Macrophage polarization toward M1 phenotype in T cell transfer colitis model.

BACKGROUND: T cell transfer colitis model is often used to study the CD4+ T cell functions in the intestine. However, the specific roles of macrophages in colitis remain unclear. In this study, we aimed to evaluate the phenotype and functions of macrophages in the colonic lamina propria (LP) in a colitis model. METHODS: Colitis was induced in scid mice via the adaptive transfer of CD4+CD45RBhi T cells. Then, flow cytometry was used to determine the number of macrophages in the colonic LP and expression of cytokines in macrophages at the onset of colitis. Moreover, M1/M2 macrophage markers were detected in the colonic LP during colitis development using high-dimensional single-cell data and gating-based analyses. Expression levels of M1 markers in macrophages isolated from the colonic LP were measured using quantitative reverse transcription-polymerase chain reaction. Additionally, macrophages were co-cultured with T cells isolated from the colon to assess colitogenic T cell activation. RESULTS: Infiltration of macrophages into the colon increased with the development of colitis in the T cell transfer colitis model. M1/M2 macrophage markers were observed in this model, as observed in the colon of patients with inflammatory bowel disease (IBD). Moreover, number of M1 macrophages increased, whereas that of M2 macrophages decreased in the colonic LP during colitis development. M1 macrophages were identified as the main source of inflammatory cytokine production, and colitogenic T cells were activated via interactions with these macrophages. CONCLUSIONS: Our findings revealed that macrophages polarized toward the M1 phenotype in LP during colitis development in the T cell transfer colitis model. Therefore, the colitis model is suitable for the evaluation of the efficacy of macrophage-targeted drugs in human IBD treatment. Furthermore, this model can be used to elucidate the in vivo functions of macrophages in the colon of patients with IBD.

Application of Piezoelectric PLLA Braided Cord as Wearable Sensor to Realize Monitoring System for Indoor Dogs with Less Physical or Mental Stress.

We attempted to realize a prototype system that monitors the living condition of indoor dogs without physical or mental burden by using a piezoelectric poly-l-lactic acid (PLLA) braided cord as a wearable sensor. First, to achieve flexibility and durability of the piezoelectric PLLA braided cord used as a sensor for indoor dogs, the process of manufacturing the piezoelectric PLLA fiber for the piezoelectric braided cord was studied in detail and improved to achieve the required performance. Piezoelectric PLLA braided cords were fabricated from the developed PLLA fibers, and the finite element method was used to realize an e-textile that can effectively function as a monitoring sensor. As a result, we realized an e-textile that feels similar to a high-grade textile and senses the complex movements of indoor dogs without the use of a complex computer system. Finally, a prototype system was constructed and applied to an actual indoor dog to demonstrate the usefulness of the e-textile as a sensor for indoor dog monitoring.

Pituitary adenylate cyclase-activating polypeptide promotes cutaneous dendritic cell functions in contact hypersensitivity.

BACKGROUND: Sensory nerves regulate cutaneous local inflammation indirectly through induction of pruritus and directly by acting on local immune cells. The underlying mechanisms for how sensory nerves influence cutaneous acquired immune responses remain to be clarified. OBJECTIVE: This study aimed to explore the effect of peripheral nerves on cutaneous immune cells in cutaneous acquired immune responses. METHODS: We analyzed contact hypersensitivity (CHS) responses as a murine model of delayed-type hypersensitivity in absence or presence of resiniferatoxin-induced sensory nerve denervation. We conducted ear thickness measurements, flow cytometric analyses, and mRNA expression analyses in CHS. RESULTS: CHS responses were attenuated in mice that were denervated during the sensitization phase of CHS. By screening neuropeptides, we found that pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA expression was decreased in the dorsal root ganglia after denervation. Administration of PACAP restored attenuated CHS response in resiniferatoxin-treated mice, and pharmacological inhibition of PACAP suppressed CHS. Flow cytometric analysis of skin-draining lymph nodes showed that cutaneous dendritic cell migration and maturation were reduced in both denervated mice and PACAP antagonist-treated mice. The expression of chemokine receptors CCR7 and CXCR4 of dendritic cell s was enhanced by addition of PACAP in vitro. CONCLUSION: These findings indicate that a neuropeptide PACAP promotes the development of CHS responses by inducing cutaneous dendritic cell functions during the sensitization phase.

A novel JAK inhibitor JTE-052 reduces skin inflammation and ameliorates chronic dermatitis in rodent models: Comparison with conventional therapeutic agents.

Janus kinases (JAKs) are required for several inflammatory cytokine signalling pathways and are implicated in the pathogenesis of chronic dermatitis, including atopic dermatitis and psoriasis. JAK inhibitors are therefore promising therapeutic candidates for chronic dermatitis. In this study, we evaluated the effects of the novel JAK inhibitor JTE-052 on inflammatory responses associated with chronic dermatitis, and compared its profile with those of conventional therapeutic agents in rodent models of chronic dermatitis. JTE-052 inhibited the Th1-, Th2- and Th17-type inflammatory responses of human T cells and mast cells in vitro. Oral administration of JTE-052 inhibited skin inflammation in hapten-induced chronic dermatitis in mice, associated with reduced levels of inflammatory cytokines in the skin and immunoglobulin (Ig) E in serum. In contrast, although ciclosporin partly inhibited skin inflammation, it did not reduce interleukin (IL)-4 production in skin, and enhanced IgE production in serum. Oral administration of JTE-052 also inhibited skin inflammation in mouse models of atopic dermatitis and psoriasis induced by a mite extract, thymic stromal lymphopoietin or IL-23. The maximal efficacy of JTE-052 in these dermatitis models was superior to the conventional therapeutic agents, ciclosporin and methotrexate. Topical application of JTE-052 ointment ameliorated hapten-induced chronic dermatitis in rats more effectively than tacrolimus ointment. Furthermore, JTE-052 ointment did not cause the thinning of normal skin associated with topical corticosteroids. These results indicate that JTE-052 is a promising candidate as an anti-inflammatory drug for various types of chronic dermatitis, with a distinctly different profile from conventional therapy following either oral or topical application.

JAK inhibitor JTE-052 regulates contact hypersensitivity by downmodulating T cell activation and differentiation.

BACKGROUND: Using JAK inhibitors to inhibit cytokine signaling is presumed to be a possible means of treating skin inflammatory disorders such as contact dermatitis. OBJECTIVE: To clarify the action site of JAK inhibitors in skin inflammatory disorders. METHODS: We analyzed the mechanism of action of the JAK inhibitor JTE-052 using murine skin inflammation models, including contact hypersensitivity (CHS) and irritant contact dermatitis. Cells isolated from ear tissue or lymph node (LN) were analyzed by flow cytometry. The amounts of cytokines in the culture medium were measured by ELISA or bead array system. Proliferation of LN cells was evaluated by measurement of tritiated thymidine incorporation. RESULTS: Oral administration of JTE-052 during both sensitization and elicitation phase attenuated CHS, but did not affect croton oil-induced irritant contact dermatitis. JTE-052 potently inhibited T cell proliferation and activation by antigen presentation in vitro, and attenuated skin inflammation in a sensitized-lymphocyte transfer model without suppressing T cell migration. JTE-052 did not affect hapten-induced cutaneous dendritic cell migration into draining lymph nodes or their costimulatory molecule expressions. CONCLUSION: The JAK inhibitor JTE-052 exerts an inhibitory effect on antigen-specific T cell activation and subsequent inflammation in acquired skin immunity, such as CHS.

Improvement of spontaneous locomotor activity with JAK inhibition by JTE-052 in rat adjuvant-induced arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to joint destruction, disability, and decreased quality of life (QOL). Inhibition of Janus kinase (JAK) signaling ameliorates articular inflammation and joint destruction in animal models of RA, but its effects on behaviors indicating well-being are poorly understood. In this study, we evaluated the effect of JAK inhibition on spontaneous locomotor activity in rats with adjuvant-induced arthritis, a rodent model of RA. METHODS: Arthritis was induced in male Lewis rats by a single subcutaneous injection of Freund's complete adjuvant. The novel JAK inhibitor JTE-052 was orally administered for 7 days after the onset of arthritis. RESULTS: Induction of arthritis suppressed the spontaneous locomotor activity of the rats. Administration of JTE-052 completely improved the spontaneous locomotor activity, with partial reductions in articular inflammation and joint destruction. Hyperalgesia and motor functions were also improved, but the efficacy was not complete. However, serum interleukin (IL)-6 levels were completely decreased at 4 h after administration of the first dose of JTE-052. CONCLUSIONS: This study demonstrated that JAK inhibition improved the spontaneous locomotor activity of rats with adjuvant-induced arthritis, in association with amelioration of pain and physical dysfunction as a consequence of suppression of joint inflammation. Moreover, although further studies are needed, there was possible participation of IL-6 downregulation in the improvement of locomotor activity by JAK inhibition.

Resolvin E1 inhibits dendritic cell migration in the skin and attenuates contact hypersensitivity responses.

Resolvin E1 (RvE1) is a lipid mediator derived from ω3 polyunsaturated fatty acids that exerts potent antiinflammatory roles in several murine models. The antiinflammatory mechanism of RvE1 in acquired immune responses has been attributed to attenuation of cytokine production by dendritic cells (DCs). In this study, we newly investigated the effect of RvE1 on DC motility using two-photon microscopy in a contact hypersensitivity (CHS) model and found that RvE1 impaired DC motility in the skin. In addition, RvE1 attenuated T cell priming in the draining lymph nodes and effector T cell activation in the skin, which led to the reduced skin inflammation in CHS. In contrast, leukotriene B4 (LTB4) induced actin filament reorganization in DCs and increased DC motility by activating Cdc42 and Rac1 via BLT1, which was abrogated by RvE1. Collectively, our results suggest that RvE1 attenuates cutaneous acquired immune responses by inhibiting cutaneous DC motility, possibly through LTB4-BLT1 signaling blockade.

The Janus kinase inhibitor JTE-052 improves skin barrier function through suppressing signal transducer and activator of transcription 3 signaling.

BACKGROUND: Barrier disruption and the resulting continuous exposure to allergens are presumed to be responsible for the development of atopic dermatitis (AD). However, the mechanism through which skin barrier function is disrupted in patients with AD remains unclear. OBJECTIVES: Taking into account the fact that the TH2 milieu impairs keratinocyte terminal differentiation, we sought to clarify our hypothesis that the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway plays a critical role in skin barrier function and can be a therapeutic target for AD. METHODS: We analyzed the mechanism of keratinocyte differentiation using a microarray and small interfering RNA targeting STATs. We studied the effect of the JAK inhibitor JTE-052 on keratinocyte differentiation using the human skin equivalent model and normal human epidermal keratinocytes. We applied topical JAK inhibitor onto NC/Nga mice, dry skin model mice, and human skin grafted to immunocompromised mice. RESULTS: IL-4 and IL-13 downregulated genes involved in keratinocyte differentiation. STAT3 and STAT6 are involved in keratinocyte differentiation and chemokine production by keratinocytes, respectively. Topical application of the JAK inhibitor suppressed STAT3 activation and improved skin barrier function, permitting increases in levels of terminal differentiation proteins, such as filaggrin, and natural moisturizing factors in models of AD and dry skin and in human skin. CONCLUSION: STAT3 signaling is a key element that regulates keratinocyte differentiation. The JAK inhibitor can be a new therapeutic tool for the treatment of disrupted barrier function in patients with AD.

Pharmacological properties of JTE-052: a novel potent JAK inhibitor that suppresses various inflammatory responses in vitro and in vivo.

OBJECTIVE: To evaluate the pharmacological properties of JTE-052, a novel Janus kinase (JAK) inhibitor. METHODS: The JAK inhibitory activity of JTE-052 was evaluated using recombinant human enzymes. The inhibitory effects on cytokine signaling pathways were evaluated using primary human inflammatory cells. The in vivo efficacy and potency of JTE-052 were examined in a mouse interleukin (IL)-2-induced interferon (IFN)-γ production model and a rat collagen-induced arthritis model. RESULTS: JTE-052 inhibited the JAK1, JAK2, JAK3, and tyrosine kinase (Tyk)2 enzymes in an adenosine triphosphate (ATP)-competitive manner and inhibited cytokine signaling evoked by IL-2, IL-6, IL-23, granulocyte/macrophage colony-stimulating factor, and IFN-α. JTE-052 inhibited the activation of inflammatory cells, such as T cells, B cells, monocytes, and mast cells, in vitro. Oral dosing of JTE-052 resulted in potent suppression of the IL-2-induced IFN-γ production in mice with an ED50 value of 0.24 mg/kg, which was more potent than that of tofacitinib (ED50 = 1.1 mg/kg). In the collagen-induced arthritis model, JTE-052 ameliorated articular inflammation and joint destruction even in therapeutic treatments where methotrexate was ineffective. CONCLUSIONS: The present results indicate that JTE-052 is a highly potent JAK inhibitor, and represents a candidate anti-inflammatory agent for suppressing various types of inflammation.

IL-17A as an inducer for Th2 immune responses in murine atopic dermatitis models.

Atopic dermatitis (AD) is generally regarded as a type 2 helper T (Th2)-mediated inflammatory skin disease. Although the number of IL-17A-producing cells is increased in the peripheral blood and in acute skin lesion of AD patients, the role of IL-17A in the pathogenesis of AD remains unclear. To clarify this issue, we used murine AD models in an IL-17A-deficient condition. In a repeated hapten application-induced AD model, skin inflammation, IL-4 production in the draining lymph nodes (LNs), and hapten-specific IgG1 and IgE induction were suppressed in IL-17A-deficient mice. Vγ4(+) γδ T cells in the skin-draining LNs and Vγ5(-) dermal γδ T cells in the skin were the major sources of IL-17A. Consistently, in flaky-tail (Flg(ft/ft) ma/ma) mice, spontaneous development of AD-like dermatitis and IgE induction were attenuated by IL-17A deficiency. Moreover, Th2 differentiation from naive T cells was promoted in vitro by the addition of IL-17A. Taken together, our results suggest that IL-17A mediates Th2-type immune responses and that IL-17A signal may be a therapeutic target of AD.

An aggregate-prone mutant of human glyceraldehyde-3-phosphate dehydrogenase augments oxidative stress-induced cell death in SH-SY5Y cells.

Glycerladehyde-3-phosphate dehydrogenase (GAPDH), a classic glycolytic enzyme, also has a role in mediating cell death under oxidative stress. Our previous reports suggest that oxidative stress-induced GAPDH aggregate formation is, at least in part, a mechanism to account for the death signaling. Here we show that substitution of cysteine for serine-284 of human GAPDH (S284C-GAPDH) leads to aggregate-prone GAPDH, and that its expression in SH-SY5Y human neuroblastoma results in greater dopamine-induced cell death than expression of wild type-GAPDH. Treatment of purified recombinant S284C-GAPDH in vitro with the nitric oxide donor NOR3 led to greater aggregation than wild type-GAPDH. Several lines of structural analysis revealed that S284C-GAPDH was amyloidogenic. Overexpression of doxycycline-inducible S284C-GAPDH in SH-SY5Y cells accelerated dopamine treatment-induced death and increased formation of GAPDH aggregates, compared to cells expressing wild type-GAPDH. These results suggest that aggregate-prone mutations of GAPDH such as S284C-GAPDH may confer risk of oxidative stress-induced cell death.

Glyceraldehyde-3-phosphate dehydrogenase aggregate formation participates in oxidative stress-induced cell death.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(2) is a classic glycolytic enzyme that also mediates cell death by its nuclear translocation under oxidative stress. Meanwhile, we previously presented that oxidative stress induced disulfide-bonded GAPDH aggregation in vitro. Here, we propose that GAPDH aggregate formation might participate in oxidative stress-induced cell death both in vitro and in vivo. We show that human GAPDH amyloid-like aggregate formation depends on the active site cysteine-152 (Cys-152) in vitro. In SH-SY5Y neuroblastoma, treatment with dopamine decreases the cell viability concentration-dependently (IC(50) = 202 microM). Low concentrations of dopamine (50-100 microM) mainly cause nuclear translocation of GAPDH, whereas the levels of GAPDH aggregates correlate with high concentrations of dopamine (200-300 microM)-induced cell death. Doxycycline-inducible overexpression of wild-type GAPDH in SH-SY5Y, but not the Cys-152-substituted mutant (C152A-GAPDH), accelerates cell death accompanying both endogenous and exogenous GAPDH aggregate formation in response to high concentrations of dopamine. Deprenyl, a blocker of GAPDH nuclear translocation, fails to inhibit the aggregation both in vitro and in cells but reduced cell death in SH-SY5Y treated with only a low concentration of dopamine (100 microM). These results suggest that GAPDH participates in oxidative stress-induced cell death via an alternative mechanism in which aggregation but not nuclear translocation of GAPDH plays a role. Moreover, we observe endogenous GAPDH aggregate formation in nigra-striatum dopaminergic neurons after methamphetamine treatment in mice. In transgenic mice overexpressing wild-type GAPDH, increased dopaminergic neuron loss and GAPDH aggregate formation are observed. These data suggest a critical role of GAPDH aggregates in oxidative stress-induced brain damage.

The active site cysteine of the proapoptotic protein glyceraldehyde-3-phosphate dehydrogenase is essential in oxidative stress-induced aggregation and cell death.

Recent studies have revealed that the redox-sensitive glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is involved in neuronal cell death that is triggered by oxidative stress. GAPDH is locally deposited in disulfide-bonded aggregates at lesion sites in certain neurodegenerative diseases. In this study, we investigated the molecular mechanism that underlies oxidative stress-induced aggregation of GAPDH and the relationship between structural abnormalities in GAPDH and cell death. Under nonreducing in vitro conditions, oxidants induced oligomerization and insoluble aggregation of GAPDH via the formation of intermolecular disulfide bonds. Because GAPDH has four cysteine residues, including the active site Cys(149), we prepared the cysteine-substituted mutants C149S, C153S, C244A, C281S, and C149S/C281S to identify which is responsible for disulfide-bonded aggregation. Whereas the aggregation levels of C281S were reduced compared with the wild-type enzyme, neither C149S nor C149S/C281S aggregated, suggesting that the active site cysteine plays an essential role. Oxidants also caused conformational changes in GAPDH concomitant with an increase in beta-sheet content; these abnormal conformations specifically led to amyloid-like fibril formation via disulfide bonds, including Cys(149). Additionally, continuous exposure of GAPDH-overexpressing HeLa cells to oxidants produced disulfide bonds in GAPDH leading to both detergent-insoluble and thioflavin-S-positive aggregates, which were associated with oxidative stress-induced cell death. Thus, oxidative stresses induce amyloid-like aggregation of GAPDH via aberrant disulfide bonds of the active site cysteine, and the formation of such abnormal aggregates promotes cell death.

[Coronary risk factor management in primary practice in Shibuya, Tokyo].

OBJECTIVES: Many large-scale clinical trials have confirmed that coronary risk factors such as hypertension, hyperlipidemia and diabetes mellitus predict a higher incidence of cardiovascular events and that control of these risk factors reduces the incidence. However, the actual management of such risk factors and the resultant improvement of the cardiovascular events in primary practice remains unclear. The Heart Care Network Shibuya, a voluntary study group of regional primary physicians, surveyed the management of coronary risk factors and the clinical outcomes. METHODS: Behavioral patterns of the coronary risk factor was investigated in the management and resultant changes of the risk factors in 209 outpatients (mean age 65.6 +/- 11.2 years) with more than one of hypertension, hyperlipidemia, diabetes mellitus or prior myocardial infarction for 1 year. RESULTS: Prescriptions of anti-hypertensive (from 1.3 +/- 0.8 to 1.4 +/- 0.8 drugs per patient) and antihyperlipidemia drugs (from 0.70 +/- 0.4 to 0.73 +/- 0.4 drugs per patient) did not significantly increase. Patient education for diet (93% to 97%, p = 0.077), exercise (69% to 81%, p < 0.05) and nonsmoking (66% to 86%, p < 0.05) significantly increased after 1 year. Blood pressure decreased from 142 +/- 16/81 +/- 10 to 138 +/- 78/78 +/- 9 mmHg (p < 0.05), serum total cholesterol level decreased from 215 +/- 39 to 203 +/- 39 mg/dl (p < 0.05). As a result, more patients attained the treatment goals recommended in the guidelines by the Japanese Society of Hypertension (34.6% to 46.8%) and Japan Atherosclerosis Society (40.2% to 49.5%), respectively. However, none of blood hemoglobin A1c level, body mass index or smokers significantly changed. CONCLUSIONS: Regional practitioners attempted to control all coronary risk factors. Hypertension and hyperlipidemia, which are relatively more dependent on the medical management, improved. In contrast, diabetes mellitus, obesity or smoking, which require life style changes, did not improve. The main issue in the risk factor management is how physicians act rather than specific actions.