Satoshi Ebata

Contribution of Soluble Forms of Programmed Death 1 and Programmed Death Ligand 2 to Disease Severity and Progression in Systemic Sclerosis

To determine the function and serum levels of soluble forms of programmed death 1 (sPD‐1) and one of its ligands, soluble PD ligand 2 (sPD‐L2), in patients with systemic sclerosis (SSc) and in a mouse model of topoisomerase I (topo I)–induced SSc.

Nucleosome in patients with systemic sclerosis: possible association with immunological abnormalities via abnormal activation of T and B cells

OBJECTIVE To determine the serum levels of nucleosome in patients with systemic sclerosis (SSc) and relate the results to the clinical features of SSc. METHODS Serum nucleosome levels in 91 patients with SSc were examined by ELISA. The expression of Toll-like receptor (TLR) 9 in T and B cells was quantified by flow cytometric intracellular protein analysis. The effects of nucleosomes on lymphocytes were also analysed. Moreover, we assessed the effects of nucleosomes on fibrosis, using wild type and CD19-deficient bleomycin-treated mice, an experimental model for human SSc. RESULTS Serum nucleosome levels were elevated in SSc compared with healthy controls and correlated positively with the extent of skin and pulmonary fibrosis and immunological abnormalities. The retrospective longitudinal analysis showed the serum nucleosome levels to be attenuated during the follow-up period. TLR9, which can be stimulated by nucleosome expression was upregulated in the affected T and B cells of patients with SSc. Moreover, nucleosome stimulation strongly increased interleukin (IL)-4 and IL-17 expression of T cells, B-cell IgG production and proliferation of lymphocytes in SSc compared with those in healthy controls. In bleomycin-induced SSc model mice, serum nucleosome levels were elevated compared with control mice. Furthermore, nucleosomes increased IgG production and proliferation of mouse B cells. Although TLR9 expression was similar between wild type and CD19-deficient splenic B cells, CD19 deficiency reduced these nucleosome effects. CONCLUSION These results suggest that nucleosomes and its signalling in B and T cells contribute to disease development in SSc via TLR9.

Unprecedented success of rituximab therapy for prednisolone- and immunosuppressant-resistant systemic sclerosis-associated interstitial lung disease

Systemic sclerosis (SSc) is a multisystem autoimmune disease (1). Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is a frequent complication of SSc and sometimes determines the prognosis (2). To date, although only the combination therapy of cyclophosphamide (CYC) with prednisolone (PSL) has been found to be effective in stabilizing or improving lung function in randomized clinical trials which included abundant numbers of patients (3), SSc-ILD treatment is still debated because its small beneficial effect seems to be short lived (4, 5). Besides, not every patient can be treated with CYC, owing to its adverse effects. Therefore, more effective therapy with fewer unfavourable effects is needed. Some clinical studies have shown the effects of rituximab (RTX) on tissue fibrosis in SSc (6–8). Although these studies treated limited numbers of patients with SSc-ILD, RTX may be a novel candidate drug for SSc-ILD. This paper presents the results of RTX treatment in a young Japanese woman who suffered from both PSLand immunosuppressant-resistant SSc-ILD. A 21-year-old woman was admitted to our hospital with complaints of dyspnoea and skin thickness. From the age of 18, she had had skin thickening, digital ulcers, abnormal nailfold capillaries, Raynaud’s phenomenon, and dyspnoea. Skin biopsy from the right forearm revealed thickening of the dermis, caused by hyperplasia of collagen fibre, and lymphocytic infiltrate around the blood vessels. Lung fibrosis was identified by high-resolution computed tomography (HRCT). Blood analysis showed that anti-topoisomerase I antibodies were present. Taking all these results together, we diagnosed her as having diffuse cutaneous SSc. Further assessment revealed her reduced lung function: the per cent predicted forced vital capacity (%FVC) was 58% (normal ≥ 80%) and per cent predicted diffusing capacity of the lung for carbon monoxide (%DLCO) was 42% (normal ≥ 70%). Serum markers for ILD were elevated: serum levels of surfactant protein D (SP-D) were 279.4 ng/mL (normal ≤ 110.0 ng/mL) and sialylated carbohydrate antigen (KL6) levels were 682 U/mL (normal ≤ 500 U/mL). The combination therapy of CYC with PSL is a first line treatment for SSc-ILD (10). However, our patient completely refused CYC treatment, because CYC can cause irreversible amenorrhoea. Instead of CYC treatment, she was treated with 40 mg/day of PSL and 200 mg/day of cyclosporine A, but her condition worsened. One month after her hospitalization, %FVC and %DLCO had decreased to 49% and 31%, respectively, while the SP-D and KL-6 levels had increased to 381.7 ng/mL and 1179 U/mL, respectively. In addition, HRCT revealed the exacerbation of ILD. Under these circumstances, we decided to start RTX treatment, which was approved by the institutional review board of Tokyo University Hospital. We treated her with 570 mg/week (375 mg/m/week) of RTX intravenously for 4 weeks in a row. B-cell depletion was confirmed by flow-cytometric analysis. One month after the first administration of RTX, %DLCO had increased to 42% and SP-D levels had decreased to 139.0 ng/mL (Figure 1A, B). The modified Rodnan total skin thickness score had improved from 12 to 10. After 6 months, HRCT revealed that the ILD had improved (Figure 1C, D). After 12 months, %FVC and %DLCO had increased to 65% and 44%, respectively. After 13 and 22 months, we treated her again with RTX because her B cells had recovered in peripheral blood. After 23 months, %FVC finally increased to 71%. The skin thickness score had decreased to 8. Serum levels of immunoglobulin M and G and anti-topoisomerase I antibody titres had also decreased. Throughout the period of RTX therapy, no adverse effects were found. In our case, %FVC and %DLCO at the 2 year evaluation were improved by 22% and 17%, respectively. These results exceeded the improvements reported previously using not only CYC treatment, but also RTX treatment (5, 6). The reason why our patient exhibited these unprecedented improvements is unknown. However, it is likely that her background, including her ethnicity, age, and disease duration, relates to the good reactivity for RTX. It has been reported that ethnicity strongly affects the Scand J Rheumatol 2017;46:247–252 247

Rapid alteration of serum interleukin-6 levels may predict the reactivity of i.v. cyclophosphamide pulse therapy in systemic sclerosis-associated interstitial lung disease

Systemic sclerosis (SSc) is an autoimmune disorder characterized by excessive extracellular matrix deposition. Although SSc‐associated interstitial lung disease (ILD) is one of the most important complications as a cause of death in SSc, prediction factors of treatment reactivity in SSc‐ILD are still unclear. To assess relationships between interleukin (IL)‐6 and reactivity to treatment, we measured serum IL‐6 levels in 23 of active SSc‐ILD patients under i.v. cyclophosphamide (IVCY) therapy and 20 of stabilized SSc‐ILD, using the high‐sensitivity enzyme‐linked immunoassay system. Serum IL‐6 levels in active SSc‐ILD patients were significantly higher than those in stabilized SSc‐ILD patients. Among active SSc‐ILD patients, baseline serum IL‐6 levels were not significantly different between IVCY responders and non‐responders. Meanwhile, serum IL‐6 levels after three IVCY doses out of a total of six were decreased in responders but not in non‐responders. Regarding changes of parameters by the three doses of a total of six of IVCY, change in serum IL‐6 levels correlated inversely with that in values of pulmonary function test. Thus, the rapid decrease in serum IL‐6 levels during a couple of doses may predict the efficacy of IVCY therapy against SSc‐ILD.

Serum interleukin-34 levels in patients with systemic sclerosis: Clinical association with interstitial lung disease

Interleukin (IL)‐34 is a hematopoietic cytokine promoting proliferation and differentiation of macrophages. Because abnormal activation of macrophages is involved in the development of systemic sclerosis (SSc), we investigated serum IL‐34 levels in patients with SSc. Serum IL‐34 levels were significantly increased in diffuse cutaneous SSc compared with limited cutaneous SSc and healthy controls, while there were no significant differences between limited cutaneous SSc and healthy controls. In addition, SSc patients with increased serum IL‐34 levels more often had interstitial lung disease (ILD) than those with normal levels. Moreover, in SSc patients, serum IL‐34 levels negatively correlated with the percentage of predicted vital capacity, while they positively correlated with ground‐glass opacity score and fibrotic score on chest computed tomography. Collectively, increased serum IL‐34 levels were associated with greater frequency and severity of ILD in SSc patients. Serum IL‐34 levels may be a useful serological marker for SSc‐associated ILD.

Successful treatment with rituximab in a Japanese patient with systemic sclerosis-associated interstitial lung disease resistant to oral steroid and cyclophosphamide

Dear Editor, Systemic sclerosis (SSc) is an autoimmune disorder characterized by excessive extracellular matrix deposition in the skin and visceral organs. SSc-associated interstitial lung disease (ILD) is especially one of the major causes of mortality in SSc. The combination therapy of cyclophosphamide (CYC) and steroid has shown statistically significant but temporal efficacy for the treatment of SSc-ILD. Therefore, more effective therapy is needed. A 38-year-old Japanese man was admitted to our hospital with complaints of dyspnea and scleroderma lasting for 15 months. A skin biopsy from his right forearm revealed thickening of the dermis. The blood analysis showed he had anti-topoisomerase I antibodies. We diagnosed him as having diffuse cutaneous SSc and started 20 mg/day of oral steroid. Eight months later, his pulmonary function test showed percent predicted of forced vital capacity (%FVC) of 72.5% (normal, ≥80%) and percent predicted of diffusing capacity of the lung for carbon monoxide (%DLco) of 38.7% (normal, ≥70%). These results were 17.7% and 17% each lower than values tested before steroid treatment. Furthermore, Krebs von den Lungen-6 (KL-6; cut-off level, 500 U/mL) levels increased to 2153 U/mL. Also, high-resolution computed tomography (HRCT) showed new ground-glass appearances in bilateral lungs. Therefore, oral CYC (2 mg/kg per day) treatment was added for SSc-ILD. In spite of continuous CYC treatment, his pulmonary function got worse. Twelve months after commencement of CYC treatment, %FVC and %DLco decreased to 64% and 32%, respectively. The surfactant protein D (SP-D; cut-off level, 110 ng/mL) and KL-6 levels increased to 184.8 ng/mL and 1239 U/mL, respectively. The modified Rodnan total skin thickness score was 10, which remained unchanged during CYC treatment. Therefore, we considered he had oral steroidand CYC-resistant SSc-ILD and decided to start rituximab (RTX)

Poromas with large lumens histopathologically mimicking syringocystadenoma papilliferum: Report of three cases

We report on three patients exhibiting tumours with exophytic pedunculated structures with eroded surfaces. All cases showed the basic histopathological features of poroma accompanied by large, invaginated ductal structures lined by multiple layers of columnar or cuboidal cells. The columnar cells of invaginated ductal/cystic structures focally exhibited subtle features reminiscent of decapitation secretion along with dense infiltration of plasma cells in the surrounding stroma, mimicking syringocystadenoma papilliferum.

OP0207 B cell depletion increases regulatory t cells and ameliorates skin and lung fibrosis in a bleomycin-induced systemic sclerosis model mouse

Background B cells play a critical role in systemic autoimmunity and disease expression through various functions such as cytokine production and induction of other immune cell activation. Recently, some clinical studies have shown that the efficacy of B cell depletion therapy with rituximab, a chimeric monoclonal antibody against human CD20, in systemic sclerosis (SSc) patients. However, it still remains unclear why B cell depletion can be an effective treatment for SSc. Objectives The purpose of this study is to assess the role of B cell depletion in SSc. We evaluated the skin and lung fibrosis of bleomycin (BLM)-induced SSc model mice treated with B cell depletion. Furthermore, we investigated the effect of B cell depletion on T cell cytokine profile. Methods To generate BLM-induced SSc model mice, 300 μg of BLM was injected subcutaneously into the shaved backs of the C57BL/6 mice every other day for 4 weeks. Anti-mouse CD20 monoclonal antibodies, which can deplete mouse B cells, were also injected every 2 weeks from 2 weeks later starting BLM treatment. After 4 weeks of BLM treatment, skin and lung fibrosis were assessed histopathologically. T cells and B cells were isolated from spleen using magnetic cell sorting system. Purified T cells (5x105 cells) were cultured with B cells (5x105 cells) in the presence of phorbol 12-myristate 13-acetate, ionomycin, and anti-CD3/CD28 antibodies. Cytokine expressions in the fibrotic skin and lung were quantified by real-time polymerase chain reaction. Cytokine production of T cells and B cells were analyzed by flow cytometric analysis. Results Dermal thickness and lung fibrosis score increased in BLM-treated mice compared with phosphate buffer saline (PBS)-treated control mice. Frequencies of interleukin (IL)-10 producing splenic B cells significantly decreased in BLM-treated mice compared with PBS-treated mice, while IL-6 producing B cell frequencies increased. Moreover, interferon (IFN)-γ, IL-4, or IL-17 producing T cell frequencies increased in BLM-treated mice. There were no significant differences in regulatory T cell frequencies between BLM-treated and PBS-treated mice. B cell depletion increased IL-10 producing regulatory T cell frequencies in BLM-treated mice. By contrast, frequencies of IFN-γ, IL-4, or IL-17 producing T cells were significantly decreased by B cell depletion in BLM-treated mice. In addition, fibrogenic cytokine mRNA expression levels of skin and lung decreased in BLM-treated mice with B cell depletion. To assess the role of B cells on T cell cytokine production, purified splenic B cells from BLM- or PBS-treated mice were cultured with naive T cells. T cells which were cultured with B cells from BLM-treated mice produced greater amounts of INF-γ, IL-4, and IL-17 than those cultured with PBS-treated mouse B cells. By contrast, B cells from PBS-treated mice induced a higher amount of IL-10 production from T cells than those from BLM-treated mice. Conclusions B cell depletion inhibited skin and lung fibrosis in BLM-treated mice. Furthermore, B cell depletion increased regulatory T cell frequencies in BLM-treated mice, though INF-γ, IL-4, and IL-17 producing T cell frequencies were decreased by B cell depletion. These results suggest that B cell depletion alters T cell cytokine profile, which results in inhibition of fibrosis in this model. Disclosure of Interest None declared