Reversal of Lupus Nephritis by SAP Treatment ALD-DNA-immunized lupus mice. n = 15. Pearson correlation analysis was used to carry out the correlation study. r = 0.76; P,0.05. (F) The ratios of SAP to DNA in SLE murine model and controls. Data are means 6 SD from 15 mice in each group. ***, P,0.001. (G) The correlation between the ratio of SAP to DNA and anti-dsDNA IgG level in SLE murine model. Pearson correlation analysis was used to carry out the correlation study. r = 0.90; P,0.001. doi:10.1371/journal.pone.0022659.g002 endosome acidification. It was found that SAP increased the intracellular fluorescence rates of treated macrophages, indicating that SAP binding to ALD-DNA promoted the uptake of ALD- DNA by macrophages (Fig. 3F). Furthermore, we performed real- time PCR to detect cytokine expression in the macrophages cultured with ALD-DNA or the complexes of purified SAP protein and ALD-DNA (SAP plus ALD-DNA), and found that mRNA levels of inflammatory cytokines including TNF-a, IL-1b, IL-6, IL- 12, and MCP-1 were notably decreased in the macrophages cultured with SAP plus ALD-DNA; however, mRNA level of IL- 10 was significantly increased in the macrophages cultured with SAP plus ALD-DNA as compared with those of macrophages cultured with ALD-DNA alone (Fig. 3G). These data suggest that pSAP plasmid could be correctly transcripted, translated and the expressed SAP protein could inhibit ALD-DNA-mediated innate immune responses in vitro. pSAP treatment in the early stage of SLE disease reverses lupus nephritis via reducing anti-dsDNA antibody production and IC deposition To evaluate the effect of pSAP treatment in mice, ALD-DNA- induced lupus mice with the onset of proteinuria (at week 4 after the initial ALD-DNA immunization) were treated with pSAP (ALD-DNA plus pSAP group). Significantly increased serum SAP levels accompanied with remarkably decreased circulating DNA levels were found in pSAP-treated lupus mice as compared with those in pcDNA3-treated lupus mice (Fig. 4A and B). The ratios of SAP to DNA were simultaneously increased in pSAP-treated lupus mice as compared with those in pcDNA3-treated lupus mice, which suggested that pSAP injection could reverse the insufficien- cy of SAP in lupus mice (Fig. 4C). 12 weeks after the initial ALD- DNA immunization, the levels of anti-dsDNA autoantibodies, IC deposition, proteinuria, renal pathology, and kidney score were analyzed. Notably reduced the levels of anti-dsDNA autoantibod- ies (Fig. 4D and E), urine protein (Fig. 4F), IC deposition (Fig. 4G and H), renal pathology (Fig. 4I), and kidney score (Fig. 4J) were found in the pSAP-treated lupus mice as compared with those of pcDNA3-treated lupus mice. These results show that pSAP treatment in the early stage of SLE disease could reverse lupus nephritis via decreasing anti-dsDNA antibody production and IC deposition in lupus mice. pSAP administration in the late stage of SLE disease alleviates lupus nephritis via reducing leukocyte infiltration and inflammatory marker production To further evaluate the protective effect of pSAP treatment in mice in the late stage of SLE disease, ALD-DNA-induced lupus mice with the established lupus nephritis (at week 8 after the initial ALD-DNA immunization) were treated with pSAP (ALD-DNA plus pSAP group). Significantly increased serum SAP levels accompanied with remarkably decreased circulating DNA levels were found in pSAP-treated lupus mice as compared with those in pcDNA3-treated lupus mice (Fig. 5A and B). The ratios of SAP to DNA were simultaneously increased in pSAP-treated lupus mice as compared with those in pcDNA3-treated lupus mice, which suggested that pSAP injection could partly improve the insuffi- ciency of SAP in lupus mice (Fig. 5C). 12 weeks after the initial ALD-DNA immunization, the levels of anti-dsDNA autoantibod- ies, IC deposition, proteinuria, renal pathology, and kidney score were analyzed in the lupus murine model receiving pSAP injection at week 8 after the initial ALD-DNA immunization when lupus mice already had the highest levels of anti-dsDNA autoantibodies and established lupus nephritis (ALD-DNA plus pSAP group). Twelve weeks after the initial immunization, notably decreased levels of urine protein (Fig. 5F) and ameliorated glomerulonephri- tis (Fig. 5I and J) but slowly reduced levels of autoantibody titers (Fig. 5D and E) and IC deposition (Fig. 5G and H) were found in pSAP-treated lupus mice as compared with pcDNA3-treated lupus mice, indicating the improved lupus nephritis was not exclusively ascribed to the decreased anti-dsDNA antibody levels and IC deposition. Other than the pathogenic anti-dsDNA autoantibody produc- tion and IC deposition, severe renal injury can be mediated by infiltrating proinflammatory leukocyte populations [17]. Flow cytometry analysis of cells extracted from kidneys of lupus mice showed a marked decrease in the number of CD45+ leukocytes in kidneys isolated from pSAP-treated lupus mice as compared with pcDNA3-treated lupus mice (Fig. 6A). Further flow cytometry analysis of infiltrating leukocyte populations revealed that pSAP- treated lupus mice displayed a decrease in renal T cells (CD4+) and B cells (CD19+) as compared to pcDNA3 treated lupus mice (Fig. 6A), but T and B cells only account for a portion of the decreased infiltrating cells in the kidneys of pSAP-treated lupus mice. Flow cytometry analysis of the presence of myeloid cells showed that pSAP-treated lupus mice exhibited a notable decrease in the number of F4/80+ macrophages (Fig. 6A), but there was no significant decrease in the number of CD11c+ dendritic cells (data not shown), suggesting that macrophages were the key cells that was influenced by pSAP treatment. As a set of inflammatory markers mainly secreted by macrophages were expressed in kidneys following glomerular immune complex deposition [8,18,19], further studies using ELISA analysis allowed us to determine several key markers in kidneys of mice. It was found that TNF-a, IL-1b, IL-6, IL-12 and MCP-1, which were upregulated in kidneys of ALD-DNA-induced lupus mice, were decreased in the pSAP-treated lupus mice (Fig. 6B). However, levels of IL-10 were notably increased in the pSAP-treated lupus mice (Fig. 6B). Analysis of the cytokine profile in serum of mice further confirmed that the inflammatory markers (including TNF- a, IL-1b, IL-6, IL-12, and MCP-1) were extensively and dramatically decreased in pSAP-treated lupus mice as compared with other control groups (Fig. 6C). These data suggest that pSAP treatment in the late stage of SLE disease could ameliorate the lupus nephritis via reducing the number of infiltrating inflamma- tory cells and decreasing the levels of inflammatory markers. Discussion We have provided here evidence that relative insufficiency of SAP played a critical role in the pathological process of the ALD-DNA- induced lupus nephritis, and verified that administration of SAP in vivo by a plasmid encoding the SAP, could significantly ameliorated the severity of SLE disease, as demonstrated by decreased levels of anti- dsDNA antibodies, reduced immune complex deposition, less proteinuria, less lupus nephritis, and decreased kidney score of PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e22659