Taku Kuwabara

CCR 7 Ligands Are Required for Development of Experimental Autoimmune Encephalomyelitis through Generating IL-23-Dependent Th17 Cells

CCL19 and CCL21 are thought to be critical for experimental autoimmune encephalomyelitis (EAE) induction, but their precise role is unknown. We examined the role of these chemokines in inducing EAE. C57BL/6 mice lacking expression of these chemokines (plt/plt mice) or their receptor CCR7 were resistant to EAE induced with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) and pertussis toxin. However, passive transfer of pathogenic T cells from wild-type mice induced EAE in plt/plt mice, suggesting a defect independent of the role of CCR7 ligands in the migration of immune cells. Examination of draining lymph node (DLN) cells from MOG35–55-immunized plt/plt mice found decreased IL-23 and IL-12 production by plt/plt dendritic cells (DCs) and a concomitant defect in Th17 cell and Th1 cell generation. In contrast, production of the Th17 lineage commitment factors IL-6 and TGF-β were unaffected by loss of CCR7 ligands. The adoptive transfer of in vitro-generated Th17 cells from DLN cells of MOG35–55-immunized plt/plt mice developed EAE in wild-type recipient mice, whereas that of Th1 cells did not. Pathogenic Th17 cell generation was restored in plt/plt DLNs with the addition of exogenous IL-23 or CCL19/CCL21 and could be reversed by inclusion of anti-IL-23 mAb in cultures. Exogenous CCL19/CCL21 induced IL-23p19 expression and IL-23 production by plt/plt or wild-type DCs. Therefore, CCR7 ligands have a novel function in stimulating DCs to produce IL-23 and are important in the IL-23-dependent generation of pathogenic Th17 cells in EAE induction.

The Role of IL-17 and Related Cytokines in Inflammatory Autoimmune Diseases

Interleukin-17 (IL-17) induces the production of granulocyte colony-stimulating factor (G-CSF) and chemokines such as CXCL1 and CXCL2 and is a cytokine that acts as an inflammation mediator. During infection, IL-17 is needed to eliminate extracellular bacteria and fungi, by inducing antimicrobial peptides such as defensin. This cytokine also plays an important role in chronic inflammation that occurs during the pathogenesis of autoimmune diseases and allergies such as human rheumatoid arthritis (RA) for which a mouse model of collagen-induced arthritis (CIA) is available. In autoimmune diseases such as RA and multiple sclerosis (MS), IL-17 is produced by helper T (Th) cells that are stimulated by IL-1β and IL-6 derived from phagocytes such as macrophages and from tissue cells. IL-17 contributes to various lesions that are produced by Th17 cells, one subset of helper T cells, and by γδ T cells and innate lymphoid cells. It strongly contributes to autoimmune diseases that are accompanied by chronic inflammation. Thus, a functional understanding of Th17 cells is extremely important. In this review, we highlight the roles of cytokines that promote the development and maintenance of pathogenic Th17 cells in autoimmune diseases.

CCR7 ligands up-regulate IL-23 through PI3-kinase and NF-κB pathway in dendritic cells

We reported previously that the production of IL‐23 is impaired in DCs from mice that lack expression of the chemokines CCL19 and CCL21, which share the receptor CCR7, suggesting that these chemokines are required for IL‐23 expression. However, the molecular mechanism of CCR7‐mediated IL‐23 production in DCs is unknown. We found that CCL19 and CCL21 stimulated DCs through CCR7 and induced transcription of IL‐23p19 mRNA and IL‐23 production in splenic and BMDC. Stimulation of DCs with CCR7 ligands induced phosphorylation of MAPK family members and of Akt, but only a specific PI3K inhibitor, LY294002, not inhibitors of ERK, JNK, or p38, decreased IL‐23p19 transcription and IL‐23 production. In DCs stimulated with CCL19 or CCL21, IκBα was degraded, and NF‐κB was translocated into the nucleus. Prevention of NF‐κB activation blocked chemokine‐mediated IL‐23p19 transcription. A PI3K inhibitor abolished NF‐κB activation and IL‐23 production. Based on these findings, we concluded that PI3K and NF‐κB signaling pathways play a critical role in CCR7‐mediated IL‐23 production in murine DCs. As IL‐23 contributes to Th17 cell generation, and Th17 cells are pathogenic in autoimmune diseases, precise elucidation of these mechanisms would contribute to the development of strategies to control autoimmune diseases.

LPS-induced apoptosis is dependent upon mitochondrial dysfunction

Bacterial infection induces apoptotic cell death in human monoblastic U937 cells that have been pretreated with interferon γ (U937IFN). Apoptosis occurs in a manner that is independent of bacterial virulence proteins. In the present study, we show that lipopolysaccharide (LPS), a membrane constituent of gram-negative bacteria, also induces apoptosis in U937IFN cells. LPS treatment led to the appearance of characteristic markers of apoptosis such as nuclear fragmentation and activation of caspases. While the caspase inhibitor Z-VAD-fmk prevented LPS-induced apoptosis as judged by its inhibition of nuclear fragmentation, it failed to inhibit cytochrome c release and loss of mitochondrial membrane potential. Transfection of peptides containing the BH4 (Bcl-2 homology 4) domain derived from the anti-apoptotic protein Bcl-XL blocked LPS-induced nuclear fragmentation and the limited digestion of PARP. These results suggest that LPS does not require caspase activation to induce mitochondrial dysfunction and that mitochondria play a crucial role in the regulation of LPS-mediated apoptosis in U937IFN cells.

SATB1 Plays a Critical Role in Establishment of Immune Tolerance

Special AT-rich sequence binding protein 1 (SATB1) is a genome organizer that is expressed by T cells. T cell development is severely impaired in SATB1 null mice; however, because SATB1 null mice die by 3 wk of age, the roles of SATB1 in T cell development have not been well clarified. In this study, we generated and analyzed SATB1 conditional knockout (cKO) mice, in which the SATB1 gene was deleted from all hematopoietic cells. T cell numbers were reduced in these mice, mainly because of a deficiency in positive selection at the CD4+CD8+ double-positive stage during T cell development in the thymus. We also found that SATB1 cKO mice developed autoimmune diseases within 16 wk after birth. In SATB1 cKO mice, the numbers of Foxp3+ regulatory T (Treg) cells were significantly reduced at 2 wk of age compared with wild-type littermates. Although the numbers gradually increased upon aging, Treg cells in SATB1 cKO mice were still less than those in wild-type littermates at adulthood. Suppressive functions of Treg cells, which play a major role in establishment of peripheral tolerance, were also affected in the absence of SATB1. In addition, negative selection during T cell development in the thymus was severely impaired in SATB1 deficient mice. These results suggest that SATB1 plays an essential role in establishment of immune tolerance.

Uterine Natural Killer Cells Severely Decrease in Number at Gestation Day 6 in Mice

ABSTRACT Uterine natural killer (uNK) cells remarkably increase in number after implantation. NK cells or their precursors migrate from the blood stream and contribute to the increase. However, the contribution of uNK cells present in the virgin uterus has been unclear. To elucidate this issue, we examined uterine leukocyte subsets during pregnancy in BALB/c mice. The most dramatic change was the massive decrease in CD11b− or Gr-1− cells at Gestation Day (gd) 6. Uterine NK cells at gd 0 were CD11b−, and severely decreased at gd 6. The decrease was selective, and the proportion of other cells examined did not decrease. Uterine NK cells almost recovered at gd 12. These cells at gd 12 were more mature and/or activated in terms of expression of CD11b, CD27, CD127, or B220 than at gd 0. CXCL12 expression was observed on uterine cells at gd 0 or 6, but not at gd 12, whereas CXCR4 was detected on uNK cells at gds 0 and 12. A much higher expression of IL-15 in uterine cells or interferon-gamma expression in uNK cells was observed at gd 12 than at gd 0. IL-15 receptor alpha chain was detected on uNK cells at gd 12, but not at gd 0. Taken together, these findings were consistent with our interpretation that uNK cells present at gd 0 do not contribute to the increase of uNK cell number after implantation, and NK cells or their precursors migrate into the uterus, mature, and produce interferon-gamma to support pregnancy.

Altered antibody production and helper T cell function in mice lacking chemokines CCL19 and CCL21-Ser

The roles of chemokines CCL19 and CCL21 in Ab production were investigated using plt mutant mice, which lack expression of CCL19 and CCL21‐ser in their lymphoid organs. In these mice, the Th response has been shown to tend towards the Th1 type because of accumulation of inflammatory dendritic cells. When plt mice were immunized with 100 μg OVA in CFA, the number of Ab‐forming cells in the draining LN, and serum concentrations of OVA‐specific IgM and IgG Ab, were very close to those of the control, yet IgG2a Ab in plt mice was increased. In vitro IFN‐γ production by the draining LN cells of plt mice was increased. In addition, the ability of helper T cells from plt mice to stimulate Ab production in vitro was prolonged. Also, in the plt mice, in vivo challenge with OVA in incomplete Freunds adjuvant elicited a stronger IgG2a response and a weaker IgG1 response, which is suggestive of a Th1‐dominant response. Similar findings were obtained when mice were immunized with 100 μg OVA in alum, except that with alum the increases observed in plt mice were IgG1 produced in vivo and IL‐4 produced in vitro by draining LN cells. Furthermore, immunization with alum adjuvant also induced a prolonged in vitro recall response of IFN‐γ and IL‐4. These findings indicate that plt mice mount an anti‐OVA Ab response, and suggest that CCL19 and CCL21 induce prompt Ab responses to antigen, and negatively regulate helper T cell responses in vivo.

Pathogenesis of lupus-like nephritis through autoimmune antibody produced by CD180-negative B lymphocytes in NZBWF1 mouse.

Toll-like receptors appear to play an important role in the pathogenesis of lupus-like nephritis in mice. In human and mouse, CD180 is a homologue of TLR4. In SLE patients, the number of CD180-negative B cells in peripheral blood changes in parallel with disease activity. In the present study using NZBWF1 mice, the population of splenic CD180-negative B cells increased with progression of renal lesions and aging. These cells produced both anti-dsDNA and histone antibodies; the peripheral blood levels of anti-dsDNA antibody increased markedly with aging. B cells infiltrating into renal lesions were CD180-negative and produced anti-dsDNA antibody. Considered together, these findings indicate that CD180-negative B cells contribute significantly to development of SLE-like morbidity in NZBWF1 mice by autoantibody production.

Acetylation Modulates IL-2 Receptor Signaling in T Cells

Ligand binding to the cognate cytokine receptors activates intracellular signaling by recruiting protein tyrosine kinases and other protein modification enzymes. However, the roles of protein modifications other than phosphorylation remain unclear. In this study, we examine a novel regulatory mechanism of Stat5, based on its acetylation. As for phosphorylation, IL-2 induces the acetylation of signaling molecules, including Stat5, in the murine T cell line CTLL-2. Stat5 is acetylated in the cytoplasm by CREB-binding protein (CBP). Acetylated Lys696 and Lys700 on Stat5 are critical indicators for limited proteolysis, which leads to the generation of a truncated form of Stat5. In turn, the truncated form of Stat5 prevents transcription of the full-length form of Stat5. We also demonstrate that CBP physically associates with the IL-2 receptor β-chain. CBP, found in the nucleus in resting CTLL-2 cells, relocates to the cytoplasm after IL-2 stimulation in an MEK/ERK pathway–dependent manner. Thus, IL-2–mediated acetylation plays an important role in the modulation of cytokine signaling and T cell fate.

Regulation of T-Cell Signaling by Post-Translational Modifications in Autoimmune Disease

The adaptive immune system involves antigen-specific host defense mechanisms mediated by T and B cells. In particular, CD4+ T cells play a central role in the elimination of pathogens. Immunological tolerance in the thymus regulates T lymphocytes to avoid self-components, including induction of cell death in immature T cells expressing the self-reactive T-cell receptor repertoire. In the periphery, mature T cells are also regulated by tolerance, e.g., via induction of anergy or regulatory T cells. Thus, T cells strictly control intrinsic signal transduction to prevent excessive responses or self-reactions. If the inhibitory effects of T cells on these mechanisms are disrupted, T cells may incorrectly attack self-components, which can lead to autoimmune disease. The functions of T cells are supported by post-translational modifications, particularly phosphorylation, of signaling molecules, the proper regulation of which is controlled by endogenous mechanisms within the T cells themselves. In recent years, molecular targeted agents against kinases have been developed for treatment of autoimmune diseases. In this review, we discuss T-cell signal transduction in autoimmune disease and provide an overview of acetylation-mediated regulation of T-cell signaling pathways.