Research ArticleFibrosis

Cadherin-11–mediated adhesion of macrophages to myofibroblasts establishes a profibrotic niche of active TGF-β

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Science Signaling  15 Jan 2019:
Vol. 12, Issue 564, eaao3469
DOI: 10.1126/scisignal.aao3469
  • Fig. 1 CDH11 is present at contacts between macrophages and MFs in fibrotic human lungs.

    (A) Representative immunohistological staining for the macrophage marker CD206 (brown) on histological sections of lung from patients with IPF in unaffected lung regions (“normal”) and regions classified by a pathologist as fibrotic from the same patient. Select regions are shown in high magnification insets. Scale bars, 50 μm. (B) Confocal fluorescence imaging corresponding to a consecutive slice of the samples shown in (A), immunolabeled for the macrophage marker CD68, the MF marker α-SMA, and CDH11. Cell nuclei are white [4′,6-diamidino-2-phenylindole (DAPI)]. Arrowheads point to CDH11 signals between CD68-positive macrophages and α-SMA–positive MFs. Scale bars, 20 μm. (C) Confocal fluorescence imaging of histological sections of two additional patients with IPF in unaffected lung regions (normal) and regions classified by a pathologist as fibrotic from the same patient. Sections are immunolabeled to show CD68, α-SMA, and CDH11. Cell nuclei are white. Scale bars, 20 μm. (D) The boxed region of the fibrotic sample from IPF patient no. 3 is shown at higher magnification with channels separated and merged. Scale bars, 20 μm. (E) Data for CDH11 mRNA expression extracted from the Gene Expression Omnibus (GEO) microarray database, comparing whole extracts of lung tissue from human patients diagnosed with early IPF (n = 8) and late IPF (n = 9) and from control subjects (n = 6) (GEO Dataset Series, GSE24206). (F) Data for CDH11 mRNA expression extracted from the GEO microarray database, comparing whole extracts of lung tissue from human patients diagnosed with IPF (n = 124) and other interstitial lung diseases (ILD) (n = 124) and from control subjects (n = 108) (GSE47460). (G) Data for CDH11 mRNA expression extracted from the GEO microarray database, comparing whole extracts of lung tissue from bleomycin-induced lung fibrosis in rat (n = 5 animals per bleomycin-treated group and n = 37 for saline controls) and mouse (n = 3 animals per group) animal studies. Gene expression value distributions are shown as means ± SD [*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, analysis of variance (ANOVA) with Kruskal-Wallis multiple comparisons test]. PBS, phosphate-buffered saline.

  • Fig. 2 Cdh11 is present between macrophages and MFs in fibrotic mouse lungs.

    (A) Western blotting of extracts of lung tissue from C57BL/6 mice 7 or 21 days after intratracheal instillation of bleomycin or saline controls for 21 days (con). Blots were probed to detect α-SMA, Cdh11, CD68, and the loading control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Protein quantifications were first normalized to GAPDH and then to saline controls. Graphs show means ± SD of n = 4 animals per time point per group (total = 12 animals; *P ≤ 0.05, Student’s t test). (B) Representative confocal immunofluorescence showing the macrophage marker CD68, the MF marker α-SMA, and Cdh11 in paraffin-embedded sections of mouse lung tissues. Cell nuclei are white (DAPI). (C) Higher magnification view of a tissue section from bleomycin-treated mice 21 days after treatment. (D) Alveolar macrophages were isolated from mice by bronchoalveolar lavage (BAL) and interstitial by digestion of lung tissue and cell sorting. (E) Flow cytometry and sorting of interstitial macrophages for CD45 and CD64 7 days after instillation of bleomycin or saline (control). The CD45/CD64 double-positive cells in the outlined region were purified. (F) Quantification of Cdh11 expression in alveolar (BAL) and sorted interstitial CD45/CD64 double-positive (digest) macrophages. The inset shows the percentages of CD45/CD64 double-positive cells among all the digested cells in box plot (median, minimum, and maximum). Cdh11 quantification is shown as means ± SD of at least three samples, each pooled from three to four mice. ***P < 0.001, ANOVA with Dunnett’s multiple comparisons test. (G) Representative confocal section showing CD45/ CD64 double-positive interstitial macrophages sorted from fibrotic mouse lung and directly seeded on top of primary lung fibroblast monolayers. Immunostaining shows the macrophage marker F4/80, α-SMA, and Cdh11. Cell nuclei are white. Scale bars, 50 μm.

  • Fig. 3 Cdh11 localizes to heterocellular junctions between mLFs and in vitro–activated macrophages.

    (A) Immunoblotting and immunofluorescence microscopy of mLFs treated with the TGF-β receptor inhibitor SB431542 on lung-soft substrates (fibroblasts, F) or with TGF-β1 on stiff substrates (MF). Blot shows Cdh2, Cdh11, α-catenin, β-catenin, vimentin, α-tubulin, and α-SMA. The abundances of α-SMA and Cdh11 were quantified. The immunofluorescence images show Cdh11, α-SMA, and nuclei (DAPI). Protein quantifications were first normalized to vimentin and then to the amount present in fibroblasts. Graphs show means ± SD (n = 3 independent cultures; *P < 0.05, Student’s t test). (B) Characterization of primary macrophages from mouse bone marrow polarized using LPS to generate M1 macrophages or IL-4 and IL-13 to generate M2 macrophages. The Western blot shows Cdh2, Cdh11, α-catenin, β-catenin, α-tubulin, CD68, the M1-specific marker iNOS, and the M2-specific markers CD206 and Arg-1 in M1 and M2 macrophages polarized in vitro. The amounts of Cdh11, iNOS, CD206, and Arg-1 in each cell type were quantified. In vitro–polarized M1 and M2 macrophages were sorted by FACS for CD80, which is high in M1 macrophages and low in M2 macrophages, and CD206. Immunofluorescence images show Cdh11, β-catenin, and the macrophage marker F4/80 in M1 and M2 in vitro–polarized macrophages. Protein quantifications were normalized to α-tubulin. Graphs show means ± SD of n = 3 independent experiments with cells from at least three animals (*P ≤ 0.05, using ANOVA followed by a post hoc Tukey’s multiple comparisons test). (C) Representative confocal micrographs of in vitro–polarized M2 macrophages seeded onto confluent monolayers of MFs. Cdh11-containing junctions (green) between macrophages (F4/80, red) and MFs (α-SMA, inset and blue) were resolved in one optical section (0.5 μm) at the cell-cell interface. (D) Representative transmission electron micrographs showing enrichment of actin at sites of intercellular adherens junctions between in vitro–polarized M2 macrophages grown on top of MFs. (E) Immunoelectron microscopy showing Cdh11 localization at heterocellular (macrophage-MF) contacts. n = 3 independent cultures from at least three different animals. Scale bars, 25 μm (A to C) and 500 nm (D to E).

  • Fig. 4 Functional Cdh11 adhesions mediate heterocellular interactions between macrophages and MFs.

    (A) Scanning electron microscopy showing M1 and M2 macrophage adhesion to and spreading on confluent MF monolayers, after jet washing of low-adherent macrophages. (B) Immunofluorescence images show the macrophage marker F4/80 and nuclei (DAPI), which were used to quantify the numbers and average spreading areas of macrophages adhering to MFs. The Western blot shows the macrophage marker CD68, the MF marker α-SMA, and the relative abundances of these proteins in M1 and M2 macrophages seeded with MFs. The ratios of CD68 to α-SMA were calculated from n = 3 independent experiments. (C) Immunofluorescence microscopy of in vitro–polarized M2 macrophages seeded onto fibroblast (F) or MF monolayers and washed to remove low-adherent macrophages. F4/80 and nuclei were used to quantify the average macrophage spreading area. (D) Immunofluorescence microscopy of aggregates formed by suspended M2 macrophages with either fibroblastic or MF cells. β-Catenin, F4/80, and α-SMA were used to quantify the percentage of cells in aggregates that were macrophages. (E) Heterocellular attachment strength was assessed by seeding M2 macrophages onto fibroblasts and MF monolayers in a fluid flow chamber, removing weakly attached macrophages using low (1.1 N/m2) shear stress, and then gradually increasing the shear stress in steps of 1 N/m2 every 30 s. The macrophages (bright and circular) remaining after each step were quantified from phase-contrast images by automated image analysis and plotted as a function of shear stress. (F and G) Fluid flow chamber adhesion tests were also performed in the presence of the Cdh11 function–blocking antibodies (Abs) 23C6 and 13C2 or immunoglobulin G (IgG) control antibodies (F) and in the presence of the calcium chelator EGTA (G). (H and I) Jet-wash (H) and aggregation (I) assays with M2 macrophages and MFs were performed in the presence of Cdh11 function–blocking antibody 23C6 or IgG control. (J) Immunofluorescence microscopy showing F-actin, Cdh11, and nuclei of MFs plated on tissue culture plastic in the presence of the Cdh11 function–blocking antibody 23C6 or the IgG control antibody. The insets show cell junctions in higher magnification. (K) Immunofluorescence microscopy showing F4/80 and nuclei of macrophages plated on plastic tissue culture dishes in the presence of the Cdh11 function–blocking antibody 23C6 or IgG control antibody. Representative images from n = 3 independent experiments. All graphs show means ± SD from n = 3 independent experiments with cells from at least three animals (*P ≤ 0.05 and **P ≤ 0.01, using ANOVA followed by a post hoc Tukey’s multiple comparisons test). Scale bars, 50 μm.

  • Fig. 5 Direct contact is required for macrophage-induced fibroblast-to-MF activation.

    (A) In plastic dishes with custom-made wells that allowed for different populations of cells to share the same medium without contacting one another, mLFs were cultured alone, in segregated cocultures with in vitro–polarized M2 macrophages, or in direct contact with in vitro–polarized M2 macrophages in the absence or presence of the TGF-β1 inhibitor TGFRII-Fc. (B) Immunofluorescence images showing α-SMA, F-actin (phalloidin), and nuclei (DAPI) in each of the culture conditions in (A). The percentages of cells that were α-SMA–positive–activated MFs in each condition were quantified by image analysis. (C) Phase-contrast microscopy of cells cultured as in (A) on a deformable silicone substrate to show and quantify high cell force exertion (contraction), as indicated by the formation of wrinkles in the substrate. (D) High magnification micrographs of mLFs and in vitro–polarized M2 macrophages on silicone substrates in direct coculture, fibroblasts alone in the presence of active TGF-β1, and in direct coculture in the presence of soluble recombinant TGFRII-Fc. (E) Triple coculture assays were performed with mLFs, in vitro–polarized M2 macrophages, and TMLC reporter cells to measure TGF-β1–dependent luciferase reporter activity. All graphs show means ± SD from n = 3 independent experiments performed with cells from at least three animals (*P ≤ 0.05, using ANOVA followed by a post hoc Tukey’s multiple comparisons test). Scale bars, 75 μm.

  • Fig. 6 Cultured macrophages produce but do not activate latent TGF-β1.

    (A) TGF-β–reporting TMLCs were used to measure the amounts of active TGF-β1 in macrophage-conditioned medium and in direct coculture with in vitro–polarized M1 and M2 macrophages. (B) Total (latent and active) TGF-β1 content of macrophages, as measured by incubating TMLCs with heat-activated macrophage culture supernatants plus cell lysates. (C) Immunofluorescence showing α-SMA, F-actin (phalloidin), and nuclei (DAPI) in fibroblasts incubated with native (not heat-activated) or heat-activated macrophage culture supernatants plus lysates. The percentage of α-SMA–positive–activated MFs was quantified for each condition. (D) In vitro–polarized M2 macrophages were cultured on substrates coated with recombinant CDH11:Fc fusion proteins and immunostained to show Cdh11, β-catenin (β-cat), and nuclei (DAPI). (E) TMLC reporter cells were used as in (A) and (B) to measure the amounts of total and active TGF-β1 in supernatants of macrophages on CDH11, fibronectin (FN), or uncoated (con) substrates. (F) Fibroblasts were cultured on substrates coated with recombinant CDH11:Fc fusion proteins and immunostained to show Cdh11 and β-catenin. (G) Western blots showing Cdh11, β-catenin (β-cat), vimentin (vim), and α-SMA in lysates from fibroblasts grown on substrates coated with poly-l-lysine (pLL), human IgG (IgG), CDH2:human IgG Fc fusion protein (CDH2), CDH11:Fc fusion protein (CDH11), tissue culture plastic (TCP), fibronectin (FN), or gelatin (gel). The ratio of the MF marker α-SMA to vimentin protein–loading control was quantified to evaluate MF activation on differently coated substrates. All graphs and Western blot quantification show means ± SD from n = 3 independent experiments performed with cells from at least three animals (*P ≤ 0.05, using ANOVA followed by a post hoc Tukey’s multiple comparisons test). All scale bars, 50 μm.

  • Fig. 7 Knockdown of Cdh11 in immortalized mouse lung MFs.

    (A) hTERT-immortalized mLFs (mLF-hT cells) cultured in the absence or presence of TGF-β1were examined by phase-contrast microscopy; by immunofluorescence staining for the MF markers α-SMA, extradomain-A fibronectin (FN), and nuclei (DAPI); and by Western blotting for Cdh11, β-catenin, and α-SMA. GAPDH is a loading control. Representative data from n = 3 independent batches of mLF-hT cells. (B) Western blot and quantification of Cdh2, Cdh11, β-catenin, vimentin, α-SMA, and TGF-β1 in mLF-hT cells that were transfected with two different Cdh11-targeting siRNAs alone (siA and siB), both CDH11-targeting siRNAs together (siA + B), a nontargeting siRNA (siNT), or no siRNA (mock). Cells that were not subjected to the transfection protocol were included as negative controls (control). Protein quantifications were first normalized to GAPDH and then to nontransfected controls. (C) Representative immunofluorescence images showing Cdh11 at junctions between mLF-hT cells that were pretreated with TGF-β1 before Cdh11 knockdown (Cdh11 siA + siB) or treatment with a nontargeting siRNA (siNT). (D) Representative immunofluorescence images showing the macrophage marker F4/80, F-actin, and nuclei (DAPI) in M2 macrophages plated on monolayers of mLF-hTs that were pretreated with TGF-β1 before Cdh11 knockdown (Cdh11 siA + siB) or transfection with the nontargeting siRNA. The fluorescence images were used to quantify the average spreading area of the macrophages. Graphs shows median, minimum, and maximum in box plots from n = 3 independent coculture experiments (*P < 0.05, two-tailed paired Student’s t test). (E) Quantitative [qRT-PCR (quantitative reverse transcription polymerase chain reaction)] assessment of cell adhesion–related gene expression in TGF-β1–pretreated mLF-hTs upon Cdh11 knockdown and controls. Quantifications were first normalized to the average value of G6pd, Gapdh, and Hmbs and then to the siNT condition. All graphs except (D) show means ± SD from n = 3 independent transfection experiments (*P < 0.05 and ***P < 0.001, ANOVA with Tukey’s multiple comparisons test).

  • Fig. 8 Proximity dependence of macrophage-activated TGF-β1 signaling in fibroblasts.

    (A) A proximity culture system was used to vary the distance between fibroblasts and macrophages in coculture. Fibroblast monolayers were grown on culture substrates of different heights below M2 macrophages adhering to the underside of membrane Transwells. Empty Transwells without macrophages were used as a negative control (con). Effective gap distances were 900, 500, and 100 μm, not taking cell heights into account. TGF-β1 signaling in fibroblasts was assessed by immunoblotting cell lysates for total Smad2 and Smad3 (Smad2/3) and phosphorylated Smad2/3 (pSmad2/3). GAPDH is a loading control. Protein quantifications were first normalized to GAPDH and then to total Smad2/3 amounts. (B and C) The experiment was repeated at 900- and 100-μm gap distances in the presence of the TGF-β1 inhibitor TGFRII-Fc (B) or the Cdh11-blocking antibody 23C6 and control IgG (C). Graphs show means ± SD from n = 3 repeats (*P ≤ 0.05, using ANOVA followed by a post hoc Tukey’s multiple comparisons test).

Supplementary Materials

  • This PDF file includes:

    • Fig. S1. Primary mLF cultures.
    • Fig. S2. Primary mouse macrophage cultures.
    • Fig. S3. Active TGF-β1 measurement using TMLC cultures.
    • Table S1. Primer list.

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