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c-Rel orchestrates energy-dependent epithelial and macrophage reprogramming in fibrosis

An Author Correction to this article was published on 10 December 2020

This article has been updated

Abstract

Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-β1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel–Pfkfb3 axis has potential for therapeutic applications in fibrotic disease.

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Fig. 1: c-Rel is a feature of chronic liver, kidney and lung disease in humans and epithelial c-Rel signalling regulates hepatic fibrogenesis and regeneration in mice.
Fig. 2: c-Rel signalling regulates epithelial inflammatory responses via regulation of Pfkfb3.
Fig. 3: c-Rel signalling in macrophages is pro-fibrogenic and regulates macrophage plasticity.
Fig. 4: c-Rel regulates pro-fibrogenic epithelial–macrophage cross-talk accelerates fibroblast activation.
Fig. 5: Epithelial- or macrophage-specific deletion of c-Rel limits renal and pulmonary fibrosis.
Fig. 6: Epithelial and macrophage c-Rel signalling synergistically promote hepatic fibrosis but antagonistically regulate hepatic regeneration in mice.
Fig. 7: Pharmacological inhibition of c-Rel limits fibrogenesis in murine models of liver, kidney and lung injury.
Fig. 8: Pharmacological inhibition of c-Rel limits fibrogenesis in human precision-cut liver slices.

Data availability

Mass spectrometric raw data are available through the PRIDE repository (https://www.ebi.ac.uk/pride/archive/) and have been assigned the identifier PXD017320. Source data are provided with this paper.

Change history

  • 10 December 2020

    A Correction to this paper has been published: https://doi.org/10.1038/s42255-020-00326-y.

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Acknowledgements

This work was funded by a UK Medical Research Council PhD studentship to J.L. and program grants MR/K0019494/1 (to D.A.M., J.M. and F.O.) and MR/R023026/1 (to D.A.M., J.M., L.A.B. and F.O.). An Arthritis Research UK grant (20812) supports F.O., D.A.M., J.L. and J.C.W. A CRUK program grant (C18342/A23390) supports J.L. and D.A.M. The cross-council Lifelong Health and Wellbeing initiative, funded by the MRC (L016354) funds D.A.M. and F.O. C.N. is supported by CRUK Beatson Institute Core funding (A171196). T.G.B. is funded by the Wellcome Trust (WT107492Z) and a CRUK/AECC/AIRC Accelerator Award (A26813). M.Y.W.Z. has a personal Ph.D award from Newton-Mosharafa fund. P.C., L.S. and L.-A.T. are financed by Methusalem funding, FWO, ERC Proof of Concept (ERC-713758) and an Advanced ERC Research Grant (EU-ERC743074). The IVIS spectrum was purchased under a Wellcome Trust Equipment Grant (087961) awarded to D.A.M. and others. We thank the Newcastle University bioimaging unit and the Newcastle University Flow cytometry core facility for technical assistance.

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Authors

Contributions

J.L. performed the majority of the laboratory-based work and analyses. S.L., A.K., C.B., G.C., L.A.B., J.C.W., A.L.C., R.A.B., L.M.G., R.C., S.M., M.T., B.S.B., X.X., M.Y.W.Z., W.J.R., H.L.P., C.N., T.B., C.B., M.G.M., L.S., L.-A.T., M.K., L.S., A.D.B. and C.B.N. performed a portion of the laboratory experiments and their related analyses. S.M.R., D.M.M., G.S., J.F., S.A.W., J.L.Z., U.K., R.F.S. and I.M. contributed materials and/or analysis tools. L.A.B., A.F., N.S., P.C., J.M. and D.A.M. provided advice and/or contributed to the experimental design and writing. J.L., D.A.M. and F.O. conceived the studies, designed the experiments and wrote the manuscript. All authors read and commented on the final manuscript.

Corresponding authors

Correspondence to Jack Leslie or Fiona Oakley.

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Competing interests

F.O., D.A.M., J.M. and L.A.B. are directors of Fibrofind. J.L., H.P., F.O., D.A.M., J.M. and L.A.B. are shareholders in Fibrofind. C.B.N. is shareholder in GSK. M.K. is a stock owner of Nordic Bioscience.

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Extended data

Extended Data Fig. 1 c-Rel correlates with disease progression in chronic kidney disease and is elevated in chronic lung disease. Investigation of the cell specific actions of c-Rel in preclinical models of liver injury.

a, Graph showing average percentage area of c-Rel stained tissue in (n=5) normal lung and (n=7) chronic obstructive pulmonary disorder (COPD) (P value = 0.0003). b, Graph showing average percentage area of c-Rel stained tissue in (n=5) normal human kidney and patients with stable (n=4, p value =0.0185) or progressive kidney disease (n=8, p value = 0.003). Normal vs progressive p value <0.0001) (c) Graphs showing the Mean Fluorescence Intensity (MFI) of GFP in hepatocytes (p value = 0.0026), Cholangiocytes (EPCAM+, p value = 0.0026) and non-parenchymal (EPCAM-) cells from the livers of Relfl/fl and RelΔAlb mice. Data are mean ± s.e.m of 3 mice/group. d, Heatmap showing gene expression of Cxcl1, Cxcl2, Ccl2, Cxcl9, Cxcl10, Il1b, Il6 and Tnfα in olive oil vehicle control and acute CCl4 injured Relfl/fl and RelΔAlb mice. Asterisk denotes significance between CCl4 injured Relfl/fl and RelΔAlb mice; there is no significant difference between olive oil treated groups. (e) Histological assessment of αSMA, PCNA and CD68 stained liver sections in acute CCl4 injured Relfl/fl and RelΔLrat mice. Data in graphs are mean ± s.e.m of n=5. a, c, e, P values were calculated using unpaired two-sided T test. b, P value was calculated using a one-way ANOVA with Tukey post-hoc t-test (* P <0.05, ** P <0.01, *** P <0.001 and **** P <0.0001).

Extended Data Fig. 2 c-Rel regulates epithelial dedifferentiation and fibrogenic gene expression.

a, Representative images of c-Rel staining in CCl4 injured Relflfl and RelΔHep mice. Representative of n=7 mice/group. Scale bar represents 100 μm. b, Graph shows mRNA levels of inflammatory cytokines Tnfa, Il-1b (p=0.00038) and Il6 (p value = 0.022)and the inflammatory chemokines Cxcl1 (p value = 0.034), Cxcl2 (p=0.054) and Ccl2 (p=0.029), in CCl4 injured livers of Relfl/fl and RelΔHep mice. Data are mean ± s.e.m of 7 mice/group. c, Heatmap showing relative mRNA expression of Il-6, Cxcl1, Cxcl2, Ccl2, Ccl4 and Ccl5 in WT and Rel−/− proximal tubular epithelial cells (PTEC) stimulated with or without TGFβ1. d, Graphs show relative levels of Ctgf (p value = 0.034), Ctsd (p= 0.039), Serpine1 (p=0.04) and Bmp1 (p=0.0054) protein expressed as Intensity in control and TGFβ1 treated WT and Rel-/- hepatocytes. Data are from 4 independent cell isolations/group. e, Quantification of soluble collagen (pg/ml) released from precision cut liver slices (PCLS) generated from WT and Rel-/- liver, stimulated ± TGFβ1 ± CTGF where (control p value = 0.012 and TGFβ1 p value = 0.045). Data are from PCLS generated 3 different donors/genotype. b, d, P values were calculated using unpaired two-sided t-test. (d) P values were calculated using the R package LIMMA (* P <0.05, ** P <0.01 and ***P<0.001).

Extended Data Fig. 3 c-Rel regulates metabolic enzymes to induce epithelial dedifferentiation and fibrogenic gene expression.

a, Seahorse analysis of basal (p<0.0001) and maximal (p<0.0001) glycolysis (extracellular acidification rate, ECAR) and basal (p<0.0007) and maximal (p<0.0004) mitochondrial respiration (oxygen consumption rate, OCR) in WT and Rel-/- hepatocytes stimulated ± TGFβ1. b, Graph shows mRNA expression of Pfkfb1 in WT and Rel-/- hepatocytes stimulated ± TGFβ1. c, Graph shows mRNA expression of Pfkfb3 in WT and Rel-/- hepatocytes stimulated ± IL-1β. (p value=0.025) (d) Schematic representation of RelA, NF-κB1 and NF-κB binding sites in the murine Pfkfb1 and Pfkfb3 promoters. e, ChIP analysis of c-Rel at the proximal and distal regions of the Pfkfb1 promoter and the proximal (p<0.0001) and distal (p=0.0185) regions of the Pfkfb3 promoter in WT hepatocytes treated ± TGFβ1. f, Representative images show PFKFB3 immunohistochemical staining in liver sections from acute CCl4 injured Pfkfb3flfl and Pfkfb3Δhep mice. Images are representative of n=5 mice/group. Scale bar is 100μm. g, Graphs show media lactate in control and TGFβ1 (p=0.0064) stimulated and glucose levels in control (p=0.0227) and TGFβ1 (p=0.00284) stimulated in hepatocytes isolated from Pfkfb3flfl and Pfkfb3Δhep mice and stimulated ± TGFβ1. h, Heatmap showing secreated Il-6, Cxcl1, Cxcl2, Cxcl10, Ccl2, Ccl3, Ccl4 and Ccl5, measured by MSD in the media of hepatocytes isolated from Pfkfb3flfl and Pfkfb3Δhep mice and stimulated ± TGFβ1. (i) Quantification of connective tissue growth factor (CTGF) in pg/ml in the culture media of hepatocytes isolated from Pfkfb3flfl and Pfkfb3Δhep mice and stimulated ± TGFβ1 (p=0.0027) (j) Heatmap showing secreated Il-6, Cxcl1, Cxcl2, Cxcl10, Ccl2, Ccl3, Ccl4 and Ccl5, measured by MSD in the media of WT hepatocytes stimulated ± TGFβ1 ± the Pfkfb3 inhibitor 3PO. k, Quantification of connective tissue growth factor (CTGF) in pg/ml in the culture media of WT hepatocytes stimulated ± TGFβ1 ± the Pfkfb3 inhibitor 3PO (p=0.0013). Data in graphs are mean ± s.e.m. in n=3 (g, i, k), n=4 (a, b, c) or n=5 (e) independent cell isolations/condition. All p values were calculated using a two-way ANOVA with Tukey post-hoc t-test (* P <0.05, ** P <0.01 and *** P <0.001).

Extended Data Fig. 4 c-Rel dependent Snail regulation in chronic liver and kidney disease.

a, Representative images show expression of Snail in normal and chronic CCl4 injured liver. b, Representative images show Snail in normal and UUO injured kidney. c, Representative images of Snail transcript in epithelial cells detected by RNAScope in fibrotic chronic CCl4 injured fibrotic mouse liver. d, Representative images of Snail transcript in epithelial cells detected by RNAScope in fibrotic UUO mouse kidney. All representative images are representative of n=5 mice/group. a, b, Scale bars equal 100 microns (c, d) Scale bars equal 50 microns. e, Graph showing mRNA levels of Snail in TGFβ1 treated hepatocytes (p=0.0001) and proximal tubular epithelial cells (PTEC) (p=0.02) isolated from WT and Rel-/- mice. Data in graphs are mean ± s.e.m. in n=3 independent cell isolations. f, Graph showing ChIP analysis of c-Rel binding to distal (p=0.0074) and proximal (p=0.0063) regions of the Snail promoter in WT hepatocytes stimulated ± TGFβ1. Data in graphs are mean ± s.e.m. in n=5 independent cell isolations. P values were calculated using unpaired two-sided t-test (e) and a ratio paired t-test (f) (* P <0.05, ** P <0.01 and *** P <0.001).

Extended Data Fig. 5 c-Rel regulates macrophages polarisation to drive tissue fibrosis.

a, Histological assessment and representative images of αSMA stained liver sections in acute CCl4 injured in (n=13) Relflfl, (n=7) RelΔAlb (p=0.0116), and (n=10) RelΔLysM (p=0.0051) mice. Data are mean ± s.e.m. Scale bar equals 100 microns. P values were calculated using a one-way ANOVA with Tukey post- hoc t-test. b, Representative low power immuno-fluorescence images show c-Rel (red), CD68 (green) and nuclear (blue) staining in diagnosed idiopathic pulmonary fibrosis lung sections. Yellow arrows denote co-localisaton of c-Rel and CD68. Scale bar equals 50 microns. Images are representative of n=8 IPF stained sections. (c) Heat map showing relative mRNA expression of fibrogenic markers; Tgfb1, Tgfb2, Tgfb3, TIMP1, Fn1, Pdgfa, Pdgfb, Mmp3, Mmp9 and Mmp13 in M1 and M2 polarised WT and Rel-/- BMDMs. d, Graphs show, seahorse analysis of basal (p<0.0001) and maximal (p=0.0004) glycolysis (extracellular acidification rate, ECAR) and basal (p=0.0002) and maximal (p=0.0024) mitochondrial respiration (oxygen consumption rate, OCR) in M1 and M2 polarised WT and Rel-/- BMDMs. Data are mean ± s.e.m. from 3 independent cell isolations/group. e, ChIP analysis of c-Rel binding to distal and proximal (p=0.0004) regions of the Pfkfb3 promoter and to distal and proximal (p=0.003) regions of the Pfkfb1 promoters in WT BMDM in response to M1 and M2 polarisation. Data are mean ± s.e.m. from 3 independent cell isolations/group. d-e, P values were calculated using two-way ANOVA with Tukey post-hoc t-test. Denoted significance refers to comparisons between WT and Rel-/- macrophages polarised to either an M1 or M2 phenotype. f, Volcano plots show differentially expressed proteins detected by proteomic analysis of the secretome of M0, M1 and M2 polarised WT and Rel-/- BMDMs. g, Graphs show relative levels of Galectin 1 (p=0.031), Galectin 3 (p=0.013), Vimentin (p=0.0128) and Matrix Metalloproteinase 12 (MMP12) (p<0.001) expressed as Intensity x108 in M0, M1 and M2 polarised WT and Rel-/- BMDMs. Data are mean ± s.e.m. from 4 independent cell isolations/group generated 4 different donors/genotype. P values were calculated using the R package LIMMA (* P <0.05, ** P <0.01 and *** P <0.001).

Extended Data Fig. 6 Validation of epithelial specific deletion of c-Rel in kidney and lung fibrosis models.

a, Flow cytometry gating strategy to identify immune cells (CD45+), epithelial cells (EPCAM+) and endothelial cells (CD31+) isolated from the kidney or lungs of Relflfl, RelΔTEC or RelΔAEC mice respectively. b-c, Ex vivo images of GFP fluorescence signal in the left and right kidneys, heart, liver, lungs and spleen of RelΔTEC mice (b) or RelΔAEC mice (c) imaged using an In Vivo Imaging System (IVIS). Graph and flow cytometry histograms show the Mean Fluorescence Intensity (MFI) of GFP in EpCAM+, CD31+, CD45+ and EpCAM-CD31-CD45- cells from the kidney of RelΔTEC and Relfl/fl control mice (b) or from the lung of RelΔAEC and Relfl/fl control mice (c), n=5 mice/group. d, Heatmap showing mRNA levels of Tgfb1, Col1a1, Col1a2, Acta2 and Timp1 in control versus UUO kidney of Relflfl, RelΔTEC and RelΔLysM mice (left) or control versus bleomycin lung of Relflfl, RelΔAEC and RelΔLysM mice (right). Heatmap data are from 2 mice/group in control kidney or lung and 5 mice/group in the injured kidney or lung. e, Quantification of hydroxyproline levels in nM per left lobe of lung tissue from bleomycin injured in Relflfl, RelΔAEC (p=0.0105), and RelΔLysM (p=0.0006) mice. f, Histological assessment and representative images of TUNEL stained lung sections in day 3 bleomycin injured in Relflfl, RelΔAEC, and RelΔLysM mice. Scale bar is 50 microns. Data are mean ± s.e.m. in n=5 mice/group. g, Quantification of neutrophil numbers in UUO injured kidneys of Relflfl, RelΔTEC (p=0.0014) and RelΔLysM (p<0.0001) mice and macrophage numbers in UUO injured kidneys of Relflfl, RelΔTEC (p=0.00045) and RelΔLysM (p=0.0004) mice. h, Quantification of neutrophil numbers in Bleomycin injured lungs of Relflfl, RelΔAEC and RelΔLysM mice and macrophage numbers in Bleomycin injured lungs of Relflfl, RelΔAEC (p=0.0018) and RelΔLysM (p=0.0327) mice. e, g, h, Data are mean ± s.e.m. in a minimum of 7 mice/group for the kidney and 10 mice/group for the lung. P values were calculated using one-way ANOVA with Tukey post-hoc t-test (* P <0.05, **P <0.01 and ***P <0.001).

Extended Data Fig. 7 Validation of single myeloid or dual hepatocyte- and myeloid- specific deletion of c-Rel in mice and analysis of fibrogenic gene expression in these mice during chronic liver injury.

a, Flow cytometry plot and graphs show the Mean Fluorescence Intensity (MFI) of GFP expression in hepatocytes, macrophages (CD45+F4/80+CD11b+) (p=0.011), T-cells (CD45+CD3+) and non-parenchymal cells (CD45-) from the liver of Relflfl versus RelΔLysM mice, n=3 mice/group. b, Flow cytometry plot and graphs show the Mean Fluorescence Intensity (MFI) of GFP expression in hepatocytes (p=0.007), macrophages (CD45+F4/80+CD11b+) (p=0.007), T-cells (CD45+CD3+) and non-parenchymal cells (CD45-) from the liver of Relflfl versus RelΔHep/ΔLysM mice, n=3 mice/group. (c) Heatmap showing mRNA levels of Tgfb1, Col1a1, Col1a2, Acta2 and Timp1 in the CCl4 injured liver of Relflfl, RelΔHep, RelΔLysM and RelΔHep/ΔLysM mice. P values were calculated using two-sided t-test (* P <0.05).

Extended Data Fig. 8 c-Rel signaling in hepatocyte and macrophages differentially regulate liver regeneration via regulation of cell cycle genes and mitogenic factors.

a, Histological assessment and representative images of PCNA positive hepatocytes in 48h partial hepatectomy injured Relflfl, RelΔAlb (p=0.0003) and RelΔLysM (p=0.0096) mice. Scale bar is 100 microns. b, Graph shows average liver/body weight ratio 48h post partial hepatectomy in Relflfl, RelΔAlb (p=0.0318) and RelΔLysM (p=0.0178) mice. c, Heat map showing relative hepatic mRNA expression of cell cycle genes; Ccna1, Ccnd1, Ccne1, Cdk2, Cdk4 and mitogenic proteins; Hgf and Egf at 48h post partial hepatectomy in Relflfl, RelΔAlb and RelΔLysM mice. Data are mean ± s.e.m. in n=5 mice/group. P values were calculated using one-way ANOVA with Tukey post-hoc t-test (* P <0.05 and ** P <0.01).

Extended Data Fig. 9 IT-603 attenuates fibrogenesis in murine models of fibrosis.

a, U937 cells stably expressing 3xNF-κB-Luc reporter were transiently transfected with RelA or c-Rel expression plasmids. Graph shows RelA and c-Rel induced NF-κB luciferase reporter activity ± IT-603 therapy. Data are mean ± s.e.m. P value = 0.00039. P value was calculated using an unpaired two-sided t-test (***P<0.001) of 3 independent experiments. b, c, Histological quantification of αSMA stained kidney (p=0.0004) or lungs (p=0.009) following their respective injury. Data are mean ± s.e.m. in 7 and 10 mice/group for kidney and lung respectively. P values were calculated using an unpaired two-sided t-test. d, Histological quantification of αSMA stained chronic MCD diet injured livers at 2 weeks (pre-treatment) and 5 weeks ± therapeutic administration of IT-603 (p=0.0044). e, Graphs showing relative hepatic expression of the fibrogenic genes; Tgfb1 (p=0.039), Acta2 (p=0.028), Col1a1 (p=0.036), Col1a2, and Timp1(p=0.015) in 2-week (pre-treatment) and 5-week methionine choline deficient diet (MCD) fed mice ± therapeutic administration of IT-603. Data are mean ± s.e.m. in n=4 control mice 8 vehicle and n=7 IT-603 treated mice/group. f, Histological quantification of αSMA stained chronic CCl4 injured livers at 3 weeks (pre-treatment) or 8 weeks ± therapeutic administration of IT-603 (p=0.0105). g, Graphs showing relative hepatic expression of the fibrogenic genes; Tgfb1 (p=0.029), Acta2 (p=0.032), Col1a1 (p=0.04), Col1a2 (p=0.016), and Timp1 (p=0.09) in chronic CCl4 injured livers at 3 week (pre-treatment), 8 week and ± therapeutic administration of IT-603 (from weeks 3-8). Data are mean ± s.e.m. in n=4 control mice 7 experimental mice/group. c, e, P values were calculated using one-way ANOVA with Tukey post-hoc t-test. d, f, P values were calculated using two-way ANOVA with Tukey post-hoc t-test (*P<0.05, ***P<0.001).

Extended Data Fig. 10 IT-603 attenuates fibrosis in ex vivo human tissues slice models of liver and kidney fibrosis.

a, Diagrams show the experimental timelines of TGFβ1 induced fibrosis in ex vivo normal human liver and kidney precision cut slices (PCS). b, Representative images of CD68 stained liver and kidney tissue slices. c, Representative images and histological quantification of Picrosirius red stained fibrotic kidney slices ± IT-603 therapy (p<0.0001). Representative images and histological quantification of αSMA stained fibrotic kidney slices ± IT-603 therapy (p<0.0001). Red line denotes the value for the T=0 slice. e, Quantification of soluble collagen released from fibrotic kidney slices ± IT-603 therapy (p=0.0015). f, Quantification of the neo-epitope pro C3 released from fibrotic kidney slices ± IT-603 therapy (p=0.0493). g, Graph showing average LDH release in the media expressed as a percentage (%) of positive control (LDH levels in media from a PCS where maximal death was induced by multiple freeze/thaws – normalized to media volume). (IT-603 p=0.023 and IT-603+TGFβ1 p=0.02). Images representative of n=3 independent slice experiments. Data are mean ± s.e.m. and representative of slices generated from 3 independent donors performed in duplicate. Scale bars equal 100 microns. P values were calculated using two-way ANOVA with Tukey post- hoc t-test (*P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001).

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Leslie, J., Macia, M.G., Luli, S. et al. c-Rel orchestrates energy-dependent epithelial and macrophage reprogramming in fibrosis. Nat Metab 2, 1350–1367 (2020). https://doi.org/10.1038/s42255-020-00306-2

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