Fn14 expression was assessed by real-time PCR and confocal staining in the spleen

Fn14 expression was assessed by real-time PCR and confocal staining in the spleen. antibodies against CD4, B220, GL-7, CD138, and PD-1. Kidneys were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). Results Administration of TWEAK increased the mRNA levels of gene results in excessive formation of Tfh and GC [5]. RoquinSan/San mice were selected as a SLE model in this study because the sanroque gene mutation causes lupus-like features through regulating Tfh and GC. TNF-like weak inducer of apoptosis (TWEAK) is a proinflammatory cytokine that mediates several cellular and inflammatory responses by binding to fibroblast growth factor-inducible 14 (Fn14, also known as the TWEAK receptor). Recently, a link has been identified between the pathogenesis of several autoimmune disorders including autoimmune encephalitis, rheumatoid arthritis, and SLE with the TWEAK/Fn14 pathway [6, 7]. Xia et al. [8] demonstrated that the TWEAK/Fn14 pathway has a crucial role in the pathogenesis of Ab-induced nephritis, and disrupting the TWEAK/Fn14 pathway is a potential treatment for Ab-induced nephritides, including lupus nephritis. Recent studies revealed that the TWEAK/Fn14 interaction has an important role in the pathogenesis of several SLE manifestations [7, 9]. The TWEAK/Fn14 pathway contributes to the pathogenesis of SLE by modulating the local environment of the target organ [7, 10]. However, the TWEAK/Fn14 pathway activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) signaling and the dysregulation of NF-B signaling can induce autoimmune disorders by altering B and T cell immunity [11]. Therefore, the TWEAK/Fn14 interaction may have systemic effects on the pathogenesis of SLE in addition to local pathological effects. We hypothesized that blocking the TWEAK/Fn14 pathway via administration of Fn14-Fc would attenuate the autoimmune response in a mouse model of SLE. To identify the mechanisms involved, we explored the effects of Fn14-Fc on Ab secretion, B cell maturation, Tfh cell development, GC formation and kidney damage. In addition, the pathologic role of TWEAK was investigated in sanroque mice by administration of TWEAK to B cells. Methods Animals Roquinsan/san (sanroque) mice in a C57BL/6 background were Neridronate obtained from the National Institutes of Health (Bethesda, MD, USA). The mice were maintained under specific pathogen-free conditions at the Catholic Research Institute of Medical Science at Rabbit Polyclonal to ERCC1 the Catholic University of Korea and were fed standard mouse chow (Ralston Purina, St. Louis, MO, USA) and water ad libitum. All experimental procedures were examined and approved by the Animal Research Ethics Committee of the Catholic University of Korea; the procedures conformed to all the USA National Institutes of Health guidelines. Preparation of Fn14-Fc The Fn14-Fc and control-Fc used in the experiments (the Neridronate hinge-CH2-CH3 form of IgG1) were bought from A&RT Therapeutics (Daejeon, South Korea). Murine B cell isolation and stimulation Spleen cells were washed with phosphate-buffered saline (PBS; pH 7.2). After centrifugation at 1300?rpm and at 4?C, the cells were incubated with CD19-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and isolated on MACS separation columns (Miltenyi Biotec). Positively selected CD19+ B cells were stimulated with TWEAK (0.1?ng/ml; R&D Systems, Minneapolis, MN, USA) for 3?days. Total RNA was extracted using the TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). Treatment with Fn14-Fc To assess the influence of Fn14-Fc on the severity of symptoms in the SLE model, sanroque mice were treated with 100?g/mouse Fn14-Fc in saline or control-Fc via intraperitoneal injections Neridronate three times weekly for 3?weeks. Treatment was started in 12-week-old sanroque mice. The groups were sacrificed 21? days Neridronate after the first injection and the spleen and kidney were obtained at the time of sacrifice. Measurement of immunoglobulin (Ig) G subtypes and autoAbs Blood was obtained from the orbital sinus of Fn14-Fc and control-Fc-treated mice and the serum was stored at ?20?C until use. Total IgG, IgG1,.