The m6A reader IMP2 directs autoimmune inflammation through an IL-17- and TNFα-dependent C/EBP transcription factor axis

Abstract

Excessive cytokine activity underlies many autoimmune conditions, particularly through the interleukin-17 (IL-17) and tumor necrosis factor-α (TNFα) signaling axis. Both cytokines activate nuclear factor κB, but appropriate induction of downstream effector genes requires coordinated activation of other transcription factors, notably, CCAAT/enhancer binding proteins (C/EBPs). Here, we demonstrate the unexpected involvement of a posttranscriptional "epitranscriptomic" mRNA modification [N6-methyladenosine (m⁶A)] in regulating C/EBPβ and C/EBPδ in response to IL-17A, as well as IL-17F and TNFα. Prompted by the observation that C/EBPβ/δ-encoding transcripts contain m⁶A consensus sites, we show that Cebpd and Cebpb mRNAs are subject to m⁶A modification. Induction of C/EBPs is enhanced by an m⁶A methylase "writer" and suppressed by a demethylase "eraser." The only m⁶A "reader" found to be involved in this pathway was IGF2BP2 (IMP2), and IMP2 occupancy of Cebpd and Cebpb mRNA was enhanced by m⁶A modification. IMP2 facilitated IL-17-mediated Cebpd mRNA stabilization and promoted translation of C/EBPβ/δ in response to IL-17A, IL-17F, and TNFα. RNA sequencing revealed transcriptome-wide IL-17-induced transcripts that are IMP2 influenced, and RNA immunoprecipitation sequencing identified the subset of mRNAs that are directly occupied by IMP2, which included Cebpb and Cebpd Lipocalin-2 (Lcn2), a hallmark of autoimmune kidney injury, was strongly dependent on IL-17, IMP2, and C/EBPβ/δ. Imp2^-/- mice were resistant to autoantibody-induced glomerulonephritis (AGN), showing impaired renal expression of C/EBPs and Lcn2 Moreover, IMP2 deletion initiated only after AGN onset ameliorated disease. Thus, posttranscriptional regulation of C/EBPs through m⁶A/IMP2 represents a previously unidentified paradigm of cytokine-driven autoimmune inflammation.

Fig 1.. Cebpd and Cebpb mRNAs are subject to m⁶A modification

(A) MEFs were transfected with siRNAs targeting Cebpd and Cebpb or control and treated ± IL-17 for 8 h. qPCR of the indicated mRNAs is shown. Data are normalized to untreated samples with control siRNA, from 3 independent experiments ± SEM. (B) Diagram of m⁶A modification by the m⁶A methyltransferase (writer) METTL3, and removal by the m⁶A demethylase (eraser) FTO. (C, D) MEFs were transfected with siRNAs targeting Mettl3 or Fto or non-targeting controls and treated ± IL-17 for 8 h. qPCR of the indicated mRNAs is shown. Data are normalized to untreated samples with control siRNA ± SEM, from 3 independent experiments. (E) MEFs were transfected with the indicated siRNAs and treated ± IL-17 for 6 h. C/EBPδ and C/EBPβ (isoforms LAP*, LAP and LIP (16) were assessed in nuclear extracts by immunoblotting. Blots are representative of 3-4 independent experiments. (F) Band intensity values were quantified from immunoblots. Means ± SEM from all experiments are shown. (G) MEFs transfected with the indicated siRNAs and treated ± IL-17 for 6 h. C/EBPδ and C/EBPβ were assessed in nuclear extracts by immunoblotting. Blots are representative of 4 independent experiments. (H) Band intensity values quantified from immunoblots. Means ± SEM from pooled experiments. (I) MEFs were treated with IL-17 for 3 h and subjected to RIP with m⁶A or IgG control Abs. qPCR of the indicated mRNAs is presented as % input. Data show mean ± SEM from 4 experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by ANOVA with post hoc Tukey’s test. N.D, not detected.

Fig. 2. IMP2 but not other m⁶A readers promote expression of IL-17 target genes

(A) MEFs were transfected with the indicated siRNAs and treated ± IL-17 for 8 h. qPCR of the indicated mRNAs is shown. Data are normalized to untreated samples with control siRNA ± SEM, from 3 independent experiments. (B) MEFs were transfected with the indicated siRNAs. qPCR and immunoblot of IMP2 is shown. Data are normalized to untreated samples with control siRNA ± SEM, from 3 independent experiments. (C) MEFs were transfected with siRNAs targeting Imp2 or non-targeting controls and treated ± IL-17 for 8 h. RNA-seq (n=3) was performed on the Illumina platform. Ingenuity Pathway Analysis of RNASeq showing top 10 predicted upstream regulators. Volcano plots showing the transcriptional response induced by IL-17 ± siImp2. In red are selected transcripts that were significantly changed (P value <0.05 and fold change > 2 or < −2; based on 3 experiments). (D) MEFs were transfected with siRNAs targeting Imp2 or non-targeting control and treated ± IL-17 for 8 h. qPCR of the indicated mRNAs is shown. Data are normalized to untreated samples with control siRNA ± SEM, from 3 independent experiments. (E) Imp2^+/+ or Imp2^−/− MEFs were treated ± IL-17 for 4 h qPCR of the indicated mRNAs is shown. Data are normalized to untreated samples with control siRNA ± SEM, 3 independent experiments. (F) HK-2 cells were transfected with siRNAs targeting IMP2 or non-targeting control and treated ± IL-17 for 8 h. qPCR of the indicated mRNAs is shown. Data are normalized to untreated samples with control siRNA ± SEM, from 4 independent experiments *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by ANOVA with post hoc Tukey’s test.

Fig 3.. IMP2 regulates IL-17-induced genes via C/EBPδ and C/EBPβ

(A, B) MEFs (A) or HK-2 cells (B) transfected with pooled siRNAs targeting Imp2 or non-targeting control and treated ± IL-17 for 8 h. qPCR of the indicated mRNAs is shown. Data are normalized to untreated samples with control siRNA ± SEM, from 3–4 independent experiments. (C) Imp2^+/+ or Imp2^−/− MEFs treated with IL-17. C/EBPδ, C/EBPβ, IMP2 and YY1 in nuclear lysates were assessed by immunoblotting. Blots are representative of 3 experiments. (D) Imp2^+/+ MEFs treated ± indicated doses of IL-17 or TNFα. C/EBPδ and YY1 in nuclear lysates were assessed by immunoblotting. Blots are representative of 2 experiments. (E) Imp2^+/+ or Imp2^−/− MEFs were treated ± 10 ng/ml TNFα. C/EBPβ and YY1 in nuclear lysates were assessed by immunoblotting. Blots represent 2 experiments. (F) MEFs transfected with siRNAs targeting Imp2 or non-targeting control and treated ± IL-17 for 6 h. C/EBPδ, C/EBPβ, IMP2 and YY1 in nuclear lysates were assessed by immunoblotting. Blots are representative of 4 experiments. (G) Pooled band intensity values with mean ± SEM from 4 experiments. (H) Imp2^+/+ or Imp2^−/− MEFs were transfected with Luc reporters driven by Il6 or Lcn2 promoters (6, 51). Cells were treated ± IL-17 for 8 h and Luc activity assessed. Data show fold-change relative to untreated Imp2^+/+ cells. (I) Imp2^−/− MEFs were co-transfected with C/EBPδ or C/EBPβ (7) with Il6- or Lcn2-Luc reporters. Cells were treated ± IL-17 for 8 h and Luc activity assessed. Data show fold-change relative to EV-transfected cells. *P<0.05, **P<0.01, ***P<0.001; ****P<0.0001 by ANOVA with post hoc Tukey’s test.

Fig 4.. IMP2 binds to Cebpd and Cebpb mRNAs and mediates post-transcriptional regulation

(A) Imp2^+/+ MEFs were treated ± IL-17 for 3 h and subjected to RIP with anti-IMP2 or IgG Abs. qPCR of the indicated mRNAs was normalized to input. Data show mean ± SEM of 4 independent experiments. Inset; IMP2 immunoprecipitates from Imp2^+/+ and Imp2^−/− MEFs was assessed by immunoblotting. (B) MEFs were treated with IL-17 for 3 h and subjected to RIP-seq with anti-IMP2 Abs. Diagram indicates the intersection between mRNAs that are IMP2-occupied based on RIPSeq compared to mRNAs that are IMP2-influenced at baseline or after IL-17 treatment. (C) Top: Diagram of Cebpd 3’UTR and predicted m⁶A site mutants (sequence, fig S8). Dashed line indicates sequence in the biotinylated synthetic mRNA. Bottom: HEK293T cells were co-transfected with IMP2-FLAG and a Luc reporter fused to WT Cebpd-3’UTR or sequences in which putative m⁶A sites were mutated. Lysates were subjected to RIP with anti-FLAG Abs and Luc mRNA assessed by qPCR. Data are normalized to input and show mean ± SEM of 3 independent experiments. (D) Lysates from HEK293T cells transfected with FLAG-tagged IMP2 were incubated with the biotinylated mRNAs corresponding to the Cebpd 3’ UTR (863-982, dashed line in panel C) in which indicated adenosines were m⁶A-modified or unmodified. RNA pulldowns were performed with streptavidin beads. FLAG-tagged IMP2 analyzed by immunoblotting. Blots are representative of 3 experiments. Pooled band intensity values with mean ± SEM. (E) MEFs were treated with TNFα for 3 h, given actinomycin D ± IL-17 for the indicated times, and Cebpd assessed by qPCR. Left: Data normalized to time=0 (100%), representative of 3 independent experiments. Right: Half-life was estimated by linear regression (17). (F) MEFs were treated ± IL-17 for 3 h and subjected to RIP with anti-eIF4G Abs. mRNAs were assessed by qPCR. Data normalized to untreated Imp2^+/+ samples precipitated with IgG. Data are representative of 2 independent experiments. Inset; eIF4G in cytoplasmic RIP fractions from Imp2^+/+ MEFs. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 by ANOVA with post hoc Tukey’s test or t-test.

Fig 5.. IMP2 forms a complex with HuR to regulate C/EBPs

(A) Imp2^+/+ MEFs were treated ± IL-17 and immunoprecipitated with anti-IMP2 Abs or IgG controls. Blots are representative of 2 independent experiments. (B) MEFs were transfected with pooled siRNAs targeting HuR (Elavl1) or control and treated ± IL-17 for 8 h. Data show mean ± SEM of 3 experiments normalized to untreated. (C) MEFs were transfected with siRNAs targeting HuR or non-targeting control and treated ± IL-17 for 6 h. C/EBPδ, C/EBPβ, HuR and YY1 were assessed by immunoblotting. Blots are representative of 3 experiments. (D) Pooled band intensity values with mean ± SEM from all experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by ANOVA with post hoc Tukey’s test.

Fig 6.. IMP2 deficient mice are resistant to IL-17-driven renal autoimmune inflammation

(A-B) Imp2^+/+, Imp2^−/− and Act1^−/− mice were subjected to AGN (71). Renal dysfunction was assessed at day 14 by ELISA of serum BUN and creatinine. Data pooled from 2 experiments. (B) Left: Representative images of H&E-stained kidney sections at day 14 of AGN (400×). Right: Slides were scored for abnormal glomeruli in Imp2^+/+ and Imp2^−/− mice (n=3) at day 14 post-AGN. Bars show mean ± SEM. (C) Kidney homogenates were prepared on day 7. Live inflammatory monocytes were determined by staining for CD11b, Ly6C and Ly6G, gated on the live CD45⁺ population. Graphs show percent of live CD45⁺CD11b⁺Ly6C^highLy6G⁻ cells. Data were pooled from 2 independent experiments. (D) The indicated mice were subjected to AGN (Imp2^+/+ control n=10; Imp2^+/+ AGN n=11, Imp2^−/− control n=8, Imp2^−/− AGN n=10, Act1^−/− control n=6 and Act1^−/− AGN n=6). Total RNA was extracted at day 7 and subjected to qPCR. Data were pooled from 2 experiments. (E) Imp2^+/+ or Imp2^−/− mice were lethally irradiated (900 rad) and reconstituted with BM from the indicated donor mice. After 6 weeks, mice were subjected to AGN. On day 14, BUN and creatinine levels were measured by ELISA. Data pooled from 2 experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, by ANOVA with post-hoc Tukey’s test.

Fig. 7.. Impaired C/EBP in Imp2^−/− mice during AGN

(A) The indicated mice were subjected to AGN (Imp2^+/+ control n=10; Imp2^+/+ AGN n=11, Imp2^−/− control n=8, Imp2^−/− AGN n=10, Act1^−/− control n=6 and Act1^−/− AGN n=6). RNA at day 7 was subjected to qPCR ± SEM. (B) Kidney homogenates from the indicated mice day 7 post-AGN were stained for CD45, CD133 and intracellular C/EBPδ. Data show live CD45⁻CD133⁺ cells. Representative FACS plot is shown. (C, D) Left: IF staining of C/EBPδ and C/EBPβ on day 7 post-AGN. Arrows indicate glomeruli. Cortex and medulla in a representative image are indicated. (4X). White boxes indicate sites of 40X images, fig S9. Right: Fluorescence intensity in non-glomerular regions of kidney was quantified by Image J, normalized to Imp2^+/+ control. Data are from two regions per slide and 2-3 independent slides (2 mice). *P<0.05, **P<0.01 by ANOVA with post hoc Tukey’s test. Scale bars = 1 mm.

Fig. 8.. Imp2 deletion post AGN induction decreased autoimmune renal dysfunction.

(A-C) Indicated mice were administered 2 mg tamoxifen (TAM) i.p. for 5 days starting day 1 after AGN induction. (B) Imp2 was assessed by qPCR at day 14. (C) BUN and creatinine were assessed at day 14. Data pooled from 2 experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, by ANOVA with post-hoc Tukey test. (D) Model of signaling through IMP2. Cebpb and Cebpd transcripts are subject to m⁶A modifications, which facilitate IMP2 mediated-post-transcriptional regulation. Imp2 deficiency impaired mRNAs whose expression is dependent on C/EBPs. Imp2-deficient mice are resistant to IL-17-driven renal inflammation. Diagram created on Biorender.com.