MBL modulates ALD-DNA–induced macrophage M2b polarization.

<p>ALD-DNA was pre-incubated with indicated mouse MBL for 2 h. RAW264.7 cells or peritoneal macrophages were cultured with PBS, ALD-DNA or ALD-DNA/MBL complexes for 24 h. (A and C) mRNA levels of TNF-α, MCP-1, IL-6 and IL-10 in RAW264.7 cells or peritoneal macrophages were analyzed by real-time PCR. (B and D) Protein levels of TNF-α, MCP-1, IL-6 and IL-10 in the supernatants of RAW264.7 cells or peritoneal macrophages were measured by ELISA. (E and F) RAW264.7 cells were cultured with PBS, ALD-DNA or ALD-DNA/MBL complexes for 24 h. Then cell lysates were prepared to measure the protein levels of iNOS by western blot analysis. All values are given relative to the expression level of the β-actin. Data are means ± SD of three independent experiments. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

Mannose-Binding Lectin Blunts Macrophage Polarization and Ameliorates Lupus Nephritis

<div><p>Background</p><p>Deficiency in clearance of self nuclear antigens, including DNA, is the hallmark of systemic lupus erythematosus (SLE), a chronic autoimmnue disease characterized by the production of various autoantibodies, immune complex deposition and severe organ damage. Our previous studies revealed that administration of syngeneic BALB/c mice with activated lymphocyte-derived DNA (ALD-DNA) could induce SLE disease. Mannose-binding lectin (MBL), a secreted pattern recognition receptor with binding activity to DNA, has been proved to be a modulator of inflammation, but whether MBL takes responsibility for DNA clearance, modulates the DNA-mediated immune responses, and is involved in the development of DNA-induced SLE disease remain poorly understood.</p><p>Methodology/Principal Findings</p><p>The levels of serum MBL significantly decreased in lupus mice induced by ALD-DNA and were negatively correlated with SLE disease. MBL blunted macrophage M2b polarization by inhibiting the MAPK and NF-κB signaling while enhancing the activation of CREB. Furthermore, MBL suppressed the ability of ALD-DNA–stimulated macrophages to polarize T cells toward Th1 cells and Th17 cells. Importantly, MBL supplement <i>in vivo</i> could ameliorate lupus nephritis.</p><p>Conclusion/Significance</p><p>These results suggest MBL supplement could alleviate SLE disease and might imply a potential therapeutic strategy for DNA-induced SLE, which would further our understanding of the protective role of MBL in SLE disease.</p></div

MBL treatment blunts macrophage M2b polarization and alleviates lupus nephritis.

<p>BALB/c mice were immunized subcutaneously with ALD-DNA (50 µg/mouse) or PBS for total 3 times in 4 weeks. Mice immunized with ALD-DNA were pre-treated intramuscularly with pMBL (100 µg/mice) or pcDNA3.1 (100 µg/mice), and injected every 2 weeks for total 5 times. (A) mRNA levels of TNF-α, MCP-1, IL-6 and IL-10 in renal macrophages purified from mice were evaluated by real-time PCR. Data are means ± SD of three independent experiments. **<i>p</i><0.01; ***<i>p</i><0.001. n = 6. (B) Levels of TNF-α, MCP-1, IL-6 and IL-10 in serum were detected by ELISA assay. ***<i>p</i><0.001. n = 6. (C) Serum anti-dsDNA IgG levels of the mice were measured by ELISA assay every 2 weeks. **<i>p</i><0.01; ***<i>p</i><0.001. (D) Urine protein levels of the mice were assessed by BCA Protein Assay Kit every 2 weeks. **<i>p</i><0.01; ***<i>p</i><0.001. (E) The deposition of IgG-containing IC in glomeruli at week 12 after initial immunization. Imagines (×200) are representative of 10 mice in each group. (F) Mean glomerular fluorescence intensity (arbitrary units) was determined for IgG in ALD-DNA immunized lupus mice and control mice at week 12 after initial immunization. n = 10. ***<i>p</i><0.001. (G) 12 weeks after initial immunization, nephritic pathological changes were shown by H&E staining of renal tissues surgical resected from the mice. Imagines (×200) are representative of 10 mice in each group. (H) The kidney score was assessed using paraffin sections stained with H&E. n = 10. ***<i>p</i><0.001.</p

Serum MBL levels decrease in lupus mice and are negatively correlated with SLE disease.

<p>6-week-old female BALB/c mice were immunized subcutaneously with ALD-DNA, UnALD-DNA, or PBS for 3 times in 4 weeks. n = 10. (A and B) Serum MBL levels in mice immunized with ALD-DNA, UnALD-DNA, or PBS were determined by western blot every 2 weeks and quantitative analysis of western blot of serum MBL levels was reflected as mean intensity. **<i>p</i><0.01. n = 10. Data are representative of results obtained from 10 mice in each group. (C and D) The levels of serum circulating DNA were determined using a PicoGreen DNA detection kit and the ratios of MBL to DNA in SLE murine model and controls were presented. Data are means ± SD from 10 mice in each group. ***<i>p</i><0.001. (E) The correlation between serum MBL and anti-dsDNA IgG level in SLE murine model (n = 20) was presented. Pearson correlation analysis was used to carry out the correlation study. r = −0.9369; <i>p</i><0.001. (F) The correlation between serum MBL and urine protein level in SLE murine model (n = 20) was presented. Pearson correlation analysis was used to carry out the correlation study. r = −0.8524; <i>p</i><0.001.</p

The signaling pathway of regulation of macrophage M2b polarization by MBL.

<p>(A) RAW264.7 cells were treated with a given inhibitor for 1 h before PBS or ALD-DNA stimulation and 24 h later the supernatants were collected for ELISA. (B) RAW264.7 cells were treated with ALD-DNA (50 µg/ml) or ALD-DNA (50 µg/ml) plus MBL (10 µg/ml) for the indicated times. The cells were harvested and β-actin, phosphorylated IκB, and the total and phosphorylated p38, ERK1/2, JNK and CREB were detected by western blot. Data are representative of results obtained in three independent experiments. **<i>p</i><0.01.</p

MBL alters the ability of ALD-DNA–stimulated macrophages to promote T cell differentiation.

<p>Peritoneal macrophages were incubated with PBS, ALD-DNA (50 µg/ml) or ALD-DNA (50 µg/ml) plus MBL (10 µg/ml) for 48 h. (A) The expression of MHC II, CD80 and CD86 was examined by flow cytometry and the levels of them were represented as mean fluorescent intensity (MFI). (B and C) These peritoneal macrophages and allogeneic T cells isolated from female BALB/c mice were cultured at a 1∶5 ratio in the presence of ALD-DNA (50 µg/ml) for 6 days. For intracellular analysis of cytokine production, T cells were pre-stimulated with 10 ng/ml phorbol myristate acetate and 1 µg/ml ionomycin in the presence of 10 µg/ml brefeldin A for 5 to 6 hours. Cells were then fixed, permeabilized, and stained for IFN-γ and IL-17 and analyzed by flow cytometry. Average of percentage of IFN-γ⁺ CD4⁺ or IL-17⁺CD4⁺ T cells was presented in the bar charts. Data are means ± SD of three independent experiments. **<i>p</i><0.01.</p

pSAP administration in the late stage of SLE disease ameliorates ALD-DNA-induced lupus nephritis.

<p>BALB/c mice were immunized subcutaneously with ALD-DNA (50 µg/mouse) or PBS for total 3 times in 4 weeks. Mice were administrated intramuscularly with pSAP (100 µg/mice) from week 8 after initial immunization (when sever lupus nephritis has been established) and injected every 2 weeks for total 4 times. (A) The dynamics of SAP level in serum of lupus mice injected with pSAP (ALD-DNA plus pSAP) or pcDNA3 (ALD-DNA plus pcDNA3) were determined by ELISA assay every 2 weeks. (B) The dynamics of circulating DNA level in serum of lupus mice injected with pSAP (ALD-DNA plus pSAP) or pcDNA3 (ALD-DNA plus pcDNA3) were determined by ELISA assay every 2 weeks. (C) The ratios of SAP to DNA in SLE murine model injected with pSAP (ALD-DNA plus pSAP) or pcDNA3 (ALD-DNA plus pcDNA3) at week 12 after initial immunization. Data are means ± SD from 10 mice in each group. ***, <i>P</i><0.001. (D) Serum anti-dsDNA IgG levels were measured by ELISA assay every 2 weeks. (E) Anti-dsDNA IgG antibody titers in serum of pSAP-treated lupus mice (ALD-DNA plus pSAP) or pcDNA3-treated lupus mice (ALD-DNA plus pcDNA3) were detected by ELISA assay at week 14 after the initial ALD-DNA immunization. n = 10. (F) Urine protein levels of the mice were assessed by BCA Protein Assay Kit (Thermo Fisher Scientific) every 2 weeks. n = 10. (G) The deposition of IgG-containing IC in glomeruli at week 12 after initial immunization. Imagines (×200) are representative of at least 10 mice in each group. (H) Mean glomerular fluorescence intensity (arbitrary units) was determined for IgG in ALD-DNA-immunized lupus mice and control mice at week 12 after initial immunization. n = 10. NS, not significant. (I) 12 weeks after initial immunization, nephritic pathological changes were shown by H&E staining of renal tissues surgical resected from the mice. Imagines (×200) are representative of at least 10 mice in each group. (J) The kidney score was assessed using paraffin sections stained with H&E. n = 10. ***, <i>P</i><0.001.</p

Efficiently expressed SAP could inhibit ALD-DNA-induced innate immune response.

<p>(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 ± SD of three independent experiments. (B–D) 6-week-old female BALB/c mice were injected intramuscularly with pSAP (100 µg/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 (×400) 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 ± 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 ± SD of three independent experiments. **, <i>P</i><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 µg/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-α, IL-1β, IL-6, IL-10, IL-12, and MCP-1 in the RAW264.7 cells were measured by real-time PCR. Data are means ± SD of three independent experiments. * <i>P</i><0.05; ***, <i>P</i><0.001.</p

pSAP administration in the early stage of SLE disease reverses ALD-DNA-induced lupus nephritis.

<p>BALB/c mice were immunized subcutaneously with ALD-DNA (50 µg/mouse) or PBS for total 3 times in 4 weeks. Mice immunized with ALD-DNA were administrated intramuscularly with pSAP (100 µg/mice) from week 4 after initial immunization (with the onset of proteinuria) and injected every 2 weeks for total 5 times. (A) The dynamics of SAP level in serum of lupus mice injected with pSAP (ALD-DNA plus pSAP) or pcDNA3 (ALD-DNA plus pcDNA3) were determined by ELISA assay every 2 weeks. (B) The dynamics of circulating DNA level in serum of lupus mice injected with pSAP (ALD-DNA plus pSAP) or pcDNA3 (ALD-DNA plus pcDNA3) were determined by ELISA assay every 2 weeks. (C) The ratios of SAP to DNA in SLE murine model injected with pSAP (ALD-DNA plus pSAP) or pcDNA3 (ALD-DNA plus pcDNA3) at week 12 after initial immunization. Data are means ± SD from 10 mice in each group. ***, <i>P</i><0.001. (D) Serum anti-dsDNA IgG levels of the mice were measured by ELISA assay every 2 weeks. (E) Anti-dsDNA IgG antibody titers in serum of pSAP-treated lupus mice (ALD-DNA plus pSAP) or pcDNA3-treated lupus mice (ALD-DNA plus pcDNA3) were detected by ELISA assay at week 8 after the initial ALD-DNA immunization. n = 10. (F) Urine protein levels of the mice were assessed by BCA Protein Assay Kit (Thermo Fisher Scientific) every 2 weeks. n = 10. (G) The deposition of IgG-containing IC in glomeruli at week 12 after initial immunization. Imagines (×200) are representative of at least 10 mice in each group. (H) Mean glomerular fluorescence intensity (arbitrary units) was determined for IgG in ALD-DNA-immunized lupus mice and control mice at week 12 after initial immunization. n = 10. **, <i>P</i><0.01. (I) 12 weeks after initial immunization, nephritic pathological changes were shown by H&E staining of renal tissues surgical resected from the mice. Imagines (×200) are representative of at least 10 mice in each group. (J) The kidney score was assessed using paraffin sections stained with H&E. n = 10. ***, <i>P</i><0.001.</p

ALD-DNA immunization induces high levels of anti-dsDNA antibody and lupus nephritis.

<p>(A) Schematic diagram of animal immunization. 6- to 8-week old female BALB/c mice were immunized subcutaneously with ALD-DNA (50 µg/mice) plus CFA at week 0, followed by two booster immunizations of ALD-DNA (50 µg/mice) emulsified with IFA at week 2 and week 4 after initial immunization. (B) Serum anti-dsDNA IgG levels were measured by ELISA every 2 weeks after initial immunization. Data are means ± SD from 10 mice in each group. (C) Urine protein levels of the mice were assessed by BCA Protein Assay Kit every 2 weeks. Data are means ± SD from 10 mice in each group. (D) 8 weeks after initial immunization, nephritic pathology was evaluated by H&E staining of renal tissues. Imagines (magnification×200) are representative of at least 10 mice in each group. (E) The kidney score was assessed using paraffin sections stained with H&E in (D). ***, <i>p</i><0.001. (F) Glomerular immune deposition were detected by direct immunofluorescence for IgG in frozen kidney section from ALD-DNA-immunized lupus mice or control mice. Representative images (magnification×200) of 10 mice are shown for each group. (G) Mean glomerular fluorescence intensity (arbitrary units) was determined for IgG in ALD-DNA-immunized lupus mice (n = 10) and control mice (n = 10). ***, <i>p</i><0.001.</p