Megumi Hirabayashi

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.

The impact of transcription factor Fli1 deficiency on the regulation of angiogenesis

The insufficiency of Friend leukaemia virus integration 1 (Fli1), a member of the Ets family transcription factors, is implicated in the pathogenesis of vasculopathy associated with systemic sclerosis (SSc). Fli1 deficiency accelerates early steps of angiogenesis, including detachment of pre‐existing pericytes and extracellular matrix degradation by endothelial proteinases, but the impact of Fli1 deficiency on the other steps of angiogenesis has not been investigated. Therefore, we evaluated the effect of Fli1 deficiency on migration, proliferation, cell survival and tube formation of human dermal microvascular endothelial cells (HDMECs). HDMECs transfected with FLI1 siRNA exhibited a greater migratory property in scratch assay and transwell migration assay and a higher proliferation rate in BrdU assay than HDMECs transfected with non‐silencing scrambled RNA. In flow cytometry‐based apoptosis assay, FLI1 siRNA‐transduced HDMECs revealed the decreased number of annexin and propidium iodide‐double‐positive apoptotic cells compared with control cells, reflecting the promotion of cell survival. On the other hand, tubulogenic activity on Matrigel was remarkably suppressed in Fli1‐deficient HDMECs relative to control cells. These results indicate that Fli1 deficiency promotes migration, proliferation and cell survival, while abating tube formation of endothelial cells, suggesting that Fli1 deficiency is potentially attributable to the development of both proliferative obliterative vasculopathy (occlusion of arterioles and small arteries) and destructive vasculopathy (loss of small vessels) characteristic of SSc vasculopathy.

Soluble form of PD-1 and PD-L2 contributes 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.

Dermatomyositis with anti-OJ antibody

A subset of patients with polymyositis (PM)/dermatomyositis (DM) has autoantibodies to aminoacyl-tRNA synthetases (ARS). The most common anti-ARS antibody is anti-Jo-1 (histidyl-tRNA synthetase) antibody, which is positive in up to 20% of patients with PM/DM [1]. In addition to anti-Jo-1 antibody, seven autoantibodies to ARS have been identified, including anti-OJ (isoleucyltRNA synthetase) antibody [1]. A 49-year-old Japanese woman presented with a 2-month history of erythema involving her eyelids, shoulders, hands, upper chest and back, and lateral thighs with associated fatigue and fever. On examination, proximal muscle weakness in her neck and lower limbs was detected. Dermatological assessment was notable for heliotrope rash involving periorbital skin, Gottron’s lesions over both extensor and flexor surfaces of metacarpophalangeal, proximal interphalangeal and distal interphalangeal joints (Fig. 1), mechanic’s hands, periungual erythema, nail fold bleeding, edematous erythema across shoulders, anterior chest and back, and thighs with some ulceration. Laboratory examinations showed increases in aldolase (11.4 U/ ml, normal: \6.1) and KL-6 (622 U/ml, normal: \500). Creatine kinase (CK; 149 IU/l) and SP-D (33.5 ng/ml) were within normal ranges. Anti-nuclear antibodies (ANA) were negative by indirect immunofluorescence. Anti-Jo-1 antibody was not detected by enzyme-linked immunosorbent assay. Electromyographic examination demonstrated myogenic pattern on iliopsoas muscle, compatible with myositis. Pulmonary function tests were within normal limits. Chest computed tomography showed ground glass opacities in bilateral lower lung fields. Histological examination of skin biopsy specimens demonstrated epidermal atrophy, liquefaction degeneration, dermal edema, and a perivascular lymphocytic infiltrate in the superficial dermis (Fig. 2). An extensive search detected no underlying malignancy. Based on these findings, she was diagnosed with DM and interstitial lung disease (ILD). Oral prednisone 60 mg/day (1 mg/kg per day) was administered. Fever shortly resolved, and her skin lesions and muscle weakness were gradually improved. Immunoprecipitation assay was conducted, and the antibody recognizing OJ antigens was identified in the serum of the present case (Fig. 3). Since DM patients with anti-ARS antibody often fail to fully respond to oral corticosteroid and manifest recurrent flares during the tapering of corticosteroid, the patient was administered with oral cyclosporine 150 mg/day. At 3 months after the initiation of cyclosporine, prednisone was successfully tapered to 20 mg/day and aldolase and KL-6 were decreased to normal ranges. S. Noda Y. Asano (&) Z. Tamaki M. Hirabayashi M. Yamamoto T. Takekoshi T. Hoashi M. Sugaya S. Sato Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan e-mail: yasano-tky@umin.ac.jp

Fli1-haploinsufficient dermal fibroblasts promote skin-localized transdifferentiation of Th2-like regulatory T cells

BackgroundFriend leukemia virus integration 1 (Fli1) deficiency, a predisposing factor of systemic sclerosis (SSc), induces SSc-like phenotypes in various cell types. A recent study demonstrated the transdifferentiation of T helper type 2 cell (Th2)-like regulatory T cells (Tregs) in SSc lesional skin through interleukin (IL)-33 produced by fibroblasts. Therefore, we investigated the role of Fli1 deficiency in dermal fibroblast-mediated transdifferentiation of Tregs.MethodsCytokine expression was assessed in Tregs by flow cytometry and in skin samples and cultivated cells by immunostaining, immunoblotting, and/or qRT-PCR. Fli1 binding to the target gene promoters was examined by chromatin immunoprecipitation. Murine dermal fibroblasts and Tregs were cocultured with or without blocking antibodies against target cytokines.ResultsTh2- and Th17-like cell proportions in skin-homing Tregs were increased in bleomycin-treated Fli1+/− mice compared with bleomycin-treated wild-type mice, whereas Th1-, Th2-, and Th17-like cell proportions in splenic Tregs were comparable. Fli1+/− fibroblasts overproduced IL-33 and IL-6, in particular IL-33, and Fli1 occupied the IL33 and IL6 promoters in dermal fibroblasts. Importantly, the IL-4-producing cell proportion was significantly higher in wild-type Tregs cocultured with Fli1+/− fibroblasts than in those cocultured with wild-type fibroblasts, which were canceled by neutralizing anti-IL-33 antibody. Under the same coculture condition, an increased tendency of IL-17A-producing cell proportion, which was possibly mediated by IL-6, was evident.ConclusionsFli1 haploinsufficiency increases the proportions of Th2- and Th17-like Tregs in bleomycin-induced profibrotic skin conditions, in which IL-33-producing dermal fibroblasts contribute to Th2-like Treg transdifferentiation, suggesting a critical role of Fli1 deficiency in the interaction of dermal fibroblasts with immune cells in pathological skin fibrosis.

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

A potential contribution of psoriasin to vascular and epithelial abnormalities and inflammation in systemic sclerosis

Antimicrobial peptides have attracted much attention as a member of disease‐associated molecules in systemic sclerosis (SSc), which is pathologically characterized by immune abnormalities, vasculopathy and tissue fibrosis.

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.

Possible pro‐inflammatory role of heparin‐binding epidermal growth factor‐like growth factor in the active phase of systemic sclerosis

Heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) is a member of the EGF family growth factors, which affects multiple aspects of the wound healing process such as epithelialization, wound contraction and angiogenesis. In our study, we measured the serum HB‐EGF levels of 51 systemic sclerosis (SSc) patients, which showed a significant increase compared with those of 20 normal subjects. Further analysis revealed a positive correlation between the HB‐EGF level and pulmonary ground‐glass score but no correlation between the former and pulmonary fibrosis score. Other findings include: a significant increase of serum sialylated carbohydrate antigen KL‐6 levels and significant shortness of disease duration in the diffuse cutaneous SSc patients with elevated HB‐EGF levels; and significantly higher HB‐EGF levels in the presence of Raynauds phenomenon, in that of telangiectasia, and in the absence of contracture of phalanges in all SSc patients. We then evaluated HB‐EGF mRNA levels of fibroblasts harvested from skin samples of the SSc patients and those of foreskin‐derived fibroblasts treated with transforming growth factor‐β, both of which were significantly higher than each control. In conclusion, we speculate that HB‐EGF plays a pro‐inflammatory role in the active skin and lung lesions of SSc.