Sayaka Shibata

Selective Inhibitors of Methionyl-tRNA Synthetase Have Potent Activity against Trypanosoma brucei Infection in Mice

ABSTRACT Human African trypanosomiasis continues to be an important public health threat in extensive regions of sub-Saharan Africa. Treatment options for infected patients are unsatisfactory due to toxicity, difficult administration regimes, and poor efficacy of available drugs. The aminoacyl-tRNA synthetases were selected as attractive drug targets due to their essential roles in protein synthesis and cell survival. Comparative sequence analysis disclosed differences between the trypanosome and mammalian methionyl-tRNA synthetases (MetRSs) that suggested opportunities for selective inhibition using drug-like molecules. Experiments using RNA interference on the single MetRS of Trypanosoma brucei demonstrated that this gene product was essential for normal cell growth. Small molecules (diaryl diamines) similar to those shown to have potent activity on prokaryotic MetRS enzymes were synthesized and observed to have inhibitory activity on the T. brucei MetRS (50% inhibitory concentration, <50 nM) and on bloodstream forms of T. brucei cultures (50% effective concentration, as low as 4 nM). Twenty-one compounds had a close correlation between enzyme binding/inhibition and T. brucei growth inhibition, indicating that they were likely to be acting on the intended target. The compounds had minimal effects on mammalian cell growth at 20 μM, demonstrating a wide therapeutic index. The most potent compound was tested in the murine model of trypanosomiasis and demonstrated profound parasite suppression and delayed mortality. A homology model of the T. brucei MetRS based on other MetRS structures was used to model binding of the lead diaryl diamine compounds. Future studies will focus on improving the pharmacological properties of the MetRS inhibitors.

Adiponectin Regulates Cutaneous Wound Healing by Promoting Keratinocyte Proliferation and Migration via the ERK Signaling Pathway

Diabetic patients are at high risk of developing delayed cutaneous wound healing. Adiponectin plays a pivotal role in the pathogenesis of diabetes and is considered to be involved in various pathological conditions associated with diabetes; however, its role in wound repair is unknown. In this study, we elucidated the involvement of adiponectin in cutaneous wound healing in vitro and in vivo. Normal human keratinocytes expressed adiponectin receptors, and adiponectin enhanced proliferation and migration of keratinocytes in vitro. This proliferative and migratory effect of adiponectin was mediated via AdipoR1/AdipoR2 and the ERK signaling pathway. Consistent with in vitro results, wound closure was significantly delayed in adiponectin-deficient mice compared with wild-type mice, and more importantly, keratinocyte proliferation and migration during wound repair were also impaired in adiponectin-deficient mice. Furthermore, both systemic and topical administration of adiponectin ameliorated impaired wound healing in adiponectin-deficient and diabetic db/db mice, respectively. Collectively, these results indicate that adiponectin is a potent mediator in the regulation of cutaneous wound healing. We propose that upregulation of systemic and/or local adiponectin levels is a potential and very promising therapeutic approach for dealing with diabetic wounds.

Adiponectin as an anti-inflammatory factor in the pathogenesis of psoriasis: induction of elevated serum adiponectin levels following therapy

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Prolactin enhances basal and IL-17-induced CCL20 production by human keratinocytes.

Psoriasis vulgaris is an autoimmune dermatosis with Th17 infiltration. Prolactin (PRL) may participate in the pathogenesis of psoriasis. The chemokine CCL20 recruits Th17 cells, and CCL20 production by epidermal keratinocytes is enhanced in psoriatic lesions. We examined the in vitro effects of PRL on CCL20 production in human keratinocytes. PRL increased basal and IL‐17‐induced CCL20 secretion, and mRNA expression in keratinocytes. CCL20 production by PRL was suppressed by antisense oligonucleotides against the AP‐1 components c‐Fos and c‐Jun, whereas that by IL‐17 was suppressed by antisense NF‐κB p50 and p65. CCL20 production induced by PRL plus IL‐17 was suppressed by antisense c‐Fos, c‐Jun, p50, and p65. PRL alone increased the transcriptional activity of AP‐1, and c‐Fos and c‐Jun expression; moderately enhanced NF‐κB activity and IκBα phosphorylation; and potently increased IL‐17‐induced NF‐κB activity. MEK and JNK inhibitors suppressed PRL‐ or PRL‐plus‐IL‐17‐induced CCL20 production and AP‐1 activities. MEK inhibitor suppressed PRL‐induced c‐Fos expression, whereas JNK inhibitor suppressed c‐Jun expression. PRL induced ERK and JNK phosphorylation. These results suggest that PRL may enhance basal and IL‐17‐induced CCL20 production in keratinocytes by AP‐1 and NF‐κB activation, which is partially mediated via MEK/ERK and JNK. PRL may promote Th17 infiltration into psoriatic lesions via CCL20.

Adiponectin regulates psoriasiform skin inflammation by suppressing IL-17 production from γδ-T cells

Accumulating epidemiologic evidence has revealed that metabolic syndrome is an independent risk factor for psoriasis development and is associated with more severe psoriasis. Adiponectin, primarily recognized as a metabolic mediator of insulin sensitivity, has been newly drawing attention as a mediator of immune responses. Here we demonstrate that adiponectin regulates skin inflammation, especially IL-17-related psoriasiform dermatitis. Mice with adiponectin deficiency show severe psoriasiform skin inflammation with enhanced infiltration of IL-17-producing dermal Vγ4+γδ-T cells. Adiponectin directly acts on murine dermal γδ-T cells to suppress IL-17 synthesis via AdipoR1. We furthermore demonstrate here that the adiponectin level of skin tissue as well as subcutaneous fat is decreased in psoriasis patients. IL-17 production from human CD4- or CD8-positive T cells is also suppressed by adiponectin. Our data provide a regulatory role of adiponectin in skin inflammation, which would imply a mechanism underlying the relationship between psoriasis and metabolic disorders.

Urea-based inhibitors of Trypanosoma brucei methionyl-tRNA synthetase: selectivity and in vivo characterization.

Urea-based methionyl-tRNA synthetase inhibitors were designed, synthesized, and evaluated for their potential toward treating human African trypanosomiasis (HAT). With the aid of a homology model and a structure-activity-relationship approach, low nM inhibitors were discovered that show high selectivity toward the parasite enzyme over the closest human homologue. These compounds inhibit parasite growth with EC(50) values as low as 0.15 μM while having low toxicity to mammalian cells. Two compounds (2 and 26) showed excellent membrane permeation in the MDR1-MDCKII model and encouraging oral pharmacokinetic properties in mice. Compound 2 was confirmed to enter the CNS in mice. Compound 26 had modest suppressive activity against Trpanosoma brucei rhodesiense in the mouse model, suggesting that more potent analogues or compounds with higher exposures need to be developed. The urea-based inhibitors are thus a promising starting point for further optimization toward the discovery of orally available and CNS active drugs to treat HAT.

Regulatory B cells suppress imiquimod-induced, psoriasis-like skin inflammation

Psoriasis is an inflammatory cutaneous disorder characterized by marked epidermal thickening and Th1 and Th17 cell infiltration. At present, the contribution of B cells to the pathogenesis of psoriasis is unclear. In mice, topical application of imiquimod induces inflamed skin lesions and serves as an experimental animal model for human psoriasis. In this study, we showed that imiquimod‐induced skin inflammation was more severe in CD19−/− than WT mice. These inflammatory responses were negatively regulated by a unique IL‐10‐producing CD1dhiCD5+ regulatory B cell subset (B10 cells) that was absent in CD19−/− mice and represented only 1–2% of splenic B220+ cells in WT mice. Splenic B10 cells entered the circulation and migrated to draining LNs during imiquimod‐induced skin inflammation, thereby suppressing IFN‐γ and IL‐17 production. Furthermore, adoptive transfer of these B10 cells from WT mice reduced inflammation in CD19−/− mice. The present findings provide direct evidence that B10 cells regulate imiquimod‐induced skin inflammation and offer insights into regulatory B cell‐based therapies for the treatment of psoriasis.

IL-27 Activates Th1-Mediated Responses in Imiquimod-Induced Psoriasis-Like Skin Lesions

IL-27, a member of the IL-12 cytokine family, primes Th1 cell differentiation, whereas it suppresses Th17 cell development. We have previously reported that serum IL-27 levels are elevated in psoriatic patients and that IL-27 greatly induces in vitro production of Th1-type chemokines through STAT1 activation. In this study, to further investigate the in vivo role of IL-27 in the pathogenesis of psoriasis, we induced psoriasis-like inflammation on mouse back skin with topical application of imiquimod (IMQ), and continuously injected IL-27 or PBS subcutaneously. IMQ-treated skin showed an increase of IL-27 mRNA levels and the infiltration of IL-27-producing cells in the papillary dermis. The injection of IL-27 to the IMQ-treated skin exacerbated the disease compared with PBS injection. The IL-27 injection further augmented mRNA levels of IFN-γ, CXCL9, CXCL10, CXCL11, and TNF-α, without altering those of IL-17A, IL-17F, IL-22, and CCL20. Finally, IL-27 antagonism attenuated the upregulation of IFN-γ, CXCL9, CXCL10, CXCL11, and TNF-α mRNA levels, and induced clinical and histological improvement in the IMQ-treated skin. These results indicate that IL-27 would act in a proinflammatory manner, and thereby exacerbate the psoriasis-like skin inflammation induced by IMQ.

Serum adiponectin levels inversely correlate with the activity of progressive skin sclerosis in patients with diffuse cutaneous systemic sclerosis

Backgrounds  Adiponectin has been demonstrated to be one of anti‐inflammatory and anti‐fibrotic factors, suggesting the potential of this cytokine to be involved in the developmental process of systemic sclerosis (SSc).

Serum lipocalin-2 levels are increased in patients with psoriasis.

The protein lipocalin (LCN)‐2 is known to be related to insulin resistance, obesity and atherosclerotic diseases. Psoriasis is an inflammatory skin disease related to metabolic syndrome. The aim of this study was to examine the relationship between serum LCN2 levels and indicators for metabolic syndrome and inflammatory cytokine levels in patients with psoriasis. Serum LCN2 levels were measured in patients with psoriasis, atopic dermatitis (AD) or bullous pemphigoid (BP), and compared with those of healthy controls. Serum LCN2 levels were also compared with several indicators for metabolic syndrome, and with serum levels of interleukin (IL)‐6 and tumour necrosis factor (TNF)‐α, two markers of inflammation. Serum LCN2 levels in patients with psoriasis were significantly higher than those of healthy controls, but there was no significant correlation between serum LCN2 and body mass index. Serum LCN2 levels also correlated with serum IL‐6 and TNF‐α levels in patients with psoriasis. Serum LCN2 levels are a general indicator for increased inflammation in the patients with psoriasis.