METTL3 facilitates kidney injury through promoting IRF4-mediated plasma cell infiltration via an m6A-dependent manner in systemic lupus erythematosus

Abstract

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause. N6-methyladenosine (m6A) is the most common mRNA modification and participates in various immune processes such as interferon production and immune cell regulation. However, the role of m6A in dysregulated immune response of SLE remains unknown.

Methods: PBMCs from SLE patients were collected to compare the m6A modification profile by methylated RNA immunoprecipitation sequencing (MeRIP-seq). Interferon regulatory factor 4 (IRF4) was identified by combination with MeRIP-seq and RNA-Seq. IRF4 and methyltransferase 3 (METTL3) were detected using qRT-PCR and WB. Clinical significance of IRF4 in SLE patients was explored subsequently. IRF4 expression in B cell subsets of female MRL/lpr mice was detected by flow cytometry. Adeno-associated viruses (AAV) including AAV9-METTL3-OE and/or AAV9-IRF4-sh were treated with female MRL/lpr mice. Autoantibody levels and kidney injury were tested by ELISA, pathological staining, and immunofluorescence. m6A level of IRF4 was detected by MeRIP-qPCR. The downstream effectors of IRF4 contributing to renal pathology were explored by RNA-seq and verified by qRT-PCR.

Results: m6A methylation features were obviously aberrant in SLE patients, and IRF4 was the upregulated gene modified by m6A. METTL3 and IRF4 expressions were elevated in SLE patients and kidney of MRL/lpr mice. Clinical analysis indicated that SLE patients with high IRF4 level were more prone to kidney damage. IRF4 expression was especially increased in plasma cells of MRL/lpr mice. METTL3 induced renal IRF4 expression, plasma creatinine, ANA and urine ALB levels, IgG and C3 deposition, and renal damage and plasma cell infiltration were aggravated in MRL/lpr mice. However, IRF4 depletion could partially reduce METTL3-induced kidney damage. Meanwhile, m6A level of IRF4 elevated with METTL3 overexpression. Also, the expression of Cxcl1, Bcl3, and Fos mRNA were significantly reduced after knockdown of IRF4, which were mainly involved in TNF signaling pathway.

Conclusions: Our study confirmed that upregulated METTL3 promoting IRF4 expression in an m6A-dependent manner, thus causing plasma cell infiltration-mediated kidney damage of SLE. This provides new evidence for the role of m6A in SLE kidney injury.

Keywords: Interferon regulatory factor; Kidney injury; N6-methyladenosine; Plasma cell; Systemic lupus erythematosus.

Fig. 1

m6A methylation pattern was aberrant in SLE patients. The differences of m6A modification in PBMCs of SLE and healthy controls were detected by MeRIP-seq; A Venn diagram of different m6A peaks in SLE patients and healthy controls; B Location of m6A differential peaks in genes. C m6A specific motifs in SLE patients and healthy controls. D GO analysis of m6A peak differentially expressed genes; E KEGG analysis of m6A peak differentially expressed genes; F Volcano plot of differentially expressed genes in SLE patients and healthy controls, with low expression in green and high expression in red; G Venn diagram, H Square chart, I GO analysis, and (J) KEGG analysis of m6A peak differentially expressed genes and differentially expressed genes between SLE patients and healthy controls; K Square chart, and (L) Heat map of m6A peak differentially expressed genes and differentially expressed genes between SLE patients in active period (SLE-AP) and SLE patients in stable period (SLE-SP)

Fig. 2

Upregulation of IRF4 was related to kidney damage in SLE patients. A Schematic diagram of MeRIP-seq detecting the m6A level of IRF4; B qRT-PCR for IRF4 mRNA expression in PBMCs of healthy controls (HC), SLE-AP, and SLE-SP; C Western blotting for IRF4 protein expression in PBMCs of HC, SLE-AP and SLE-SP; Correlation between IRF4 expression and the proportions of peripheral T lymphocytes (D), T helper cells (E), suppressor T cells (F), NK cells (G) and B lymphocytes (H) in SLE patients (n=12); I Correlation between IRF4 expression and the proportions of peripheral B lymphocytes in SLE-AP (n=5). *P<0.05, **P<0.01, ***P<0.001

Fig. 3

IRF4 expression was elevated in plasma cells of MRL/lpr mice. Flow cytometry for the proportion of spleen B-cell subsets (A, B), IRF4 expression of spleen B cell subsets (C), and the proportion of IRF4+ cells of spleen B cell subsets (D) in MRL/lpr mice; E qRT-PCR for IRF4 mRNA expression in kidneys of C57BL/6 (n=6) and MRL/lpr mice (n=7); F Western blotting for IRF4 protein expression in the kidneys of C57BL/6 (n=6) and MRL/lpr mice (n=7). Flow cytometry for the proportion (G), and mean fluorescence intensity of IRF4 (H) in kidney plasma cells of C57BL/6 (n=7) and MRL/lpr mice (n=7). I Immunofluorescence staining for IRF4 expression and its co-localization with kidney plasma cells of C57BL/6 and MRL/lpr mice, the Scale bar was 50 μm; * P<0.05, **P<0.01, ***P<0.001

Fig. 4

IRF4 depletion alleviated kidney damage of MRL/lpr mice. A Western blotting for IRF4 protein expression in kidneys of MRL/lpr mice injected with control and AAV9-IRF4-sh virus; ELISA or biochemical kit for levels of plasma creatinine (B), urine ALB (C), plasma ANA (D) and plasma urea nitrogen (E) in MRL/lpr mice injected with control and AAV9-IRF4-sh virus. F Immunofluorescence staining for kidney IgG and C3 deposition in kidneys MRL/lpr mice injected with control and AAV9-IRF4-sh virus, Scale bar, 50 μm; G HE, PAS and MASSON staining for pathological condition in kidneys of MRL/lpr mice injected with control and AAV9-IRF4-sh virus, Scale bar, 50 μm; H Flow cytometry for proportion of kidney plasma cells in MRL/lpr mice injected with control and AAV9-IRF4-sh virus. *P<0.05, **P<0.01, ***P<0.001

Fig. 5

METTL3 was increased in SLE patients and MRL/lpr mice. A Western blotting for expression of three m6A-related proteins, including METTL3, ALKBH5, and FTO in PBMCs of healthy controls (n=5) and SLE patients (n=8); B Immunohistochemistry staining for METTL3 expression in renal tissues of patients with minimal change nephropathy (Ctrl) or lupus nephritis (LN), Scale bar, 100 μm; C Western blotting for METTL3 protein expression in kidneys of C57BL/6 (n=6) and MRL/lpr mice (n=7); D Flow cytometry for METTL3 expression in kidney plasma cells of C57BL/6 (n=5) and MRL/lpr mice (n=5). E Immunofluorescence staining for METTL3 expression and its co-localization with kidney plasma cells in C57BL/6 and MRL/lpr mice, Scale bar, 50 μm; * P<0.05, **P<0.01, ***P<0.001.

Fig. 6

IRF4 depletion partially reversed METTL3-aggravated kidney damage of MRL/lpr mice. A Western blotting for METTL3 and IRF4 protein expression in kidneys of MRL/lpr mice injected with AAV9-METTL3-OE or/and AAV9-IRF4-sh virus; ELISA or biochemical kit for levels of plasma creatinine (B), urine ALB (C), plasma ANA (D), and plasma urea nitrogen (E) in MRL/lpr mice injected with AAV9-METTL3-OE or/and AAV9-IRF4-sh virus. F Immunofluorescence staining for kidney IgG and C3 deposition in MRL/lpr mice injected with AAV9-METTL3-OE or/and AAV9-IRF4-sh virus, Scale bar, 50 μm; G HE, PAS and MASSON staining for pathological condition in kidneys of MRL/lpr mice injected with AAV9-METTL3-OE or/and AAV9-IRF4-sh virus, Scale bar, 50 μm; H Flow cytometry for proportion of kidney plasma cells in MRL/lpr mice injected with AAV9-METTL3-OE or/and AAV9-IRF4-sh virus;*P<0.05, **P<0.01, ***P<0.001

Fig. 7

METTL3 promoted m6A RNA modifications on IRF4 mRNA, inducing IRF4 expression. Romas cells were transduced by lentiviruses of METTL3 overexpression and its control virus; A Western blotting for METTL3 and IRF4 protein expression in (n=3); B qRT-PCR for IRF4 mRNA expression (n=3); C MeRIP-qPCR assays, along with (D) agarose gel images for detection of m6A enrichment in IRF4 mRNA. RNA-sequencing on the kidneys of mice injected with AAV9-IRF4-sh and the control virus were conducted; E Volcano plot and (F) Heatmap of differentially expressed genes in kidneys of MRL/lpr mice injected with control and AAV9-IRF4-sh virus; G KEGG analysis of differentially expressed genes; H qRT-PCR for verification of Cxcl1, Bcl3, Fos, Socs3, and Jun mRNA expression. I Mechanism diagram of METTL3-IRF4 axis promoting kidney injury during SLE. *P<0.05, **P<0.01, ***P<0.001.