Role Of TWEAK-P38 MAPK Signaling Pathway In The Pathogensis Of Lupus Nephritis

Background:Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by the presence of autoantibodies directed against nuclearantigens.Renal involvement in SLE, known as lupus nephritis (LN), is a major complication of SLE and is associated with high rates of morbidity and mortality. Although clinical signs of renal involvement appear in only 50-80% of patients, the disease involves the kidney in almost all patients from whom sufficient tissue can be obtained for analysis.Immune dysfunction is the core mechanism of lupus nephritis. It is well known that activation of T and B cells plays a key role in the pathogenesis of this disease in innate immunity and inflammatory responses via production of various cytokines and chemical mediators, such as MCP-l,IL-10. it is intriguing that these cytokines are crucial in the pathogenesis of SLE. The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor (TNF) superfamily (TNFSF) initially described in 1997. The expression of TWEAK is relatively low in normal tissue, however, it is increased in both acute or chronic inflammatory processes in numerous tissues and peritoneal macrophages. TWEAK may be expressed as a membrane-bound protein (mTWEAK) and as a 156-aa, 18kDa soluble protein in mmune cells such as monocytes-macrophages, dendritic cells, NK cells and activated T cells. Many studies showed that TWEAK and its cognate receptor play an important role in angiogenesis, cell proliferation, apoptosis, cytokines, and immune inflammatory diseases, such as lupus nephritis, experimental autoimmune encephalomyelitis, autoimmune arthritis, multiple sclerosis, myositis, lupus nephritis. Cell-surface expression of TWEAK has also been reported in human monocytes after IFN-rexposure and in T cells from patients with systemic lupus erythematosus. Treatment with an anti-TWEAK Ab or deficiency of the TWEAK receptor Fn14 can decrease renal damage of cGVH-induced lupus mice as indicated by significantly reduced proteinuria, glomerular Ig deposition and kidney cytokine expression.Recent study showed that biological effects of TWEAK may be mainly through the induction of downstream signaling pathways, including prolonged of NF-KB activation and activating the canonical (or alternative) pathway of NF-KB. p38 MAPK (mitogen-activated protein kinase) is an important upstream regulatory factors of NF-KB, some scholars have found that TWEAK can activate p38 MAPK signaling pathway involved in the pathogenesis of myositis. Studies have found that P38 MAPK expression increased significantly, its specific inhibitor SB203580 could significantly reduce the renal tissue T cells macrophages infiltration, reduce the immunoglobulin deposition, and decrease renal pathological damage, anti-dsDNA antibodies inflammatory mediators and chemokine also decreased significantly. P38 MAPK signaling pathway plays an important role in the pathogenesis of LN.Thus, We carried out serious research to explore the following questions:(1) To detect the expression of TWEAK in peripheral blood mononuclear cells and analyze the correlation between TWEAK and disease activity and renal damage of SLE.(2) To support a possible role of TWEAK in PBMCs in the pathogenesis of LN and to find whether TWEAK influences the activation of p38 MAPK signalling in activated PBMCs.(3) To study the expression of TWEAK in renal tissue in MRL/lpr mice and analysis its possible mechanism. 1. The expression of TWEAK in peripheral blood mononuclear cells and its correlation to disease activity and renal damage of SLE.Methods:Subjects comprised 48 patients with SLE including 25 patients with ranal damage and 23 without,20 patients with rheumatoid arthrithis(RA) and 15 healthy controls.The expression of TWEAK in PBMCs was determined by RT-PCR and western blot. SLE disease activity was evaluated by Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) 2000 score. Next were analyzed the correlations of TWEAK mRNA and protein to serum IL-10, MCP-1 and some laboratory parameters of SLE disease activity.Results:The results showed that TWEAK expressions in PBMCs from SLE patients were significantly higher than that in RA patients or healthy controls, especially higher in those patients with renal disease. Elevated production of TWEAK is correlated positively and significantly with SLEDAI, proteinuria, serum anti-dsDNA,IL-10 and MCP-1,but inversely associated with serum complements.Conclusion:Our results suggested that TWEAK in PBMCs is positively related to SLE disease activity and might be involved in the pathogenesis of LN. 2. Tumor Necrosis Factor-Like Weak Inducer of Apoptosis (TWEAK) mediates p38 Mitogen-Activated Protein Kinase Activation (P38 MAPK) Signal Transduction in Peripheral Blood Mononuclear Cells from Patients with Lupus nephritisMethods:42 patients with SLE including 26 patients with ranal damage and 16 without, and 20 healthy controls were included in. The isolated PBMCs were treated with p38 inhibitor, SB203580 or anti-TWEAK mAb, with or without PHA/PMA stimulation. Immunofluorescence technique and western blot experiments were uesd to evaluate the protein expression of TWEAK in PBMCs and to identify the subcellular distribution of TWEAK and P38 MAPK.Next the contents of Interleukin (IL-10) monocyte chemoattractant protein (MCP-1) and anti-double stranded (DNA) (anti-dsDNA) in the supernatant were measured by ELISA.Results:The results showed that expression of TWEAK protein in PBMCs from LN patients was significantly higher than that from healthy controls. PHA/PMA simulation could up-regulate the productions of TWEAK and p38MAPK in PBMCs from LN. Anti-TWEAK mAb treatment down-regulated both TWEAK and p38 MAPK expression in PBMCs,as well as anti-dsDNA,IL-10 and MCP-1 in the supernatant, SB203580 had the same effect on cytokine production in PBMC, but have no effect in the expression of TWEAK.Conclusion:TWEAK-p38 MAPK-IL-10?MCP-1 signaling pathway in PBMC played an important pathogenic role in lupus nephritis.3. Study of the therapeutic efficacy and mechanism of TWEAK-siRNA in the MRL/lpr murine model of lupus nephritis Methods:12-week-old female MRL/lpr mice were divided into 4 groups: group A (MRL/lpr mice without intervention)(n=9); (2)group B (liposome intraperitoneal injection)(n=9); (3) group C (Negative control siRNA)(n=9); (4) group D (TWEAK-siRNA group),(n=9); Another 9 C57BL/6J were included in as the normal group(group E). 1mg/kg synthetic siRNAs dissolved in liposomes or empty liposomes were intravenous injected per week in 1000 ul volume. Urine protein, serum anti-dsDNA,IgQIgM were determined;RT-PCR, Immunofluores-cence stain, Western blotting were used to detect TWEAK, P38 MAPK expression. RT-PCR was used to detect IL-10 and MCP-1.Results:Urine protein excretion,serum anti-dsDNA,IgG,IgM increased and renal pathology showed pathological changes in MRL/lpr mice.Compared with the C57BL/6J group, there was significant increase in local TWEAK and P38 MAPK level in kidney tissue. TWEAK-siRNA significantly down-regulated of TWEAK?P38 MAPK and IL-10?MCP-1 expression in MRL/lpr mice. Urine protein excretion,serum anti-dsDNA,IgG,IgM were also significantly down-regulated. Injection with TWEAK-siRNA could improve the damage of vessels and nephric tubules, and decrease immune complex deposit in glomerulus.Conclusion:TWEAK take part in the renal damage of MRL/lpr lupus model through P38 MAPK signalling pathway, TWEAK-siRNA is expected to become one of the therapeutic targets of LN.