Kumiko Hashimoto

Antagonizing dopamine D1-like receptor inhibits Th17 cell differentiation: preventive and therapeutic effects on experimental autoimmune encephalomyelitis.

Five types of dopamine receptors, termed D1 to D5, have been identified to date. The D1 and D5 receptors form the D1-like group that couples with the Galphas class of G proteins, while D2, D3 and D4 form the D2-like group that couples with the Galphai class of G proteins. A D2-like-receptor (D2-like-R) antagonist L750667 induced dendritic cell (DC)-mediated Th17 differentiation. In contrast, a D1-like-R antagonist SCH23390 inhibited DC-mediated Th17 differentiation. The D1-like-Rs were expressed on both DCs and T cells, whereas D2-like-Rs were marginally expressed on CD4+CD45RA+ naïve T cells. In addition, SCH23390 had the ability to prevent experimental autoimmune encephalomyelitis (EAE) in mice. Spleen cells from EAE mice showed decreased IL-17 production, when SCH23390 was administered. Adoptive transfer of DCs treated with SCH23390 successfully prevented EAE. These findings indicate that antagonizing D1-like-Rs on DCs inhibits Th17 differentiation, thereby leading to an amelioration of EAE.

B Cell Chemoattractant CXCL13 Is Preferentially Expressed by Human Th17 Cell Clones

Th 17 cells represent a novel subset of CD4+ T cells that have a protective effect against extracellular microbes, while they are also responsible for autoimmune disorders in mice. However, the protein expression profile of Th17 cells remains to be clarified. In this study, we report an effective method to establish human allo-reactive Th17 cell clones and demonstrate that human Th17, but not Th1 or Th2, cells express B cell chemoattractant CXCL13, by using DNA chips, RT-PCR, and ELISA. Such a pattern was also the case in Candida albicans-specific Th17 clones and synovial fluid specimens obtained from patients with rheumatoid arthritis. The biological implication of this finding is discussed.

Dopamine D1-Like Receptor Antagonist Attenuates Th17-Mediated Immune Response and Ovalbumin Antigen-Induced Neutrophilic Airway Inflammation

Allergic airway inflammation is generally considered a Th2-type immune response. Recent studies, however, demonstrated that Th17-type immune responses also play important roles in this process, especially in the pathogenesis of neutrophilic airway inflammation, a hallmark of severe asthma. We previously reported that dendritic cells release dopamine to naive CD4+ T cells in Ag-specific cell–cell interaction, in turn inducing Th17 differentiation through dopamine D1-like receptor (D1-like-R). D1-like-R antagonist attenuates Th17-mediated diseases such as experimental autoimmune encephalomyelitis and autoimmune diabetes. However, the effect of antagonizing D1-like-R on Th17-mediated airway inflammation has yet to be studied. In this study, we examined whether D1-like-R antagonist suppresses OVA-induced neutrophilic airway inflammation in OVA TCR-transgenic DO11.10 mice and then elucidated the mechanism of action. DO11.10 mice were nebulized with OVA or PBS, and some mice received D1-like-R antagonist orally before OVA nebulization. D1-like-R antagonist significantly suppressed OVA-induced neutrophilic airway inflammation in DO11.10 mice. It also inhibited the production of IL-17 and infiltration of Th17 cells in the lung. Further, D1-like-R antagonist suppressed the production of IL-23 by lung CD11c+ APCs. In contrast, D1-like-R antagonist did not increase Foxp3+ regulatory T cells in the lung. D1-like-R antagonist neither suppressed nonspecific LPS-induced neutrophilic airway inflammation nor OVA-induced eosinophilic airway inflammation. These results indicate that D1-like-R antagonist could suppress Th17-mediated neutrophilic airway inflammation, raising the possibility that antagonizing D1-like-R serves as a promising new strategy for treating neutrophil-dominant severe asthma.

Curdlan Induces DC-Mediated Th17 Polarization via Jagged1 Activation in Human Dendritic Cells

BACKGROUND Th17-inducing activity is carried by certain polysaccharides such as beta-glucan derived from Candia albicans. Our previous studies have shown that Th1- and Th2-inducing activities can be qualitatively evaluated by the expression patterns of Notch ligand isoforms, using human monocyte-derived dendritic cells (Mo-DCs) and some leukemic cell lines such as THP-1. The association of Th17-inducing activities with Notch ligand expression patterns has been unclear. METHODS Mo-DCs from healthy volunteers were co-cultured with HLA-DR-nonshared allogeneic CD4+ naïve T cells to induce a mixed lymphocyte reaction, in the presence of adjuvants, such as curdlan. Culture supernatants were assayed for IFNgamma, IL-5 and IL-17 by an enzyme-linked immunosorbent assay (ELISA). Notch ligand expression on Mo-DCs and THP-1 cells was evaluated by using RT-PCR. RESULTS The present study shows that curdlan, one of the beta-glucans, has the ability to induce DC-mediated Th17 differentiation. It is also interesting to note that Jagged1 mRNA in Mo-DCs and THP-1 cells is up-regulated by curdlan. Furthermore, polyclonal anti-Jagged1 antibody inhibited such DC-mediated Th17 differentiation. CONCLUSIONS This study suggests that curdlan induces human DC-mediated Th17 polarization via Jagged1 activation in DCs.

Dopamine D1-like receptor antagonist, SCH23390, exhibits a preventive effect on diabetes mellitus that occurs naturally in NOD mice.

Dopamine receptors have five isoforms, termed D1-D5. The D1 and D5 receptors form the D1-like group that couples with the Galphas class of G proteins, while D2, D3 and D4 form the D2-like group that couples with the Galphai class of G proteins. In our previous studies, a D1-like-R antagonist, SCH23390, inhibited DC-mediated Th17 differentiation and exhibited preventive and therapeutic effects on experimental autoimmune encephalomyelitis (EAE) in mice. We herein demonstrate in the current study that in the pancreas obtained from NOD mice, islet infiltrates appear to be composed of mononuclear cells positive for IL-23R, one of the specific markers for Th17. Thereafter, NOD mice were orally administered SCH23390 from week 6 to week 26. At week 26, 67% and 25% of mice developed diabetes in the control and the SCH23390 groups, respectively (p<0.05). A histological examination of SCH23390-treated mice exhibited a typical normal islet structure with no signs of periductal and perivascular infiltrates, whereas the islets from vehicle controls showed insulitis. In week 26, spleen cells were re-stimulated with anti-CD3 and anti-CD28 antibodies in vitro and exhibited an augmentation of IFNgamma induction and the suppression of IL-17 induction in the SCH23390-treated mice. These findings indicate that antagonizing D1-like-R suppresses IL-17 expression, thereby leading to a decreased occurrence of NOD.

D1-Like Receptor Antagonist Inhibits IL-17 Expression and Attenuates Crescent Formation in Nephrotoxic Serum Nephritis

Background/Aims: A dopamine type 1-like receptor (D1-like-R) expressed on dendritic cells was involved in Th17 cell differentiation of naive CD4+ T cells. Thus, we treated mice with nephrotoxic serum nephritis (NTN) with a D1-like-R antagonist to test whether Th17 cells play a role in this kidney disease. Methods: The SCH group of nephritic mice was administered with a D1-like-R antagonist, SCH23390, from day 0 to day 13. The kidney and spleen were collected at sacrifice on day 14. Glomerular pathology and CCR6+ cell infiltration were quantitatively evaluated. IL-4, IFN-γ and IL-17 mRNA levels in the kidney, and IFN-γ and IL-17 concentrations in the supernatants from splenocytes activated in vitro were measured. Results: Crescent formation had significantly developed in the positive control (PC) group of untreated, nephritic mice, which was suppressed in the SCH group. Glomerular infiltration of CCR6+ cells was also noted in the PC group, which was abolished in the SCH group. Renal IL-17 and IFN-γ mRNA levels were significantly reduced in the SCH group compared with the PC group. Additionally, in vitro activation augmented IFN-γ induction, but suppressed IL-17 induction by splenocytes from the SCH group compared with those from the PC group. Conclusion: We identified treatment with a D1-like-R antagonist inhibited IL-17 expression and attenuated crescent formation in NTN mice.

Evaluation of Adjuvant Activities Using Human Antigen Presenting Cells in Vitro

BACKGROUND Immunomodulators such as lipopolysaccharides (LPS) and forskolin change the nature of dendritic cells (DCs) to induce Th1 and Th2 cells, respectively, thereby designated Th1 or Th2 adjuvants. Our previous study showed that such activities can be qualitatively evaluated by expression patterns of Notch ligand isoforms, using human monocyte-derived DCs and some leukemic cell lines, such as THP-1 or KG-1. However, quantitative evaluation of the adjuvant activities was not fully established. METHODS PMA-treated human monocytic cell line THP-1 was used as a target. Cells were stimulated with various adjuvants, and the intracellular levels of cAMP were determined. RESULTS Th2 adjuvant forskolin was qualitatively evaluated by an increased expression of Delta1 in PMA-treated THP-1 cells, as reported in our previous study. In PMA-treated THP-1 cells, intracellular cAMP levels increased after stimulation with forskolin, in a dose-dependent manner. On the other hand, LPS, one of the known Th1 adjuvants, suppressed the increased cAMP level in a dose-dependent manner. CONCLUSIONS Th2- and Th1-adjuvant activities can be quantitatively evaluated by using PMA-treated THP-1 cells and PMA-treated/forskolin-stimulated THP-1 cells, respectively.

Qualitative evaluation of adjuvant activities and its application to Th2/17 diseases.

Dendritic cells (DCs) are antigen-presenting cells specialized to activate naive T lymphocytes and initiate primary immune responses. The different classes of specific immune responses are driven by the biased development of antigen-specific helper T cell subsets – that is, Th1, Th2, and Th17 cells – that activate different components of cellular and humoral immunity. DCs reside in an immature state in many nonlymphoid tissues such as the skin or airway mucosa which are highly exposed to allergens, pathogens, and chemicals. T cell receptor stimulation with costimulation allows naive Th cells to develop into effector cells, normally accompanied by high-level expression of selective sets of cytokines. The balance of these cytokines and the resulting class of immune responses depend on the conditions under which DCs are primed. Immunomodulators such as lipopolysaccharides/forskolin/curdlan change the nature of DCs to induce Th1/Th2/Th17 cells thereby designated Th1/Th2/Th17 adjuvants. We have recently found that such activities can be scrutinized by using mixed lymphocyte reaction, cAMP, and differential expression of Notch ligand isoforms. Application of these methods for the analyses of atopic dermatitis and experimental autoimmune encephalomyelitis will be discussed.

Wogonin Attenuates Ovalbumin Antigen-Induced Neutrophilic Airway Inflammation by Inhibiting Th17 Differentiation

Allergic airway inflammation is generally considered to be a Th2-type immune response. Recent studies, however, have demonstrated that Th17-type immune responses also play important roles in this process, particularly in the pathogenesis of neutrophilic airway inflammation, a hallmark of severe asthma. We scrutinized several Kampo extracts that reportedly exhibit anti-inflammatory activity by using in vitro differentiation system of human and mouse naïve T cells. We found that hange-shashin-to (HST) and oren-gedoku-to (OGT) possess inhibitory activity for Th17 responses in vitro. Indeed, wogonin and berberine, major components common to HST and OGT, exhibit Th17-inhibitory activities in both murine and human systems in vitro. We therefore evaluated whether wogonin suppresses OVA-induced neutrophilic airway inflammation in OVA TCR-transgenic DO11.10 mice. Consequently, oral administration of wogonin significantly improved OVA-induced neutrophilic airway inflammation. Wogonin suppressed the differentiation of naïve T cells to Th17 cells, while showing no effects on activated Th17 cells.