Novel Bruton's tyrosine kinase inhibitor TAS5315 suppresses the progression of inflammation and joint destruction in rodent collagen-induced arthritis.

Rheumatoid arthritis is an inflammatory autoimmune disease, characterized by autoantibody production, synovial inflammation, and joint destruction. Its pathogenesis is due to environmental factors and genetic backgrounds. Bruton's tyrosine kinase is a cytoplasmic non-receptor tyrosine kinase, expressed in most hematopoietic cell lineages, except T cells and plasma cells, and regulates various immune-related signaling pathways, thereby playing a crucial role in pathogenesis. Thus, inhibiting Bruton's tyrosine kinase may prove beneficial in treating autoimmune diseases. In the present study, we characterized Bruton's tyrosine kinase inhibitor, TAS5315, in vitro and evaluated its therapeutic effects in experimental arthritis models. TAS5315 markedly inhibited Bruton's tyrosine kinase enzyme activity and suppressed the B-cell receptor signaling pathway in Ramos cells. Moreover, it suppressed the expression of CD69, CD86, and MHC class II in mouse B lymphocytes and the production of TNF-α and MIP-1α in mouse macrophages and decreased bone resorption activity in mouse osteoclasts. Furthermore, it ameliorated the pathological changes in two rodent models of collagen-induced arthritis in vivo. TAS5315 improved bone mineral density and bone intensity. Thus, these results suggest that TAS5315 could be a promising therapeutic option for the treatment of rheumatoid arthritis.

Activation of Invariant NKT cells with glycolipid ligand α-galactosylceramide ameliorates glucose-6-phosphate isomerase peptide-induced arthritis.

OBJECTIVE: Invariant natural killer T (iNKT) cells regulate collagen-induced arthritis (CIA) when activated by their potent glycolipid ligand, alpha-galactosylceramide (α-GalCer). Glucose-6-phosphate isomerase (GPI)-induced arthritis is a closer model of human rheumatoid arthritis based on its association with CD4+ T cells and cytokines such as TNF-α and IL-6 than CIA. Dominant T cell epitope peptide of GPI (GPI325-339) can induce arthritis similar to GPI-induced arthritis. In this study, we investigated the roles of activation of iNKT cells by α-GalCer in GPI peptide-induced arthritis. METHODS: Arthritis was induced in susceptible DBA1 mice with GPI peptide and its severity was assessed clinically. The arthritic mice were treated with either the vehicle (DMSO) or α-GalCer. iNKT cells were detected in draining lymph nodes (dLNs) by flow cytometry, while serum anti-GPI antibody levels were measured by enzyme-linked immunosorbent assay. To evaluate GPI peptide-specific cytokine production from CD4+ T cells, immunized mice were euthanized and dLN CD4+ cells were re-stimulated by GPI-peptide in the presence of antigen-presenting cells. RESULTS: α-GalCer induced iNKT cell expansion in dLNs and significantly decreased the severity of GPI peptide-induced arthritis. In α-GalCer-treated mice, anti-GPI antibody production (total IgG, IgG1, IgG2b) and IL-17, IFN-γ, IL-2, and TNF-α produced by GPI peptide-specific T cells were significantly suppressed at day 10. Moreover, GPI-reactive T cells from mice immunized with GPI and α-GalCer did not generate any cytokines even when these cells were co-cultured with APC from mice immunized with GPI alone. In vitro depletion of iNKT cells did not alter the suppressive effect of α-GalCer on CD4+ T cells. CONCLUSION: α-GalCer significantly suppressed GPI peptide-induced arthritis through the suppression of GPI-specific CD4+ T cells.

Involvement of NK 1.1-positive γδT cells in interleukin-18 plus interleukin-2-induced interstitial lung disease.

Interstitial lung disease (ILD) is induced by various factors in humans. However, the exact mechanism of ILD remains elusive. This study sought to determine the role of natural killer (NK) 1.1(+) γδT cells in ILD. The injection of IL-18 plus IL-2 (IL-18/IL-2) into C57BL6 (B6) mice induced acute ILD that resembled early-stage human ILD. An accumulation of NK1.1(+) γδT cells similar to NK cells was evident in the lungs. The T Cell Receptor (TCR) Vγ and Vδ repertoires of NK1.1(+) γδT cells indicated polyclonal expansion. The expression of IL-2 receptor ß (Rß) and IL-18Rß in NK1.1(+) γδT cells was higher than in NK1.1(-) γδT cells. IL-18/IL-2 stimulated the proliferation of NK1.1(+) γδT cells, but not NK1.1(-) γδT cells. The IL-18/IL-2-stimulated NK1.1(+) γδT cells produced higher concentrations of IFN-γ than did NK1.1(-) γδT cells. Moreover, NK1.1(+) γδT and NK1.1(-) γδT cells constituted completely different cell populations. The IL-18/IL-2-induced ILD was milder in TCRδ(-/-) and IFN-γ(-/-) mice, compared with B6 mice. Furthermore, cell-transfer experiments demonstrated that NK1.1(+) γδT cells could induce the expansion of NK cells and IFN-γ mRNA in the lung by IL-18/IL-2. Our results suggest that NK1.1(+) γδT cells function as inflammatory mediators in the early phase of IL-18/IL-2-induced ILD.

Induction of Th1-biased cytokine production by alpha-carba-GalCer, a neoglycolipid ligand for NKT cells.

NKT cells are characterized by their production of both T(h)1 and T(h)2 cytokines immediately after stimulation with alpha-galactosylceramide (alpha-GalCer), which is composed of alpha-galactopyranose linked to ceramide (itself composed of sphingosine and fatty-acyl chains); the chain length of the ceramide varies and this affects the ability of alpha-GalCer to stimulate cytokine production. However, the contribution of its galactopyranose sugar moiety remains unclear. We synthesized alpha-carba-GalCer, which has an alpha-linked carba-galactosyl moiety; here, the 5a'-oxygen atom of the D-galactopyranose ring of alpha-GalCer is replaced by a methylene group. The alpha-carba-GalCer was more stable and showed higher affinity to the NKT receptor. It thus enhanced and prolonged production of IL-12 and IFN-gamma compared with alpha-GalCer, resulting in augmented NKT cell-mediated adjuvant effects in vivo. The alpha-carba-GalCer, which has an ether linkage, was more resistant to degradation by liver microsomes than was alpha-GalCer, which has an acetal bond. Modulation of the sugar moiety in glycolipids might therefore provide optimal therapeutic reagents for protective immune responses against tumor or pathogens.

Adjuvant activity mediated by iNKT cells.

Invariant natural killer T (iNKT) cells have adjuvant activity due to their ability to produce large amounts of IFN-gamma, which activates other cells in innate and acquired systems, and orchestrates protective immunity. Based on these adjuvant mechanisms, we developed iNKT cell-targeted adjuvant therapy and carried out a phase I/IIa trial on advanced lung cancer patients. The patient group with increased numbers of IFN-gamma-producing cells showed prolonged survival with a median survival time of 31.9 months. Sixty percent of the patients in this group survived for more than 2 years with only a primary treatment and without tumor progression and metastasis.

Altered peptide ligands inhibit arthritis induced by glucose-6-phosphate isomerase peptide.

INTRODUCTION: Immunosuppressants, including anti-TNFalpha antibodies, have remarkable effects in rheumatoid arthritis; however, they increase infectious events. The present study was designed to examine the effects and immunological change of action of altered peptide ligands (APLs) on glucose-6-phosphate isomerase (GPI) peptide-induced arthritis. METHODS: DBA/1 mice were immunized with hGPI325-339, and cells of draining lymph node (DLN) were stimulated with hGPI325-339 to investigate the T-cell receptor (TCR) repertoire of antigen-specific CD4+ T cells by flow cytometry. Twenty types of APLs with one amino acid substitution at a TCR contact site of hGPI325-339 were synthesized. CD4+ T cells primed with human GPI and antigen-presenting cells were co-cultured with each APL and cytokine production was measured by ELISA to identify antagonistic APLs. Antagonistic APLs were co-immunized with hGPI325-339 to investigate whether arthritis could be antigen-specifically inhibited by APL. After co-immunization, DLN cells were stimulated with hGPI325-339 or APL to investigate Th17 and regulatory T-cell population by flow cytometry, and anti-mouse GPI antibodies were measured by ELISA. RESULTS: Human GPI325-339-specific Th17 cells showed predominant usage of TCRVbeta8.1 8.2. Among the 20 synthesized APLs, four (APL 6; N329S, APL 7; N329T, APL 12; G332A, APL 13; G332V) significantly reduced IL-17 production by CD4+ T cells in the presence of hGPI325-339. Co-immunization with each antagonistic APL markedly prevented the development of arthritis, especially APL 13 (G332V). Although co-immunization with APL did not affect the population of Th17 and regulatory T cells, the titers of anti-mouse GPI antibodies in mice co-immunized with APL were significantly lower than in those without APL. CONCLUSIONS: We prepared antagonistic APLs that antigen-specifically inhibited the development of experimental arthritis. Understanding the inhibitory mechanisms of APLs may pave the way for the development of novel therapies for arthritis induced by autoimmune responses to ubiquitous antigens.

Low levels of soluble CD1d protein alters NKT cell function in patients with rheumatoid arthritis.

CD1d molecules on the cell surface play a critical role in the presentation of glycolipid antigens to natural killer T (NKT) cells. We previously showed that the human CD1d gene has 8 splice variants, one of which is a soluble form lacking the beta2-m and transmembrane domains. This study focused on soluble CD1d (sCD1d) by generating recombinant sCD1d proteins and assaying them in plasma using a newly established ELISA method. The amount of sCD1d proteins in plasma was significantly decreased in rheumatoid arthritis (RA) patients (55.2+/-13.3 years, mean +/-SD) compared with healthy donors (31.2+/-7.4 years). Plasma sCD1d protein levels correlated with the number of NKT cells (TCR V alpha 24+ V beta 11+CD3+) in peripheral blood mononuclear cells (r(2)=0.061). Furthermore, sCD1d proteins induced IFN-gamma production from NKT cells, but neither IL-4 nor IL-10. These findings suggest that the low plasma levels of sCD1d protein in RA patients reduce the number and thus activation of peripheral NKT cells. It is therefore hypothesized that sCD1d stimulates NKT cells and low plasma sCD1d levels in RA reflect a pathogenic mechanism associated with a decrease in NKT cells.

Altered peptide ligands regulate type II collagen-induced arthritis in mice.

We reported that peripheral blood mononuclear cells from HLA-DRB1*0101 Japanese patients with rheumatoid arthritis (RA) were highly reactive to 256-271 peptide of type II collagen (CII). Similar to RA, T cells reactive to CII (AA256-271) play a crucial role in the generation of arthritis in CII-induced arthritis mouse (I-A(q)). In the present study, we regulated the CII reactivity of T cells from CIA mouse with I-A(q) by altered peptide ligand (APL). Eight different APLs were designed and screened for their antagonistic activity using CII reactive cytokine production assay. Four APLs of CII 256-271 exhibited antagonistic activity in CII-reactive T cells. Moreover, intraperitoneally injected APL-5 (G262A) significantly suppressed CII-induced arthritis in mice, whereas the other three APLs did not. Compared with the control, APL-5 suppressed interleukin (IL)-17 production by T cells from CII-induced arthritis mice. These results suggest that CII APL is a potentially suitable therapeutic strategy for the control of RA.