Stable form of galectin-9, a Tim-3 ligand, inhibits contact hypersensitivity and psoriatic reactions: A potent therapeutic tool for Th1- and/or Th17-mediated skin inflammation
Introduction
T cell Ig- and mucin domain-containing molecule (Tim)-3 belongs to the TIM family, which has an Ig V-like domain and a mucin-like domain. Tim-3 was first identified to be specifically expressed in CD4 (+) Th1 cells, but not in Th2 cells and the interaction of Tim-3 with Tim-3 ligand negatively regulates Th1 immune response [1], [2]. Treatment with anti-Tim-3 Ab exacerbates experimental autoimmune encephalomyelitis, a Th1-dependent autoimmune disease [1]. Tim-3-Ig fusion proteins given to immunized mice with myelin proteolipid protein causes hyperproliferation of Th1 cells and Th1 cytokine release [3]. Blockade of the Tim-3 pathway by either Tim-3 Ab or Tim-3-Ig fusion protein accelerates diabetes in the diabetic NOD mouse model and prevents acquisition of transplantation tolerance [2]. Anti-Tim-3 treatment in an acute graft-versus-host disease mouse model augments the activation of effector T cells expressing IFN-γ [4].
Galectins constitute a family of animal lectins that have binding specificity for β-galactoside. Among this family, galectin-9 belongs to a tandem-repeat type of galectins that consist of two homologous carbohydrate recognition domains connected by a linker peptide [5]. Galectin-9 is known to have a variety of physiological and pathological functions, such as cell differentiation, adhesion, aggregation and cell death [5], [6], [7]. A recent finding is that galectin-9 is a physiological ligand of Tim-3 [8]. Galectin-9 bound to Tim-3 on Th1 cells induces calcium influx and cell death.
Galectin-9 expression is regulated by IFN-γ, a major cytokine produced by Th1 cells. Surface expression of galectin-9 in fibroblasts and endothelial cells is up-regulated by IFN-γ treatment [9], [10]. Thus, it is conceivable that galectin-9 induced by IFN-γ from Th1 cells eliminates Tim-3 (+) Th1 cells, thereby preventing exacerbation of Th1 immunity, i.e., a negative feedback mechanism, and that dysregulation of the galectin-9-Tim-3 network causes an autoimmune disease [11]. Indeed, impaired expression of Tim-3 on T cells has been demonstrated in patients with multiple sclerosis [12].
Contact dermatitis is a common skin disease. Murine contact hypersensitivity (CHS) is a widely used model of contact dermatitis. It is generally thought that the Th1 type response is involved in elicitation of this reaction [13], [14], [15]. However, recent studies have demonstrated the critical involvement of Th17/Tc17 cells in CHS. Mice deficient in IL-17 exhibit reduced CHS responses to DNFB [16]. CD8 (+) IL-17-producing T cells (Tc17) have been shown to be essential for the development of CHS [17].
Psoriasis vulgaris is another common skin disease that has a chronic clinical course and is resistant to treatment. IFN-γ-producing Th1 cells, the differentiation of which is promoted by IL-12, are thought to be key players in the pathogenesis of psoriasis [18], [19], [20]. In the lesional skin of psoriasis, IL-12p40 subunit levels are elevated [20], [21], while administration of anti-IL-12p40 Ab significantly improves clinical score in human psoriasis [22], [23]. Subsequently, however, it was found that levels of IL-12p35, another subunit of IL-12, were not elevated in psoriatic skin lesions [21]. On the other hand, lesional levels of the p19 subunit, which constitutes IL-23 via a heterodimer with IL-12p40, were elevated [21]. It is now considered that Th17 cells induced by IL-23, rather than Th1 cells induced by IL-12, are a prerequisite for the development of psoriatic lesions. Indeed, local administration of IL-23 induced dermal inflammation and acanthosis mimicking human psoriatic skin [21].
A recent finding demonstrated that Tim-3 is expressed on Th17 cells in addition to Th1 cells [24], and that galectin-9 represses differentiation of Th17 cells and induces cell apoptosis [25]. Considering the apoptotic signals mediated by Tim-3, it can be hypothesized that administration of galectin-9, the Tim-3 ligand, exerts therapeutic effects on Th1 and/or Th17-mediated skin inflammation via inhibition of these cells. The natural form of galectin-9, however, is easily inactivated by proteases, such as elastase and metalloproteinase-3, due to the labile nature of the linker peptide [26]. In this study, we elucidated the therapeutic potential and mode of action of the stable form of galectin-9 (sGal-9), which we have recently developed by partial deletion of the linker peptide [26], in CHS and a psoriatic model of inflammation.
Section snippets
Mice
BALB/c and C57BL/6 mice were purchased from Sankyo Labo Service Co. (Tokyo, Japan). Mice were maintained under specific pathogen-free conditions in our animal facility. The use of animals was in full compliance with the guidelines of the Committee for Animal Experiments of Tokyo Medical and Dental University.
Antibodies
FITC-conjugated anti-mouse CD4 Ab (CD4-FITC) (GK1.5), CD8-FITC (53-6.7), PE-conjugated anti-mouse Tim-3 Ab (Tim-3-PE) (8B.2C12), IL-13-PE (eBio 13A), DX5-FITC, PE-Cy5-conjugated anti-mouse
Effect of sGal-9 on allergic CHS
Mice sensitized with DNFB were intraperitoneally treated with various doses of sGal-9 on days 3, 4, and 5. Ear swelling responses were markedly inhibited by sGal-9 (Fig. 1A). Histological examination revealed reduced dermal edema and cellular infiltrate comprising mononuclear cells, eosinophils and neutrophils (Fig. 1B). In addition, the number of dermal mast cells decreased. The inhibitory effects of sGal-9 were also seen in CHS in response to TNCB, FITC, and Ox (Figs. 1A and C), although the
Discussion
In this study, we demonstrated that sGal-9 successfully inhibits elicitation of the CHS response to DNFB. Diminished skin responses were accompanied by reduced local levels of IFN-γ, IL-17 and Th1 chemokines, such as RANTES (CCL5) and IP-10 (CXCL-10). sGal-9-treated mice also showed selective loss of CD4(+) or CD8(+) cells expressing IFN-γ and CD4(+) or CD8(+) cells expressing IL-17 in regional LNs, which were probably mediated by sGal-9-Tim-3 interaction.
Unlike CHS responses to DNFB, which are
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