Takemichi Fukasawa

The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin modulates radiosensitivity by downregulating serine/threonine kinase 38 via Sp1 inhibition

The ansamycin-based HSP90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin) combats tumors and has been shown to modulate cellular sensitivity to radiation, prompting researchers to use 17-AAG as a radiosensitizer. 17-AAG causes the degradation of several oncogenic and signaling proteins. We previously demonstrated that oxidative stress activates serine/threonine kinase 38 (STK38), a member of the protein kinase A (PKA)/PKG/PKC-like family. In the present study, we investigated how 17-AAG affects STK38 expression, and evaluated STK38s role in the regulation of radiosensitivity. We found that 17-AAG depleted cellular STK38 and reduced STK38s kinase activity. Importantly, 17-AAG downregulated the stk38 gene expression. Deletion analysis and site-directed mutagenesis experiments demonstrated that Sp1 was required for the stk38 promoter activity. Treatment with 17-AAG inhibited Sp1s binding to the stk38 promoter by decreasing the amount of Sp1 and knocking down Sp1 reduced STK38 expression. Moreover, 17-AAG treatment or STK38 knockdown enhanced the radiosensitivity of HeLa cells. Our data provide a novel mechanism, mediated by stk38 downregulation, by which 17-AAG radiosensitizes cells.

Critical contribution of the interleukin-6/signal transducer and activator of transcription 3 axis to vasculopathy associated with systemic sclerosis

Reflecting the critical role of interleukin (IL)‐6 in systemic sclerosis (SSc), tocilizumab, an anti‐IL‐6 receptor antibody, is currently under a global phase III trial against skin sclerosis of this disease. We here demonstrate that the IL‐6/signal transducer and activator of transcription 3 axis is broadly activated in various cell types in the lesional skin of SSc patients irrespective of disease subtypes, especially in endothelial cells. Importantly, 12 monthly infusions of tocilizumab improved nailfold capillary changes as well as skin sclerosis in a patient with diffuse cutaneous SSc. The present findings suggest a potential disease‐modifying effect of tocilizumab on SSc vasculopathy.

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.

Serine-Threonine Kinase 38 regulates CDC25A stability and the DNA damage-induced G2/M checkpoint.

Cells respond to DNA damage by activating protein kinase-mediated signaling pathways that promote cell-cycle arrest, DNA repair, or apoptosis. A key regulator of cell-cycle arrest is the CDC25A (cell division cycle 25 homologue A) phosphatase. CDC25A normally plays a pivotal role in regulating the G1/S and G2/M transitions by dephosphorylating and activating cyclin/cyclin-dependent kinase (CDK) complexes; however, CDC25A is specifically degraded in response to DNA damage. Here, we demonstrate that the depletion of serine-threonine kinase 38 (STK38) prevents the DNA-damage-induced degradation of CDC25A and subsequent G2 arrest, and that STK38 directly phosphorylates CDC25A at Ser-76, resulting in CDC25As degradation. Taken together, these results indicate that the STK38-mediated phosphorylation of CDC25A at Ser-76 and the subsequent degradation of CDC25A are required to promote DNA damage-induced G2/M checkpoint activation.

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

Exacerbated Immune Complex-Mediated Vascular Injury in Mice with Heterozygous Deficiency of Aryl Hydrocarbon Receptor through Upregulation of Fcγ Receptor III Expression on Macrophages

Aryl hydrocarbon receptor (AhR), which was discovered as a receptor for environmental concomitants, plays an important role widely in the immune system. In this study, we assessed AhR involvement in immune-complex-mediated vascular injury by examining the reverse-passive Arthus reaction in AhR heterozygous knockout (AhR+/-) mice. In the cutaneous Arthus reaction, dermal edema was severer in AhR+/- mice than in wild-type mice. The number of infiltrating neutrophils and mRNA expression levels of CXC chemokine ligand 1 and IL-6 were also increased in AhR+/- mice. Similarly, in the peritoneal Arthus reaction, infiltration of neutrophils was increased in AhR+/- mice. Peritoneal macrophages from AhR+/- mice expressed higher levels of Fcγ receptor III and produced higher levels of CXC chemokine ligand 1 and IL-6 after immune complex treatment. In addition, AhR occupied the promoter regions of Fcγ receptor III gene in peritoneal macrophages in a ligand-dependent manner. Depletion of macrophages reduced the cutaneous Arthus reaction in AhR+/- mice, and adoptive transfer of AhR+/- mice macrophages into wild-type mice exacerbated the peritoneal Arthus reaction. Furthermore, AhR expression was decreased and Fcγ receptor III expression was increased in CD14+ monocytes in peripheral blood from patients with immune-complex-mediated vasculitis compared with those from healthy controls. These results suggest that downregulation of AhR is associated with the exacerbation of immune-complex-mediated vascular injury.

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)