Kazuki Fujioka

Myeloid-Derived Suppressor Cells Play Crucial Roles in the Regulation of Mouse Collagen-Induced Arthritis

Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. The role of MDSCs in autoimmune diseases remains controversial, and little is known about the function of MDSCs in autoimmune arthritis. In this study, we clarify that MDSCs play crucial roles in the regulation of proinflammatory immune response in a collagen-induced arthritis (CIA) mouse model. MDSCs accumulated in the spleens of mice with CIA when arthritis severity peaked. These MDSCs inhibited the proliferation of CD4+ T cells and their differentiation into Th17 cells in vitro. Moreover, MDSCs inhibited the production of IFN-γ, IL-2, TNF-α, and IL-6 by CD4+ T cells in vitro, whereas they promoted the production of IL-10. Adoptive transfer of MDSCs reduced the severity of CIA in vivo, which was accompanied by a decrease in the number of CD4+ T cells and Th17 cells in the draining lymph nodes. However, depletion of MDSCs abrogated the spontaneous improvement of CIA. In conclusion, MDSCs in CIA suppress the progression of CIA by inhibiting the proinflammatory immune response of CD4+ T cells. These observations suggest that MDSCs play crucial roles in the regulation of autoimmune arthritis, which could be exploited in new cell-based therapies for human rheumatoid arthritis.

The rapid efficacy of abatacept in a patient with rheumatoid vasculitis

We report a case of rheumatoid vasculitis (RV) that responded well to abatacept, a cytotoxic T lymphocyte-associated antigen 4 (CTLA4)-immunoglobulin fusion protein. A 38-year-old woman developed RV despite treatment with methotrexate and tumor necrosis factor (TNF) inhibitors. The effects of steroid therapy, immunoabsorption plasmapheresis, and interleukin-6 inhibitor were insufficient, however, administration of abatacept rapidly improved her clinical symptoms with almost normalization of the immunological findings. This is the first published case report of the successful treatment of RV with abatacept.

Inhibition of osteoclastogenesis by osteoblast-like cells genetically engineered to produce interleukin-10

Bone destruction at inflamed joints is an important complication associated with rheumatoid arthritis (RA). Interleukin-10 (IL-10) may suppress not only inflammation but also induction of osteoclasts that play key roles in the bone destruction. If IL-10-producing osteoblast-like cells are induced from patient somatic cells and transplanted back into the destructive bone lesion, such therapy may promote bone remodeling by the cooperative effects of IL-10 and osteoblasts. We transduced mouse fibroblasts with genes for IL-10 and Runx2 that is a crucial transcription factor for osteoblast differentiation. The IL-10-producing induced osteoblast-like cells (IL-10-iOBs) strongly expressed osteoblast-specific genes and massively produced bone matrix that were mineralized by calcium phosphate in vitro and in vivo. Culture supernatant of IL-10-iOBs significantly suppressed induction of osteoclast from RANKL-stimulated Raw264.7 cells as well as LPS-induced production of inflammatory cytokine by macrophages. The IL-10-iOBs may be applicable to novel cell-based therapy against bone destruction associated with RA.

Allograft inflammatory factor-1 in the pathogenesis of bleomycin-induced acute lung injury

Allograft inflammatory factor-1 (AIF-1) is a protein expressed by macrophages infiltrating the area around the coronary arteries of rats with an ectopic cardiac allograft. Some studies have shown that expression of AIF-1 increased in a mouse model of trinitrobenzene sulfonic acid-induced acute colitis and in acute cellular rejection of human cardiac allografts. These results suggest that AIF-1 is related to acute inflammation. The current study used bleomycin-induced acute lung injury to analyze the expression of AIF-1 and to examine its function in acute lung injury. Results showed that AIF-1 was significantly expressed in lung macrophages and increased in bronchoalveolar lavage fluid from mice with bleomycin-induced acute lung injury in comparison to control mice. Recombinant AIF-1 increased the production of IL-6 and TNF-α from RAW264.7 (a mouse macrophage cell line) and primary lung fibroblasts, and it also increased the production of KC (CXCL1) from lung fibroblasts. These results suggest that AIF-1 plays an important role in the mechanism underlying acute lung injury.

Roles of high-mobility group box 1 and thrombin in murine pulmonary fibrosis and the therapeutic potential of thrombomodulin

Cross talk between inflammation and coagulation plays important roles in acute or subacute progressive pulmonary fibrosis characterized by diffuse alveolar damage. Thrombomodulin is a physiological inhibitor of high-mobility group box 1 (HMGB1), and thrombin and may be effective for this condition. This study investigated the roles of HMGB1 and thrombin in the pathophysiology of bleomycin-induced pulmonary fibrosis and the efficacy of recombinant human soluble thrombomodulin (rhTM). Pulmonary fibrosis was induced in wild-type C57BL/6 mice by intratracheal instillation of bleomycin. We first assessed HMGB1, thrombin, transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA) levels in bronchoalveolar lavage fluid and lung tissue sections over time. Expression of HMGB1 and thrombin was elevated before that of TGF-β1 and α-SMA and remained high during the fibrotic phase after bleomycin instillation. We next examined whether in vitro stimulation with HMGB1 and thrombin induced expression of TGF-β1 and α-SMA in cultured alveolar macrophages and lung fibroblasts, respectively, by performing quantitative PCR, enzyme-linked immunosorbent assay, Western blot, and immunofluorescence analyses. HMGB1 and thrombin stimulation induced TGF-β1 production by alveolar macrophages, and thrombin stimulation also induced α-SMA expression in lung fibroblasts. Finally, we evaluated the effect of rhTM on bleomycin-induced pulmonary fibrosis. Compared with the vehicle control, both early and late-phase administration of rhTM suppressed the fibrotic process. Our results suggest that HMGB1 and thrombin were involved in the pathophysiology of pulmonary fibrosis via production of profibrotic proteins and that rhTM attenuated bleomycin-induced pulmonary fibrosis. rhTM may be a therapeutic option for acute or subacute pulmonary fibrosis.

THU0112 Myeloid-Derived Suppressor Cells have Regulatory Roles in Mouse Collagen-Induced Arthritis

Background Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. MDSCs are characterized by the co-expression of the myeloid differentiation antigens Gr-1 and CD11b in mice (1). MDSCs in cancer have been studied in detail and are known to play roles in tumor associated immune suppression. However, the roles of MDSCs in autoimmune disease remain controversial and little is known about MDSCs in autoimmune arthritis. Objectives We investigate the roles of MDSCs in autoimmune arthritis using collagen-induced arthritis (CIA) mouse models. Methods We determined the number of Gr-1+ CD11b+ MDSCs in the spleens of CIA mice by flow cytometry. Next, we isolated MDSCs from CIA mice by magnetic cell sorting and cultured with CD4+ T cells to analyze the functions of MDSCs. We investigated the proliferation of CD4+ T cells by CFSE dye dilution assay and estimated cytokine levels produced by CD4+ T cells using ELISA. Furthermore, we investigated CD4+ T cell differentiation into Th17 cells by flow cytometry. Finally, we performed adoptive transfer of MDSCs into CIA mice and investigated the severity of arthritis. Results MDSCs significantly accumulated in the spleens of mice with CIA when arthritis severity peaked. These MDSCs inhibited CD4+ T cell proliferation and differentiation into Th17 cells. Moreover, MDSCs inhibited the production of IFNγ, IL-2, TNFα, and IL-6 by CD4+ T cells in vitro, whereas they promoted the production of IL-10. Adoptive transfer of MDSCs reduced both clinical (Image A) and histological arthritis scores (Image B) in vivo, which was accompanied by a decrease in the number of CD4+ T cells and Th17 cells in the draining lymph nodes. Image/graph Conclusions MDSCs in CIA suppress the progression of CIA by inhibiting the pro-inflammatory immune response of CD4+ T cells. These observations suggest that MDSCs play crucial roles in the regulation of autoimmune arthritis, which could be exploited in new cell-based therapies for human rheumatoid arthritis (RA). References Gabrilovich, D. I., and S. Nagaraj. 2009. Myeloid-derived suppressor cells as regulators of the immune system. Nat. Rev. Immunol. 9: 162-174. Disclosure of Interest None Declared

Role of allograft inflammatory factor-1 in bleomycin-induced lung fibrosis

Allograft inflammatory factor-1 (AIF-1) is a protein expressed by macrophages infiltrating the area around the coronary arteries in a rat ectopic cardiac allograft model. We previously reported that AIF-1 is associated with the pathogenesis of rheumatoid arthritis and skin fibrosis in sclerodermatous graft-versus-host disease mice. Here, we used an animal model of bleomycin-induced lung fibrosis to analyze the expression of AIF-1 and examine its function in lung fibrosis. The results showed that AIF-1 was expressed on lung tissues, specifically macrophages, from mice with bleomycin-induced lung fibrosis. Recombinant AIF-1 increased the production of TGF-β which plays crucial roles in the mechanism of fibrosis by mouse macrophage cell line RAW264.7. Recombinant AIF-1 also increased both the proliferation and migration of lung fibroblasts compared with control group. These results suggest that AIF-1 plays an important role in the mechanism underlying lung fibrosis, and may provide an attractive new therapeutic target.

AB0247 A New Disease Activity Biomarker Alternative To CRP under Tocilizumab Therapy for Rheumatoid Arthritis via Peptidomic Analysis

Background Tocilizumab, anti-IL-6 receptor monoclonal antibody, is widely used for patients with rheumatoid arthritis. IL-6 is essential for production of C-reactive protein (CRP). Tocilizumab fully inhibit the production of CRP. Therefore, we have difficulty in objective assessment of infection and disease activity because the level of CRP is suppressed under tocilizumab therapy. The development of new biomarker alternative to CRP is needed for daily practice. Objectives To discover new biomarkers alternative to CRP under tocilizumab therapy for rheumatoid arthritis. Methods We registered patients with rheumatoid arthritis treated with tocilizumab. We collected serum samples from those patients at baseline, 4 weeks after the first tocilizumab administration when patients CRP level is almost normal, and 1 year later. And we measured CRP, ESR and clinical disease activity index (CDAI) score.Serum peptidomic analysis was conducted by newly-established one-step direct transfer technology (BLOTCHIP-MS analysis), a rapid quantitative technology for peptidomic analysis. All sample measurements were repeated four times. Statistical analyses of MS spectral data were conducted using ClinProTools version 2.2 (Bruker Daltonics). Results We registered 14 patients and their background is shown in Table 1. The levels of CRP were 1.16±0.99 mg/dl at baseline, 0.02±0.01 mg/dl at 4 weeks and 0.01±0.01mg/dl at 1 year, respectively. Their CDAI score were 22±9.2 at baseline, 15±8.9 at 4 weeks and 3±2.6 at 1 year, respectively. CRP titer decreased to almost normal level at 4 weeks regardless whether CDAI score did not fully decrease. We detect 6 biomarkers, named as PRSJ01 to PRSJ06, by the peptidomic analysis (Table 2). The AUC of diagnostic value of these markers is from 0.742 to 0.858. For example, the level of PRSJ06 significantly decreased 4 weeks (Wilcoxon singed-rank test, p=0.02) and 1 year (Wilcoxon singed-rank test, p=0.003) after first tocilizumab administration (Figure 1). and it was inversely-correlated with CDAI score. Conclusions We detect new disease activity biomarkers alternative to CRP under tocilizumab therapy for rheumatoid arthritis. It is useful for exact evaluation of disease activity and infection during tocilizumab therapy. Disclosure of Interest None declared

AB0910 Once-Weekly Teriparatide is Effective for Glucocorticoid-Induced Osteoporosis Patients with Collagen Diseases

Background Glucocorticoid-induced osteoporosis (GIOP) patients are at very high risk of fractures. Unfortunately, only a small minority of GC users receive effective preventive, diagnostic, and therapeutic interventions. Intermittent administration of teriparatide (TPTD) is the only currently available anabolic agent to stimulate osteoblast activity. Daily TPTD was more efficacious than alendronate in increasing lumbar spine BMD in men and pre- and postmenopausal women. Recently, once-weekly TPTD was provided, which had both rapid and powerful anti-fracture efficacy. It has unique feature; serum NTx is decreasing after administration for patients with primary osteoporosis contrast to daily TPTD. Once-weekly TPTD is expected to be effective for GIOP, but it has not been reported. Objectives To examine the efficacy of once-weekly TPTD for patients with GIOP and collagen diseases. Methods We registered GIOP patients with collagen diseases treated with once-weekly TPTD at our department. They were treated with PSL for at least 6 months and had inadequate response for bisphosphonates. We measured YAM (Young Adult Mean), serum NTx (cross-linked N-terminal telopeptides of type I collagen), BAP (Bone alkaline phosphatase), Ca, FRAX at baseline, 6 months, 12months and 18 months after starting once-weekly TPTD. We calculated the change rate of those values to baseline. The continuous variables were compared using ANOVA and Wilcoxon matched-pairs single rank test. Results 12 GIOP patients with collagen diseases were registered into this study, and 9 patients completed (6 systemic lupus erythematosus, 2 rheumatoid arthritis, 1 adult onset still disease; 7 female, 2 male; Age 57.4±11.1). Only one new fracture event, that was lumbar compression fracture, occurred during the study period despite the patient had many fractures before this treatment. The change rate of lumbar spine YAM was significantly improved at 18 months (p=0.041, ANOVA; p=0.019, Wilcoxon matched-pairs single rank test). Femoral neck YAM (p=0.477, ANOVA), serum NTx (p=0.555), BAP (p=0.936), Ca and FRAX were not significantly changed. Interestingly, the change rate of serum NTx was increased during this GIOP study despite the rate was decreased in primary osteoporosis previously reported. Conclusions Our study shows that once-weekly teriparatide is effective for GIOP patients with collagen diseases, and its dosing period needs 18 months. Mean % change of serum NTx was increased in GIOP patients in spite of primary osteoporosis. References Bone 2006;39:244–52. J Bone Miner Res 2010;25:472–81. Osteoporos Int 2009;20:2095–104. J Bone Miner Metab 2004;22:569–76. Disclosure of Interest None declared

SAT0562 Directly Reprogrammed Osteoblasts Genetically Engineered to Produce Interleukin-10 Significantly Suppress Osteoclastgenesis

Background Recent reports demonstrated that somatic cells such as fibroblasts can be directly reprogrammed into other cell types including neurons and cardiomyocytes by introducing critical transcription factors involved in the differentiation of the corresponding cell lineages1,2. Such procedures in combination with co-transduction of a particular gene may allow creation of the cells with desired functions. If patient specific osteoblasts can be induced and engineered to produce IL-10, autologous transplantation of such cells may suppress inflammation and bone destruction in rheumatoid arthritis by modulating immune responses and osteoclast development. Objectives To generate IL-10-producing mouse osteoblast-like cells from fibroblasts, and estimate potential effect of the cell supernatants on osteoclast differentiation. Methods Various combinations of transcription factors were transduced into mouse embryonic fibroblasts (MEFs) with retroviral vectors and the efficiency of conversion into osteoblast-like cells were estimated by alizarine red S staining. IL-10 gene was also transduced to the cells that received the most effective combination of the transcription factors, and resultant cells were characterized by qRT-PCR, alkaline phosphatase staining, and alizarine red S staining. IL-10 production was measured by qRT-PCR and ELISA. The supernatant was added to a mouse macrophage cell line Raw264.7 cells that were induced to differentiate into osteoclasts by an addition of RANKL. Results MEFs were successfully induced to massively produce bone matrix that were mineralized by calcium phosphate. Co-transduction of the IL-10 gene by means of a retrovirus vector resulted in establishment of osteoblasts that produced IL-10 at a significant level. The culture supernatant of the cells significantly suppressed osteoclast differentiation from Raw264.7 cells. Conclusions IL-10-secreting osteoblasts were successfully generated from fibroblasts by direct reprogramming procedures. The cells may be useful in novel cell-based therapy against RA in the future. References Ieda, M. et al.: Cell, 142: 375-386, 2010. Vierbuchen T.: Nature, 463: 1035-1041, 2010. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2247