Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: Regeneration of epithelium was assessed by BrdU staining. (a) Representative image at 10X (scale bar of 50 µm) of Cdh1 fl/fl mice instilled with adeno-Cre recombinase (Ad5CMVCre-eGFP) to knockdown E-cadherin for 3 months shows decreases in BrdU as compared to Cdh1 fl/fl instilled with adeno-Ctrl (Ad-5CMVeGFP). Decreases in the fluorescence intensity of b E-cadherin and c BrdU in Cdh1 fl/fl mice instilled with adeno-Cre as compared to Cdh1 fl/fl mice instilled with adeno-Ctrl. Data is generated from eight mice. To knock down E-cadherin in the AT2 cells of mice lungs, Cdh1 fl/fl -Spc Cre mice were fed tamoxifen (TAM) for 30 days. These were compared to Cdh1 fl/fl -Spc Cre mice receiving a normal chow diet (ND). In vivo knockdown of E-cadherin in Cdh1 fl/fl -Spc Cre mice show decreases in BrdU expression as observed in the d representative images at 10X (scale bar of 25 µm), with decreases in the intensity of e E-cadherin and f BrdU in AT2 cells. Data is generated from five mice. Undifferentiated normal basal epithelial cells were transfected with Ad-GFP-U6-h-CDH1-shRNA to knock down E-cadherin, and undifferentiated COPD cells were transfected with Ad-GFP-U6-h-CDH1 to overexpress E-cadherin. We compared to respective undifferentiated Normal/COPD with Ad-GFP. g Representative image at 40× (scale bar of 25 µm) and h quantification showing decreased BrdU intensity in the normal epithelium with E-cadherin knockdown (Normal + shCDH1) and COPD at baseline (COPD + GFP) as compared to Normal + GFP. Also, COPD with overexpressed E-cadherin (COPD + CDH1) demonstrates increased BrdU intensity compared to COPD + GFP. Data are expressed as median bars and generated from three inserts from two donors.

Article Snippet: Primary non-diseased human bronchial epithelial (normal) and COPD human bronchial epithelial (COPD) cells were purchased from MatTek Life Sciences (Ashland, MA, USA) or Lonza (Basel, Switzerland) were expanded on collagen-coated T 75 flask and differentiated into pseudostratified epithelium at the air-liquid interface (ALI) on Transwells with spolyester membranes with 0.4 μm pores as described by us before , , .

Techniques: BrdU Staining, Knockdown, Fluorescence, Generated, In Vivo, Expressing, Transfection, shRNA

Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: Immunofluorescence at 40× (scale bar of 50 µm) of COPD human bronchial epithelial cells differentiated at week one to three of air–liquid interface (ALI) show decreased expression of E-cadherin and β-tubulin (ciliated cells marker) expression, increase expression of MUC5AC (goblet cell marker), without any changes in Cytokeratin 14 (Basal cell marker), as compared to non-diseased human bronchial epithelial (normal) cells.

Article Snippet: Primary non-diseased human bronchial epithelial (normal) and COPD human bronchial epithelial (COPD) cells were purchased from MatTek Life Sciences (Ashland, MA, USA) or Lonza (Basel, Switzerland) were expanded on collagen-coated T 75 flask and differentiated into pseudostratified epithelium at the air-liquid interface (ALI) on Transwells with spolyester membranes with 0.4 μm pores as described by us before , , .

Techniques: Immunofluorescence, Expressing, Marker

Journal: Communications Biology

Article Title: Loss of E-cadherin is causal to pathologic changes in chronic lung disease

doi: 10.1038/s42003-022-04150-w

Figure Lengend Snippet: COPD cells at 4 to 6 weeks ALI were transfected Ad-GFP-U6-h-CDH1 (CDH1) to overexpress E-cadherin or Ad-GFP (GFP) as control at 2 × 10 9 pfu. COPD cells transfected with Ad-CDH1 result in a increased epithelial resistance, b reduced cellular velocity of COPD cells, and c increased mRNA expression of CDH1 . Overexpression of E-cadherin in COPD cells d – g increased mRNA expression of claudins— CLDN1 , CLDN3 , CLDN7 , and CLDN8 , h decreased mRNA expression of CLDN10, i increased mRNA expression of occludin (OCLN), j increased mRNA expression of tight junction protein 1 (TJP1), and k TJP2 was not altered. Data is expressed as median bars and generated from 4 to 12 transwells per condition from two donors. To induce the over-expression of E-cadherin in Cdh1 knock-in mice, mice tracheal epithelial cells (mTECs) were transfected with adeno-Cre (Ad5CMVCre-eGFP) at 2 × 10 9 pfu and were exposed to CS for 10 days. Overexpression of E-cadherin in mTECs protects against CS-induced epithelial functional phenotypes by l decreasing monolayer permeability, m decreasing the cellular velocity, and n protecting Cdh1 mRNA downregulation due to CS exposure. Data for l – m involves three to six transwells per condition. COPD cells treated with CDDO-Me restore epithelial function by o improving the epithelial resistance, p decreasing the cellular velocity, and q increasing the CDH1 mRNA expression of COPD cells as compared to age and gender-matched non-diseased epithelium (normal controls). Similarly, cigarette smoke (CS) exposed to healthy normal cells treated with CDDO-Me r restores epithelial resistance, s decreases cellular velocity, and t protects against CDH1 mRNA downregulation due to CS exposure. (Data for o – t is generated from 3 to 6 transwells from 2 donors). For the panels the same data for normal are used for a – k , and o – t in the figure. Data are expressed as median bars. Kruskal-Wallis test, followed by Dunn’s multiple comparison test was performed . P < 0.05 were considered statistically significant.

Article Snippet: Primary non-diseased human bronchial epithelial (normal) and COPD human bronchial epithelial (COPD) cells were purchased from MatTek Life Sciences (Ashland, MA, USA) or Lonza (Basel, Switzerland) were expanded on collagen-coated T 75 flask and differentiated into pseudostratified epithelium at the air-liquid interface (ALI) on Transwells with spolyester membranes with 0.4 μm pores as described by us before , , .

Techniques: Transfection, Control, Expressing, Over Expression, Generated, Knock-In, Functional Assay, Permeability, Comparison

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: COPD epithelia demonstrate cellular plasticity with worsened monolayer barrier integrity, lower monolayer height and increased expression of mesenchymal markers. (A) FITC–dextran permeability is greater in gender- and age-matched COPD epithelial samples (CHBE) than NHBE. (B) Transepithelial resistance (TEER) is lower in gender and age-matched CHBE than NHBE. (C) Immunofluorescence staining of apical polarity (PKC-ζ, green) and basal polarity (Na + /K + -ATPase, red) shows preservation of polarity in both non-diseased epithelia (NHBE) and CHBE, although the CHBE has a much shorter monolayer height. Images representative of three donors per group (two inserts per donor). (D) The height of pseudostratified epithelium of the NHBE is greater than that of CHBE. (E­–L) There is decreased mRNA expression of epithelial (CDH1) in CHBE compared to age- and gender-matched NHBE (E), and an increase in mRNA expression of mesenchymal markers (CDH2, VIM, SNAl1, SNAl2, TWIST2, ZEB1 and ZEB2) relative to GAPDH in CHBE as analyzed by qPCR (F–L). Data is generated from three donors per group (age- and gender-matched and one to three inserts per donor). Data is expressed with median bars. Statistics determined by Mann–Whitney test, with P <0.05 considered statistically significant.

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Expressing, Permeability, Immunofluorescence, Staining, Preserving, Generated, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: Epithelial plasticity is associated with increased cell velocity. (A) Representative heat maps of cell velocity of NHBE exposed to air or cigarette smoke (CS) and CHBE exposed to clean air for 10 days, showing increase in velocity in injured and diseased cells. (B–D) CHBE typically have increased mean cell velocity (B) with CHBE having an altered distribution of spatial autocorrelation function (C) and have a higher correlation length (D) as compared to age- and gender-matched NHBE epithelia. (E–G) CS-exposed NHBE have a higher mean cell velocity (E) with an altered distribution of spatial autocorrelation function (F) and increases in the correlation length (G) in air-exposed NHBE. (H–J) Cells undergoing pEMT induced by treatment with EMT inducer supplement (EMT supp) show increased mean cell velocity (H) with an altered distribution of the spatial autocorrelation function (I) and increased correlation length (J) compared to age- and gender-matched NHBE treated with PBS Ctrl. (K) Representative images of three donors (three inserts per donor) used for cell shape factor quantification in NHBE exposed to air or CS and CHBE. (L) Comparison of quantified cell shape among NHBE exposed to air or CS and CHBE demonstrated an increased cell shape factor in CS-exposed and CHBE cells. Data is generated from three donors (three inserts per donor) and the mean±s.d. is given underneath in L. Graphs are generated with median bars for cell velocity and correlation length. Statistics determined by Mann–Whitney test (B,D,E,G,H,J) or Kruskal–Wallis test followed by Dunn's multiple comparison test (L), with P <0.05 considered statistically significant.

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Generated, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: Both cigarette smoke exposed non-diseased and COPD-derived epithelia have an increased fraction of polymerized actin. (A) Representative western blot of globular (G) actin and filamentous (F) actin (top panel) and the quantification of polymerized actin (bottom panel) demonstrates that there is a higher polymerized fraction of actin in CS-exposed cells and in cells treated with the polymerizing agent (JaspA), while treatment with actin depolymerizing agent (LatA) reduces the fraction of polymerized actin. Data is representative of three inserts from one donor. (B) Representative western blot of G and F actin (top panel) and quantification of polymerized actin (bottom panel) in age and gender matched cells demonstrate an increase in polymerized fraction of actin in CHBE. Data is representative of three donors (one to two inserts per donor). (C,D) Representative image of immunofluorescence demonstrates increased fluorescence of phalloidin in (C) NHBE exposed to CS and (D) CHBE compared to their respective controls. Data is representative of two donors (two inserts per donor) with images taken under identical exposures. Scale bars: 25 µm. (E) Cells undergoing pEMT do not have an increased fraction of polymerized actin, as shown by the representative western blot of G-actin and F-actin (top panel) and quantification of polymerized actin (bottom panel) of in NHBE treated with PBS Ctrl and EMT Supp (2× EMT inducer supplement). Data is representative of four inserts from one donor. (F) A decrease in phalloidin is seen in sections of cells undergoing pEMT (representative image of immunofluorescence staining of phalloidin in NHBE treated with PBS Ctrl and EMT Supp; images taken under identical exposures). Data is representative of two donors (two inserts per donor). Scale bars: 25 µm. Polymerized actin is calculated as the summation of F-actin to total actin (G-actin+F-actin). Statistics determined by Mann–Whitney test (B,E) or Kruskal–Wallis test followed by Dunn's multiple comparison test (A), with P <0.05 considered statistically significant.

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Derivative Assay, Western Blot, Immunofluorescence, Fluorescence, Staining, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: Inhibitors of actin polymerization improve monolayer integrity and reverse plasticity. (A–C) LatA (an actin polymerization inhibitor) and U0126 (a MAPK inhibitor) are the only drugs which reduced cellular velocity (A), TEER (B) and correlation length (C) on the CS-exposed epithelia [cells treated with pathway modulators such as LatA, the actin-polymerizing agent JaspA, U0126, the TGFβ1 neutralizer (1D11), or an antagonist of Wnt signaling pathway (DKK1) and compared to vehicle control]. Data is representative of two donors (three to six inserts per donor). (D) None of the inhibitors restored the CS-induced decrease in basal mRNA expression of the epithelial marker CDH1 . (E–J) The levels of the mesenchymal markers ( CDH2 , VIM , SNAI1 , SNAI2 , ZEB1 and ZEB2 ) were significantly reduced relative to GAPDH in CS-exposed epithelia treated with several pathway modulators (LatA, U0126, 1D11 and DKK1) as analyzed by qPCR. (K) LatA and U0126 treatment reduced mean square displacement (MSD) in CS exposed epithelia. Data is representative of three to six inserts from two donors. (L–O) LatA and U0126 treatment improved TEER in CHBE cells (L), LatA and U0126 treatment decreased cellular velocity in COPD epithelia (CHBE) (M), LatA and U0126 treatment reduced correlation length in CHBE cells (N), and LatA and U0126 treatment improved the height of pseudostratified epithelia on COPD epithelia (CHBE) (O). (P) Representative DIC image from three inserts from one donor taken with a 40× oil objective of CHBE treated with or without LatA or U0126 showing improved monolayer height. Scale bars: 25 µm. (Q) LatA and U0126 treatment reduced the MSD with time in CHBE cells approaching levels similar to that of NHBE. Of note, there is not a significant increase in MSD with time in pEMT-induced NHBE. Data is representative of nine inserts, except for cellular velocity and correlation length (six inserts) and height of pseudostratified epithelia (three inserts) from one donor of CHBE. Graphs are shown with median bars. Statistics determined by Kruskal–Wallis test followed by Dunn's multiple comparison test, with P <0.05 considered statistically significant.

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Expressing, Marker

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: Restoring cofilin-1 improves airway epithelial integrity. (A) Age- and gender-matched CHBE cells have lower cofilin-1 expression (representative blot, left panel and quantification, right panel) as detected by western blotting. GAPDH levels are shown as a loading control (data is representative of four donors, one to two inserts per donor). (B) There is reduced cofilin-1 expression as seen by immunofluorescence. Representative image from four donors (two inserts per donor) of cofilin-1 (red) and DAPI (blue) in NHBE, CS-exposed NHBE and CHBE epithelia. Taken with a 40× oil objective under identical exposures. Scale bars: 25 µm. (C) Despite having lower protein expression, CHBE express higher mRNA levels of CFL1 (encodes for cofilin-1) as analyzed by qPCR. Data is representative of three to four donors (three inserts per donor). (D) CS-exposure reduces cofilin-1 expression (representative blot, left panel and quantification, right panel) as detected by western blotting and GAPDH levels are shown as a loading control. Data is representative of three inserts from one donor. (E–G) mRNA expression of several actin-binding proteins were altered, with increases in ARPC2 , ARPC3 and PFN1 as analyzed by qPCR. Data is representative of three to four donors (three inserts per donor). (H) Cells treated with EMT Supp to induce pEMT do not have decreased cofilin-1 expression (representative blots, left panel and quantification, right panel) as detected by western blotting. GAPDH levels are shown as a loading control (data is representative of four inserts from one donor). (I–U) NHBEs were transduced with adenovirus to knockdown or overexpress cofilin-1 [Ad-GFP (GFP), Ad-GFP-U6-h-CFL1-shRNA (KD) and Ad-GFP-h-CFL1 (OE) at 1×10 10 PFU/ml]. (I) Transfection of GFP-tagged control virus, CFL1 shRNA virus, and CFL1 overexpression virus had an efficiency of ∼40–60%, as visualized by GFP fluorescence. Taken with a 20× objective. Scale bars: 100 µm. (J,K) Representative blot (left panel) and quantification (right panel) of cofilin-1 normalized to GAPDH as loading control as detected by western blotting (J), and mRNA expression of CFL1 (encodes for cofilin-1) indicating ∼50% knockdown and ∼2-fold overexpression (K). Data is representative of two to three inserts from two donors for western blotting and three inserts from one donor for qPCR. (L) Representative blot of NHBE transduced with GFP-tagged control virus (GFP), and CFL-1 shRNA virus GFP (KD) indicating that cofilin-1 manipulation did not alter E-cadherin levels. Data is representative of three inserts from one donor. (M,N) Both knockdown and overexpression of cofilin-1 increased FITC–dextran permeability (M), and knockdown increased cellular velocity, while overexpression trended towards an increase without reaching statistical significance (N). Data is generated from four inserts from one donor. (O) Cofilin-1 knockdown increased the fraction of polymerized actin (representative blot, top panel and quantification, bottom panel) of cofilin-1 expression from air and CS-exposed NHBE transduced with adenovirus as indicated, as detected by western blotting. GAPDH levels are shown as a loading control (data is representative of three inserts from one donor). (P) Cofilin-1 overexpression was sustained despite CS exposure (representative western blot, left panel and quantification, right panel) in NHBE transduced with adenovirus as indicated. Data is representative of four inserts from one donor. (Q,R) Cofilin-1 overexpression protected the monolayer from the CS-induced increase in (Q) FITC–dextran permeability, and (R) cellular velocity. Data is representative of three to four inserts from one donor. (S) Cofilin-1 over-expression protected from the CS-induced increase in MSD with time. (T) Representative images used for cell shape factor quantification in NHBE with knockdown or overexpression of cofilin-1 and in CS-exposed NHBE transduced with cofilin-1 overexpression. (U) NHBE with knockdown or overexpression of cofilin-1 demonstrated an increase in cell shape factor as compared to NHBE, whereas CS-exposed NHBE transduced with cofilin-1 overexpressing virus did not show statistical significant differences. The cell shape factor values were compared to NHBE values from Fig. 4L . Data is generated from three to four inserts from one donor. Graphs are generated with median bars. Statistics determined by Mann–Whitney test (A,C–H) or Kruskal–Wallis test followed by Dunn's multiple comparison test (J,K,M–R,U), with P <0.05 considered statistically significant.

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Expressing, Western Blot, Immunofluorescence, Binding Assay, Transduction, shRNA, Transfection, Over Expression, Fluorescence, Permeability, Generated, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin

doi: 10.1242/jcs.258513

Figure Lengend Snippet: CS-exposed epithelia and COPD epithelia have cellular energetics that are distinct from pEMT. The COPD epithelia (CHBE) and CS-exposed NHBE were treated with LatA or U0126. The NHBE were treated with 2× EMT inducer supplement serve as a model for pEMT. (A) Ensembled average one-dimensional spatial energy spectra [E(κ), κ being wavenumber] of the horizontal velocity component is greatest for CS-exposed epithelium and CHBE, both which display classic slopes characteristic of Kolmogorov–Kraichnan turbulence as modeled by the −5/3 dashed line. Treatment of CS-exposed cells with the inhibitor of actin polymerization (LatA) or MAPK kinase inhibitor (U0126), decreased the energetics and turbulence in the monolayer approaching that of NHBE. (B) COPD epithelia (CHBE) also display classic slopes characteristic of Kolmogorov–Kraichnan turbulence, as modeled by the −5/3 dashed line. Treatment of CHBE with LatA or U0126 decreased the energetics and turbulence in the monolayer approaching that of NHBE. The pEMT cells have a completely different slope. (C) In monolayers with cofilin-1 knockdown (KD) or overexpression (OE) in NHBE, analysis was performed in the entire monolayer (brightfield image, BF) or in GFP–cofilin-1-manipulated cells only (GFP). The curve of cofilin-1 KD-specific cells (GFP) demonstrate a −5/3 slope similar to that of the CHBE cells, with similar energetics. The curve of the cofilin-1 OE-specific (GFP) cells have higher energetics, but similar slope to that seen in pEMT. (D) Biphasic relationship between cofilin-1 and cell velocity. There is a biphasic relationship of cell velocity as a function of cofilin-1 (NHBE exposed to air or cigarette smoke and treated with GFP control, KD and OE at 1×10 10 pfu/ml and CHBE donors).

Article Snippet: Primary human bronchial epithelial cells from non-diseased human bronchial epithelial cells (NHBE) and COPD-derived human bronchial epithelial cells (CHBE) were either sourced from MatTek Corporation (MA, United States) or Lonza Group AG (Basel, Switzerland) or Epithelix SàRL (Geneva, Switzerland).

Techniques: Over Expression