social importance of IA as a cause of subarachnoid hemorrhage, there is no medical treatment to prevent rupture, except for surgical procedures, because the mechanisms regulating IA formation are poorly understood. Therefore, these mechanisms should be elucidated to identify a therapeutic target for IA treatment. In human IAs, the presence of inflammatory responses, such as an increase of tumor necrosis factor (TNF)-alpha, have been observed, suggesting a role for inflammation in IA formation. Recent investigations using rodent models of IAs have revealed the crucial role of inflammatory responses in IA formation, supporting the results of human studies. Thus, we identified nuclear factor (NF)-kappaB as a critical mediator of inflammation regulating IA formation, by inducing downstream pro-inflammatory genes such as MCP-1, a chemoattractant for macrophages, and COX-2. In this study, we focused on TNF-alpha signaling as a potential cascade that regulates NF-kappaB-mediated IA formation. RESULTS: We first confirmed an increase in TNF-alpha content in IA walls during IA formation, as expected based on human studies. Consistently, the activity of TNF-alpha converting enzyme (TACE), an enzyme responsible for TNF-alpha release, was induced in the arterial walls after aneurysm induction in a rat model. Next, we subjected tumor necrosis factor receptor superfamily member 1a (TNFR1)-deficient mice to the IA model to clarify the contribution of TNF-alpha-TNFR1 signaling to pathogenesis, and confirmed significant suppression of IA formation in TNFR1-deficient mice. Furthermore, in the IA walls of TNFR1-deficient mice, inflammatory responses, including NF-kappaB activation, subsequent expression of MCP-1 and COX-2, and infiltration of macrophages into the IA lesion, were greatly suppressed compared with those in wild-type mice. CONCLUSIONS: In this study, using rodent models of IAs, we clarified the crucial role of TNF-alpha-TNFR1 signaling in the pathogenesis of IAs by inducing inflammatory responses, and propose this signaling as a potential therapeutic target for IA treatment

Requirements of basic amino acid residues within the lectin-like domain of LOX-1 for the binding of oxidized low-density lipoprotein

AbstractLectin-like OxLDL receptor-1 (LOX-1) was identified as the major receptor for oxidized low-density lipoprotein (OxLDL) in aortic endothelial cells. LOX-1 is a type II membrane protein that structurally belongs to the C-type lectin family. Here, we found that the lectin-like domain of LOX-1 is essential for ligand binding, but the neck domain is not. In particular, the large loop between the third and fourth cysteine of the lectin-like domain plays a critical role for OxLDL binding as well as C-terminal end residues. Alanine-directed mutagenesis of the basic amino acid residues around this region revealed that all of the basic residues are involved in OxLDL binding. Simultaneous mutations of these basic residues almost abolished the OxLDL-binding activity of LOX-1. Electrostatic interaction between basic residues in the lectin-like domain of LOX-1 and negatively charged OxLDL is critical for the binding activity of LOX-1

The small GTP-binding protein RhoA regulates c-Jun by a ROCK-JNK signaling axis

RhoA regulates the actin cytoskeleton and the expres- sion of genes associated with cell proliferation. This includes c-fos and c-jun, which are members of the AP1 family of transcription factors that play a key role in normal and aberrant cell growth. Whereas RhoA stimulates the c-fos SRE by a recently elucidated mechanism that is dependent on actin treadmilling, how RhoA regulates c-jun is still poorly understood. We found that RhoA stimulates c-jun expression through ROCK, but independently from the ability of ROCK to promote actin polymerization. Instead, we found that ROCK activates JNK, which then phosphor- ylates c-Jun and ATF2 when bound to the c-jun pro- moter. Thus, ROCK represents a point of signal diver- gence downstream from RhoA, as it promotes actin reorganization and the consequent expression from the c-fos SRE, while a parallel pathway connects ROCK to JNK, thereby stimulating c-jun expression. Ultimately, these pathways converge in the nucleus to regulate AP1 activity

T cell–intrinsic prostaglandin E₂-EP2/EP4 signaling is critical in pathogenic T_H17 cell–driven inflammation

PGE2経路による病因細胞Th17の増殖機構を解明 --乾癬の慢性的な皮膚炎症を改善する新しい治療薬開発に向けて--. 京都大学プレスリリース. 2018-06-20.Background: IL-23 is the key cytokine for generation of pathogenic IL-17–producing helper T (TH17) cells, which contribute critically to autoimmune diseases. However, how IL-23 generates pathogenic TH17 cells remains to be elucidated. Objectives: We sought to examine the involvement, molecular mechanisms, and clinical implications of prostaglandin (PG) E2–EP2/EP4 signaling in induction of IL-23–driven pathogenic TH17 cells. Methods: The role of PGE2 in induction of pathogenic TH17 cells was investigated in mouse TH17 cells in culture in vitro and in an IL-23–induced psoriasis mouse model in vivo. Clinical relevance of the findings in mice was examined by using gene expression profiling of IL-23 and PGE2-EP2/EP4 signaling in psoriatic skin from patients. Results: IL-23 induces Ptgs2, encoding COX2 in TH17 cells, and produces PGE2, which acts back on the PGE receptors EP2 and EP4 in these cells and enhances IL-23–induced expression of an IL-23 receptor subunit gene, Il23r, by activating signal transducer and activator of transcription (STAT) 3, cAMP-responsive element binding protein 1, and nuclear factor κ light chain enhancer of activated B cells (NF-κB) through cyclic AMP–protein kinase A signaling. This PGE2 signaling also induces expression of various inflammation-related genes, which possibly function in TH17 cell–mediated pathology. Combined deletion of EP2 and EP4 selectively in T cells suppressed accumulation of IL-17A+ and IL-17A+IFN-γ+ pathogenic Th17 cells and abolished skin inflammation in an IL-23–induced psoriasis mouse model. Analysis of human psoriatic skin biopsy specimens shows positive correlation between PGE2 signaling and the IL-23/TH17 pathway. Conclusions: T cell–intrinsic EP2/EP4 signaling is critical in IL-23–driven generation of pathogenic TH17 cells and consequent pathogenesis in the skin

Association of body temperature with in-hospital mortality among paediatric trauma patients: An analysis of a nationwide observational trauma database in Japan

Okada A, Okada Y, Narumiya H, et alAssociation of body temperature with in-hospital mortality among paediatric trauma patients: an analysis of a nationwide observational trauma database in JapanBMJ Open 2020;10:e033822. doi: 10.1136/bmjopen-2019-033822