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.

Background

Systemic lupus erythematosus (SLE) is a common autoimmune disorder characterized with the production of autoantibodies, the deposition of immune complexes, and multiple system involvement [1]. Kidney damage is one of the most serious manifestations of organ involvement in SLE. About 30–70% of SLE patients present kidney damage [2], and 5–20% of lupus nephritis (LN) patients can develop end-stage renal disease within 10 years of diagnosis [3]. At present, the mechanisms of SLE and LN remain unclear, but genetic susceptibility, abnormal T and B lymphocyte phenotypes, function, and metabolism have been identified as pathogenesis factors of SLE [4, 5].

B cells and plasma cells, as producers of autoantibodies, play key roles in the pathogenesis of SLE and LN [6]. Studies have found that B cells secreting anti-glomerular antigen autoantibodies can be isolated from the kidneys of MRL/lpr mice, and the infiltration of B cells and plasma cells increased in the kidney of MRL/lpr and NZB/NZW F1 mice with lupus nephritis [7, 8]. SLE patients present an increase in peripheral plasma cells and memory B cells, but a decrease in naive B cells [9, 10]. In NZB/NZW F1 mice, the degree of infiltrated plasma cells in kidney is related to anti-dsDNA autoantibodies, and the immunoglobulin secretion capacity is similar to that of bone marrow plasma cells. In addition, the degree of plasma cell infiltration in the kidney of LN patients was also correlated with renal histological activity and chronic index [8]. Ma et al. confirmed that most plasma cells expressing interleukin-17 (IL-17) receptor in SLE patients and SLE mice could produce anti-dsDNA antibodies with the stimulation of IL-17. Transplantation of peripheral blood mononuclear cells (PBMCs) from SLE patients without helper T cell (Th) to SLE mice significantly inhibits plasma cell response and renal damage [11]. In conclusion, B cells and plasma cells are involved in the occurrence and development of kidney damage in SLE, and inhibiting the infiltration of B cells or plasma cells may alleviate kidney damage in SLE.

SLE is one of the most extensively studied autoimmune diseases with epigenetic modification. In recent years, epigenetic mechanisms, such as DNA methylation and histone modification, have been determined as key regulatory factors of cellular immunity and play the important role in the occurrence and development of SLE [12]. Various epigenetic regulated signaling molecules were dysregulated in SLE, which can regulate the expression of immune-related genes and then affect the function of immune cells and cytokine secretion, resulting in the continuous autoimmune inflammation [13]. N6-methyladenosine (m6A) is one of the most common RNA modifications in eukaryotic cells, which methylated on the adenine [14]. m6A can be installed by methyltransferase complexes (including METTL3, METTL14, and WTAP) and removed by demethyltransferase (including ALKBH5 and FTO). m6A reading proteins can specifically recognize and bind modification sites to perform corresponding biological functions [15]. Many studies have also found that the abnormal m6A modification can lead to the occurrence of immune disorders and autoimmune diseases by affecting the expression of key immune factors [16, 17]. Deletion of methyltransferase 14 (METTL14) in Treg cells can cause spontaneous colitis in mice [18], and m6A methylation of mRNA can maintain the suppressive function of Treg [19]. In addition, limited studies have shown that m6A modification can affect early B cell development. Deletion of METTL14 can significantly reduce the m6A mRNA level in B cells and inhibit B cell development, and block the IL-7-induced pro-B cell proliferation and the transformation process of large pre-B cells into small pre-B cells, leading to the dysregulation of important genes related to B cell development. This suggests that m6A methylation plays an important role in the development of bone marrow B cells [20]. Yu Z et al. found that long noncoding RNA (lncRNAs) associated with m6A modification may participate in the regulation of infiltration of naive B cells and plasma cells in gastric cancer tissues using bioinformatics methods [21]. Methyltransferase 3 (METTL3) can regulate the development of bone marrow hematopoietic stem cells and germinal center B cells by inhibiting IgH-related DNA breaks and maintaining genomic stability [22]. However, the role of m6A modification in regulating plasma cell differentiation and function has not been further studied.

Although the studies of m6A in gene expression regulation and immune response have made great progress, the current understanding of m6A modification in SLE is still limited. Luo et al. previously found that the expression of METTL14, ALKBH5, and YTHDF2 mRNA decreased in PBMCs of SLE patients [23]. They also found that METTL3, METTL14, WTAP, FTO, ALKBH5 and YTHDF2 mRNA expressions were decreased in PBMCs of SLE patients, and ALKBH5 level was associated with anti-dsDNA, rash and ulcer, which can be a risk factor for SLE [24]. Studies have found that the downregulated expression of ALKBH5 in SLE patients can affect the apoptosis and proliferation of T cells, but further studies are needed to prove this [25]. In addition, a comprehensive analysis of m6A and immune infiltration profiles of SLE using RNA sequencing has been conducted, which found that immune reading protein IGFBP3 and two key immune genes (CD14 and IDO1) may be helpful for the diagnosis and treatment of SLE [26]. m6A modification can also regulate the function process of non-coding RNA. Previous studies reported that m6A levels in PBMCs of SLE patients were reduced. The decreased expression levels of m6A-associated lncRNA (Xist and PSMB8-AS1) were correlated with various clinical manifestations, suggesting that m6A methylation and m6A-related lncRNA may be involved in the pathogenesis of SLE [27]. CircGARS, as the molecular sponge of miR-19a, can regulate the expression of YTHDF2 through the A20/NF-κB axis to promote the progression of SLE [28].

However, whether m6A is associated with the abnormal expression of immune-related genes in SLE, whether related proteins are involved in the pathogenesis of SLE, and the possible molecular mechanism is still unclear. In this study, we aimed to explore the expression and potential role of m6A modification and its regulatory protein in immune cells of SLE patients, and provide evidence for potential molecular targeted therapy in the future.

Methods

Patients and clinical data

The whole blood samples of 17 healthy volunteers and 31 SLE patients were collected. The SLE patients were admitted to the Seventh Affiliated Hospital of Sun Yat-sen University from May 2020 to June 2022 and met the clarification criteria for SLE revised by the American College of Rheumatology in 1997 [29]. There were 3 males and 28 females in SLE group, aged 18–68 years, with an average age of 33.68 (33.68 ± 13.12) years. A total of 17 healthy volunteers were selected as the control group, including 2 males and 15 females, aged 22–48 years, with an average age of 30.12 (30.12 ± 6.85) years. The two groups were comparable, for there was no statistical significance in age distribution and gender composition (P > 0.05). This study was approved by the Ethics Committee of the Seventh Affiliated Hospital of Sun Yat-sen University, and all patients and healthy volunteers signed informed consent (Certificate Number: 2019SYSUSH-037). Clinical and laboratory data of SLE patients were collected, and the clinical disease activity score was evaluated according to the Systemic lupus erythematosus disease activity index (SLEDAI) [30]. SLEDAI score > 9 was considered to be SLE patients in active period (SLE-AP), and SLEDAI score ≤ 9 was considered to be SLE patients in stable period (SLE-SP). PBMCs were separated and extracted using human lymphocyte separation medium (P8610, Solarbio, China) following the manufacturer’s steps.

Animal experiments

Ten-to-twelve-week-old female MRL/lpr mice and MRL/MPJ mice were purchased from Beijing Spafo Company and Changzhou Cavens Model Animal Co., Ltd, respectively, and raised in specific pathogen-free conditions of Laboratory Animal Center of Sun Yat-sen University for 12 h day and night with food and water provided at will. This study has been approved by the Animal Experiment Ethics Committee of Sun Yat-sen University (Certificate Number: SYSU-IACUC-2022–000413). We collected whole blood, urine, spleen and kidneys of 20-week-old female MRL/lpr mice and normal female control C57BL/6 and MRL/MPJ mice. For METTL3 overexpression or interferon regulatory factor 4 (IRF4) shRNA knockdown, we used the adeno-associated viruses (AAV9) packaged IRF4 knockdown or METTL3 overexpression plasmid, and injected female MRL/lpr mice in situ through the renal pelvis as previously described [31]. After 6–8 weeks, mice were humanely killed, and their kidneys and blood were collected for further analysis. Six to eight mice were used in each group, and mice were randomly assigned to experimental groups using a simple random sampling method, which was blind to the experimenters.

For blood collection, peripheral blood of mice was collected into the EDTA anticoagulant tube. Centrifuge at 3000 rpm/min for 15 min, collect upper plasma for detection of plasma indicators, such as anti-nuclear antibody (ANA), urea nitrogen, and creatinine. For preparation of mouse spleen and kidney single-cell suspensions, the spleen and kidney of the mice were bluntly separated and then pressed through a 75-μm cell stainer. 1 × ACK buffer to remove the red blood cell was added and washed with 1 × PBS. Cells were re-suspended for further study. Urine was collected by the method of metabolizable cage and used to measure urinary albumin (ALB).

Generation of plasmids and shRNA knockdown

Full-length METTL3 gene (NM_152758.5) was cloned into a pLV-CMV-MCS-zsgreen-puro vector purchased from Hanbio Biotechnology (Shanghai, China) to generate METTL3 overexpression plasmid. Adeno-associated virus 9-mediated METTL3 overexpression (AAV9-METTL3-OE) or IRF4 interference (AAV9-IRF4-sh) and their control virus were obtained from Shanghai Heyuan Biotechnology Company (Shanghai, China).

Cells and viruses

Romas cells were maintained in RPMI-1640 medium with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C in 5% CO₂. Lentivirus for METTL3 overexpression and its control virus were purchased by Newhelix Biotechnology Co., Ltd (Shanghai, China). Romas cells were transduced by lentiviruses with 4 mg/ml polybrene for 48 h.

MeRIP-sequencing and RNA-sequencing

Total RNA was extracted from PBMCs of 3 healthy controls and 6 SLE patients using MagZol reagent (R4801, Magen, China) according to the manufacturer’s protocol. RNA concentration and integrity were assessed using K5500 (Beijing Kaiao, China) and Agilent 2200 (Agilent Technologies, USA) and 1.5% agarose gel, respectively. Then ribosome was removed with riboNextSeqTM rRNA Remove Probe Pool, and RNA samples were fragmented into ~ 100-nucleotide-long fragments. Fragmented RNA was enriched with magnetic bead-antibody, and the immunocoprecipitated RNA was eluted and separated. The MeRIP RNA was constructed according to the instructions for using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina (NEB Corporation, USA). Library construction and quality inspection included the synthesis of the first complementary DNA (cDNA), the synthesis of the second cDNA following by adaptor ligation and enrichment with a low-cycle, and final library quality inspection using Agilent 2200 TapeStation (Life Technologies, USA). Sample preparation, manipulation, and sequencing are performed on Illumina platform instruments using the methods described in the User Guide. Data will be analyzed according to methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) bioinformatics processes. Biological information analysis mainly contained sequence gene comparison, motif prediction and annotation, peaks annotation and statistical analysis, differential peaks analysis, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Effective reads of input sample were used for RNA-seq analysis, and the reads count value of each transcript was calculated by HTSeq (version:0.6.0). Differential expressed genes were identified by edgeR R package according to the criteria of | log2(Fold Change) |≥ 1 and P-value < 0.05. KOBAS3.0 was used for enrichment analysis of KEGG and GO pathways, and P-value < 0.05 was considered to be significantly enriched.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated with TRIzol (108–95–2, Life Technologies, USA) following the manufacturer’s protocol. OD260/OD280 ratio of purified RNA was kept between 1.8 and 2.0. PrimeScript RT kit (RR047B, Takara, China) was used to generate cDNA (1 μg). Quantitative real-time PCR was performed using TB Green Premix Ex Taq II (RR820B, Takara, China) with the following primers (forward, reverse): human IRF4 (5′-GCCCAGCAGGTTCACAACTA-3′, 5′-TGTCACCTGGCAACCATTTTC-3′); human GAPDH (5′-AGAAGGCTGGGGCTCATTTG-3′, 5′-AGGGGCCATCCACAGTCTTC-3′); mouse Irf4 (5′-GGATTGTTCCAGAGGGAGCC-3′, 5′-GTTATGAACCTGCTGGGCTG-3′); mouse gapdh (5′-AAGATTGTCAGCAATGCATC-3′, 5′-CCTTCCACAATGCCAAAGTT-3′). Each well was repeated 3 times, and two-step PCR amplification was performed on a real-time fluorescence quantitative PCR apparatus (95℃ 30 s cycles: 1; 95℃ 5 s 60 ℃ 30 s cycles: 40). The relative abundance of mRNA was standardized with GAPDH or gapdh as the internal controls.

Western blotting

Cells were lysed by RIPA buffer containing 1% cocktail for 30 min. Protein lysate concentration was determined by BCA method (23227, Thermo Fisher Scientific, USA). Samples were boiled with 4 × SDS/PAGE loading buffer and separated on SDS-PAGE electrophoresis gel. Proteins were transferred onto PVDF membrane and then washed 3 times by 1 × TBST. PVDF membrane was blocked by 5% non-fat milk for 1 h and subsequently incubated by primary antibodies overnight for 4℃. Primary antibodies included anti-IRF4 antibody (1:1000, 15106S, Cell Signaling Technology, USA), anti-GAPDH antibody (1:2000, 60004–1-Ig, Proteintech, China), anti-METTL3 antibody (1:1000, ab195352, Abcam), anti-FTO antibody (1:1000, ab126605, Abcam), anti-ALKBH5 antibody (1:500, NBP3-06321, Novus, USA), anti-Vinculin antibody (1:2000, 66305–1-Ig, Proteintech, China). Then wash with 1 × TBST and incubate with secondary antibodies for 1 h at room temperature. Secondary antibodies included HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (1:5000, SA00001-2-500UL, Proteintech, China), HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (1:5000, SA00001-1-500UL, Proteintech, China). Add the ECL chemiluminescence chromogenic agent to visualize the immunoblots using Bio-Rad ChemiDoc XRS + System (Bio-Rad, USA). GAPDH or Vinculin were used as the internal controls. ImageJ was used to quantitatively analyze the expression of target bands.

Flow cytometry

For flow cytometric analysis, cells were suspended using cell staining buffer and blocked by 10% mouse serum. Stain with specific surface antibodies on ice incubation. Surface antibodies included APC/Fire™ 750 anti-mouse CD3 (1:100, 100247, Biolegend, USA), Alexa Fluor® 700 anti-mouse CD4 (1:200, 100429, Biolegend, USA), PerCP/Cyanine5.5 anti-mouse CD8a (1:100, 100733, Biolegend, USA), Brilliant Violet 785™ anti-mouse CD25 (1:100, 102051, Biolegend, USA), Alexa Fluor® 647 anti-mouse FOXP3 (1:100, 126407, Biolegend, USA), FITC anti-mouse CD19 (1:200, 152403, Biolegend, USA), Brilliant Violet 421™ anti-mouse/rat/human CD27 (1:100, 124223, Biolegend, USA), PE/Cyanine7 anti-mouse IgD (1:100, 405719, Biolegend, USA), Brilliant Violet 711™ anti-mouse CD138 (1:100, 142519, Biolegend, USA). After surface staining, cells were fixed and permeabilized using True-Nuclear™ Transcription Factor Buffer set (424401, Biolegend, USA) according to the manufacturer’s instructions. Then cells were stained with mAbs against the intracellular molecules including PE anti-IRF4 antibody (1:100, 646403, Biolegend, USA), and CoraLite®594-conjugated METTL3 Monoclonal antibody (1:100, sCL594-67733, Proteintech, China). IRF4 or METTL3 expression were evaluated by mean fluorescence intensity. All data were collected with BD LSR II, and analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA).

Distribution analysis of renal IRF4 in lupus nephritis

To explore the expression and distribution of renal IRF4 in lupus nephritis patients, we used the data from the Accelerating Medicines Partnership (AMP) SLE Phase I project at https://immunogenomics.io/ampsle/.

Immunofluorescent and immunohistochemistry staining

For preparation of frozen section, fresh mouse kidney tissues were fixed in 4% paraformaldehyde for 12–24 h. The tissues were removed and transferred into 10% sucrose, 20% sucrose, and 30% sucrose successively at 4 ℃. Then the kidney tissues were completely wrapped with OCT embedding agent, rapidly frozen, and fixed with liquid nitrogen. The frozen sections of the kidney were 6 μm. For immunofluorescence (IF) staining, frozen sections were permeabilized using 0.2% Triton X-100 for 20 min and blocked with 3% BSA for 1 h. Incubate overnight with primary antibodies at 4 ℃ and then with secondary antibodies at room temperature for 1 h. Primary antibodies included anti-IRF4 antibody (1:200, 15106S, Cell Signaling Technology, USA), anti-METTL3 antibody (1:200, ab195352, Abcam, UK), and anti-CD138 antibody (1:50, Novus Biologicals, MAB2966-SP, USA). Secondary antibodies included Goat anti-Rat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (1:200, A11007, Thermo Fisher, USA), Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488(1:500, A11008, Thermo Fisher, USA), and Goat anti-Mouse IgG(H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor™ 488 (1:200, A11029, Thermo Fisher, USA). The nucleus was then stained with DAPI buffer. For immunohistochemistry (IHC) staining of human biopsies, all samples of kidney biopsies from LN patients and minimal change nephropathy patients were obtained with informed consent. Paraffin-embedded kidney sections (4 μm) were deparaffinized, rehydrated through an alcohol series followed by antigen retrieval with sodium citrate buffer. Sections were then blocked with BSA for 30 min at room temperature and then incubated with primary antibodies at 4℃ overnight. IHC staining was performed with horseradish peroxidase (HRP) conjugates using DAB detection. Images were taken by LEICA DMi8 microscope and analyzed by LAS X.

Renal pathological staining

Hematoxylin–eosin (HE) staining, Periodic Acid-Schiff (PAS) staining, and MASSON staining were performed using HE staining set (G1005, Servicebio, China), PAS staining set (G1008, Servicebio, China), and Masson staining set (G1006, Servicebio, China) according to the manufacturer’s instructions, and histologic examinations were performed using light microscopy. Renal histology and interstitial inflammation scores of MRL/lpr mice were graded using the previously described scoring system [32]. Briefly, the kidney sections were scored for glomerular inflammation, proliferation, crescent formation, and necrosis. The score of 0 to 3 (0 = none, 1 = mild, 2 = moderate, and 3 = severe) was assigned to each of these features, which were then combined to produce a final pathology score. The interstitial inflammation was scored as follows: < 5% involvement = 0, 5% to 25% involvement = 1, 25 to 50% involvement = 2, and > 50% involvement = 3.

Detection of autoantibodies and renal function indicators

Plasma of mice was collected. Plasma ANA and urine ALB levels were detected using mouse ANA enzyme-linked immunosorbent assay (ELISA) kit (MM-1042M1, MEIMIAN, China) and mouse urine ALB ELISA kit (MM-44286M1, MEIMIAN, China), respectively. Plasma creatinine levels were measured using the creatinine test kit (E-BC-K188-M, Elabscience, China). Plasma urea nitrogen level was detected with the urea nitrogen detection kit (BC1530, Solarbio, China). Assays were carried out according to the manufacturer’s instructions.

MeRIP-qPCR

Total RNA was isolated from Romas and used for m6A immunoprecipitation (m6A-IP) with the riboMeRIP™ m6A Transcriptome Profiling Kit (R11096.3, RiboBio, China) following the manufacturer’s protocol. Input and m6A-immunoprecipitated RNAs were extracted for further enrichment analysis by quantitative real-time PCR.

Statistical analysis

IBM SPSS Statistics 23.0 software was used to analyze the data. Quantitative data were presented as mean ± standard deviation (x ± s), and normal distribution test was performed. For statistical comparisons between two groups subject to normal distribution, the two-tailed Student’s t test was used; otherwise, the two-sided Mann–Whitney U test was used. Qualitative data were presented as frequency and percentage (%), and clinical features were compared between the two groups using X² test or X² continuity correction. The correlation between IRF4 and proportion of peripheral blood immune cells in SLE patients was evaluated by Pearson correlation analysis. P-value < 0.05 was considered statistically significant. Statistical analysis and image visualization were performed using GraphPad Prism 7.0, R version 4.1.2, and IBM SPSS Statistics 23.0. Mechanism diagram was created with BioRender.com (Agreement number: LY278F6KSN).

Results

m6A methylation pattern was aberrant in SLE patients

To explore the m6A methylation pattern in SLE patients, MeRIP-seq was used to analyze the m6A modification level, location and modified motifs in PBMCs of healthy controls and SLE patients. The results showed that SLE patients contained only 773 m6A peaks the same as healthy controls, and 212 peaks were specific in SLE group (Fig. 1A). The m6A differential peaks were enriched in exon, intron, and near 3’ untranslated regions (UTR) (Fig. 1B). In addition, compared with healthy controls, m6A modified motifs were obviously aberrant in SLE patients (Fig. 1C). GO and KEGG enrichment analysis showed that the differential m6A peak associated genes were related to regulation of cellular process, and cytokine-cytokine receptor interaction signaling pathway (Fig. 1D and E). These results suggested a distinctly abnormal m6A modification features in SLE patients. RNA-seq analysis was then performed on PBMCs of the above 3 healthy controls and 6 SLE patients to reveal the gene expression profile in SLE patients. A total of 1503 differentially expressed transcripts were screened, of which 948 were upregulated and 555 were downregulated (Fig. 1F). To clarify the effect of m6A modification on gene expression regulation in SLE, the intersection of differential expression and differential peak genes was analyzed, and 15 genes were screened (Fig. 1G and H). GO analysis indicated that these genes were enriched in myeloid cell differentiation and KEGG results indicated they participated in the NF − kappa B signaling pathway (Fig. 1I and J). To further analyze the possible expression regulation of m6A in SLE patients with different disease activity, 13 genes with both differential expression and m6A modification level were also screened between SLE patients in active period (SLE-AP) and SLE patients in stable period (SLE SP) (Fig. 1K). Among them, IRF4 was the gene with both increasing expression and m6A level (Fig. 1L). Since IRF4 plays the important role in innate and adaptive immunity, we considered it as a potential molecule associated with m6A methylation in SLE for further exploration.

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)

Upregulated IRF4 was related to kidney damage of SLE patients

MeRIP-seq suggested that m6A levels of IRF4 mRNA were elevated in SLE patients (Fig. 2A). To validate IRF4 expression in SLE patients, PBMCs from 17 healthy controls and 31 SLE patients (18 stable and 13 active period) were collected, and IRF4 mRNA levels were detected by qRT-PCR. The result showed that compared with healthy controls, the expression of IRF4 mRNA in active SLE patients was significantly increased (P = 0.0355), while there was no significance in stable SLE patients (Fig. 2B). IRF4 protein expression was found to be increased both in active and stable SLE patients using Western blotting (Fig. 2C). Subsequently, the relationship between IRF4 and clinical characteristics in SLE patients were analyzed. SLE patients were divided into patients with high and low IRF4 level by the median IRF4 expression level. Qualitative clinical indicators were analyzed between the two groups, including gender, vasculitis, myositis, fever, hypocomplement, pericarditis, pleurisy, oral ulcer, pneumonia, renal involvement (urinary protein > 0.5 g/24h or +  +  + , or cylindruria), central nervous system involvement (seizures or psychosis), arthritis, red rash, anti-dsDNA, anti-Sm antibodies, AuaA antibodies, and anti-Histone antibodies. The results suggested SLE patients with high IRF4 level were more prone to kidney damage (Table 1, 56.25% vs 20%, P = 0.0385), implying that high IRF4 level was closely related to kidney damage of SLE. To explore the potential regulation of IRF4 on lymphocytes in SLE patients, we collected the percentage of lymphocytes in peripheral blood of SLE patients, and analyzed the correlation between IRF4 expression and the proportion of T, B, and NK cells. The results showed that IRF4 expression was positively correlated with proportion of peripheral B cells of SLE patients (P = 0.0373, r = 0.60), and the correlation was higher in active SLE patients (P = 0.0297, r = 0.91), but not with the proportion of T and NK cells (Fig. 2D–I). As shown above, IRF4 expression was upregulated in SLE patients, which was related with kidney damage. In addition, IRF4 expression was positively correlated with peripheral B cells. Therefore, we speculated that IRF4 might be involved in kidney damage of SLE through B cell regulation.

Table 1 Comparison of clinical manifestations and laboratory indexes between low-level and high-level IRF4 group

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

IRF4 expression was elevated in renal plasma cell of MRL/lpr mice

To further clarify that which B cell subsets is regulated by IRF4, we first detected the IRF4 expression of various B-cell subsets in spleen of female C57BL/6 and MRL/lpr mice using flow cytometry. The results suggested that compared with C57BL/6 mice, the proportion of B cells and naive B cells in the spleen of MRL/lpr mice were decreased, and plasma cells, memory B cells and nonclassical memory B cells were increased (Fig. 3A and B). In addition, the mean fluorescence intensity of IRF4 in B cells, plasma cells, memory B cells and naive B cells in MRL/lpr mice were increased, and the increase in plasma cells was the most significant, nearly 5 times that of the control group (Fig. 3C). The proportion of IRF4+ B cells, plasma cells and memory B cells were also increased obviously (Fig. 3D). Additionally, we also used public databases to explore the expression of IRF4 in various cells of LN patients. From the publicly available single-cell RNA sequencing (scRNA-seq) data of LN, we found that IRF4 was predominantly expressed in various B cell subsets, including activated B cells (CB0), plasma cells/plasmablasts (CB1), naive B cells (CB2a), pDCs (CB2b), ISG-high B cells (CB3). The expression of IRF4 was highest in plasma cells/plasmablasts (CB1), which suggested that IRF4 may mainly regulate plasma cells in LN (Additional file 1: Fig. S1). Therefore, we speculated that IRF4 may be related to kidney damage by promoting the plasma cell infiltration in SLE.

To determine the role and mechanism of IRF4 in kidney damage of SLE, we detected the renal IRF4 mRNA and protein expression in the female MRL/lpr mice and C57BL/6 mice using qRT-PCR and Western Blotting. The results suggested that there was almost no IRF4 expression in the kidney of control C57BL/6 and MRL/MPJ mice, while significantly increased in the MRL/lpr mice (Fig. 3E and F, and Additional file 1: Fig. S2A and B). In order to further explore whether IRF4 regulated plasma cell infiltration in kidney of SLE, we conducted the flow cytometry, which showed that the proportion of total and IRF4 positive plasma cells in kidney of MRL/lpr mice were both obviously increased (Fig. 3G), and the mean fluorescence intensity of IRF4 in plasma cells was also increased (Fig. 3H, P = 0.0048). We also confirmed evident plasma cell infiltration in the kidney of MRL/lpr mice, but almost no plasma cell infiltration in the C57BL/6 and MRL/MPJ mice using IF staining. In addition, IRF4 was significantly elevated and co-localized with plasma cells in the kidney of MRL/lpr mice (Fig. 3I and Additional file 1: Fig. S2C). This result supported the hypothesis that IRF4 may be involved in kidney damage of SLE by regulating plasma cell infiltration.

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

IRF4 depletion can alleviate kidney damage of MRL/lpr mice

To identify whether IRF4 is involved in kidney injury in MRL/lpr mice, we interfered IRF4 in MRL/lpr mice using an AAV9-packaged IRF4 knockdown plasmid. We confirmed the lower expression of IRF4 in kidney of mice injected with AAV9-IRF4-sh virus using Western blotting (Fig. 4A, P = 0.0030). The results showed that knockdown of IRF4 could reduce the levels of plasma creatinine, ANA, and urinary ALB in MRL/lpr mice (Fig. 4B–D), while urea nitrogen levels had no significant changes (Fig. 4E). In addition, knockdown of IRF4 lead to reduced mesangial matrix proliferation, immune complex deposition, and injury severity of mice (Fig. 4F-G). IgG and C3 deposition in the kidney of MRL/lpr mice were reduced after knockdown of IRF4 (Fig. 4H). Flow cytometry showed that kidney plasma cells in MRL/lpr mice were reduced (Fig. 4I). These results suggested that IRF4 depletion can alleviate the kidney injury and plasma cell infiltration, indicating that IRF4 plays an important role in the kidney injury of MRL/lpr mice.

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

METTL3 was increased in SLE patients and MRL/lpr mice

Considering the abnormality in m6A level of IRF4, we aim to explore the m6A-related proteins involved in IRF4 dysregulation. We examined the expression of 3 m6A-related proteins, including METTL3, ALKBH5, and FTO, in PBMCs of healthy controls and SLE patients. The results indicated that the expression of METTL3 and ALKBH5 in SLE patients were significantly increased (Fig. 5A). There were no significant differences in FTO expressions between the two groups (Fig. 5B). We also observed higher METTL3 expression in kidney of LN patients using IHC staining, which was mainly located in glomerular inflammatory cells, renal tubule cells, and rarely expressed in the glomeruli (Fig. 5B, Additional file 1: Fig. S3). To further investigate whether m6A-related proteins are involved in SLE kidney damage, we determined METTL3 and ALKBH5 protein expression in the kidney of MRL/lpr mice. Compared with the control group, renal METTL3 expression in the MRL/lpr mice was distinctly increased, while renal ALKBH5 expression showed no significant difference (Fig. 5C, Additional file 1: Fig. S2D, Additional file 1: Fig. S4). We also revealed that METTL3 expression was elevated in kidney plasma cells of MRL/lpr mice using flow cytometry (Fig. 5D, P = 0.0407). Consistent with the above results, we used IF staining to confirm the higher METTL3 expression in kidney and infiltrating plasma cells of MRL/lpr mice (Fig. 5E and Additional file 1: Fig. S2E). These results proved that the METTL3 expression was evidently upregulated in PBMCs of SLE patients and kidney plasma cells of MRL/lpr mice.

IRF4 depletion can reverse METTL3-aggravated kidney damage of MRL/lpr mice

In order to determine whether METTL3 induces plasma cell infiltration to promote kidney injury in MRL/lpr mice by increasing IRF4 expression, we interfered IRF4 or overexpressed METTL3 in MRL/lpr mice using the AAV9-packaged IRF4 knockdown or METTL3 overexpression plasmid. Compared with the control group, METTL3 and IRF4 protein levels in the kidneys of mice injected with AAV9-METTL3-OE virus were significantly increased (Fig. 6A). With METTL3 overexpression, plasma creatinine, ANA, and urinary ALB of MRL/lpr mice were significantly increased (Fig. 6B–D), while urea nitrogen levels were not significantly changed (Fig. 6E). Based on the METTL3 overexpression in the kidneys of MRL/lpr mice, we interfered with the IRF4 expression and confirmed knockdown efficiency of IRF4 by Western blotting (Fig. 6A). Knockdown of IRF4 could reverse METTL3-mediated aggravation in urinary creatinine, ANA, and ALB levels in MRL/lpr mice (Fig. 6B–D). HE, PAS, and MASSON staining confirmed that the mesangial matrix was hyperplasia, the deposition of immune complexes was increased, and the damage degree was aggravated (Fig. 6F-G). IF staining showed that METTL3 overexpression could significantly increase the IgG and C3 deposition in the kidney of mice (Fig. 6H). In addition, flow cytometry showed increased renal plasma cell infiltration after METTL3 overexpression (Fig. 6I). Based on the METTL3 overexpression, knockdown of IRF4 expression can reduce the pathological damage of the kidney (Fig. 6F-G), the IgG and C3 deposition in the kidney (Fig. 6H), and the degree of kidney plasma cell in MRL/lpr mice (Fig. 6I). These results confirmed that METTL3 overexpression can aggravate kidney injury in MRL/lpr mice, and IRF4 can mediate renal injury in MRL/lpr mice.

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

METTL3 promoted the up−regulation of IRF4 in m6A-dependent manner

According to the above research results, we focused on IRF4 as a target for METTL3. We transduced Romas cells with lentiviruses to overexpress METTL3. And we found that IRF4 mRNA and protein expression were elevated with METTL3 overexpression (Fig. 7A,B). By applying MeRIP-qPCR, we revealed that IRF4 mRNA was enriched by m6A antibody, and m6A methylation of IRF4 mRNA was induced in Romas cells transduced with METTL3-overexpression lentiviruses (Fig. 7C,D). To determine the molecular pathways by which IRF4 contributed to renal pathology, we conducted RNA-sequencing on the kidney tissues of mice injected with AAV9-IRF4-sh and the control virus. We found that there were 123 upregulated genes and 387 downregulated genes after knockdown of IRF4 (Fig. 7E,F). Then we performed KEGG enrichment analyses on differentially expressed genes to reveal potential key pathways. The results showed that IL-17 signaling pathway, cytokine-cytokine receptor interaction, TNF signaling pathway, and toll-like receptor signaling pathway were enriched (Fig. 7G). Subsequently, we verified the RNA-seq results by qPCR and found that the expression of Cxcl1, Bcl3, and Fos mRNA was significantly reduced (Fig. 7H). These genes were mainly involved in TNF signaling pathway, which may participate in the downstream pathway of IRF4-mediated renal injury in SLE. Collectively, these results indicate that IRF4 is a target of METTL3, and METTL3 can promote the abundance of IRF4 in a m6A-dependent manner (Fig. 7I).

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.

Discussion

In this study, we focused on the role of m6A modification in the pathogenesis of SLE. We revealed that the m6A modification pattern in PBMCs of SLE patients was significantly different from that of healthy controls. IRF4 presented the increased m6A modification and upregulated expression in SLE patients. SLE patients with high IRF4 level were more prone to kidney damage. In addition, IRF4 expression was significantly positively correlated with the proportion of peripheral B cells in SLE patients. Subsequently, we found that IRF4 expression in plasma cells increased most significant. Considering that m6A level of IRF4 was elevated in SLE patients, we detected the expression of m6A-related proteins in SLE and the results showed that METTL3 was distinctly upregulated. Overexpression of METTL3 can induce kidney IRF4 expression, increase kidney plasma cell infiltration, and promote kidney injury in MRL/lpr mice. Knockdown of IRF4 can alleviate this injury. We also showed METTL3 can induce IRF4 expression in an m6A-dependent mechanism in Romas cells. These results suggested that METTL3 was involved in kidney involvement of SLE by upregulating IRF4 expression.

Studies on the role of m6A modification in SLE patients and its possible regulatory mechanisms have not been reported until recently. Zhao X et al. found that m6A level in PBMCs supernatant of SLE patients was higher than that of healthy people [28]. However, Wu J et al. found that the m6A level of PBMCs mRNA in SLE patients was lower than that in healthy people, and it was closely related to lncRNA expression regulation [27], indicating that m6A modification can be tissue-specific. Luo et al. reported that the decrease of ALKBH5 mRNA level in peripheral blood was correlated with biomarkers of autoimmune response in SLE patients [24]. Another study showed that the mRNA expressions of ALKBH5, METTL14, and YTHDF2 were downregulated in SLE, and the decrease of YTHDF2 was associated with higher disease activity in SLE [23]. mRNA has the complex post-transcriptional modification, but these two studies did not detect the expression level of m6A-related proteins in SLE, so it is necessary to confirm the expression of m6A-related proteins in SLE. Our study found that compared with healthy controls, METTL3 protein expression in PBMCs of SLE patients and kidney tissues of MRL/lpr mice were upregulated. Recent studies have shown that the methyltransferase METTL3 participated in the occurrence of various kidney diseases. Abnormal activation of METTL3/TAB3 axis was one of the important mechanisms driving acute kidney injury [31], and the pathological role of methionine-METTL3-m6A axis in polycystic kidney has also been confirmed [33]. Our study revealed METTL3 overexpression could increase the levels of plasma creatinine, ANA, and urine ALB, promote deposition of IgG and C3, and exacerbate kidney damage in MRL/lpr mice. ANA is the main autoantigen of LN, and the deposition of immune complexes against the antigen in the glomerulus can cause main clinical symptoms such as proteinuria and nephrotic syndrome [34]. Also, IgG and C3 are involved in the deposition of immune complexes. Therefore, our findings suggested that METTL3 had a role in promoting kidney damage of SLE.

B cells have important immune functions associated with SLE and LN development, such as autoantibody production and proinflammatory cytokine secretion. Growing evidence showed that m6A was involved in the abnormal immune response and the onset of autoimmune diseases, and new research suggested that m6A-related proteins may affect the expression of key genes related to immune response in SLE [35]. m6A methylation has also been shown to be an important regulator of B cell development and differentiation [36]. A previous study showed that the loss of METTL14 can reduce m6A methylation level in B cells, resulting in severe inhibition of the pro-B cell proliferation [20]. Cytoplasmic m6A reading protein YT521-B homology domain family protein 2 (YTHDF2) can maintain IL-7-induced pro-B cell proliferation by inhibiting a set of transcripts [20]. METTL3 regulated early B-cell differentiation of bone marrow hematopoietic stem cells in a mRNA methylation-dependent manner [37]. However, the role of m6A in abnormal B cells in SLE and LN remains unclear. Two m6A RNA methylation modification subtypes have been identified in LN patients, which had significant differences in immune microenvironment, biological functional pathways, and clinical characteristics, suggesting that m6A methylation was closely related to the immune characteristics of LN [38]. In our study, we found METTL3 was upregulated in kidney plasma cell of MRL/lpr mice and METTL3 overexpression can induce kidney plasma cell infiltration. These results implicate that METTL3 participate the abnormal plasma cell infiltration in kidney of MRL/lpr mice.

In order to explore the possible mechanism of METTL3 promoting kidney injury in SLE, we used MeRIP-seq and RNA-seq combined analysis to screen genes with changes in m6A modification and expression, in which the m6A peak level and expression of IRF4 increased simultaneously. IRF4 is a key factor in lymphocytes, which can regulate the function of a variety of lymphocytes. IRF4 is dynamically expressed in B cells at different developmental stages, and the development of germinal B cells and plasma cells largely depends on the regulation of IRF4 [39]. Previous study has reported that there was no difference in the level of IRF4 mRNA in peripheral blood of SLE patients [40]. In our study, we found that compared with healthy controls, IRF4 mRNA expression in active SLE patients was significantly increased, while there was no significance in stable SLE patients. IRF4 protein expression was verified to be increased. The previous paper [40] studied the expression changes of IRF4 in peripheral whole blood samples of SLE patients, while our study studied peripheral blood mononuclear cells, with difference in the samples. The main components of whole blood are red blood cells and platelets, in which IRF4 expression level was relatively low. In addition, the objectives of this paper were Spanish patients, while our research objectives were Chinese patients. Another research [41] reported that the expression of IRF4 in kidney and peripheral blood T and B cells of lupus nephritis patients was significantly higher than that in healthy people. So ethnic differences and involvement of specific organs may also affect the expression of IRF4. Furthermore, the expression of IRF4 in SLE patients may be different from clinical medication, and disease activity. Lech et al. found that IRF4-deficient C57BL/6-(Fas)lpr mice lacked ANA and anti-dsDNA autoantibodies, immune complex deposition, and complement-mediated glomerulonephritis, suggesting that deficiency of IRF4 could prevent the maturation of plasma cells and effector T cells, and restrain the development of LN [42]. We found that SLE patients with high IRF4 expression were more prone to kidney damage and IRF4 expression was positively correlated with the proportion of peripheral B cells. Further study on IRF4 and B subpopulations showed that the proportion of IRF4 + B cells, plasma cells, and memory B cells in the spleen of MRL/lpr mice increased, and the mean fluorescence intensity of IRF4 in B cells, plasma cells, memory B cells, and initial B cells also elevated, among which the plasma cells was the most distinct. This suggested that IRF4 may be closely related to the abnormalities of B cells and plasma cells. For knockdown of IRF4 expression in MRL/lpr mice, we can reduce the plasma cell infiltration and kidney damage. These results indicated that IRF4 may participate in kidney damage in SLE through plasma cell infiltration.

We also conducted RNA-sequencing on the kidneys of control and IRF4-sh mice and found differentially expressed genes were mainly enriched in IL-17 signaling pathway, cytokine-cytokine receptor interaction, TNF signaling pathway, and toll-like receptor signaling pathway. Cxcl1, Bcl3, and Fos mRNA expression was verified to be decreased, which were mainly involved in TNF signaling pathway. The absence of IFN regulatory factor 4-binding (IRF-4-binding) protein (IBP) has been reported to decrease c-Fos induction and lead to the spontaneous development of a systemic autoimmune disorder [43]. Fisetin was indicated to reduce CXCL‑1 and 2 to manage the development of lupus nephritis [44]. In addition, B-Cell CLL/Lymphoma 3 (Bcl-3) was significantly upregulated by Sirt1 overexpression, which could be a potential risk factor of SLE [45]. These results suggested that TNF signaling pathway, including Cxcl1, Bcl3, and Fos, may be the downstream effectors of IRF4 during kidney injury of SLE.

Previous study suggested that DNA demethylases Tet2/3-dependent demethylation of specific CpG sites is dispensable for IRF4 expression, which is essential for plasma cell differentiation [46]. Lower DNA methylation of Irf4 was related to plasma cell differentiation [47]. Although some studies have explored the expression of IRF4 in SLE, the regulatory mechanism of its expression in SLE remains unclear. Since the high m6A peak level of IRF4, we speculated that IRF4 may be the target of METTL3. Subsequently, we overexpressed METTL3 in MRL/lpr mice using an AAV9-packaged METTL3 overexpression plasmid and found that METTL3 increased the expression of IRF4. Interfering IRF4 expression on the basis of METTL3 overexpression, levels of plasma creatinine, ANA, and urine ALB in MRL/lpr mice could be decreased, deposition of IgG and C3 were reduced, and kidney injury was alleviated. This suggested that IRF4 may be involved in kidney damage in SLE as a downstream target of METTL3. Furthermore, we proved that m6A level of IRF4 was elevated in Romas cells overexpressed METTL3. Our results provided new evidence for the role of m6A in the regulation of IRF4 expression.

Our study still has some limitations. We have not yet constructed SLE mice with plasma cell-specific knockout of METTL3 and IRF4 to further verify the in vivo functions of METTL3 and IRF4. The role of METTL3 in promoting kidney damage of SLE by affecting plasma cell functions, such as development, differentiation, maturation, remains to be determined. In addition, the number of samples in this study is still limited.

Conclusions

In summary, our results suggested that METTL3 was upregulated in the PBMCs of SLE patients and kidney of MRL/lpr mice and that METTL3 promoted plasma cell infiltration and kidney damage by increasing IRF4 expression in MRL/lpr mice. Our work reveals a possible molecular mechanism of m6A modification in SLE, providing new evidence for potential strategies to treat renal involvement. Studies related to m6A in SLE and LN are still lacking, but the existing evidence still suggests that targeting m6A regulatory proteins and modification processes has great potential in the treatment of SLE and LN.