Yoshiyuki Miyazaki

WSX-1 Is Required for Resistance to Trypanosoma cruzi Infection by Regulation of Proinflammatory Cytokine Production

WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors and is essential for resistance to Leishmania major infection. In the present study, we demonstrated that WSX-1 was also required for resistance to Trypanosoma cruzi. WSX-1-/- mice exhibited prolonged parasitemia, severe liver injury, and increased mortality over wild-type mice. WSX-1-/- splenocytes produced enhanced levels of Th2 cytokines, which were responsible for the prolonged parasitemia. Massive necroinflammatory lesions were observed in the liver of infected WSX-1-/- mice, and IFN-gamma that was overproduced in WSX-1-/- mice compared with wild-type mice was responsible for the lesions. In addition, vast amounts of various proinflammatory cytokines, including IL-6 and TNF-alpha, were produced by liver mononuclear cells in WSX-1-/- mice. Thus, during T. cruzi infection, WSX-1 suppresses liver injury by regulating production of proinflammatory cytokines, while controlling parasitemia by suppression of Th2 responses, demonstrating its novel role as an inhibitory regulator of cytokine production.

Two-Sided Roles of IL-27: Induction of Th1 Differentiation on Naive CD4+ T Cells versus Suppression of Proinflammatory Cytokine Production Including IL-23-Induced IL-17 on Activated CD4+ T Cells Partially Through STAT3-Dependent Mechanism

Recent lines of evidence have demonstrated that IL-27, a newly identified IL-12-related cytokine, has two apparently conflicting roles in immune responses: one as an initiator of Th1 responses and the other as an attenuator of inflammatory cytokine production. Although the IL-27-mediated Th1 initiation mechanism has been elucidated, little is known about the molecular basis for the suppression of cytokine production. In the present study, we demonstrated that IL-27 suppressed the production of various proinflammatory cytokines by fully activated CD4+ T cells while it had no effect on the cytokine production by CD4+ T cells at early phases of activation. IL-27 also suppressed IL-17 production by activated CD4+ T cells, thereby counteracting IL-23, another IL-12-related cytokine with proinflammatory effects. In fully activated CD4+ T cells, STAT3 was preferentially activated by IL-27 stimulation, whereas both STAT1 and 3 were activated by IL-27 in early activated CD4+ T cells. Lack of STAT3 in fully activated cells impaired the suppressive effects of IL-27. These data indicated that the preferential activation of STAT3 in fully activated CD4+ T cells plays an important role in the cytokine suppression by IL-27/WSX-1.

Interleukin 27: a double‐edged sword for offense and defense

Cytokine‐mediated immunity plays a crucial role in the pathogenesis of various diseases including infection and autoimmune diseases. IL‐27, along with IL‐12, −23, and −35, belongs to the IL‐12 cytokine family. These family members play roles in regulation of Th cell differentiation. IL‐27 is unique in that although it induces Th1 differentiation, the same cytokine suppresses immune responses. In the absence of IL‐27‐mediated immunosuppression, hyperproduction of various proinflammatory cytokines concomitant with severe inflammation is observed. The immunosuppressive effects of IL‐27 depend on IL‐2 suppression, inhibition of Th17 development, and induction of IL‐10 production. Administration of IL‐27 suppresses some diseases of autoimmune or allergic origin, demonstrating its potential in therapy of diseases mediated by inflammatory cytokines. In this review, we discuss recent studies about the role of IL‐27 in immune regulation in view of its pro‐ and anti‐inflammatory properties and possible therapeutic application.

Exacerbation of Experimental Allergic Asthma by Augmented Th2 Responses in WSX-1-Deficient Mice

WSX-1 (IL-27R) is a class I cytokine receptor with homology to gp130 and IL-12 receptors and is typically expressed on CD4+ T lymphocytes. Although previous reports have clarified that IL-27/WSX-1 signaling plays critical roles in both Th1 differentiation and attenuation of cell activation and proinflammatory cytokine production during some bacterial or protozoan infections, little is known about the importance of WSX-1 in cytokine-mediated diseases of allergic origin. To this aim, we took advantage of WSX-1-deficient (WSX-1−/−) mice and induced experimental asthma, in which Th2 cytokines are central modulators of the pathology. OVA-challenged WSX-1−/− mice showed marked enhancement of airway responsiveness with goblet cell hyperplasia, pulmonary eosinophil infiltration, and increased serum IgE levels compared with wild-type mice. Production of Th2 cytokines, which are largely responsible for the pathogenesis of asthma, was augmented in the lung or in the culture supernatants of peribronchial lymph node CD4+ T cells from WSX-1−/− mice compared with those from wild-type mice. Surprisingly, IFN-γ production was also enhanced in WSX-1−/− mice, albeit at a low concentration. The cytokine overproduction, thus, seems independent from the Th1-promoting property of WSX-1. These results demonstrated that IL-27/WSX-1 also plays an important role in the down-regulation of airway hyper-reactivity and lung inflammation during the development of allergic asthma through its suppressive effect on cytokine production.

Membranous Glomerulonephritis Development with Th2-Type Immune Deviations in MRL/lpr Mice Deficient for IL-27 Receptor (WSX-1)

MRL/lpr mice develop spontaneous glomerulonephritis that is essentially identical with diffuse proliferative glomerulonephritis (World Health Organization class IV) in human lupus nephritis. Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. Diffuse proliferative glomerulonephritis is associated with autoimmune responses dominated by Th1 cells producing high levels of IFN-γ. The initial mounting of Th1 responses depends on the function of the WSX-1 gene, which encodes a subunit of the IL-27R with homology to IL-12R. In mice deficient for the WSX-1 gene, proper Th1 differentiation was impaired and abnormal Th2 skewing was observed during infection with some intracellular pathogens. Disruption of the WSX-1 gene dramatically changed the pathophysiology of glomerulonephritis developing in MRL/lpr mice. WSX-1−/− MRL/lpr mice developed disease resembling human membranous glomerulonephritis (World Health Organization class V) with a predominance of IgG1 in glomerular deposits, accompanied by increased IgG1 and IgE in the sera. T cells in WSX-1−/− MRL/lpr mice displayed significantly reduced IFN-γ production along with elevated IL-4 expression. Loss of WSX-1 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of human lupus nephritis development.

IL-17 Is Necessary for Host Protection against Acute-Phase Trypanosoma cruzi Infection

IL-17A is a key cytokine that induces inflammatory responses through the organized production of inflammatory cytokines, such as IL-6, TNF-α, and GM-CSF, and induces neutrophil migration. The roles of IL-17A in infection of intracellular protozoan parasites have not been elucidated, although augmented immune responses by IL-17A are important for the resolution of some bacterial and fungal infections. Therefore, we experimentally infected IL-17A–deficient (IL-17A−/−) mice with Trypanosoma cruzi. IL-17A−/− mice had a lower survival rate and prolonged worse parasitemia compared with control C57BL/6 wild-type (WT) mice postinfection. In the infected IL-17A−/− mice, multiple organ failure was observed compared with WT mice, as reflected by the marked increase in serologic markers of tissue injury, such as aspartate aminotransferase, which resulted in increased mortality of IL-17A−/− mice. Expression of cytokines, such as IFN-γ, IL-6, and TNF-α, was lower in liver-infiltrating cells from the IL-17A−/− mice compared with WT mice. A similar defect was observed in the expression of neutrophil enzymes, such as myeloperoxidase and lipoxygenase, whereas cellular infiltration into the infected tissues was not affected by IL-17A deficiency. These results suggested that the efficient activation of immune-related cells critical for the killing of T. cruzi was impaired in the absence of IL-17A, resulting in the greater susceptibility of those mice to T. cruzi infection. From these results, we conclude that IL-17A is important for the resolution of T. cruzi infection.

Hyperproduction of Proinflammatory Cytokines by WSX-1-Deficient NKT Cells in Concanavalin A-Induced Hepatitis

Administration of Con A induces liver injury that is considered to be an experimental model for human autoimmune or viral hepatitis, where immunopathology plays roles mediated by activated lymphocytes, especially NK1.1+ CD3+ NKT cells, and inflammatory cytokines, including IFN-γ and IL-4. In the present study we investigated the role of WSX-1, a component of IL-27R, in Con A-induced hepatitis by taking advantage of WSX-1 knockout mice. WSX-1-deficient mice were more susceptible to Con A treatment than wild-type mice, showing serum alanine aminotransferase elevation and massive necrosis in the liver. Although the development of NKT cells appeared normal in WSX-1 knockout mice, purified NKT cells from the knockout mice produced more IFN-γ and IL-4 than those from wild-type mice in response to stimulation with Con A both in vitro and in vivo. In addition, hyperproduction of proinflammatory cytokines, including IL-1, IL-6, and TNF-α, was observed in the knockout mice after Con A administration. These data revealed a novel role for WSX-1 as an inhibitory regulator of cytokine production and inflammation in Con A-induced hepatitis.

Augmentation of Antigen-Presenting and Th1-Promoting Functions of Dendritic Cells by WSX-1(IL-27R) Deficiency

WSX-1 is the α subunit of the IL-27R complex expressed by T, B, NK/NKT cells, as well as macrophages and dendritic cells (DCs). Although it has been shown that IL-27 has both stimulatory and inhibitory effects on T cells, little is known on the role of IL-27/WSX-1 on DCs. LPS stimulation of splenic DCs in vivo resulted in prolonged CD80/CD86 expression on WSX-1-deficient DCs over wild-type DCs. Upon LPS stimulation in vitro, WSX-1-deficient DCs expressed Th1-promoting molecules higher than wild-type DCs. In an allogeneic MLR assay, WSX-1-deficient DCs were more potent than wild-type DCs in the induction of proliferation of and IFN-γ production by responder cell proliferation. When cocultured with purified NK cells, WSX-1-deficient DCs induced higher IFN-γ production and killing activity of NK cells than wild-type DCs. As such, Ag-pulsed WSX-1-deficient DCs induced Th1-biased strong immune responses over wild-type DCs when transferred in vivo. WSX-1-deficient DCs were hyperreactive to LPS stimulation as compared with wild-type DCs by cytokine production. IL-27 suppressed LPS-induced CD80/86 expression and cytokine production by DCs in vitro. Thus, our study demonstrated that IL-27/WSX-1 signaling potently down-regulates APC function and Th1-promoting function of DCs to modulate overall immune responses.

Generation and Characterization of B7-H4/B7S1/B7x-Deficient Mice

ABSTRACT Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.

Tumor‐specific cytotoxic T cell generation and dendritic cell function are differentially regulated by interleukin 27 during development of anti‐tumor immunity

Interleukin (IL‐) 27 is a member of IL‐12 cytokine family with Th1‐promoting and anti‐inflammatory effects. IL‐27 has been shown to facilitate tumor‐specific cytotoxic T lymphocyte (CTL) induction against various tumors. However, IL‐27 suppresses cytokine production of lymphocytes and antigen‐presenting function of dendritic cells (DCs). To examine the in vivo role of IL‐27 in generation of anti‐tumor immunity, we examined IL‐27‐mediated antitumor‐effects using WSX‐1 (IL‐27 receptor α chain)‐deficient (WSX‐1−/−) mice. In WSX‐1−/− mice inoculated with B16 melanoma cells, tumor growth was higher than in wild‐type (WT) mice. Accordingly, tumor‐specific CTL generation was lower in WSX‐1−/− mice than in WT mice. CTL induction in WSX‐1−/− mice was not restored by transfer of WT DCs pulsed with TRP2 peptide, indicating that IL‐27 is directly required for generation of tumor‐specific CTLs. However, when transferred into tumor‐bearing mice, WSX‐1−/− DCs pulsed with TRP2 peptide was more potent than WT DCs in tumor growth inhibition and generation of CTLs, indicating suppressive effects of IL‐27 on DC function. Finally, the combination of WT CD8+ T cells and KO DCs is more potent in generation of antigen‐specific CTLs than any other combinations. Expression of perforin gene and percentages of tumor‐specific CD8+ T cells were also the highest in the combination of WT CD8+ T cells and WSX‐1−/− DCs. It was thus revealed that IL‐27 promotes CTL generation while suppressing DC function during generation of tumor immunity. The combination of WT T cells and IL‐27 signal‐defective DCs may have therapeutic potential against tumors.