Immunophenotypic and structural signatures of severe granulation tissue in airway implant patients

Figures

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  Overview of the study design and main findings on the immunological and structural characterisation of granulation tissue following endobronchial valve (EBV) or stent implantation. Granulation tissue was obtained by forceps biopsy during bronchoscopy. Scoring was performed according to severity, followed by histological and immunofluorescence analysis for immune cell infiltration, immunophenotyping by flow cytometry, and assessment of extracellular matrix (ECM) remodelling. Increased granulation grade was associated with clinical complications including EBV dislocation, dysfunction, and need for revision procedures. Key immune cell types [neutrophils, type 2 T helper (Th2) cells, regulatory T cells (Treg), central memory cells (CM), CD16high natural killer cells (NK), CD163+ macrophages], goblet cells, fibroblasts, endothelial cells (endoglin, ICAM), and ECM components (β-Catenin, fibronectin, collagen IV, tenascin C, pan-laminin).

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  Fig. 1. Study design. A Retrospective analysis in a discovery cohort (n = 56) of endobronchial valve (EBV) patients to develop an optical scaling and scaling sheet. B Categorisation based on the scaling sheet in an independent replication cohort (= 91) and re-categorisation of discovery cohort. C Prospective analysis of bronchoscopic biopsies from EBV patients EBV (n = 41) or stent (n = 11) grouped by the advanced score to assess the degree of granulation

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  Fig. 2. Scoring system of EBV patients by optical scaling including other risk factors. A The visual scaling has been developed from the discovery cohort and representative examples for each granulation tissue degree are shown. The groups were originally divided into none, minimal, moderate, strong, or severe granulation tissue. B Then, a categorisation was developed for EBV patients including additional influencing factors: Grade 0 = none (0 points), Grade 1 = moderate (1–2 points), Grade 2 = severe (3–6 points). C Relative distribution of optical scaling of granulation tissue after valve implantation of the discovery cohort (n = 56). D Relative distribution of granulation tissue degree after categorizing the discovery and the replication cohort (Grades 0–2, n = 147)

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  Fig. 3. Immunophenotyping of inflammatory cells in biopsies from valve (Grade 0, Grade 1, Grade 2) and stent patients. Percentage of A granulocytes in CD45+, neutrophils from granulocytes, and eosinophils from granulocytes, B natural killer (NK) cells (CD56+) in CD45+ and NK cells positive for CD16high, C CD4+ T helper cells from CD3+, cytotoxic CD8+ T cells from CD3+, CD4+ central memory cells (CD45RA−/CD62L+) from CD4+, regulatory T cells (Treg; CD25+/CD127low) from CD4+, and central memory cells (CD45RA−/CD62L+) in regulatory T cells D monocytes (CD14+/CD16+) in CD45+, macrophages in CD45+ and their activation markers CD86, CD197, CD206, CD80, and CD163. Results are expressed as mean ± standard deviation (plot: min to max, all points). Statistical analyses were performed by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Grade 0: n = 13–15, Grade 1: n = 13–14, Grade 2: n = 12, stent: n = 9

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  Fig. 4. Heat map summarising key findings of immune and structural cell populations in tissue samples from patients with different grades of granulation tissue and from stent patients. Immune subsets include neutrophils, CD16high NK cells, type 2 T helper (Th2) cells, CD4+ central memory cells (CM), regulatory T cells (Treg) and Treg central memory cells, as well as macrophages (total and polarised M1 and M2 macrophages). Structural components comprise mesenchymal cells, epithelial cells, goblet cells, fibroblasts, as well as markers of angiogenesis (endoglin) and cell adhesion (ICAM). Colour intensity represents the mean proportion (%) of each cell population within the respective group, with darker blue indicating higher frequency of the respective cell population. Columns represent mean values from Grade 0, Grade 1, Grade 2 and stent samples

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  Fig. 5. Tissue composition of biopsies from EBV patients (Grade 0, Grade 1, Grade 2) or stents analysed by flow cytometry. Percentage of A leukocytes (CD45+) and mesenchymal cells (CD45−) cells, B epithelial cells (CD326+) and goblet cells (TSPAN8+) C fibroblasts (CD90+ or PDGFRα+), D endothelial cells (CD31+) and the mean fluorescence intensity [geometric mean] of endoglin on endothelial cells. Results are expressed as mean ± standard deviation (plot: min to max, all points). Statistical analyses were performed by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Grade 0: n = 13–15, Grade 1: n = 13–14, Grade 2: n = 12, stent: n = 6–9

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  Fig. 6. Immunofluorescence staining of epithelial cell and structural markers in tissue samples from patients with different grades of granulation tissue and from stent patients. A total of three patients per group (Grade 0, Grade 1, Grade 2, stents) were analysed (supplementary Figure 12-23). Representative images of one patient per group are shown. Pan-cytokeratin (green) for epithelial cells, CCSP (green) for club cells, and TP63 (orange) for basal cells. β-Catenin (green) for cellular adhesion and proliferation, and MUC5AC (orange) for goblet cells. ZO-1 (green) for tight junctions and α-Tubulin (orange) for ciliated cells. FOXJ (green) is a transcription factor for ciliogenesis and vimentin (orange) for mesenchymal cells. VWF (green) for endothelial cells and α-SMA for myofibroblasts or smooth muscle cells. Scale bar: 100 µm; images acquired at 20× magnification

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  Fig. 7. Immunofluorescence staining of extracellular matrix remodelling markers across granulation tissue with different severity grades and stent biopsies. A total of three patients per group (Grade 0, Grade 1, Grade 2, stents) were analysed (supplementary Fig. 12–23). Representative images of one patient per group are shown. Collagen I (green) extracellular matrix in wound healing and collagen IV (orange) for basal membrane during wound healing. Pan-laminin (green) for basal membrane during wound healing and collagen III (orange) for tissue regeneration and stiffness. Tenascin C (green) extracellular matrix in early wound healing or fibrosis and fibronectin (orange) for acute wound healing or fibrosis. In all images, cell nuclei (Hoechst 3342) are depicted in blue. Scale bar: 100 μm; images acquired at 20× magnification