Reversal of Lupus Nephritis by SAP Treatment Figure 3. Efficiently expressed SAP could inhibit ALD-DNA-induced innate immune response. (A) NIH 3T3 cells were transfected with pcDNA3 or pcDNA3-SAP plasmid (pSAP) using Lipofectamine 2000 transfection reagent, and cell supernatants were collected and subjected to ELISA assay for the expression of SAP protein at 24 h, 48 h, and 72 h post transfection. Data are means 6 SD of three independent experiments. (B–D) 6- week-old female BALB/c mice were injected intramuscularly with pSAP (100 mg/mice). (B) Immunohistochemistry examination was performed to determine the expression of SAP protein with anti-mouse SAP Ab in the muscle received pSAP injection. Imagines (6400) are representative of at least 10 mice in each group. (C) ELISA assay was performed to determine the levels of SAP protein in the serum of mice received pSAP injection. Data are means 6 SD of three independent experiments. n = 10. (D) The serum was collected on day 10 after pSAP injection. Western blot analysis was performed to detect the expression of SAP in serum of mice received pSAP injection. Data are representative of at least 10 mice in each group. (E–G) SAP protein was purified from the serum of mice received pSAP injection. (E) The binding ability of the purified mouse SAP protein to DNA was detected by dot blot analysis. Quantitative analysis of blots was reflected as mean intensity. Data are means 6 SD of three independent experiments. **, P,0.01. (F) Alexa Fluor 488 labeled ALD-DNA (AF488-ALD-DNA) was incubated with or without the purified SAP protein for 2 h (SAP plus ALD- DNA). BMDMs were treated with or without chloroquine (100 mg/ml) before DNA incubation. The intracellular Alexa Fluor 488 labeled ALD-DNA (AF488-ALD-DNA) in chloroquine-treated or untreated macrophages was determined by flow cytometry. Data are representative of results obtained in three independent experiments. (G) The purified SAP protein was incubated with ALD-DNA (SAP plus ALD-DNA) for 2 h. RAW264.7 cells were treated with PBS, ALD-DNA, or SAP plus ALD-DNA. 12 h later, levels of TNF-a, IL-1b, IL-6, IL-10, IL-12, and MCP-1 in the RAW264.7 cells were measured by real-time PCR. Data are means 6 SD of three independent experiments. * P,0.05; ***, P,0.001. doi:10.1371/journal.pone.0022659.g003 glomerulonephritis. This therapeutic effect was closely associated with reduced production of anti-dsDNA antibodies in the early stage of the disease and significantly decreased infiltrating lymphocytes and reduced levels of inflammatory markers in kidneys of pSAP-treated mice in the late stage of the disease. In previous study, the crucial and versatile functions of SAP in autoimmune disease have been well established [9,10]. SAP2/2 mice spontaneously develop antinuclear autoimmunity and severe glomerulonephritis, a phenotype resembling human SLE [15]. However, people doubt if SAP deficiency or strain combination PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e22659