Macrophage Migration Inhibitory Factor deficiency attenuates kidney Injury by downregulating cytokines via CYR61 in lupus-prone mice induced by pristane. CURRENT

Background Lupus nephritis (LN) is one of the most serious manifestations of systemic lupus erythematous (SLE) and accounts for significant mortality and morbidity. Previous research has demonstrated that macrophage migration inhibitory factor (MIF) is involved in the pathogenesis of lupus nephritis, but the detailed mechanism is not elucidated. The aim of this study was to explore the pathogenesis of MIF in lupus nephritis with the pristane-induced mouse model of SLE. Methods Mif-/- mice and Wild type mice in the C57BL/6 (B6) background were used to induce Lupus model by pristane. 24 hour urine and sera were collected in the sixth month and mice were sacrificed to harvest tissues. Serum ANA, anti-dsDNA antibodies, C3, urinary creatinine and albumin were detected by ELISA. Related inflammatory cytokines were detected by Bio-Plex Pro™ assays and ELISA. CYR61 mRNA expression was detected by RT-qPCR and CYR61 protein expression were detected by Western blot. Immunofluorescence was used to detect the expression of MIF, ICs and C3 deposition and related cytokines expression in the kidneys. Immunohistochemical staining was used to detect macrophage infiltration and periodic acid-Schiff (PAS) staining were used for kidney histology. The Mann-Whitney test and Student’s t test were used to compare multiple group differences. The correlation were analyzed by Spearman correlation. Results Mif -/- mice with pristane-induced SLE have less inflammatory cytokines expression in sera. The Mif-/- mice have reduced renal injury, less macrophage infiltration, CYR61 and inflammatory cytokines expression in the kidneys. MIF induced the expression of CYR61, which can induce the expression of IL-1β, IL-6 and MCP-1 in bone marrow-derived macrophages (BMDM) in a cell-based assay. Conclusions The results suggest that MIF plays an important role in kidney injury by inducing macrophages infiltration and inflammatory cytokines expression in situ. Our finding support the pathogenic contribution of high expression MIF alleles in SLE and suggest that MIF antagonism might offer an effective therapeutic option in lupus nephritis.


Introduction
Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore). The membranes were blocked with 8% skim milk for 1 hr, washed with TBST buffer, and the membranes incubated with primary antibodies directed against tubulin (purchased from Cell Signaling Technology, Catalog 5335T), CYR61 (CST, Catalog 14479T) at 4℃ overnight. After incubation with HRP-conjugated secondary antibody (1: 2000, CST, Catalog 14708S), the membranes were washed and the bands were visualized by chemiluminescence detection kit (Thermo). Anti-tubulin antibody was used as internal control. Protein expression was quantified by density-analysis software (Image J).

Statistical analysis
Data obtained from three independent experiments were analyzed by Graphpad Version 7, and multiple group differences were compared using one-way ANOVA with the Mann-Whitney test or Student's t test and presented as mean± SD (standard deviation). Correlations were analyzed by Spearman correlation. P <0.05 was considered significant.

The kidney injury is alleviated in Mif-/-mice with pristane induced lupus
To explore whether kidney injury is alleviated in Mif-/-mice with pristane-induced lupus, we first compared the urinary albumin/creatinine ratio between Mif-/-C57BL/6 and Mif+/+ C57BL/6 (wild-type) mice, and found it to be significantly decreased in the Mif -/-pristane group (2.408 ± 0.332mg/mg creatinine) compared with the pristine-treated wild-type group (4.496 ± 0.697mg/mg creatinine) (P 0.01, Fig. 1A). Kidneys were harvested and the pathology of the kidneys examined. There was clear pathological damage in kidneys consistent with nephritis in mice that received pristine, and MIF expression was significantly increased in the wild-type mice (Fig. 1B, Fig 1C). To examine whether MIF exacerbates immune complex (IC) and complement 3 (C3) deposition in glomeruli, we examined the expression of ICs (IgG) and C3 in the kidneys. There were less IC and C3 deposition in the kidneys from Mif -/-mice than in wild-type mice (Fig. 1B, D, E). The PAS-positive area in wild-type mice treated with pristane was significantly larger than in corresponding Mif -/-mice (P 0.05, Fig. 1B, F), indicating that Mif -/-mice treated with pristane had less histological injury in the kidney. Previous studies have demonstrated that macrophage infiltration plays an important role in SLE-induced kidney injury and that intrarenal macrophage content is a prognosticator for severe glomerular damage [9]. The expression of the macrophage marker MAC-2 in glomerulus was detected in all groups and there was little macrophage infiltration detectable in mice not treated with pristine. Macrophage infiltration increased significantly after pristane treatment,and there was less macrophage infiltration in Mif-/pristane-treated mice compared with pristane-treated wild-type mice (P 0.01, Fig. 1B, G). These results indicate that MIF aggravates kidney injury in C57BL/6 mice with pristane induced lupus model. To explore more closely the mechanism of MIF action in lupus nephritis, we examined autoantibodies and complement C3 expression in sera. After pristane treatment, the expression of ANA and anti-dsDNA antibodies were significantly increased, and sera concentrations of C3 were significantly decreased. However, no significant differences were observed between the pristane-treated Mif-/-and wild-type groups ( Fig. 2A, B, C). Under control (untreated) conditions, the spleen weights in Mif-/-mice also similar to that of wild-type C57BL/6 mice, however, the volume and weights of spleens in Mif-/mice were significantly decreased after treatment with pristane (P 0.01, Fig. 2D, E).

Mif -/-mice with pristane-induced SLE have less inflammatory cytokine expression in
sera when compared to wild type mice with pristane-induced SLE.
To confirm the results of the Bio-Plex Pro™ assays, the expression of IL-1β, IL-6 and MCP-1 also was measured by ELISA. IL-1β, IL-6 and MCP-1 were significantly increased in the sera from wild type C57BL/6 mice when compared with Mif-/-mice (P 0.01, Fig. 3B, C, D). To assess whether the relative decrease in inflammatory cytokine expression was associated with reduced renal injury, we examined the relationship between cytokine expression in sera and PAS-positive areas in kidneys. We found the expression of IL-1β, IL-6, and MCP-1 to be positively associated with the PAS-positive areas in kidney (P 0.05, Fig. 3E, F, G). These results indicate that decreased systemic inflammatory cytokines expression contribute to the reduction in kidney injury observed in lupus mice.

MIF promotes the expression of inflammatory cytokines IL-1β, IL-6 and MCP-1 in pristane-induced SLE.
We next examined the expression of IL-1β, IL-6 and MCP-1 in the renal glomeruli and quantitated the fluorescence intensity by image J. We found that there were less IL-1β, IL-6 and MCP-1 expression in the glomerulus in Mif-/-mice when compared to wild-type mice (Fig. 4A, B, C, D).

MIF promotes the expression of IL-1β, IL-6 and MCP-1 and CYR61 in BMDMs.
To further verify that MIF can promote inflammatory cytokines expression in macrophages in experimentally-induced SLE, we generated BMDM cells both from wild-type and Mif-/-mice, and stimulated them with recombinant MIF. The expression of IL-1β, IL-6 and MCP-1 by BMDMs from Mif-/mice was significantly decreased when compared with wild type mice (P 0.001, Fig. 5A, B, C), and MIF treatment significantly increased the expression of IL-1β, IL-6 and MCP-1 (P 0.001, Fig. 5A, B, C).
These results showed that MIF promotes the expression of IL-1β, IL-6 and MCP-1 in macrophages, which participate in the pathogenesis of kidney injury.
To explore potential mechanisms by which MIF increases the expression of inflammatory cytokines, we analyzed a GEO datasets (GSE10241) for the whole-genome expression in MIF knockdown HEK293 cells and the control HEK293 cells. Among the 15 genes identified to show the greatest reduction in expression in MIF gene deficient cells (Fig. 5D), 3 genes: CYR61, ANKRD1 and CTGF, are key molecules involved in the Hippo signaling pathway. Emerging evidence has demonstrated that the Hippo signaling pathway is a key regulator in organ size control, tissue regeneration, tumor suppression and the immune system in multiple organisms [16,17]. We therefore examined the expression of CYR61, ANKRD1 and CTGF in the kidneys, and found that there was reduced CYR61 expression in Mif-/-mice (P 0.001, Fig. 5E), and no significant differences in the expression of ANKRD1 and CTGF (data were not shown). We also examined the CYR61 mRNA and protein expression in BMDM, and we found that both the CYR61 mRNA and protein expression in BMDM were significantly reduced in BMDM cells (P 0.01, Fig. 5F, G). MIF treatment also significantly increased the expression of CYR61 mRNA and protein in vitro (P 0.05, Fig. 5F, G).

MIF induces the expression of IL-1β, IL-6 and MCP-1 via CYR61
Recent studies have supported a critical role for Hippo signaling in autoimmunity [18], and that CYR61 blockade attenuates renal inflammation [19]. We examined the hypothesis that MIF may induce the expression of IL-1β, IL-6 and MCP-1 via CYR61 by overexpressing CYR61 in WT and Mif-/-BMDM cells. The increased expression of CYR61 was evident under our experimental conditions (P 0.01, Fig. 6A), and was associated with a coincident increase in the expression of IL-1β, IL-6 and MCP-1 (P 0.01, Fig. 6B, C, D). We also knocked down CYR61 expression by CYR61 siRNA (P 0.001, Fig. 6E) and observed a significant reduction in the expression of IL-1β, IL-6 and MCP-1 (P 0.05, Fig. 6F, G, H).

Discussion
The biological activity of MIF as a soluble immunologic mediator was      Figure A, B, C, E, F and G were analyzed by Mann-Whitney test. Data were shown as mean ± SD. * P <0.05, ** P