Model of Chronic Itch in Aged Mice: Beneficial Effects of Drugs Affecting Descending Modulatory Systems.

Pruritus in the elderly, particularly those cases without skin dryness or other identifiable causes, makes treatment challenging due to the lack of evidence regarding the therapeutic effects of antipruritics. This study proposes an age-related alloknesis mouse model for an evaluation system for such cases, and aimed to investigate the effectiveness and mechanisms of action of several drugs commonly used as antipruritics in Japan, utilizing this model. Mice 69-80 weeks old were used as aged mice, and the level of mechanical alloknesis was counted as the number of scratching behaviours in response to innocuous stimuli. Bepotastine, neurotropin, pregabalin, baricitinib, and abrocitinib were used as antipruritics, and yohimbine and methysergide as inhibitors of the descending inhibitory pathway. The findings suggest that mechanical alloknesis in aged mice is a suitable animal model for assessing pruritus in the elderly without xerosis, and pregabalin, neurotropin, baricitinib, and abrocitinib may be effective antipruritics in the elderly through activating both the noradrenergic and serotonergic descending inhibitory pathways. These findings may be useful for the selection of antipruritics for pruritus in the elderly without skin lesions or dryness.

Effects of Psychological Stress on Spontaneous Itch and Mechanical Alloknesis of Atopic Dermatitis.

Atopic dermatitis (AD), a chronic inflammatory skin disease, manifests as an intractable itch. Psychological stress has been suggested to play a role in the onset and worsening of AD symptoms. However, the pathophysiological relationships between psychological stressors and cutaneous manifestations remain unclear. To elucidate the mechanisms underlying the stress-related exacerbation of itch, we investigated the effects of water stress, restraint stress and repeated social defeat stress on itch-related scratching behaviour, mechanical alloknesis and dermatitis in male NC/Nga mice with AD-like symptoms induced by the repeated application of ointment containing Dermatophagoides farina body. NC/Nga mice with AD-like symptoms were subjected to water stress, restraint stress and repeated social defeat stress, and their scratching behaviour, sensitivity to mechanical stimuli (mechanical alloknesis) and severity of  dermatitis were evaluated. Social defeat stress+ Dermatophagoides farina body-treated mice exposed to stress showed slower improvements in or the exacerbation of AD-like symptoms, including dermatitis and itch. In the mechanical alloknesis assay, the mechanical alloknesis scores of social defeat stress+ Dermatophagoides farina body-treated mice exposed to stress were significantly higher than those of non-exposed social defeat stress+ Dermatophagoides farina body- and social defeat stress-treated mice. These results suggest that psychological stress delays improvements in dermatitis by exacerbating itch hypersensitivity in AD.

Peripheral endomorphins drive mechanical alloknesis under the enzymatic control of CD26/DPPIV.

BACKGROUND: Mechanical alloknesis (or innocuous mechanical stimuli-evoked itch) often occurs in dry skin-based disorders such as atopic dermatitis and psoriasis. However, the molecular and cellular mechanisms underlying mechanical alloknesis remain unclear. We recently reported the involvement of CD26 in the regulation of psoriatic itch. This molecule exhibits dipeptidyl peptidase IV (DPPIV) enzyme activity and exerts its biologic effects by processing various substances, including neuropeptides. OBJECTIVE: The aim of the present study was to investigate the peripheral mechanisms of mechanical alloknesis by using CD26/DPPIV knockout (CD26KO) mice. METHODS: We applied innocuous mechanical stimuli to CD26KO or wild-type mice. The total number of scratching responses was counted as the alloknesis score. Immunohistochemical and behavioral pharmacologic analyses were then performed to examine the physiologic activities of CD26/DPPIV or endomorphins (EMs), endogenous agonists of µ-opioid receptors. RESULTS: Mechanical alloknesis was more frequent in CD26KO mice than in wild-type mice. The alloknesis score in CD26KO mice was significantly reduced by the intradermal administration of recombinant DPPIV or naloxone methiodide, a peripheral µ-opioid receptor antagonist, but not by that of mutant DPPIV without enzyme activity. EMs (EM-1 and EM-2), selective ligands for µ-opioid receptors, are substrates for DPPIV. Immunohistochemically, EMs were located in keratinocytes, fibroblasts, and peripheral sensory nerves. Behavioral analyses revealed that EMs preferentially provoked mechanical alloknesis over chemical itch. DPPIV-digested forms of EMs did not induce mechanical alloknesis. CONCLUSION: The present results suggest that EMs induce mechanical alloknesis at the periphery under the enzymatic control of CD26/DPPIV.

Exposure of female NZBWF1 mice to imiquimod-induced lupus nephritis at an early age via a unique mechanism that differed from spontaneous onset.

Systemic lupus erythematosus (SLE) is a chronic inflammatory and representative autoimmune disease. Extremely complicated and multifactorial interactions between various genetic factors and individual susceptibility to environmental factors are involved in the pathogenesis of SLE. Several studies have reported that mutation and activation of toll-like receptor (TLR) 7 are involved in the onset of autoimmunity, including SLE. Thus, we investigated the response of SLE-prone mice to continuous environmental factors, particularly TLR7 agonist exposure, and changes in their phenotypes. Female and male NZBWF1 (BWF1) mice were treated from 20 weeks of age with a TLR7 agonist, imiquimod (IMQ), 3 times weekly for up to 12 weeks. IMQ-exposed female BWF1 mice showed worsened lupus nephritis. However, autoantibody production was not enhanced in IMQ-exposed female BWF1 mice. The Th1 cytokine expression was upregulated in the kidney of IMQ-treated mice. In IMQ-exposed BWF1 mice, neutralization of IFN-γ suppressed early-phase lupus nephritis. Additionally, in male BWF1 mice IMQ exposure induced minor aggravation of lupus nephritis. These results suggest that the induction of aggravated lupus nephritis by TLR7 agonist exposure was related to the expression of IFN-γ via acute TLR7 signal-induced renal inflammation, and that the involvement of genetic factors associated with a predisposition to SLE is also essential. Thus, the activation of TLR7 signaling by exposure to environmental factors may upset the balance of factors that maintain SLE remission. We hypothesize that the inhibition of TLR7 signaling and IFN-γ signaling is effective for preventing the onset and flare and maintaining remission of lupus nephritis.

Skin Treatment with Detergent Induces Dermatitis with H1-Antihistamine-Refractory Itch and Upregulates IL-4 and Th17/Th22 Cytokine Gene Expression in C57BL/6 Mice.

INTRODUCTION: Repeated skin contact to detergents causes chronic irritant contact dermatitis (ICD) associated with itch sensation and eczema. However, the mechanisms of detergent-induced ICD are poorly understood. Here, we established a new murine model of detergent-induced ICD with H1-antihistamine-refractory itch. METHODS: Ear skin of wild-type and mast cell-deficient mice on the C57BL/6 genetic background was treated with a detergent, sodium dodecyl/lauryl sulfate (SDS), daily for approximately 2 weeks with or without administration of an H1-antihistamine, fexofenadine. Skin inflammation, barrier dysfunction, and itching were analyzed. Quantitative PCR for earlobe gene expression and flow cytometry analysis for draining lymph node cells were conducted. RESULTS: SDS treatment induced skin inflammation with ear swelling, increased transepidermal water loss, and hind-paw scratching behaviors in the wild-type and mast cell-deficient mice. The peak value of scratching bouts was retained for at least 48 h after the last SDS treatment. H1-antihistamine administration showed no or little reduction in the responses. SDS treatment upregulated gene expression for a Th2 cytokine IL-4 and Th17/Th22 cytokines, IL-17A, IL-17F, and IL-22, and increased cell numbers in draining lymph nodes of CD4+ T, CD8+ T, and γδT cells with enhanced expression of GATA3, RORγt, T-bet, or FOXP3 compared with untreated mice. CONCLUSIONS: The present study showed that SDS treatment of ear skin in C57BL/6 mice induces mast cell-independent skin inflammation with H1-antihistamine-refractory itch and suggested a possible Th cytokine- and/or lymphocyte-mediated regulation of the model. The model would be useful for elucidation of mechanisms for inflammation with H1-antihistamine-refractory itch in detergent-induced ICD.

Experimental and Clinical Evidence Suggests That Treatment with Betacellulin Can Alleviate Th2-Type Cytokine-Mediated Impairment of Skin Barrier Function.

Betacellulin (BTC) is a peptide ligand that belongs to the epidermal growth factor family, the members of which have been implicated in skin morphogenesis, homeostasis, repair, and angiogenesis; however, the role of BTC in the regulation of the skin barrier remains unknown. To examine the role of BTC in skin barrier function, we analyzed atopic dermatitis (AD) transcriptomic data from Gene Expression Omnibus (GEO) datasets, performed BTC immunohistochemistry using human skin tissues, and evaluated the effects of BTC on primary human keratinocytes by real-time PCR, Western blotting, and assay of the transepidermal electrical resistance (TER), a functional parameter to monitor the tight junction barrier. We found that the gene expression of BTC was downregulated in skin lesions from patients with AD, and this downregulated expression recovered following biological treatments. Consistently, the BTC protein levels were downregulated in the lesional skin of AD patients compared with the normal skin of healthy participants, suggesting that the BTC levels in skin might be a biomarker for the diagnosis and therapy of AD. Furthermore, in human keratinocytes, BTC knockdown reduced the levels of skin-derived antimicrobial peptides and skin barrier-related genes, whereas BTC addition enhanced their levels. Importantly, in human skin equivalents, BTC restored the increased tight junction permeability induced by Th2 cytokine IL-4/IL-13 treatment. In addition, specific inhibitors of epidermal growth factor receptor (EGFR) and protein kinase C (PKC) abolished the BTC-mediated improvement in skin barrier-related proteins in keratinocyte monolayers. Collectively, our findings suggest that treatment with BTC might improve the Th2-type cytokine-mediated impairment of skin barrier function through the EGFR/PKC axis and that BTC might be a novel potential biomarker and therapeutic target for the treatment of skin conditions characterized by the overproduction of Th2 cytokines and dysfunctional skin barriers, such as AD.

Peripheral itch sensitization in atopic dermatitis.

Atopic dermatitis is a skin disorder caused by skin dryness and barrier dysfunction, resulting in skin inflammation and chronic itch (or pruritus). The pathogenesis of atopic dermatitis is thought to be initiated by a lowering of the itch threshold due to dry skin. This lowering of the itch threshold is at least partially due to the increase in intraepidermal nerve fibers and sensitization of sensory nerves by interleukin (IL)-33 produced and secreted by keratinocytes. Such skin is easily prone to itch due to mechanical stimuli, such as rubbing of clothing and chemical stimuli from itch mediators. In patients with atopic dermatitis, once itch occurs, further itch is induced by scratching, and the associated scratching breaks down the skin barrier. Disruption of the skin barrier allows entry into the epidermis of external foreign substances, such as allergens derived from house dust mites, leading to an increased induction of type 2 inflammatory responses. As a result, type 2 cytokines IL-4, IL-13, and IL-31 are mainly secreted by Th2 cells, and their action on sensory nerve fibers causes further itch sensitization. These sequences of events are thought to occur simultaneously in patients with atopic dermatitis, leading to a vicious itch-scratch cycle. This vicious cycle becomes a negative spiral that leads to disease burden. Therefore, controlling itch is essential for the treatment of atopic dermatitis. In this review, we summarize and discuss advances in the mechanisms of peripheral itch sensitization in atopic dermatitis, focusing on skin barrier-neuro-immune triadic connectivity.

Lysophosphatidylglucoside is a GPR55 -mediated chemotactic molecule for human monocytes and macrophages.

Neutrophils undergo spontaneous apoptosis within 24-48 h after leaving bone marrow. Apoptotic neutrophils are subsequently phagocytosed and cleared by macrophages, thereby maintaining neutrophil homeostasis. Previous studies have demonstrated involvement of lysophosphatidylglucoside (lysoPtdGlc), a degradation product of PtdGlc, in modality-specific repulsive guidance of spinal sensory axons, via its specific receptor GPR55. In the present study, using human monocytic cell line THP-1 as a model, we demonstrated that lysoPtdGlc induces monocyte/macrophage migration with typical bell-haped curve and a peak at concentration 10-9 M. Lysophosphatidylinositol (lysoPtdIns), a known GPR55 ligand, induced migration at higher concentration (10-7 M). LysoPtdGlc-treated cells had a polarized shape, whereas lysoPtdIns-treated cells had a spherical shape. In EZ-TAXIScan (chemotaxis) assay, lysoPtdGlc induced chemotactic migration activity of THP-1 cells, while lysoPtdIns induced random migration activity. GPR55 antagonist ML193 inhibited lysoPtdGlc-induced THP-1 cell migration, whereas lysoPtdIns-induced migration was inhibited by CB2-receptor inverse agonist. SiRNA experiments showed that GPR55 mediated lysoPtdGlc-induced migration, while lysoPtdIns-induced migration was mediated by CB2 receptor. Our findings, taken together, suggest that lysoPtdGlc functions as a chemotactic molecule for human monocytes/macrophages via GPR55 receptor, while lysoPtdIns induces random migration activity via CB2 receptor.

Metformin repositioning in rheumatoid arthritis.

OBJECTIVES: Metformin is a known therapeutic agent for diabetes. Recently, several reports suggested the possibility of improvement in autoimmune disease and malignancy conditions through the effect of metformin on the immune system. Although there have been reports on the therapeutic effects of metformin on mouse models of collagen-induced arthritis, simulating human rheumatoid arthritis (RA), the effect of metformin on human RA remains unknown. Therefore, we investigated the inhibitory effect of metformin on the pathogenesis of human RA in vitro. METHODS: Osteoclastogenesis was evaluated with or without metformin. through tartrate-resistant acid phosphatase staining, osteoclast-specific enzyme expression analysis, and a bone resorption assay. Human fibroblast-like synoviocyte MH7A cells were stimulated with TNF-α, and the expression of proinflammatory cytokines and protease and growth factor genes was evaluated with or without metformin. Metformin has been used to evaluate their potential modulatory effects on cells treated with TNF-α. Moreover, we examined angiogenesis by performing a tube formation assay using human umbilical vein endothelial cells (HUVECs) with or without metformin. RESULTS: Osteoclastogenesis was suppressed in the presence of metformin, and the expression of osteoclast-specific genes was reduced. The TNF-α-induced expression of inflammatory cytokines and protease and growth factor genes in MH7A cells was downregulated by metformin. Additionally, the induced formation of tubular networks in HUVECs was also disrupted following treatment with metformin. CONCLUSIONS: These results suggest that metformin might improve the pathogenesis of RA, including joint inflammation and destruction. Thus, metformin might be utilised as a potential therapeutic agent in the treatment of RA.

Role of Eosinophil Relative Count and Neutrophil-to-Lymphocyte Ratio in the Assessment of Severity of Atopic Dermatitis.

The aim of this study is to elucidate the relationship between 2 different types of severity-indicating parameters (i.e. between subjective and objective severity-indicating parametersin patients with atopic dermatitis. The disease severity of 55 patients with atopic dermatitis was assessed using 7 subjective parameters indicating severity, including visual analogue scale for itch, Patient-Oriented Eczema Measure, 5-D itch scale, Dermatology Life Quality Index, Eczema Area and Severity Index, body surface area, and Investigator Global Assessment, and 8 objective parameters indicating severity, including eosinophil relative count, neutrophil-to-lymphocyte ratio, lactate dehydrogenase, and thymus and activation-regulated chemokine. Five subjective parameters reflecting itch correlated significantly with eosinophil relative count, but not with neutrophil-to-lymphocyte ratio. In contrast, 2 subjective parameters, mainly reflecting the degree of inflammation and area of affected regions, correlated significantly with neutrophil-to-lymphocyte ratio. The eosinophil relative count may correlate with the degree of itch, while the neutrophil-to-lymphocyte ratio may correlate with the degree of inflammation and the area of the affected region. The eosinophil relative count and neutrophil-to-lymphocyte ratio may thus be stand-alone parameters from each other in the assessment of the severity of atopic dermatitis.

Connections between Immune-Derived Mediators and Sensory Nerves for Itch Sensation.

Although histamine is a well-known itch mediator, histamine H1-receptor blockers often lack efficacy in chronic itch. Recent molecular and cellular based studies have shown that non-histaminergic mediators, such as proteases, neuropeptides and cytokines, along with their cognate receptors, are involved in evocation and modulation of itch sensation. Many of these molecules are produced and secreted by immune cells, which act on sensory nerve fibers distributed in the skin to cause itching and sensitization. This understanding of the connections between immune cell-derived mediators and sensory nerve fibers has led to the development of new treatments for itch. This review summarizes current knowledge of immune cell-derived itch mediators and neuronal response mechanisms, and discusses therapeutic agents that target these systems.

Regulatory T Cells Exhibit Interleukin-33-Dependent Migratory Behavior during Skin Barrier Disruption.

Regulatory T cells (Tregs) suppress immune responses and maintain immunological self-tolerance and homeostasis. We currently investigated relationships between skin barrier condition and Treg behavior using skin barrier-disrupted mice. Skin barrier disruption was induced by repeated topical application of 4% sodium dodecyl sulfate (SDS) on mice. The number of CD4+ forkhead box protein P3 (Foxp3)+ Tregs was higher in 4% SDS-treated skins than in controls. This increasing was correlated with the degree of acanthosis. The numbers of interleukin (IL)-10+ and transforming growth factor (TGF)-ß+ Tregs also increased in 4% SDS-treated skins. Localization of IL-33 in keratinocytes shifted from nucleus to cytoplasm after skin barrier disruption. Notably, IL-33 promoted the migration of Tregs in chemotaxis assay. The skin infiltration of Tregs was cancelled in IL-33 neutralizing antibody-treated mice and IL-33 knockout mice. Thus, keratinocyte-derived IL-33 may induce Treg migration into barrier-disrupted skin to control the phase transition between healthy and inflammatory conditions.

A Novel In Vitro Assay Using Human iPSC-Derived Sensory Neurons to Evaluate the Effects of External Chemicals on Neuronal Morphology: Possible Implications in the Prediction of Abnormal Skin Sensation.

Neuronal morphological changes in the epidermis are considered to be one of causes of abnormal skin sensations in dry skin-based skin diseases. The present study aimed to develop an in vitro model optimised for human skin to test the external factors that lead to its exacerbation. Human-induced pluripotent stem cell-derived sensory neurons (hiPSC-SNs) were used as a model of human sensory neurons. The effects of chemical substances on these neurons were evaluated by observing the elongation of nerve fibers, incidence of blebs (bead-like swellings), and the expression of nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2). The nerve fiber length increased upon exposure to two common cosmetic preservatives-methylparaben and phenoxyethanol-but not to benzo[a]pyrene, an air pollutant at the estimated concentrations in the epidermis. Furthermore, the incidence of blebs increased upon exposure to benzo[a]pyrene. However, there was a decrease in the expression of NMNAT2 in nerve fibers, suggesting degenerative changes. No such degeneration was found after methylparaben or phenoxyethanol at the estimated concentrations in the epidermis. These findings suggest that methylparaben and phenoxyethanol promote nerve elongation in hiPSC-SNs, whereas benzo[a]pyrene induces nerve degeneration. Such alterations may be at least partly involved in the onset and progression of sensitive skin.

Social defeat stress exacerbates atopic dermatitis through downregulation of DNA methyltransferase 1 and upregulation of C-C motif chemokine receptor 7 in skin dendritic cells.

DNA methylation is an epigenetic modification that regulates gene transcription. DNA methyltransferase 1 (DNMT1) plays an important role in DNA methylation. However, the involvement of DNMT1 and DNA methylation in the pathogenesis of atopic dermatitis (AD) remains unclear. In this study, microarray analysis revealed that peripheral blood mononuclear cells of AD patients with low DNMT1 expression (DNMT1-low) highly expressed dendritic cell (DC) activation-related genes. Also, DNMT1-low AD patients exhibited a higher itch score compared to AD patients with high DNMT1 expression (DNMT1-high). By using an AD-like mouse model induced by the application of Dermatophagoides farinae body ointment, we found that Dnmt1 expression was decreased, while the expression of C-C chemokine receptor type 7 (Ccr7) was upregulated in mouse skin DCs. Furthermore, mice exposed to social defeat stress exhibited Dnmt1 downregulation and Ccr7 upregulation in skin DCs. Additionally, dermatitis and itch-related scratching behavior were exacerbated in AD mice exposed to stress. The relationship between low DNMT1 and itch induction was found in both human AD patients and AD mice. In mouse bone marrow-derived DCs, Ccr7 expression was inhibited by 5-aza-2-deoxycytidine, a methylation inhibitor. Furthermore, in mouse skin DCs, methylation of CpG sites in Ccr7 was modified by either AD induction or social defeat stress. Collectively, these findings suggest that social defeat stress exacerbates AD pathology through Dnmt1 downregulation and Ccr7 upregulation in mouse skin DCs. The data also suggest a role of DNMT1 downregulation in the exacerbation of AD pathology.

Mechanisms and Management of Itch in Dry Skin.

Chronic itch is a burdensome clinical problem that often accompanies pathological dry skin-based conditions, such as atopic dermatitis, and systemic disorders, such as kidney diseases, with an unclear pathomechanism and treatments. One of the basic mouse models to investigate mechanisms of itch associated with dry skin is a mixture of acetone and ether followed by water. Animal studies using the acetone and ether followed by water model have revealed that many mediators and receptors, e.g. mas-related G protein-coupled receptor family, transient receptor potential, and chemokines, are responsible for itch and its hypersensitivity, supporting the hypothesis that dry skin-induced itch is a histamine-independent pathway. New insights have been acquired into the interplay between neurones and non-neuronal cells in the initiation, modulation, and sensitization of itch. Several thera-peutic options for itching have thus been developed. This review summarizes the updated pathogenesis and therapeutic strategies for itch in dry skin conditions.

Effects of 308 nm excimer light treatment on the skin microbiome of atopic dermatitis patients.

BACKGROUND: The skin microbiome has been implicated in the pathophysiology of atopic dermatitis (AD). Although 308 nm excimer light treatment is an effective phototherapy for AD, its effects on the skin microbiome currently remain unclear. Therefore, we investigated the effects of the excimer light treatment on the skin bacterial and fungal microbiome of lesional skin of AD. METHODS: Swab samples were collected from 11 healthy controls, non-lesional and lesional skin of 11 AD patients. The excimer light treatment was administered to the lesional skin. The composition of the skin microbiome, the clinical score and skin barrier function of the lesional skin were examined before and after the treatment. The composition of the skin microbiome was determined by sequencing bacterial 16S and fungal internal transcribed spacer regions. RESULTS: The excimer light treatment significantly changed the composition of the bacterial microbiome in the lesional skin of AD, as well as improved the clinical score and skin barrier function. The treatment increased the relative abundance of the phylum Cyanobacteria and decreased that of the phylum Bacteroidetes in lesional skin. At the species level, the treatment significantly decreased the relative abundance of Staphylococcus aureus (S aureus) in lesional skin. There was also a significant correlation between the reduction of S aureus and improvement of the clinical outcomes. CONCLUSION: Our findings suggest that alterations of the skin microbiome with excimer light treatment, specifically the decrease in the abundance of S aureus, are partly involved in the improvement of AD lesions.

Molecular and Cellular Mechanisms of Itch in Psoriasis.

Itch (or pruritus) was not previously recognized as a serious symptom of psoriasis. However, approximately 60-90% of psoriatic patients with pruritus have stated that it deteriorates their quality of life. Since conventional antipruritic therapies, such as antihistamines, only exert limited effects, the establishment of a treatment option for itch in psoriasis is urgently needed. Although a definitive drug is not currently available, various itch mediators are known to be involved in pruritus in psoriasis. In this review, we describe the clinical features of pruritus in psoriasis, classify a wide range of itch mediators into categories, such as the nervous, immune, endocrine, and vascular systems, and discuss the mechanisms by which these mediators induce or aggravate itch in the pathophysiology of psoriasis.

NUP62: the target of an anti-sperm auto-monoclonal antibody during testicular development.

Ts4, an autosperm-monoclonal antibody (mAb), reacts with a specific oligosaccharide (OS) of glycoproteins containing bisecting N-acetylglucosamine residues. Ts4 reactivity was observed against epididymal spermatozoa, testicular germ cells, and the early embryo, but not against major organs in adult mice. In mature testis, Ts4 exhibits immunoreactivity with a germ cell-specific glycoprotein, TEX101, whereas the mAb immunoreacts with alpha-N-acetylglucosaminidase in the acrosomal region of cauda epididymal spermatozoa. Thus, Ts4 seems to react against different molecules throughout spermiogenesis via binding to its OS epitope. Since the Ts4-epitope OS is observed only in reproduction-related regions, the Ts4-reactive OS may play a role in the reproductive process. The aim of this study is to investigate the characteristics of the Ts4-reactive molecule(s) during testicular development. Ts4 reactivity was observed in testes from the prenatal period; however, its distribution changed according to the stage of maturation and was identical to that of the adult testes after 29-day-postpartum (dpp). Ts4 immunoreactivity was detected against a protein with 63 kDa in testis from 1 to 29 dpp. In contrast, Ts4 showed reactivity against some other glycoproteins after 29 dpp, including TEX101 at the 5-week-old stage and onward. To identify the Ts4-reactive 63 kDa molecule, we identified NUP62 as the target of Ts4 in 22 dpp testis using liquid chromatography-tandem mass spectrometry analysis. Because NUP62 has been known to play active roles in a variety of cellular processes including mitosis and cell migration, the bisecting GlcNAc recognized by Ts4 on NUP62 may play a role in regulating the early development of germ cells in male gonadal organs.

Effect of Propofol on the Production of Inflammatory Cytokines by Human Polarized Macrophages.

Macrophages are key immune system cells involved in inflammatory processes. Classically activated (M1) macrophages are characterized by strong antimicrobicidal properties, whereas alternatively activated (M2) macrophages are involved in wound healing. Severe inflammation can induce postoperative complications during the perioperative period. Invasive surgical procedures induce polarization to M1 macrophages and associated complications. As perioperative management, it is an important strategy to regulate polarization and functions of macrophages during inflammatory processes. Although propofol has been found to exhibit anti-inflammatory activities in monocytes and macrophages, it is unclear whether propofol regulates the functions of M1 and M2 macrophages during inflammatory processes. This study therefore investigated the effects of propofol on human macrophage polarization. During M1 polarization, propofol suppressed the production of IL-6 and IL-1ß but did not affect TNF-α production. In contrast, propofol did not affect the gene expression of M2 markers, such as IL-10, TGF-ß, and CD206, during M2 polarization. Propofol was similar to the GABAA agonist muscimol in inducing nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and inhibiting IL-6 and IL-1ß, but not TNF-α, production. Knockdown of Nrf2 using siRNA significantly reduced the effect of propofol on IL-6 and IL-1ß production. These results suggest that propofol prevents inflammatory responses during polarization of human M1 macrophages by suppressing the expression of IL-6 and IL-1ß through the GABAA receptor and the Nrf2-mediated signal transduction pathway.

MicroRNA-766-3p Contributes to Anti-Inflammatory Responses through the Indirect Inhibition of NF-κB Signaling.

MicroRNA (miRNA) is small RNA of 20 to 22 nucleotides in length and is stably present in plasma. Regulating the expression of miRNA taken into cells has been suggested as a general therapeutic approach. We identified the novel anti-inflammatory miRNA hsa-miR-766-3p and investigated its biological function in human rheumatoid arthritis (RA) fibroblast-like synoviocyte MH7A cells. To verify the function of the miRNA present in the plasma of RA patients, we performed a comprehensive analysis of the miRNA expression during abatacept treatment and identified eight miRNAs with significantly altered expression levels. Among these eight miRNAs, miR-766-3p was found to have a clear function. The expression of inflammatory genes in response to inflammatory stimuli was suppressed in MH7A transduced with miR-766-3p. We showed that miR-766-3p indirectly reduced the activation of NF-κB and clarified that this mechanism was partially involved in the reduction of the mineralocorticoid receptor expression. In addition, the inflammatory responses were suppressed in other types of cells. These results indicate the novel function of miR-766-3p, findings that may aid in the development of therapies to suppress inflammation, not only in RA but also in other diseases.