NF-κB-inducing kinase maintains T cell metabolic fitness in antitumor immunity.

Metabolic reprograming toward aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show that deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8+ effector T cells hypofunctional in the tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8+ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via a NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular reactive oxygen species levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme that mediates production of the antioxidant NADPH. We show that the G6PD-NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a post-translational mechanism of metabolic regulation.

NIK signaling axis regulates dendritic cell function in intestinal immunity and homeostasis.

Dendritic cells (DCs) play an integral role in regulating mucosal immunity and homeostasis, but the signaling network mediating this function of DCs is poorly defined. We identified the noncanonical NF-κB-inducing kinase (NIK) as a crucial mediator of mucosal DC function. DC-specific NIK deletion impaired intestinal immunoglobulin A (IgA) secretion and microbiota homeostasis, rendering mice sensitive to an intestinal pathogen, Citrobacter rodentium. DC-specific NIK was required for expression of the IgA transporter polymeric immunoglobulin receptor (pIgR) in intestinal epithelial cells, which in turn relied on the cytokine IL-17 produced by TH17 cells and innate lymphoid cells (ILCs). NIK-activated noncanonical NF-κB induced expression of IL-23 in DCs, contributing to the maintenance of TH17 cells and type 3 ILCs. Consistent with the dual functions of IL-23 and IL-17 in mucosal immunity and inflammation, NIK deficiency also ameliorated colitis induction. Thus, our data suggest a pivotal role for the NIK signaling axis in regulating DC functions in intestinal immunity and homeostasis.

The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses.

Metabolic fitness of T cells is crucial for immune responses against infections and tumorigenesis. Both the T cell receptor (TCR) signal and environmental cues contribute to the induction of T cell metabolic reprogramming, but the underlying mechanism is incompletely understood. Here, we identified the E3 ubiquitin ligase Peli1 as an important regulator of T cell metabolism and antitumor immunity. Peli1 ablation profoundly promotes tumor rejection, associated with increased tumor-infiltrating CD4 and CD8 T cells. The Peli1-deficient T cells display markedly stronger metabolic activities, particularly glycolysis, than wild-type T cells. Peli1 controls the activation of a metabolic kinase, mTORC1, stimulated by both the TCR signal and growth factors, and this function of Peli1 is mediated through regulation of the mTORC1-inhibitory proteins, TSC1 and TSC2. Peli1 mediates non-degradative ubiquitination of TSC1, thereby promoting TSC1-TSC2 dimerization and TSC2 stabilization. These results establish Peli1 as a novel regulator of mTORC1 and downstream mTORC1-mediated actions on T cell metabolism and antitumor immunity.

Enteric Viruses Ameliorate Gut Inflammation via Toll-like Receptor 3 and Toll-like Receptor 7-Mediated Interferon-ß Production.

Metagenomic studies show that diverse resident viruses inhabit the healthy gut; however, little is known about the role of these viruses in the maintenance of gut homeostasis. We found that mice treated with antiviral cocktail displayed more severe dextran sulfate sodium (DSS)-induced colitis compared with untreated mice. DSS-induced colitis was associated with altered enteric viral abundance and composition. When wild-type mice were reconstituted with Toll-like receptor 3 (TLR3) or TLR7 agonists or inactivated rotavirus, colitis symptoms were significantly ameliorated. Mice deficient in both TLR3 and TLR7 were more susceptible to DSS-induced experimental colitis. In humans, combined TLR3 and TLR7 genetic variations significantly influenced the severity of ulcerative colitis. Plasmacytoid dendritic cells isolated from inflamed mouse colon produced interferon-ß in a TLR3 and TLR7-dependent manner. These results imply that recognition of resident viruses by TLR3 and TLR7 is required for protective immunity during gut inflammation.

Stretchable and Skin-Mountable Temperature Sensor Array Using Reduction-Controlled Graphene Oxide for Dermatological Thermography.

Since thermometry of human skin is critical information that provides important aspects of human health and physiology, accurate and continuous temperature measurement is required for the observation of physical abnormalities. However, conventional thermometers are uncomfortable because of their bulky and heavy features. In this work, we fabricated a thin, stretchable array-type temperature sensor using graphene-based materials. Furthermore, we controlled the degree of graphene oxide reduction and enhanced the temperature sensitivity. The sensor exhibited an excellent sensitivity of 2.085% °C-1. The overall device was designed in a wavy meander shape to provide stretchability for the device so that precise detection of skin temperature could be performed. Furthermore, polyimide film was coated to secure the chemical and mechanical stabilities of the device. The array-type sensor enabled spatial heat mapping with high resolution. Finally, we introduced some practical applications of skin temperature sensing, suggesting the possibility of skin thermography and healthcare monitoring.

Development of Non-Invasive miRNA Markers for Assessing the Quality of Human Induced Pluripotent Stem Cell-Derived Retinal Organoids.

Human retinal organoids (ROs) have emerged as valuable tools for studying retinal development, modeling human retinal diseases, and screening drugs. However, their application is limited primarily due to time-intensive generation, high costs, and low reproducibility. Quality assessment of RO differentiation is crucial for their application in research. However, traditional methods such as morphological evaluation and immunohistochemical analysis have limitations due to their lack of precision and invasiveness, respectively. This study aims to identify non-invasive biomarkers for RO differentiation quality using exosomal microRNAs (miRNAs), which are known to reflect cell-specific functions and development in the retina. We differentiated ROs from human induced pluripotent stem cells (hiPSCs) and classified them into 'superior' and 'inferior' groups based on morphological and immunohistochemical criteria. Exosomes from the conditioned media were isolated and analyzed for miRNA content. Our findings revealed distinct miRNA profiles between superior and inferior ROs, with superior ROs exhibiting higher miRNA diversity and specifically up- or down-regulated miRNAs. Gene ontology and pathway enrichment analyses indicated that the target genes of these miRNAs are involved in neuron proliferation and differentiation. The study suggests the potential of exosomal hsa-miR-654-3p and hsa-miR-451a as non-invasive biomarkers for real-time monitoring of RO quality, facilitating the development of standardized, efficient, and cost-effective culture methods.

Early Developmental Characteristics and Features of a Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis.

X-linked juvenile retinoschisis (XLRS) is a hereditary retinal degeneration affecting young males caused by mutations in the retinoschisin (RS1) gene. We generated human induced pluripotent stem cells (hiPSCs) from XLRS patients and established three-dimensional retinal organoids (ROs) for disease investigation. This disease model recapitulates the characteristics of XLRS, exhibiting defects in RS1 protein production and photoreceptor cell development. XLRS ROs also revealed dysregulation of Na/K-ATPase due to RS1 deficiency and increased ERK signaling pathway activity. Transcriptomic analyses of XLRS ROs showed decreased expression of retinal cells, particularly photoreceptor cells. Furthermore, relevant recovery of the XLRS phenotype was observed when co-cultured with control ROs derived from healthy subject during the early stages of differentiation. In conclusion, our in vitro XLRS RO model presents a valuable tool for elucidating the pathophysiological mechanisms underlying XLRS, offering insights into disease progression. Additionally, this model serves as a robust platform for the development and optimization of targeted therapeutic strategies, potentially improving treatment outcomes for patients with XLRS.

DYRK1a mediates BAFF-induced noncanonical NF-κB activation to promote autoimmunity and B-cell leukemogenesis.

B-cell-activating factor (BAFF) mediates B-cell survival and, when deregulated, contributes to autoimmune diseases and B-cell malignancies. The mechanism connecting BAFF receptor (BAFFR) signal to downstream pathways and pathophysiological functions is not well understood. Here we identified DYRK1a as a kinase that responds to BAFF stimulation and mediates BAFF-induced B-cell survival. B-cell-specific DYRK1a deficiency causes peripheral B-cell reduction and ameliorates autoimmunity in a mouse model of lupus. An unbiased screen identified DYRK1a as a protein that interacts with TRAF3, a ubiquitin ligase component mediating degradation of the noncanonical nuclear factor (NF)-κB-inducing kinase (NIK). DYRK1a phosphorylates TRAF3 at serine-29 to interfere with its function in mediating NIK degradation, thereby facilitating BAFF-induced NIK accumulation and noncanonical NF-κB activation. Interestingly, B-cell acute lymphoblastic leukemia (B-ALL) cells express high levels of BAFFR and respond to BAFF for noncanonical NF-κB activation and survival in a DYRK1a-dependent manner. Furthermore, DYRK1a promotes a mouse model of B-ALL through activation of the noncanonical NF-κB pathway. These results establish DYRK1a as a critical BAFFR signaling mediator and provide novel insight into B-ALL pathogenesis.

Intravitreal Administration of Retinal Organoids-Derived Exosomes Alleviates Photoreceptor Degeneration in Royal College of Surgeons Rats by Targeting the Mitogen-Activated Protein Kinase Pathway.

Increasing evidence suggests that exosomes are involved in retinal cell degeneration, including their insufficient release; hence, they have become important indicators of retinopathies. The exosomal microRNA (miRNA), in particular, play important roles in regulating ocular and retinal cell functions, including photoreceptor maturation, maintenance, and visual function. Here, we generated retinal organoids (ROs) from human induced pluripotent stem cells that differentiated in a conditioned medium for 60 days, after which exosomes were extracted from ROs (Exo-ROs). Subsequently, we intravitreally injected the Exo-RO solution into the eyes of the Royal College of Surgeons (RCS) rats. Intravitreal Exo-RO administration reduced photoreceptor apoptosis, prevented outer nuclear layer thinning, and preserved visual function in RCS rats. RNA sequencing and miRNA profiling showed that exosomal miRNAs are mainly involved in the mitogen-activated protein kinase (MAPK) signaling pathway. In addition, the expression of MAPK-related genes and proteins was significantly decreased in the Exo-RO-treated group. These results suggest that Exo-ROs may be a potentially novel strategy for delaying retinal degeneration by targeting the MAPK signaling pathway.

Characterization of Edwardsiella piscicida CK108 flagellin genes and evaluation of their potential as vaccine targets in the zebrafish model.

The control of bacterial pathogens, including Edwardsiella piscicida, in the aquaculture industry has high economic importance. This study aimed to identify a potential live vaccine candidate against E. piscicida infection to minimize the side effects and elicit immunity in the host. This study evaluated the virulence factors of E. piscicida CK108, with a special focus on the flagella. E. piscicida has two important homologous flagellin genes, namely flagellin-associated protein (fap) and flagellin domain-containing protein (fdp). CK226 (Δfap), CK247 (Δfdp) and CK248 (Δfap, fdp) mutant strains were constructed. Both CK226 and CK247 displayed decreased length and thickness of flagellar filaments, resulting in reduced bacterial swimming motility, while CK248 was non-motile as it lacked flagella. The loss of flagella and decreased motility was expected to decrease the pathogenicity of CK248. However, the median lethal dose (LD50 ) of CK248 against zebrafish was lower than those of the wild-type, CK226 and CK247 strains. The protective immunity and cytokine gene expression levels in the CK248-infected zebrafish were lower than those in the wild type-infected zebrafish. In conclusion, Fap and Fdp are essential for flagella formation and motility, and for stimulating fish immune response, which can be utilized as a potential adjuvants for E. piscicida vaccination.

Combined Treatment of Monopolar and Bipolar Radiofrequency Increases Skin Elasticity by Decreasing the Accumulation of Advanced Glycated End Products in Aged Animal Skin.

It is well known that skin aging is related to the destruction of collagen and elastin fibers by metalloproteinases (MMPs). Aged fibroblasts have a decreased ability to synthesize collagen and elastin. Nuclear factor erythroid 2-related factor 2 (NRF2) involves glyoxalase (GLO) activation, which inhibits the production of advanced glycated end products (AGE) and the expression of its receptor (RAGE). RAGE increases nuclear transcription factor-kappa B (NF-κB), which upregulates MMPs and decreases skin elasticity. NRF2 also decreases M1 macrophages, which secrete tumor necrosis factor-alpha (TNF-α), thereby decreasing AGE production. It is well known that radiofrequency (RF) decreases skin elasticity by increasing collagen synthesis. We evaluated whether RF increases skin elasticity via NRF2/GLO and whether they decrease AGE and RAGE expression in aged animal skin. We also compared the effects of RF based on the modes (monopolar or bipolar) or the combination used. In aged skin, NRF2, GLO-1, and M2 macrophage expression was decreased, and their expression increased when RF was applied. M1 and TNF-α demonstrated increased expression in the aged skin and decreased expression after RF application. AGE accumulation and RAGE, NF-κB, and MMP2/3/9 expression were increased in the aged skin, and they were decreased by RF. The papillary and reticular fibroblast markers showed decreased expression in young skin and increased expression in aged skin. The densities of collagen and elastin fiber in the aged skin were low, and they were increased by RF. In conclusion, RF leads to increased collagen and elastin fibers by increasing NRF2/GLO-1 and modulating M1/M2 polarization, which leads to decreased AGE and RAGE and, consequently, decreased NF-κB, which eventually slows collagen and elastin destruction. RF also leads to increased collagen and elastin fiber synthesis by increasing papillary and reticular fibroblast expression.

Radiofrequency Irradiation Mitigated UV-B-Induced Skin Pigmentation by Increasing Lymphangiogenesis.

Dermal macrophages containing melanin increase skin pigmentation since dermal melanin removal is slower than epidermal melanin removal. Lymphatic vessels are also involved in melanin clearance. We evaluated whether radiofrequency (RF) irradiation induced an increase in HSP90, which promotes lymphangiogenesis by activating the BRAF/MEK/ERK pathway and decreasing tyrosinase activity, in the UV-B exposed animal model. The HSP90/BRAF/MEK/ERK pathway was upregulated by RF. Tyrosinase activity and the VEGF-C/VEGFR 3/PI3K/pAKT1/2/pERK1/2 pathway, which increase lymphangiogenesis, as well as the expression of the lymphatic endothelial marker LYVE-1, were increased by RF. Additionally, the number of melanin-containing dermal macrophages, the melanin content in the lymph nodes, and melanin deposition in the skin were decreased by RF. In conclusion, RF increased HSP90/BRAF/MEK/ERK expression, which decreased tyrosinase activity and increased lymphangiogenesis to eventually promote the clearance of dermal melanin-containing macrophages, thereby decreasing skin pigmentation.

A Mixture of Topical Forms of Polydeoxyribonucleotide, Vitamin C, and Niacinamide Attenuated Skin Pigmentation and Increased Skin Elasticity by Modulating Nuclear Factor Erythroid 2-like 2.

It is well-known that increased oxidative stress caused by ultraviolet B (UV-B) radiation induces melanogenesis and activates metalloproteinases (MMPs), which degrade collagen and elastin fibers, leading to decreased skin elasticity. Various antioxidant agents, such as vitamin C and niacinamide, have been evaluated for use as treatments for photoaging or skin pigmentation. In this study, we evaluated the ability of a topical liquid formula of polydeoxyribonucleotide (PDRN), vitamin C, and niacinamide (PVN) delivered via a microneedling therapy system (MTS) to attenuate photoaging and pigmentation by increasing nuclear factor erythroid 2-like 2 (NRF2)/heme oxygenase-1 (HO-1) and decreasing MMP expression in a UV-B-radiated animal model. The effects of the PVN were compared with those of individual PDRN and hydroquinone (HQ) compounds. The expression of NRF2/HO-1 significantly increased in response to HQ, PDRN, and PVN in UV-B-radiated animal skin. The activity of nicotinamide adenine dinucleotide phosphate hydrogen oxidase decreased in response to HQ, PDRN, and PVN, and the superoxide dismutase activity increased. The expression of tumor protein p53 and microphthalmia-associated transcription factor and tyrosinase activity decreased in response to HQ, PDRN, and PVN, and this decrease was accompanied by decreased melanin content in the skin. The expression of nuclear factor kappa-light-chain enhancer of activated B cells and MMP2/3/9 decreased in response to HQ, PDRN, and PVN in UV-B-radiated skin. However, the expression of collagen type I α1 chain and the amount of collagen fibers that were evaluated by Masson's trichrome staining increased in response to HQ, PDRN, and PVN. The contents of elastin fibers, fibrillin 1/2 and fibulin 5 increased in response to HQ, PDRN, and PVN. In conclusion, PVN delivered via MTS led to decreased melanogenesis and destruction of collagen and elastin fibers by MMPs, and, thus, PVN decreased skin pigmentation and increased skin elasticity.

Intestinal Epithelial TBK1 Prevents Differentiation of T-helper 17 Cells and Tumorigenesis in Mice.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) regulate intestinal immune cells, particularly development of T-helper 17 (Th17) cells. Deregulation of this process leads to intestinal inflammation and tumorigenesis, via unknown mechanisms. TANK-binding kinase 1 (TBK1) is expressed by IECs and cells in the innate immune system. We studied the functions of TBK1 in the intestinal immune response and tumorigenesis in mice. METHODS: We performed studies of wild-type mice, mice with conditional disruption of Tbk1 (Tbk1IEC-KO), Tbk1IEC-KO mice crossed with ApcMin/+ mice, and Mt-/- mice crossed with ApcMin/+ mice. Some mice were given intraperitoneal injections of a neutralizing antibody against interleukin 17 (IL17) or IL1ß. Intestine tissues were collected from mice and analyzed by histology, for numbers of adenomas and Th17 cells, and expression of inflammatory cytokines by real-time PCR. IECs were isolated from wild-type and Tbk1IEC-KO mice, stimulated with lipopolysaccharide, co-cultured for with bone marrow-derived macrophages, and analyzed by RNA sequencing and biochemical analyses. RESULTS: Compared to ApcMin/+Tbk1WT mice, ApcMin/+Tbk1IEC-KO mice had significant increases in number and size of intestinal polyps, and significantly more Th17 cells in lamina propria. Administration of an antibody against IL17 reduced the number of intestinal polyps in ApcMin/+Tbk1IEC-KO mice to that observed in ApcMin/+Tbk1WT mice. In culture, TBK1-deficient IECs promoted expression of IL1ß by macrophages, which induced differentiation of naïve CD4+ T cells into Th17 cells. RNA sequencing analysis revealed that the TBK1-deficient IECs had increased expression of metallothionein 1 (MT1), an immune regulator that promotes intestinal inflammation. Intestine tissues from ApcMin/+Mt-/- mice had significant fewer Th17 cells than ApcMin/+Mt+/+ mice, and a significantly lower number of polyps. Analyses of colorectal tumors in the Cancer Genome Atlas found colorectal tumors with high levels of MT1 and IL17 mRNAs to be associated with reduced survival times of patients. CONCLUSIONS: Expression of TBK1 by IECs suppresses expression of MT1 and prevents expression of IL1ß by macrophages and differentiation of Th17 cells, to prevent inflammation and tumorigenesis. Strategies to block this pathway might be developed for colorectal tumorigenesis.

mTOR-dependent dysregulation of autophagy contributes to the retinal ganglion cell loss in streptozotocin-induced diabetic retinopathy.

BACKGROUND: Neurodegeneration, an early event in the pathogenesis of diabetic retinopathy (DR), precedes clinically detectable microvascular damage. Autophagy dysregulation is considered a potential cause of neuronal cell loss, however underlying mechanisms remain unclear. The mechanistic target of rapamycin (mTOR) integrates diverse environmental signals to coordinate biological processes, including autophagy. Here, we investigated the role of mTOR signaling in neuronal cell death in DR. METHODS: Diabetes was induced by a single intraperitoneal injection of streptozotocin and tissue samples were harvested at 1, 2, 3, 4, and 6 months of diabetes. Early-stage of DR was investigated in 1-month-diabetic mice treated with phlorizin (two daily subcutaneous injections at a dose of 200 mg/kg of body weight during the last 7 full days of the experiment and the morning of the 8th day, 3 h before sacrifice) or rapamycin (daily intraperitoneal injections, at a dose of 3 mg/kg for the same period as for phlorizin treatment). The effect of autophagy modulation on retinal ganglion cells was investigated in 3-months-diabetic mice treated with phlorizin (two daily subcutaneous injections during the last 10 full days of the experiment and the morning of the 11th day, 3 h before sacrifice) or MHY1485 (daily i.p. injections, at a dose of 10 mg/kg for the same period as for phlorizin treatment). Tissue samples obtained from treated/untreated diabetic mice and age-matched controls were used for Western blot and histologic analysis. RESULTS: mTOR-related proteins and glucose transporter 1 (GLUT1) was upregulated at 1 month and downregulated in the following period up to 6 months. Diabetes-induced neurodegeneration was characterized by an increase of apoptotic marker-cleaved caspase 3, a decrease of the total number of cells, and NeuN immunoreactivity in the ganglion cell layer, as well as an increase of autophagic protein. Insulin-independent glycemic control restored the mTOR pathway activity and GLUT1 expression, along with a decrease of autophagic and apoptotic proteins in 3-months-diabetic mice neuroretina. However, blockade of autophagy using MHY1485 resulted in a more protective effect on ganglion cells compared with phlorizin treatment. CONCLUSION: Collectively, our study describes the mechanisms of neurodegeneration through the hyperglycemia/ mTOR/ autophagy/ apoptosis pathway. Video Abstract.

Fucoidans and Bowel Health.

Fucoidans are cell wall polysaccharides found in various species of brown seaweeds. They are fucose-containing sulfated polysaccharides (FCSPs) and comprise 5-20% of the algal dry weight. Fucoidans possess multiple bioactivities, including antioxidant, anticoagulant, antithrombotic, anti-inflammatory, antiviral, anti-lipidemic, anti-metastatic, anti-diabetic and anti-cancer effects. Dietary fucoidans provide small but constant amounts of FCSPs to the intestinal tract, which can reorganize the composition of commensal microbiota altered by FCSPs, and consequently control inflammation symptoms in the intestine. Although the bioactivities of fucoidans have been well described, there is limited evidence to implicate their effect on gut microbiota and bowel health. In this review, we summarize the recent studies that introduce the fundamental characteristics of various kinds of fucoidans and discuss their potential in altering commensal microorganisms and influencing intestinal diseases.

Evaluating Whether Radiofrequency Irradiation Attenuated UV-B-Induced Skin Pigmentation by Increasing Melanosomal Autophagy and Decreasing Melanin Synthesis.

Autophagy is involved in the degradation of melanosomes and the determination of skin color. TLR4 and tumor necrosis factor (TNF) signaling upregulates NF-kB expression, which is involved in the upregulation of mTOR. The activation of mTOR by UV-B exposure results in decreased autophagy, whereas radiofrequency (RF) irradiation decreases TLR4 and TNF receptor (TNFR) expression. We evaluated whether RF decreased skin pigmentation by restoring autophagy by decreasing the expression of TLR4 or TNFR/NF-κB/mTOR in the UV-B-irradiated animal model. UV-B radiation induced the expressions of TNFR, TLR, and NF-κB in the skin, which were all decreased by RF irradiation. RF irradiation also decreased phosphorylated mTOR expression and upregulated autophagy initiation factors such as FIP200, ULK1, ULK2, ATG13, and ATG101 in the UV-B-irradiated skin. Beclin 1 expression and the expression ratio of LC3-I to LC3-II were increased by UV-B/RF irradiation. Furthermore, melanin-containing autophagosomes increased with RF irradiation. Fontana-Masson staining showed that the amount of melanin deposition in the skin was decreased by RF irradiation. This study showed that RF irradiation decreased skin pigmentation by restoring melanosomal autophagy, and that the possible signal pathways which modulate autophagy could be TLR4, TNFR, NF-κB, and mTOR.

Attenuation Effect of Radiofrequency Irradiation on UV-B-Induced Skin Pigmentation by Decreasing Melanin Synthesis and through Upregulation of Heat Shock Protein 70.

Excess melanin deposition in the skin causes cosmetic problems. HSP70 upregulation decreases microphthalmia-associated transcription factor (MITF) expression, which eventually decreases tyrosinase activity and melanogenesis. Ultraviolet (UV) radiation upregulates p53, which increases the melanocortin receptor (MC1R) and MITF. Furthermore, HSP70 decreases p53 and radiofrequency irradiation (RF) increases HSP70. We evaluated whether RF increased HSP70 and decreased p53, consequently decreasing the MITF/tyrosinase pathway and melanogenesis in UV-B radiated animal skin. Various RF combinations with 50, 100, and 150 ms and 5, 10, and 15 W were performed on the UV-B radiated mouse skin every 2 d for 28 d. When RF was performed with 100 ms/10 W, melanin deposition, evaluated by Fontana-Masson staining, decreased without skin crust formation in the UV-B radiated skin. Thus, we evaluated the effect of RF on decreasing melanogenesis in the HEMn and UV-B radiated skin at a setting of 100 ms/10 W. HSP70 expression was decreased in the UV-B radiated skin but was increased by RF. The expression of p53, MC1R, and MITF increased in the UV-B radiated skin but was decreased by RF. The expression of p53, MC1R, and MITF increased in the α-MSH treated HEMn but was decreased by RF. The decreasing effects of RF on p53, MC1R, CREB and MITF were higher than those of HSP70-overexpressed HEMn. The decreasing effect of RF on p53, MC1R, CREB, and MITF disappeared in the HSP70-silenced HEMn. MC1R, CREB, and MITF were not significantly decreased by the p53 inhibitor in α-MSH treated HEMn. RF induced a greater decrease in MC1R, CREB, and MITF than the p53 inhibitor. Therefore, RF may have decreased melanin synthesis by increasing HSP70 and decreasing p53, thus decreasing MC1R/CREB/MITF and tyrosinase activity.

mTORC1 and mTORC2 are differentially engaged in the development of laser-induced CNV.

BACKGROUND: The mechanistic target of rapamycin (mTOR) pathway is a potential target to inhibit pathologic processes in choroidal neovascularization. However, the exact role of mTOR signaling in the development of CNV remains obscure. In this study, we assessed the role of mTORC1 and mTORC2 as well as the effect of rapamycin (sirolimus) on choroidal neovascularization (CNV) in a laser-induced mouse model. METHODS: In experiment A, we observed the natural course of CNV development and the dynamics of mTOR-related proteins during the 12 days after the laser injury. The expression of mTOR-related proteins was evaluated using Western blot (WB). Cryosections of CNV-induced mice were immunostained for the visualization of the vascular and extravascular components of the CNV. Experiment B was performed to confirm the critical period of mTOR signaling in the development of laser-induced CNV, we administered rapamycin before and/or during the active period of mTOR complexes. WB and immunofluorescence staining was performed to evaluate the mode of action and the effect of mTOR inhibition on CNV development. RESULTS: In experiment A, we detected high levels of p-mTOR S2448 and p-mTOR S2481 from the 5th to 12th day of laser injury. Immunofluorescence imaging of cryosections of mice sacrificed on day 7 revealed greater co-immunoreactivity of p-mTOR S2448 positive cells with CD11b and F4/80, while p-mTOR S2481 positive cells showed colocalization with CD31, α-SMA, and cytokeratin. In experiment B, rapamycin injection during the active period of mTOR signaling demonstrated near-complete inhibition of CNV lesion as well as significant induction of autophagy. CONCLUSION: Our study suggests the mTOR as a critical player during CNV development in laser-induced mouse model through differentially acting with the mTORC1 and mTORC2. mTORC1 activity was high predominantly in inflammatory cells in CNV lesion, while mTORC2 activity was higher in vascular components and the RPE.