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. 2024 May 23:15:1361343.
doi: 10.3389/fimmu.2024.1361343. eCollection 2024.

Macrophage-derived macrophage migration inhibitory factor mediates renal injury in anti-glomerular basement membrane glomerulonephritis

Hui Yang #  1   2 , Jinhong Li #  3 , Xiao-Ru Huang  2   4 , Richard Bucala  5 , Anping Xu  1 , Hui-Yao Lan  2   4
Affiliations

Macrophage-derived macrophage migration inhibitory factor mediates renal injury in anti-glomerular basement membrane glomerulonephritis

Hui Yang et al. Front Immunol. .

Abstract

Macrophages are a rich source of macrophage migration inhibitory factor (MIF). It is well established that macrophages and MIF play a pathogenic role in anti-glomerular basement membrane crescentic glomerulonephritis (anti-GBM CGN). However, whether macrophages mediate anti-GBM CGN via MIF-dependent mechanism remains unexplored, which was investigated in this study by specifically deleting MIF from macrophages in MIFf/f-lysM-cre mice. We found that compared to anti-GBM CGN induced in MIFf/f control mice, conditional ablation of MIF in macrophages significantly suppressed anti-GBM CGN by inhibiting glomerular crescent formation and reducing serum creatinine and proteinuria while improving creatine clearance. Mechanistically, selective MIF depletion in macrophages largely inhibited renal macrophage and T cell recruitment, promoted the polarization of macrophage from M1 towards M2 via the CD74/NF-κB/p38MAPK-dependent mechanism. Unexpectedly, selective depletion of macrophage MIF also significantly promoted Treg while inhibiting Th1 and Th17 immune responses. In summary, MIF produced by macrophages plays a pathogenic role in anti-GBM CGN. Targeting macrophage-derived MIF may represent a novel and promising therapeutic approach for the treatment of immune-mediated kidney diseases.

Keywords: MIF; T cells; anti-GBM crescentic glomerulonephritis; inflammation; macrophages.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Characterization of MIFf/f-lysM-cre mice. (A) Bone marrow-derived macrophages (BMDMs) were isolated from MIFf/f, MIFf/f-lysM-cre and MIF KO mice. Enzymelinked immunosorbent assay (ELISA) show that MIFf/f-lysM-cre and MIF KO mice inhibit MIF expression by BMDMs. (B, C) RNA was isolated from cells for quantitative reverse transcription-PCR (qRT-PCR) quantitation of MIF and CD74. (D) Immunofluorescence staining for MIF(green) with nuclear DAPI (blue) counterstain in macrophages stimulated with TNF-a (10ng/ml for 24hours). Each bar represents mean±SEM. Each dot represents one mouse. **P < 0.01, ***P < 0.001 compared with MIFf/f mice; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 2
Figure 2
Selective MIF depletion in macrophages ameliorates experimental anti-GBM GN. (A) Representative images in PAS sections (magnification X200). (B) Semi-quantitative analysis of histology. (C) Urine albumin creatinine ration (ACR) over the disease course. (D) plasma creatinine. (E) Creatinine clearance. Each bar represents mean±SEM. Each dot represents one mouse. *p <0.05, **p <0.01, ***p< 0.001, ****p< 0.0001 versus corresponding control; #p<0.05, ##p < 0.01, ###p< 0.001 versus corresponding MIFf/f.
Figure 3
Figure 3
Selective MIF depletion in macrophages reduces macrophage and T cell recruitment in experimental anti-GBM GN. (A) Immunohistochemical staining for F4/80-positive macrophages in the kidney with anti-GBM crescentic GN on Day 14 after disease induction. (B, C) Summary data for macrophages in glomerulus and tubulointerstitium. (D) Immunohistochemical staining for CD3-positive T cells in the kidney with anti-GBM crescentic GN on Day 14 after disease induction. (E, F) Summary data for T cells in glomerulus and tubulointerstitium. Original magnification 200X. Each bar represents mean±SEM. Each dot represents one mouse. **p <0.01, ***p< 0.001, ****p< 0.0001 versus corresponding control; #p<0.05, ##p < 0.01, ###p< 0.001, ####p<0.0001 versus corresponding MIFf/f.
Figure 4
Figure 4
Selective MIF depletion in macrophages attenuates plasma levels of mouse anti-sheep IgG antibody and serum MIF production, which does not affect immune complex deposition in inflamed glomeruli. (A) Immunofluorescence staining for the deposition of sheep IgG, mouse IgG and mouse C3 in the kidney glomeruli with anti-GBM crescentic GN on Day 14 after disease induction. Plasma mouse anti-sheep IgG (B) and serum MIF (C) were determined using ELISA kits according to the manufacturer's protocol. Original magnification 200X. Each bar represents mean±SEM. Each dot represents one mouse. **p <0.01, ****p< 0.0001 versus corresponding control; #p<0.05, ##p < 0.01, ###p< 0.001 versus corresponding MIFf/f.
Figure 5
Figure 5
Selective MIF depletion in macrophages promotes macrophage polarization from M1 towards M2. (A) Representative flow cytometry plots and quantification of renal singlets analyzed for M1 (F4/80+CD86+). (B) Representative flow cytometry plots and quantification of renal singlets analyzed for M2 (F4/80+CD206+). Reverse transcription-PCR (RT-PCR) was performed on whole mouse kidney total RNA with anti-GBM crescentic GN on Day 14 after disease induction. (C–E) IL-1 β, monocyte chemoattractant protein-1 (MCP-1) and IL-10mRNA expression were normalized with GAPDH mRNA. Each bar represents mean±SEM. Each dot represents one mouse. ****p< 0.0001 versus corresponding control; #p<0.05, ##p < 0.01 versus corresponding MIFf/f.
Figure 6
Figure 6
Selective MIF depletion in macrophages downregulates renal Th1/Th17 response and upregulates renal Treg response in experimental anti-GBM GN. Flow cytometry analysis of renal infiltrated CD4+IFNy+Th1 cells (A), CD4+ IL-4+Th2 cells (B), CD4+ IL-17+Th17 cells (C) and CD4+CD25+ Foxp3+Treg (D). CD4+CD25+ Foxp3+Treg is Gated on CD4+ T cells. Each bar represents mean±SEM. Each dot represents one mouse. #p<0.05 versus corresponding MIFf/f.
Figure 7
Figure 7
Selective MIF depletion in macrophages ameliorates experimental anti- GBM GN by inactivating NF-kB and p38/MAPK signaling and inhibiting M1 macrophages activation. (A) Western blots. (B–E) Statistics data of the protein expression (iNOS, CD74, pp65/p65 and pp38/p38). Each bar represents mean ± SEM. Each dot represents one mouse. *p <0.05, ***p<0.001 versus corresponding control; #p<0.05, ##p < 0.01, ###p< 0.001 versus corresponding MIFf/f.
Figure 8
Figure 8
Bone marrow-derived macrophages (BMDM) lacking MIF show inhibition of MIF signaling and M1 macrophage activation in response to TNFa in vitro. (A) Western blots; (B–E) Statistics data of the protein expression (CD74, pp65/p65, pp38/p38, INOS). Results show that bone marrow-derived macrophages (BMDMs) isolated MIF KO mice inhibit TNF-α (10ng/ml)-induced upregulation of MIF signaling by downregulating CD74 expression and activation of NF-kB/p65 and p38/MAPK, there by inhibiting M1 macrophage activation by suppressing iNOS expression. Each bar represents mean±SEM. Each dot represents one mouse. ***P < 0.001 compared with MIF WT mice; ##P < 0.01, ###p< 0.001 compared with MIF WT mice treated with TNF-a 10ng/ml.
Figure 9
Figure 9
Mechanisms of MIF mediate experimental anti-GBM GN.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the Research Grants Council of Hong Kong (GRF 14104019, 14101121, and R4012-18); the High-Level Hospital Construction Project from Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Science (KJ012019108), the Guangdong-Hong Kong-Macao-Joint Labs Program (2019B121205005), the Lui Che Woo Institute of Innovative Medicine (CARE program), NIH 1R01-AR078334 (RB), Shenzhen Technology Project (JCYJ20190809120801655, JCYJ20180307150634856), the National Natural Science Funds of China (81870481), the Guangdong Provincial Natural Science Foundation (2021A1515011625) and the Guangdong Provincial Natural Science Foundation (2022A1515012308).