Koichi Hirose

IL-23 and Th17 Cells Enhance Th2-Cell–mediated Eosinophilic Airway Inflammation in Mice

RATIONALEnThe IL-23-IL-17A-producing CD4(+) T-cell (Th17 cell) axis plays an important role in the development of chronic inflammatory diseases, including autoimmune diseases. However, the role of the IL-23-Th17 cell axis in the regulation of allergic airway inflammation is still largely unknown.nnnOBJECTIVESnTo determine the role of IL-23 and Th17 cells in allergic airway inflammation.nnnMETHODSnWe examined the effect of anti-IL-23 antibody on antigen-induced airway inflammation. We also investigated the effect of enforced expression of IL-23 on allergic airway inflammation by generating lung-specific IL-23 transgenic mice. Moreover, we examined the effect of adoptive transfer of antigen-specific Th17 cells on allergic airway inflammation.nnnMEASUREMENTS AND MAIN RESULTSnIL-23 mRNA was expressed in the lung of sensitized mice upon antigen inhalation, and the neutralization of IL-23 decreased antigen-induced eosinophil recruitment and Th2 cytokine production in the airways. The enforced expression of IL-23 in the airways significantly enhanced antigen-induced eosinophil and neutrophil recruitment into the airways; Th2 cytokine, IL-17A, and tumor necrosis factor (TNF)-alpha production in the airways; goblet cell hyperplasia; and airway hyperresponsiveness. Moreover, IL-23-mediated enhancement of antigen-induced Th2 cytokine production and eosinophil recruitment in the airways was still observed in the mice lacking IL-17A. Furthermore, although adoptive transfer of antigen-specific Th17 cells alone induced neutrophil but not eosinophil recruitment into the airways upon antigen inhalation, cotransfer of Th17 cells with Th2 cells significantly enhanced antigen-induced Th2-cell-mediated eosinophil recruitment into the airways and airway hyperresponsiveness.nnnCONCLUSIONSnIL-23 and Th17 cells not only induce Th17-cell-mediated neutrophilic airway inflammation but also up-regulate Th2-cell-mediated eosinophilic airway inflammation.

Development and characterization of IL-21–producing CD4+ T cells

It has recently been shown that interleukin (IL)-21 is produced by Th17 cells, functions as an autocrine growth factor for Th17 cells, and plays critical roles in autoimmune diseases. In this study, we investigated the differentiation and characteristics of IL-21–producing CD4+ T cells by intracellular staining. Unexpectedly, we found that under Th17-polarizing conditions, the majority of IL-21–producing CD4+ T cells did not produce IL-17A and -17F. We also found that IL-6 and -21 potently induced the development of IL-21–producing CD4+ T cells without the induction of IL-4, IFN-γ, IL-17A, or IL-17F production. On the other hand, TGF-β inhibited IL-6– and IL-21–induced development of IL-21–producing CD4+ T cells. IL-2 enhanced the development of IL-21–producing CD4+ T cells under Th17-polarizing conditions. Finally, IL-21–producing CD4+ T cells exhibited a stable phenotype of IL-21 production in the presence of IL-6, but retained the potential to produce IL-4 under Th2-polarizing conditions and IL-17A under Th17-polarizing conditions. These results suggest that IL-21–producing CD4+ T cells exhibit distinct characteristics from Th17 cells and develop preferentially in an IL-6–rich environment devoid of TGF-β, and that IL-21 functions as an autocrine growth factor for IL-21–producing CD4+ T cells.

c-Maf activates the promoter and enhancer of the IL-21 gene, and TGF-β inhibits c-Maf-induced IL-21 production in CD4+ T cells

Previous studies have shown that IL‐6 potently induces IL‐21 production in CD4+ T cells, whereas TGF‐β inhibits IL‐6‐induced IL‐21 production in CD4+ T cells. In this study, we addressed the mechanisms underlying the transcriptional regulation of IL‐21 production in CD4+ T cells. We found that IL‐6 induced c‐Maf expression in CD4+ T cells and that the enforced expression of c‐Maf induced IL‐21 production in CD4+ T cells without IL‐6, IL‐4/STAT6 signaling, or an autocrine effect of IL‐21. Moreover, we found that c‐Maf directly bound to and activated IL‐21P and the CNS‐2 enhancer through MARE sites. On the other hand, we also found that although TGF‐β up‐regulated IL‐6‐induced c‐Maf expression in CD4+ T cells, TGF‐β inhibited c‐Maf‐induced IL‐21 production in CD4+ T cells. Finally, we found that Foxp3 bound to IL‐21P and the CNS‐2 enhancer and inhibited c‐Maf‐induced IL‐21 production modestly but significantly in CD4+ T cells. Taken together, these results suggest that c‐Maf induces IL‐21 production directly in CD4+ T cells by activating IL‐21P and the CNS‐2 enhancer and that TGF‐β suppresses c‐Maf‐induced IL‐21 production in CD4+ T cells.

Development of autoimmune hepatitis–like disease and production of autoantibodies to nuclear antigens in mice lacking B and T lymphocyte attenuator

OBJECTIVEnB and T lymphocyte attenuator (BTLA), a coreceptor expressed on lymphocytes, was recently described as an inhibitory coreceptor that negatively regulates lymphocyte activation. The purpose of this study was to investigate the role of BTLA in the regulation of immune homeostasis and the pathogenesis of autoimmunity.nnnMETHODSnWe examined the levels of immunoglobulins and autoantibodies to nuclear antigens and the activation status of T cells in BTLA(-/-) mice. We also examined histopathologic changes in the organs of BTLA(-/-) mice.nnnRESULTSnWe observed that BTLA(-/-) mice gradually developed hypergammaglobulinemia, antinuclear antibodies, anti-SSA antibodies, anti-double-stranded DNA antibodies, and an increased number of activated CD4+ T cells in the periphery with age. Lack of BTLA led to spontaneous development of autoimmune hepatitis-like disease characterized by an elevation in the level of transaminases, interface hepatitis, and spotty necrosis of the liver. BTLA(-/-) mice also showed inflammatory cell infiltration of multiple organs, including the salivary glands, lungs, and pancreas; these features are similar to those of Sjögrens syndrome, which is a frequent complication of autoimmune hepatitis. Furthermore, the survival rate of BTLA(-/-) mice was significantly reduced after the age of 7 months.nnnCONCLUSIONnOur results indicate that BTLA plays an important role in the maintenance of immune tolerance and the prevention of autoimmune diseases.

Role of Th2 Cells in IgG4-Related Lacrimal Gland Enlargement

Background: Lacrimal gland enlargement (LGE) is one of the characteristics of Mikulicz’s disease (MD). Recently, marked serum IgG4 elevation and infiltration of IgG4-positive plasmacytes in the enlarged exocrine glands have been reported in MD patients. However, little is known about the role of CD4+ T cells and their cytokines in IgG4-related diseases. The aim of this study was to evaluate the characteristics of CD4+ T cells in patients with IgG4-related diseases. Methods: We investigated the clinical characteristics of 9 patients with LGE and elevated serum IgG4 levels (named IgG4-related LGE). We also examined mRNA expression of cytokines and transcription factors of peripheral blood CD4+ T cells in patients with IgG4-related LGE. Results: All patients with IgG4-related LGE showed elevated serum IgE levels. In addition, 5 of 9 patients with IgG4-related LGE exhibited eosinophilia and asthma-like symptoms. In patients with IgG4-related LGE, mRNA expression of IL-4, IL-5, IL-10 and GATA-3 but not IFN-γ or T-bet was enhanced on CD4+ T cells compared with that in healthy controls. Conclusions: Th2 cells may be involved in the pathogenesis of IgG4-related diseases.

Role of IL-23 and Th17 Cells in Airway Inflammation in Asthma

Asthma is characterized by chronic airway inflammation with intense eosinophil and lymphocyte infiltration, mucus hyperproduction, and airway hyperresponsiveness. Accumulating evidence indicates that antigen-specific Th2 cells and their cytokines such as IL-4, IL-5, and IL-13 orchestrate these pathognomonic features of asthma. In addition, we and others have recently shown that IL-17-producing CD4+ T cells (Th17 cells) and IL-23, an IL-12-related cytokine that is essential for survival and functional maturation of Th17 cells, are involved in antigen-induced airway inflammation. In this review, our current understanding of the roles of IL-23 and Th17 cells in the pathogenesis of allergic airway inflammation will be summarized.

Protein geranylgeranylation regulates the balance between Th17 cells and Foxp3+ regulatory T cells

Recent studies have suggested that statins, the inhibitors for 3-hydroxy-3-methyglutaryl (HMG)-CoA reductase in the mevalonate pathway, exhibit anti-inflammatory effects. However, the immune modulatory effects of statins on the differentiation of CD4(+) T cells and their underlying mechanisms are still largely unknown. To address these issues, we examined the effect of simvastatin and inhibitors for protein farnesylation and geranylgeranylation on the differentiation of IL-17-producing T cells (T(h)17 cells) and Foxp3(+) CD4(+) T cells. Simvastatin inhibited the differentiation of T(h)17 cells through the inhibition of HMG-CoA reductase activity but enhanced the differentiation of Foxp3(+) CD4(+) T cells. Geranylgeranyltransferase I inhibitor, GGTI-298, but not farnesyltransferase inhibitor, FTI-277, mimicked the effects of simvastatin, indicating that the inhibition of protein geranylgeranylation is responsible for the effects. Moreover, Foxp3(+) CD4(+) T cells developed in the presence of transforming growth factor-beta and GGTI-298 functioned as regulatory T cells (Tregs) in in vitro T cell proliferation assay as well as in an autoimmune colitis model. Finally, GGTI-298 induced SOCS3 expression and inhibited IL-6-induced signal transducers and activators of transcription3 phosphorylation in CD4(+) T cells. Taken together, these results indicate that protein geranylgeranylation enhances the differentiation of T(h)17 cells and inhibits the differentiation of Foxp3(+) Tregs partly via the inhibition of SOCS3 expression.

B and T lymphocyte attenuator suppresses IL-21 production from follicular Th cells and subsequent humoral immune responses.

We recently showed that mice lacking B and T lymphocyte attenuator (BTLA), a third inhibitory coreceptor expressed on B cells and T cells, exhibit an increased Ag-specific IgG response and gradually develop hyper-γ–globulinemia and autoantibody production. Recent studies revealed that follicular Th (Tfh) cells, which are non-Th1, non-Th2 effector T cells that express CXCR5 and provide help for B cells to produce Ig, also express BTLA. However, the role of BTLA in Tfh cell function remains unknown. In this study, we examined the regulatory role of BTLA in the development and function of Tfh cells. We found that CXCR5+ Tfh cells expressed higher levels of BTLA than did CXCR5− conventional CD4+ T cells. We also found that adoptive transfer of BTLA−/− CD4+ T cells, stimulated under Tfh cell-inducing conditions (Tfh-like cells), to wild-type (WT) mice induced more Ag-specific IgG2a and IgG2b production compared with that of WT Tfh-like cells. By contrast, another adoptive-transfer experiment using BTLA−/− mice as recipients showed that the expression of BTLA on B cells was not involved in the regulation of Tfh-like cell-mediated Ag-specific IgG responses. Moreover, the development of IL-21–producing CXCR5+ Tfh-like cells was significantly increased in BTLA−/− CD4+ T cells compared with WT CD4+ T cells. Furthermore, Tfh-like cell-mediated IgG responses were abolished when IL-21R−/− mice were used as recipients. These results suggest that BTLA signaling suppresses IL-21 production from Tfh cells and subsequent Tfh cell-mediated IgG responses.

Overlapping and Distinct Roles of STAT4 and T-bet in the Regulation of T Cell Differentiation and Allergic Airway Inflammation

T-bet and STAT4 play critical roles in helper T cell differentiation, especially for Th1 cells. However, it is still unknown about the relative importance and redundancy of T-bet and STAT4 for Th1 differentiation. It is also unknown about their independent role of T-bet and STAT4 in the regulation of allergic airway inflammation. In this study, we addressed these issues by comparing T-bet-deficient (T-bet−/−) mice, STAT4−/− mice, and T-bet- and STAT4-double-deficient (T-bet−/−STAT4−/−) mice on the same genetic background. Th1 differentiation was severely decreased in T-bet−/− mice and STAT4−/− mice as compared with that in wild-type mice, but Th1 differentiation was still observed in T-bet−/− mice and STAT4−/− mice. However, Th1 cells were hardly detected in T-bet−/−STAT4−/− mice. In contrast, the maintenance of Th17 cells was enhanced in T-bet−/− mice but was reduced in STAT4−/− mice and T-bet−/−STAT4−/− mice. In vivo, Ag-induced eosinophil and neutrophil recruitment into the airways was enhanced in T-bet−/− mice but was attenuated in STAT4−/− mice and T-bet−/−STAT4−/− mice. Ag-induced IL-17 production in the airways was also diminished in STAT4−/− mice and T-bet−/−STAT4−/− mice. These results indicate that STAT4 not only plays an indispensable role in T-bet-independent Th1 differentiation but also is involved in the maintenance of Th17 cells and the enhancement of allergic airway inflammation.

Protective Roles of B and T Lymphocyte Attenuator in NKT Cell-Mediated Experimental Hepatitis

Although B and T lymphocyte attenuator (BTLA) was originally identified as an inhibitory coreceptor selectively expressed on Th1 cells and B cells, recent studies have revealed that BTLA is expressed on a variety of cells, including macrophages, dendritic cells, and NK cells, and modulates their functions. However, the role of BTLA in the regulation of NKT cell function remains unknown. In this study, we found that BTLA was expressed on NKT cells at the levels similar to those on T cells and that BTLA-deficient (BTLA−/−) NKT cells produced larger amounts of IL-4 and IFN-γ upon α-glactosylceramide stimulation as compared with wild-type (WT) NKT cells. In vivo, BTLA−/− mice produced larger amounts of IL-4 and IFN-γ upon Con A injection and were more susceptible to Con A-induced hepatitis than WT mice. In addition, the augmentation of Con A-induced hepatitis in BTLA−/− mice was not observed in BTLA/NKT-double deficient mice. Moreover, NKT−/− mice reconstituted with BTLA−/− NKT cells were significantly more susceptible to Con A-induced hepatitis as compared with NKT −/− mice reconstituted with WT NKT cells. These results suggest that BTLA functions as the inhibitory coreceptor of NKT cells and plays a critical role in the prevention of NKT cell-mediated liver injury.