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Chronic inflammation imposes aberrant cell fate in regenerating epithelia through mechanotransduction

Abstract

Chronic inflammation is associated with a variety of pathological conditions in epithelial tissues, including cancer, metaplasia and aberrant wound healing. In relation to this, a significant body of evidence suggests that aberration of epithelial stem and progenitor cell function is a contributing factor in inflammation-related disease, although the underlying cellular and molecular mechanisms remain to be fully elucidated. In this study, we have delineated the effect of chronic inflammation on epithelial stem/progenitor cells using the corneal epithelium as a model tissue. Using a combination of mouse genetics, pharmacological approaches and in vitro assays, we demonstrate that chronic inflammation elicits aberrant mechanotransduction in the regenerating corneal epithelium. As a consequence, a YAP–TAZ/β-catenin cascade is triggered, resulting in the induction of epidermal differentiation on the ocular surface. Collectively, the results of this study demonstrate that chronic inflammation and mechanotransduction are linked and act to elicit pathological responses in regenerating epithelia.

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Figure 1: Corneal squamous cell metaplasia (CSCM) in Notch1Δ mice is associated with an augmented and chronic inflammatory response.
Figure 2: Chronic inflammation is necessary and sufficient to induce CSCM.
Figure 3: CSCM is induced in limbal and peripheral cells during repair.
Figure 4: Chronic inflammation promotes CSCM through elevated β-catenin signalling.
Figure 5: Increased ECM deposition in the corneal stroma in response to aberrant inflammation.
Figure 6: Activation of mechanotransduction in the corneal epithelium in response to aberrant inflammation.
Figure 7: CSCM is associated with increased tissue stiffness and mechanical stimuli.
Figure 8: Manipulation of mechanotransduction affects corneal cell fate.

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Acknowledgements

This work was supported in part by OptiStem, the Swiss National Science Foundation, the Swiss Cancer League, the Marie Curie Foundation and EuroSystem. P.D.O. and G.E.F. acknowledge financial support from the Swiss National Science Foundation under award number 205321_134786 and 205320_152675 and European Union FP7/2007-2013 ERC under Grant Agreement No. 307338-NaMic. Work performed in the laboratory of S.P. was supported by grants from AIRC (5x1000 and PI) and the ERC. We thank R. Kemler (Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany) for providing the conditional β-catenin mice, M. Taketo (Centre for Frontier Medicine, Kyoto University, Japan) for the Ctnnb1ΔEx3 mice and P. Chambon and D. Metzger (Institute of Genetics and Molecular and Cellular Biology, Strasbourg, France) for the K5CreERT and K14CreERT2 mice. We thank J. Huelsken (Swiss Institute for Experimental Cancer Research, EPFL, Lausanne, Switzerland) for providing the TCF–luciferase reporter cells and the anti-periostin antibody. We would like to thank A. Radenovic, J. Artacho, J. Sordet-Dessimoz and M. Garcia for technical assistance with microscopy, histology and flow cytometry. We would like to thank G. Ferrand for guidance and advice concerning animal experiments.

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Authors and Affiliations

Authors

Contributions

C.S.N. designed and performed experiments, analysed data and wrote the manuscript. P.D.O. performed nanomechanical measurement experiments and analysed data. L.A. performed experiments and analysed data. S.H. performed experiments and analysed data. E.F.W., G.E.F., M.P.L., Y.B. and S.P. analysed data and provided conceptual and experimental guidance throughout the study. F.R. conceived the study and analysed data.

Corresponding authors

Correspondence to Craig S. Nowell or Freddy Radtke.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Inflammation in Notch1Δ mice following corneal injury.

(a) Immunofluorescent staining for K12, K1 and CD45 in unwounded WT (Notch1lox/lox) and Notch1Δ corneas. Data are representative of 6 corneas over three independent experiments. (b) Flow cytometric analysis showing the proportion of total CD45+ cells (upper plots) and CD45+CD11b+Gr1+ cells (lower plots) in WT (Notch1lox/lox) and Notch1Δ cornea 21 days after the repeated injury procedure (see Fig. 1a). Data are representative of 6 individual analyses performed on cells pooled from 4 corneas of each genotype over three independent experiments. (c) Flow cytometric analysis showing proportion of total CD45+ cells (upper plots) and CD45+CD11b+Gr1+ cells (lower plots) in WT (Notch1lox/lox) and Notch1Δ cornea 24 h after a single injury. Data are representative of 6 individual analyses performed on cells pooled from 4 corneas of each genotype over three independent experiments. Scale bars represent 500 μm.

Supplementary Figure 2 Chronic inflammation is associated with CSCM in Notch1Δ mice.

(a) Immunohistochemistry for pS73cjun following repeated corneal injury. (b) QRT-PCR analysis for the indicated cytokines in WT (Notch1lox/lox:cjunlox/lox), Notch1Δ and Notch1Δ:cjunΔ corneal epithelial cells 24 h after a single corneal injury (n = 6 biological replicates for each genotype over three independent experiments. Each replicate consists of corneal epithelial tissue pooled from 6 corneas isolated from 3 mice of each genotype). Data are expressed relative to the expression in WT unwounded corneal epithelial cells. (c,d) Quantification of the proportion of CD45+ cells in WT (Notch1lox/lox:cjunlox/lox), Notch1Δ and Notch1Δ:cjunΔ cornea 21 days after repeated corneal injury (c) or 24 h after a single corneal injury (d). Proportions were measured by performing flow cytometry on dissociated corneas. (n = 6 biological replicates for each genotype over three independent experiments. Each replicate consists of cells pooled from 4 corneas isolated from 2 mice of each genotype). (e) QRT-PCR analysis of Notch1 in the corneal epithelium of WT (Notch1lox/lox:cjunlox/lox), Notch1Δ:cjunΔ and Notch1Δ mice following repeated injury (n = 6 biological replicates for each genotype over three independent experiments. Each replicate consists of corneal epithelial tissue pooled from 6 corneas isolated from 3 mice of each genotype). (f) Representative immunofluorescent staining for K12, K1 and CD45 in Notch1Δ:cjunΔ and Notch1Δ corneas following repeated corneal injury. Large panels are low magnification tiled images. White outlined insets show high magnification images of the indicated regions. (g) Proportion of WT (Notch1lox/lox:cjunlox/lox) (n = 10 corneas), Notch1Δ (n = 20 corneas) and Notch1Δ:cjunΔ (n = 16 corneas) corneal tissue exhibiting corneal or epidermal identity after repeated corneal injury. (h) Immunfluorescent staining for K12, K1, cjun (pS73) and CD45 on corneal tissue isolated from WT (Notch1lox/lox:cjunlox/lox), Notch1Δ:cjunΔ and Notch1Δ mice after repeated corneal injury. Data are representative of 16 corneas. (i) QRT-PCR analysis of Notch1 in the corneal epithelium of WT (Notch1lox/lox) and Notch1Δ mice treated with ophthalmic lubricating gel or Tobradex following repeated injury (n = 6 biological replicates for each treatment over three independent experiments. Each replicate consists of corneal epithelial tissue isolated during corneal wounding of a single eye). Scale bars represent 500 μm on tiled images and 5 μm on all other images. St – Stroma. P < 0.01, P < 0.05 (unpaired, two tailed t-tests). Error bars represent standard deviation.

Supplementary Figure 3 Spatial and temporal kinetics of CSCM.

(a) Representative immunofluorescent staining for K12 and K1 on wholemount corneal epithelial tissue isolated from WT and Notch1Δ corneas. Upper panels show unwounded corneas, middle panels show corneal epithelial tissue 24 h after the first injury, lower panels show corneal epithelial tissue 24 h after the second injury. Data are representative of 6 corneal wholemounts over three independent experiments. (b) XY scatter plots showing K12 and K1 expression in limbus, peripheral cornea and central cornea in unwounded corneas and in corneas isolated 24 h after the second corneal injury. Each data point represents mean fluorescence intensity measured from an individual cornea. Grey boxes = WT (Notch1lox/lox), black boxes = Notch1Δ (n = 6 corneas for each genotype over three independent experiments). (c) Immunofluorescent staining for Ki67 on WT and Notch1Δ corneas. Data are representative of 6 corneas. Upper panels show unwounded corneas, middle panels show corneal tissue 24 h after the first injury, lower panels show corneal tissue 24 h after the second injury. Large panels are low magnification tiled images. Insets outlined in green, red and yellow show high magnification images of the limbus, peripheral cornea and central cornea respectively. (d) Quantification of the proportion of Ki67+ cells in the limbus, peripheral cornea and central cornea in unwounded corneas (upper panels), corneas 24 h after the first injury (middle panels) and corneas 24 h after the second injury (lower panels). Black boxes = WT (Notch1lox/lox), grey boxes = Notch1Δ (n = 6 corneas for each genotype over three independent experiments). Scale bars represent 500 μm on tiled images and 5 μm on all other images. St – Stroma, Limb – limbus, Per – Periphery, Cen – Centre. Error bars represent standard deviation.

Supplementary Figure 4 Elevated β-catenin is necessary and sufficient to induce CSCM.

(ab) Quantification of relative β-catenin expression in limbus, peripheral cornea and central cornea in unwounded eyes (a) and 24 h after the first injury (b). Black bars = WT (Notch1lox/lox), grey bars = Notch1Δ (n = 6 corneas for each genotype over three independent experiments). Values for expression levels are relative values normalised to the expression level in the conjunctiva of each sample, determined by mean fluorescence intensity. (c) Immunofluorescent staining for β-catenin, K12 and K1 on Notch1Δ and Notch1Δ:Ctnnb1Δ corneas after repeated injury. Data are representative of 8 corneas for each genotype over 3 independent experiments. (d) Immunofluorescent staining for β-catenin, K12 and K1 on WT (Ctnnb1lox(Ex3)/lox(Ex3)) and Ctnnb1ΔEx3 corneas 7 days after a single injury. Data are representative of 8 WT corneas and 10 Ctnnb1ΔEx3 corneas over 4 independent experiments. (e) Immunofluorescent staining for K12 and K1 on wholemount corneal epithelial tissue isolated from or WT (Ctnnb1lox(ex3)/lox(ex3)) or Ctnnb1ΔEx3 corneas 7 days after a single corneal injury. Data are representative of 6 WT and 8 Ctnnb1ΔEx3 corneas over 3 independent experiments. Scale bars represent 500 μm on tiled wholemount images and 5 μm on all other images. Error bars represent standard deviation.

Supplementary Figure 5 Inflammation induced CSCM is associated with increased ECM deposition in the corneal stroma.

(a) QRT-PCR analysis of Wnt ligands that exhibit expression in corneal tissue after repeated injury. Analysis was performed on whole corneal tissue isolated from WT (Notch1lox/lox) and Notch1Δ mice 21 days after the 3rd corneal injury (n = 5 biological replicates for each genotype over four independent experiments. Each replicate consists of corneal epithelial tissue pooled from 4 corneas isolated from 2 mice of each genotype). Data are expressed relative to the expression in whole corneal tissue isolated from unwounded mice. Error bars represent standard deviation. (b) Immunofluorescence for Periostin, CD45 and DAPI on chronically inflamed Notch1Δ corneal tissue after repeated injury. Single fluorescent images and merges are shown as indicated. Data are representative of 4 corneas analysed over two independent experiments. Scale bars represent 5 μm. (c) Immunofluorescent staining for K14 and Tenascin C (upper panels) or K14 and Periostin (lower panels) in Notch1Δ mice treated with either ophthalmic gel (control) or Tobradex during the repeated injury procedure. Data are representative of 6 corneas analysed over three independent experiments. Scale bars represent 500 μm.

Supplementary Figure 6 CSCM is associated with mechanotransduction.

(a,b) Quantification of FAK phosphorylation (upper panels) and nuclear:cytoplasmic ratio of ROCK2 (middle panels) and YAP/TAZ (lower panels) in the limbus, peripheral cornea and central cornea isolated from unwounded eyes (a) or eyes isolated 24 h after the first corneal injury (b). Values for expression levels are relative values normalised to the expression level in the conjunctiva of each sample, determined by mean fluorescence intensity. Black bars = WT (Notch1lox/lox), grey bars = Notch1Δ (n = 6 corneas for each genotype over three independent experiments). Error bars represent standard deviation. (c) Immunofluorescence for pFAK, ROCK2 and YAP/TAZ on corneal tissue isolated from Notch1Δ mice treated with ophthalmic gel (control) or Tobradex during the repeated injury procedure. Data are representative of 6 corneas for each treatment analysed over three independent experiments. Scale bars represent 5 μm.

Supplementary Figure 7 Manipulation of mechanotransduction affects β-catenin expression in corneal epithelial cells.

(a,b) Immunofluorescence for β-catenin, K12 and K1 (a) or YAP/TAZ, K12 and K1 (b) on PCESCs cultured on stiff (glass– >2 GPa) substrates and maintained in vehicle (PBS—upper panels) or 10 μM Y27632 (lower panels). Data are representative of 8 individual cultures for each condition. (ce) Quantification of β-catenin expression (c), YAP/TAZ nuclear:cytoplasmic ratio (d) and the proportion of K12+K1 and K12K1+ cells (e) in PCESCs maintained in vehicle (PBS) or 10 μM Y27632 (n = 8 individual cultures over four independent experiments). For (c) β-catenin expression is determined by mean fluorescence intensity. (f) Schematic depicting the transfection of TCF-Luc 293 T cells with pR2KD or pEGFP. (g) Western blot performed on TCF-Luc 293T cells 48 h after transfection with pR2KD or pEGFP. (h) Relative luciferase activity in TCF-Luc 293 T cells 48 hours after transfection (n = 6 individual transfections over three independent experiments). P < 0.01, P < 0.05, (unpaired, two tailed t-tests). Error bars represent standard deviation. Scale bars represent 20 μm.

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Nowell, C., Odermatt, P., Azzolin, L. et al. Chronic inflammation imposes aberrant cell fate in regenerating epithelia through mechanotransduction. Nat Cell Biol 18, 168–180 (2016). https://doi.org/10.1038/ncb3290

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