Phenazine methosulfate (PMS), a generator of superoxide, evoked the transcription of cas2 and cefF, ultimately resulting in the enhanced biosyntheses of clavulanic acid (CA) and cephamycin C (CMC) in Streptomyces clavuligerus. The transcriptional activation of cas2 and cefF was accompanied with that of ccaR, a regulatory gene for biosyntheses of CA and CMC. PMS or H2O2 in cell-free extract exerted a positive regulation on in vitro protein phosphorylation. The PMS-mediated activation of protein phosphorylation was significantly offset by butylated hydroxyanisole, a radical scavenger. Staurosporine, a protein kinase inhibitor, was shown to have a negative effect on PMS-promoted CA accumulation. Therefore, it is suggestive that PMS-activated transcription of cas2 and cefF is mediated by protein phosphorylation and the expression of a pathway-specific transcriptional activator as found in other streptomycetes. These experimental results present an example of the functional relationship between oxidative stimuli and secondary metabolite production in streptomycetes.

Background:Semaphorin 4D (SEMA4D) / CD100, known as a subfamily of axonal guidance proteins, has also been reported to act as an immunoregulator in several infectious and inflammatory diseases [1]. Sjögren’s syndrome (SS) is a systemic autoimmune disease that primarily affects the exocrine glands by infiltrated lymphocytes resulting in dryness of mouth and eyes. IL-17 was reported to impair the integrity of tight junction barrier and attenuate the expression of aquaporin 5 (AQP5), causing salivary gland dysfunction in SS [2].Objectives:This study was aimed to evaluate the role of SEMA4D in patients with SS and investigate the effect of SEMA4D on human salivary gland epithelial cell (SGEC) and T cell.Methods:Soluble SEMA4D levels in plasma were measured by enzyme-linked immunosorbent assay (ELISA) from patients with SS, non-SS sicca and healthy controls. Immortalized human SGECs, originated from acini (NS-SV-AC) and duct (NS-SV-DC), were used to evaluate the effects of SEMA4D. CD4+T cells from human peripheral blood were isolated to determine the secretion of cytokines in response to SEMA4D. IFN-γ and IL-17 were used to determine the effects on AQP5 expression of SGEC.Results:The levels of soluble SEMA4D in plasma were increased in patients with SS (median [interquartile range]: 1221.3 [393.5] pg/mL) compared to non-SS sicca (940.2 [355.1] pg/mL,p= 0.006) or healthy controls (909.5 [108.0] pg/mL,p <0.0001). The levels of soluble SEMA4D in plasma were correlated with the levels of several autoantibodies including anti-SSA (Spearman’s rho = 0.358,p= 0.006), anti-SSB (rho = 0.350,p= 0.007), and anti-muscarinic receptor 3 (M3R) Ab (rho = 0.495,p< 0.001), and also correlated with total IgG (rho = 0.431,p= 0.002). SEMA4D-stimulated SGECs showed decreased expression of tight junctions such as occludin and Zo-1. CD4+T cells secreted IFN-γ (p= 0.025), IL-17 (p= 0.028), and IL-21 (p= 0.007) with SEMA4D stimulation. IFN-γ and IL-17 decreased AQP5 expression in SGECs.Conclusion:SEMA4D contributed to decreased expression of tight junction in SGECs. SEMA4D induced production of IFN-γ and IL-17 in CD4+T cells and these cytokine decreased AQP5 expression in SGECs.References:[1]Worzfeld T, Offermanns S. Nat Rev Drug Discov. 2014;13(8):603-21.[2]Bhattarai KR, Junjappa R, Handigund M, Kim HR, Chae HJ. Autoimmun Rev. 2018;17(4):376-90.Disclosure of Interests:None declared

Background:Polymyositis (PM) is a chronic inflammatory myopathy that impairs muscle functions. While the treatment with glucocorticoids (GC) has been the cornerstone of the treatment for PM to suppress immune-mediated muscle injury, some patients suffer from glucocorticoid-induced myopathy during the treatment, which further deteriorates the muscle weakness. It has been reported that significant disability and muscle weakness persist in a quarter of the patients even after successful treatment with the immunosuppressive therapy1. Ultimately, new therapeutic strategies to preserve and recover muscle strength as well as to suppress immune-mediated muscle injury are needed. Glucagon-like peptide-1 (GLP-1) is a peptide hormone with a variety of functions. Although GLP-1 receptor (GLP-1R) agonists have been developed as an anti-diabetic therapy to promote insulin secretion, emerging data suggest that they have pleiotropic actions including anti-inflammatory effects and suppression of muscle wasting2. We presumed that GLP-1R agonists have beneficial effect on PM to preserve and recover muscle strength.Objectives:To examine the effect of a GLP-1R agonist on C protein-induced myositis (CIM), a murine model of polymyositis3, in monotherapy or in combination with prednisolone (PSL).Methods:Muscle specimens of PM patients and CIM were examined with immunohistological staining for the expression of GLP-1R. The therapeutic effect of PF1801 (ImmunoForge), a GLP-1R agonist (5 mg/kg body weight (BW)/day), in monotherapy or in combination with PSL (20 mg/kg BW/day) on CIM was examined for grip strength, muscle weight and histological muscle inflammation.Results:GLP-1R was expressed on the plasma membrane of muscle cells of PM patients and CIM. The expression levels were high in the area where inflammatory infiltrates were observed. The treatment of CIM with PF1801 in monotherapy or in combination with PSL suppressed the CIM-induced decrease in grip strength on day 14. The combination therapy with PF1801 and PSL ameliorated the CIM-induced muscle weight loss in quadriceps, while the monotherapy with PF1801 or PSL did not. The histological analysis of muscle specimens on day 14 of CIM revealed that the muscle inflammation was suppressed by the treatments with PF1801, PSL, or the combination of PF1801 and PSL. None of the mice in the combination therapy group developed histologically evident myositis, while the myositis was observed in 90%, 40% and 40 % of the mice in vehicle treated group, PF1801 treated group, and PSL treated group, respectively. The necrotic area of the muscle in CIM was also reduced in the mice treated with PF1801, PSL, or the combination of PSL and PF1801. The CIM-induced increase in spleen weight was suppressed by PF1801, PSL, or the combination of PSL and PF1801. The additive effect of PSL and PF1801 on the suppression of CIM-induced increase in spleen weight was observed.Conclusion:PF1801 ameliorated CIM-induced muscle weakness and muscle inflammation in CIM. The combination therapy with PF1801 and PSL ameliorated CIM-induced muscle weight loss. PF1801 could be a novel therapy to recover muscle weakness and to suppress muscle inflammation in PM.References:[1]Bronner IM, et al.Ann Rheum Dis.2006;65:1456–61.[2]Hong Y, et al.J Cachexia Sarcopenia Muscle. 2019;10:903-18.[3]Sugihara T, et al.Arthritis Rheum. 2007;56:1304-14.Disclosure of Interests:Mari Kamiya: None declared, Seon Uk Kim: None declared, Jeong Yeon Kim: None declared, Yeong Wook Song: None declared, Eun Young Lee: None declared, Fumitaka Mizoguchi Grant/research support from: ImmunoForge, Consultant of: ImmunoForge

BackgroundIdiopathic inflammatory myopathy (IIM) is a rare autoimmune disease characterized by proximal muscle weakness, elevated muscle enzymes, inflammation in the muscle. If the muscle is already damaged in IIM, it may not regain full strength even after treatment. Also the mechanisms that contribute to muscle weakness in myositis are currently unknown. Nox is the main source of ROS production in various cells types, and is also the source of mitochondria ROS. Excessive expression of Nox4 is characteristic of sclerosis, fibrosis, and cardiac related diseases, and it had been reported that knock down of Nox4 lowers the production of superoxide and hydrogen peroxide. However, the effect of Nox4 in myositis has not been reported.ObjectivesWe investigated the expression and role of Nox4 in damaged myoblasts of myositis.MethodsMyoblasts were isolated from IIM patients who underwent muscle biopsy in Seoul National University Hospital between February 2019 and February 2021. Skeletal muscle cell(SMC) was purchased. Myoblasts and SMCs were treated with cytokine mixture {interferon gamma(IFN-γ), interleukin(IL)-6, IL-15} or PBS for 30 minutes. The expression of Nox4, MyoD and myosin heavy chain(MYH) in myoblast and myotube was analysed by Western blot. For inhibition of Nox4, GKT137831(inhibitor of Nox4) was treated before the cytokine stimulation.ResultsMyoblasts from IIM patients did not differentiate into myotubes like healthy myoblasts suggesting impaired muscle regeneration. Myoblasts from IIM patients express higher level of Nox4 compared to healthy myoblasts. When treated with cytokine mixture(IFN-r, IL-6, IL-15), healthy myoblasts or SMC mimicked inflammatory myositis and showed increased expression of Nox4 and decreased expression of MYH similar to myositis phenotype. Nox4 inhibitor suppressed Nox4 expression and MyoD overexpression in inflammatory myoblasts.ConclusionWe showed that Nox4 expression is increased in myoblasts from IIM patients and is associated with impaired muscle regeneration. Nox4 inhibition attenuated Nox4 and MyoD overexpression in inflamed myoblasts.References[1]Bi Y, Lei X, Chai N,et al.NOX4: a potential therapeutic target for pancreatic cancer and its mechanism.J Transl Med19, 515 (2021)[2]Rayavarapu S, Coley W, Kinder T.B.,et al.Idiopathic inflammatory myopathies: pathogenic mechanisms of muscle weakness.Skeletal Muscle3, 13 (2013)[3]Dalakas MC, Hohlfeld R. Polymyositis and dermatomyositis.Lancet. 2003 Sep 20;362(9388):971-82.[4]Piera-Velazquez S, Makul A, Jiménez SA. Increased expression of NAPDH oxidase 4 in systemic sclerosis dermal fibroblasts: regulation by transforming growth factor β.Arthritis Rheumatol. 2015 Oct;67(10):2749-58.Acknowledgements:NIL.Disclosure of InterestsNone Declared.