Excessive nucleic acid R-loops induce mitochondria-dependent epithelial cell necroptosis and drive spontaneous intestinal inflammation

Abstract

Oxidative stress, which can be activated by a variety of environmental risk factors, has been implicated as an important pathogenic factor for inflammatory bowel disease (IBD). However, how oxidative stress drives IBD onset remains elusive. Here, we found that oxidative stress was strongly activated in inflamed tissues from both ulcerative colitis patients and Crohn's disease patients, and it caused nuclear-to-cytosolic TDP-43 transport and a reduction in the TDP-43 protein level. To investigate the function of TDP-43 in IBD, we inducibly deleted exons 2 to 3 of Tardbp (encoding Tdp-43) in mouse intestinal epithelium, which disrupted its nuclear localization and RNA-processing function. The deletion gave rise to spontaneous intestinal inflammation by inducing epithelial cell necroptosis. Suppression of the necroptotic pathway with deletion of Mlkl or the RIP1 inhibitor Nec-1 rescued colitis phenotypes. Mechanistically, disruption of nuclear TDP-43 caused excessive R-loop accumulation, which triggered DNA damage and genome instability and thereby induced PARP1 hyperactivation, leading to subsequent NAD⁺ depletion and ATP loss, consequently activating mitochondrion-dependent necroptosis in intestinal epithelial cells. Importantly, restoration of cellular NAD⁺ levels with NAD⁺ or NMN supplementation, as well as suppression of ALKBH7, an α-ketoglutarate dioxygenase in mitochondria, rescued TDP-43 deficiency-induced cell death and intestinal inflammation. Furthermore, TDP-43 protein levels were significantly inversely correlated with γ-H2A.X and p-MLKL levels in clinical IBD samples, suggesting the clinical relevance of TDP-43 deficiency-induced mitochondrion-dependent necroptosis. Taken together, these findings identify a unique pathogenic mechanism that links oxidative stress to intestinal inflammation and provide a potent and valid strategy for IBD intervention.

Keywords: R-loop; TDP-43; inflammatory bowel disease; necroptosis.

Fig. 1.

Oxidative stress induces nuclear-to-cytosolic TDP-43 export. (A) Representative immunohistochemical images of COX2 and NOX2 in UC tissues and paired adjacent normal tissues. n = 20. (Scale bar: 100 µm.) (B) Double immunofluorescence for TDP-43 and G3BP1 in UC tissues and paired adjacent normal tissues. n = 20. (Scale bar: 100 µm.) (C) Immunofluorescence for TDP-43 in NCM460 cells with or without H₂O₂ treatment. Arrowheads indicate TDP-43 signal in cytoplasm. The percentage of TDP-43 nuclear export was quantified. n = 12. (Scale bar: 25 µm.) (D) Double immunofluorescence for TDP-43 and G3BP1 in NCM460 cells with or without H₂O₂ treatment. n = 12. (Scale bar: 25 µm.) (E) Western blotting for TDP-43 in nuclear and cytoplasmic proteins isolated from NCM460 cells treated with different concentrations of H₂O₂. Histone H3 and GAPDH were used as loading control for nuclear and cytoplasmic proteins, respectively. (F) Representative immunohistochemical images of TDP-43 in UC tissues and paired adjacent normal tissues. Scores reflecting TDP-43 expression levels were quantified. n = 20. (Scale bar: 100 µm.) (G) Schematic of DSS-induced mouse acute colitis model. (H) Immunofluorescence for Tdp-43 in colon at different time points after DSS induction. n = 6. (Scale bar: 50 µm.) (I) Western blotting for Tdp-43 in nuclear and cytoplasmic proteins isolated from colonic epithelium at different time points after DSS induction. Histone H3 and β-Tubulin were used as loading control for nuclear and cytoplasmic proteins, respectively. (J) Double immunofluorescence for Tdp-43 and G3BP1 in colon from chronic TNBS-induced colitis mouse model. n = 6. (Scale bar: 50 µm.) (K) Western blotting for Tdp-43 in nuclear and cytoplasmic proteins isolated from colonic epithelium of control and chronic TNBS-induced colitis mice. Histone H3 and β-Tubulin were used as loading control for nuclear and cytoplasmic proteins, respectively. (L) Immunofluorescence for Tdp-43 in colon from wild-type (WT) and Il10^−/− mice at 20 wk of age. n = 6. (Scale bar: 50 µm.) (M) Western blotting for Tdp-43 in nuclear and cytoplasmic proteins isolated from colonic epithelium of WT and Il10^−/− mice. Histone H3 and β-Tubulin were used as loading control for nuclear and cytoplasmic proteins, respectively. Data are presented as the mean ± SD. ***P < 0.001.

Fig. 2.

Disruption of nuclear Tdp-43 in intestinal epithelium triggers spontaneous colitis. (A) Immunofluorescence for Tdp-43 (C-term, applied to detect Tdp-43 cytosolic localization) in colon from Tardbp^fl/fl and Tardbp^iIEC-KO mice at 7 dpi. n = 7. (Scale bar: 50 µm.) (B) Quantification of body weight change in Tardbp^fl/fl and Tardbp^iIEC-KO mice after TAM injection. n = 10. (C) Kaplan–Meier survival curves of Tardbp^fl/fl and Tardbp^iIEC-KO mice after TAM injection. n = 10. (D) Hematoxylin and eosin (H&E) staining, immunohistochemical staining for Ki67 and PAS in colon from Tardbp^iIEC-KO mice at different time points after TAM injection. n = 7. (Scale bar: 100 µm.) (E) Representative colonoscopy images of control and Tardbp^iIEC-KO mice at 10 dpi. Colonoscopy score was quantified. n = 7. (F) ELISA analysis of CRP, IL-6 and TNF-α in serum from control and Tardbp^iIEC-KO mice at 10 dpi. n = 6. (G) Immunohistochemistry for p-Stat3 in colon from Tardbp^fl/fl and Tardbp^iIEC-KO mice at 11 dpi. n = 7. (Scale bar: 100 µm.) (H) Western blotting for Il-1β, Nlrp3, p-Stat3, and Stat3 in colon from Tardbp^fl/fl and Tardbp^iIEC-KO mice at 11 dpi. β-Tubulin was used as loading control. (I) qRT–PCR analysis for chemokines in colon from Tardbp^fl/fl and Tardbp^iIEC-KO mice at 11 dpi. n = 7. (J and K) Immunofluorescence for CD45 (J) and F4/80 (K) in colon from Tardbp^fl/fl and Tardbp^iIEC-KO mice at 11 dpi. CD45⁺ cells and F4/80⁺ cells were quantified. n = 77 fields from seven mice. (Scale bar: 100 µm.) Data are presented as the mean ± SD. **P < 0.01; ***P < 0.001.

Fig. 3.

Disruption of nuclear Tdp-43 triggers necroptosis. (A and B) Immunohistochemical staining for TUNEL and cleaved Caspase-3 in colon from Tardbp^fl/fl and Tardbp^iIEC-KO mice at 10 dpi (A). Numbers of TUNEL⁺ and cleaved Caspase-3⁺ cells per crypt were quantified (B). n = 297 crypts from 6 mice. (Scale bar: 100 µm.) (C) KEGG pathway analysis of differentially expressed genes in transcriptome profiles from Tardbp^fl/fl and Tardbp^iIEC-KO mice at 10 dpi. n = 3. (D) GSEA of TNF-related genes in Tardbp^fl/fl and Tardbp^iIEC-KO mice. (E) Heatmap showing altered TNF-related genes in Tardbp^fl/fl and Tardbp^iIEC-KO mice. n = 3. (F) qRT–PCR analysis validated altered expression of necroptosis-related genes in Tardbp^fl/fl and Tardbp^iIEC-KO mice at 10 dpi. n = 7. (G) Western blotting for necroptosis-related genes Ripk1, p-Ripk3, p-Mlkl, and Mlkl in colon from Tardbp^fl/fl and Tardbp^iIEC-KO mice at 11 dpi. β-Tubulin was used as loading control. (H) Immunohistochemical staining for Mlkl in colon from Tardbp^iIEC-KO mice at indicated time points. n = 6. (Scale bar: 100 µm.) (I) Electron microscopy images of necrotic-like colon crypt cells in 10-dpi Tardbp^iIEC-KO mice. Arrowheads indicate fragmented nuclei, dotted red lines encircle the swelling mitochondria, and a red asterisk marks low electronic condensation of cytoplasm. n = 4. TEM, transmission electron microscopy. (Scale bar: 1 µm.) (J) Representative images of organoids from Tardbp^fl/fl and Tardbp^iIEC-KO mice before and after 4-OHT induction. Red lines mark cells that are undergoing anoikis. n = 6. (Scale bar: 100 µm.) (K) Cell viability analysis of organoids from Tardbp^fl/fl and Tardbp^iIEC-KO mice 48 h after 4-OHT induction. n = 8. (L) Time-series images of organoids from Tardbp^fl/fl and Tardbp^iIEC-KO mice 48 h after 4-OHT induction in the presence of PI (red). n = 6. Data are presented as the mean ± SD. ***P < 0.001.

Fig. 4.

TDP-43 disruption causes genome instability and DNA damage. (A) Heatmap showing altered genes of the intrinsic apoptotic signaling pathway in response to DNA damage in transcriptome profiles of Tardbp^fl/fl and Tardbp^iIEC-KO mice. n = 3. (B) Left panel: immunohistochemical staining for Tdp-43 in colon from Tardbp^fl/fl and Tardbp^iIEC-KO mice at 8 dpi. Arrowheads indicate nuclei with light hematoxylin staining of Tdp-43⁻ cells. n = 7. (Scale bar: 25 µm.) Right panel: Western blotting for Tdp-43 and γ-H2A.X in colon from Tardbp^fl/fl and Tardbp^iIEC-KO mice at 8 dpi. β-Tubulin was used as loading control. (C and D) Double immunofluorescence for Tdp-43 and γ-H2A.X in colon from Tardbp^iIEC-KO mice at indicated time points (C). The number of γ-H2A.X ⁺ cells per field was quantified (D). n = 30 fields from six mice. (Scale bar: 100 µm.) (E) DNA damage levels of NCM460 cells infected with sh-NC or sh-TDP43 were evaluated by the comet assay. Representative images (Left) and quantification of mean olive tail moment (Right) (n = 104 nuclei) are shown. (Scale bar: 50 µm.) (F) Western blotting for TDP-43, γ-H2A.X, PARP-1, and PAR in NCM460 cells infected with sh-NC or sh-TDP43. β-Tubulin was used as loading control. (G) Double immunofluorescence for TDP-43 and γ-H2A.X in NCM460 cells infected with sh-NC or sh-TDP43. Dotted white lines encircled locations of the hidden nuclei. (Scale bar: 25 µm.) (H) Double immunofluorescence for TDP-43 and PAR in NCM460 cells infected with sh-NC or sh-TDP43. Dotted white lines encircled locations of hidden nuclei. (Scale bar: 25 µm.) (I) Number of γ-H2A.X⁺ foci per cell in panel G (n = 65 cells for sh-NC and n = 83 cells for sh-TDP43) and relative mean fluorescence intensity of PAR per field in panel H (n = 21 fields for sh-NC and n = 23 fields for sh-TDP43) were quantified. (J) Double immunofluorescence for TDP-43 and γ-H2A.X in colon from healthy people and IBD patients. Arrowheads indicate TDP-43⁻ and γ-H2A.X⁺ intestinal epithelial cells. n = 20. (Scale bar: 50 µm.) (K) Double immunofluorescence for TDP-43 and PAR in colon from healthy people and IBD patients. Arrowheads indicate TDP-43⁻ and PAR⁺ intestinal epithelial cells. n = 20. (Scale bar: 50 µm.) Data are presented as the mean ± SD. ***P < 0.001.

Fig. 5.

TDP-43 nuclear clearance activates mitochondrion-dependent necroptosis. (A) NAD⁺ level analysis of NCM460 cells infected with sh-NC or sh-TDP43. n = 7. (B) ATP level analysis of NCM460 cells infected with sh-NC or sh-TDP43. n = 7. (C) NAD⁺ level analysis of colonic epithelial cells from Tardbp^fl/fl and Tardbp^iIEC-KO mice at 10 dpi. n = 6. (D) ATP level analysis of colonic epithelial cells from Tardbp^fl/fl and Tardbp^iIEC-KO mice at 10 dpi. n = 7. (E) AcAc/β-OHB ratio of NCM460 cells infected with sh-NC or sh-TDP43. n = 7. (F) Western blotting for TDP-43, HA, γ-H2A.X, PARP-1, and PAR in NCM460 cells infected with sh-NC or sh-TDP43 with and without NAD⁺ treatment. β-Tubulin was used as loading control. (G) Cell viability analysis of NCM460 cells infected with sh-NC or sh-TDP43 with and without NAD⁺ treatment. n = 8. (H) Western blotting for TDP-43 and ALKBH7 in wild type (WT) or ALKBH7 knockout (ALKBH7^Δ) 293T cell lines infected with sh-NC or sh-TDP43. β-Tubulin was used as loading control. (I) NAD⁺ level analysis of WT or ALKBH7^Δ cells infected with sh-NC or sh-TDP43. n = 7. (J and K) Cell viability analysis (J, n = 8) and LDH cytotoxicity analysis (K, n = 6) of WT or ALKBH7^Δ cells infected with sh-NC or sh-TDP43. (L and M) Mitochondrial calcein-AM retention assay in WT or ALKBH7^Δ cells infected with sh-NC or sh-TDP43 (L). The relative mean fluorescence intensity of calcein was quantified (M). n = 16. (Scale bar: 25 µm.) (N) Spearman correlation analysis of TDP-43 and γ-H2A.X (Left, P = 0.001; r = 0.684) or TDP-43 and p-MLKL (Right, P = 0.007; r = 0.586) in clinical UC patient samples. n = 20. (O) Spearman correlation analysis of TDP-43 and γ-H2A.X (Left, P = 0.015; r = 0.522) or TDP-43 and p-MLKL (Right, P < 0.0001; r = 0.846) in clinical CD patient samples. n = 21. (P) NAD⁺ level analysis of healthy control tissues (n = 9) and pathological tissues from clinical UC (n = 7) and CD (n = 5) patients. Data are presented as the mean ± SD. **P < 0.01; ***P < 0.001.

Fig. 6.

TDP-43 deficiency causes the excessive accumulation of R-loops. (A and B) Representative immunofluorescence images (A) and quantification of nuclear S9.6 intensity signal (B) of NCM460 cells depleted for TDP-43 (sh-TDP43) and treated with RNase H nuclease probed with R-loop-specific (S9.6) and nucleolin antibodies. n = 80 fields for seven independent biological replicates. (Scale bar: 20 µm.) (C and D) Dot blot analysis (C) and quantification of relative S9.6 levels (D) in nuclear fractions of NCM460 cells treated with sh-TDP43 and/or RNase H nuclease. n = 6. DNA was probed with dsDNA-specific antibody as loading control. (E and F) Dot blot analysis (E) and quantification of relative S9.6 levels (F) in nuclear fractions from colonic epithelial cells of Tardbp^fl/fl and Tardbp^iIEC-KO mice at 6 dpi. n = 6. DNA was probed with dsDNA-specific antibody as loading control. (G) DRIP-qPCR analysis of NCM460 cells that were treated with sh-NC or sh-TDP43 with or without RNase H nuclease. The SNRPN gene sequence was added as known negative control. n = 6. (H and I) Dot blot analysis (H) and quantification of relative S9.6 levels (I) in nuclear fractions of pathological tissues and paired non-pathological tissues from clinical CD patients. n = 5. Data are presented as the mean ± SD. *P < 0.05; ***P < 0.001.

Fig. 7.

NAD⁺ supplementation can alleviate Tardbp deficiency-induced spontaneous colitis. (A) Quantification of body weight change in Tardbp^fl/fl or Tardbp^iIEC-KO mice with NAD⁺ or Nec-1 treatment after the first TAM injection. n = 8. (B) Kaplan–Meier survival curves of Tardbp^fl/fl or Tardbp^iIEC-KO mice with NAD⁺ or Nec-1 treatment after the first TAM injection. n = 8. (C) Representative colonoscopy images of Tardbp^fl/fl and Tardbp^iIEC-KO mice with NAD⁺ or Nec-1 treatment at 11 dpi. Colonoscopy score was quantified. n = 6. (D and E) H&E staining (D) and double immunofluorescence for Ki67 and β-catenin (E) in colon from Tardbp^fl/fl or Tardbp^iIEC-KO mice with NAD⁺ or Nec-1 treatment at 11 dpi. n = 10. (Scale bar: 100 µm.) (F) Quantification of the clinical scores in Tardbp^fl/fl or Tardbp^iIEC-KO mice treated with NAD⁺ or Nec-1 at 11 dpi. n = 10. (G) ELISA analysis of CRP, IL-6 and TNF-α in serum from Tardbp^fl/fl and Tardbp^iIEC-KO mice with NAD⁺ or Nec-1 treatment at 11 dpi. n = 6. (H) Western blotting of Tdp-43, γ-H2A.X, Il-1β, p-Stat3, Stat3, and necroptosis related genes p-Ripk3, p-Mlkl, and Mlkl in colon from Tardbp^fl/fl or Tardbp^iIEC-KO mice treated with NAD⁺ or Nec-1 at 11 dpi. β-Tubulin was used as loading control. (I and J) qRT–PCR analysis of TNF-related genes Tnfα, Tnfrsf1a, Tnfrsf1b (I) and validation of altered chemokines Cxcl1, Cxcl2, Cxcl5, Cxcl16 (J) in colon from Tardbp^fl/fl or Tardbp^iIEC-KO mice treated with NAD⁺ or Nec-1 at 11 dpi. n = 7. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.