Cytokine signaling converging on IL11 in ILD fibroblasts provokes aberrant epithelial differentiation signatures

Abstract

Introduction: Interstitial lung disease (ILD) is a heterogenous group of lung disorders where destruction and incomplete regeneration of the lung parenchyma often results in persistent architectural distortion of the pulmonary scaffold. Continuous mesenchyme-centered, disease-relevant signaling likely initiates and perpetuates the fibrotic remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area.

Methods: With the aim of identifying functional mediators of the lung mesenchymal-epithelial crosstalk with potential as new targets for therapeutic strategies, we developed a 3D organoid co-culture model based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells that form alveolar organoids in presence of lung fibroblasts from fibrotic-ILD patients, in our study referring to cases of pulmonary fibrosis, as well as control cell line (IMR-90).

Results: While organoid formation capacity and size was comparable in the presence of fibrotic-ILD or control lung fibroblasts, metabolic activity was significantly increased in fibrotic-ILD co-cultures. Alveolar organoids cultured with fibrotic-ILD fibroblasts further demonstrated reduced stem cell function as reflected by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. To screen for key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used mass spectrometry and characterized the secretome of end stage fibrotic-ILD lung fibroblasts in comparison to non-chronic lung disease (CLD) patient fibroblasts. Out of the over 2000 proteins detected by this experimental approach, 47 proteins were differentially abundant comparing fibrotic-ILD and non-CLD fibroblast secretome. The fibrotic-ILD secretome profile was dominated by chemokines, including CXCL1, CXCL3, and CXCL8, interfering with growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating alveolar organoids with IL11, we recapitulated the co-culture results obtained with primary fibrotic-ILD fibroblasts including changes in metabolic activity.

Conclusion: We identified mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD. In our alveolar organoid co-cultures, we were able to highlight the importance of fibroblast-initiated aberrant epithelial differentiation and confirmed IL11 as a key player in fibrotic-ILD pathogenesis by unbiased fibroblast secretome analysis.

Keywords: IL11; co-culture model; cytokine; disease modeling; human pluripotent stem cells; interstitial lung disease; organoids; secretome.

Figure 1

(A) Representative maximum intensity projections from high-content images of iAT2s growing as alveolar organoids at day 12. Scale bar 500µm. (B) Immunofluorescence of 3D co-culture of iAT2s with ILD fibroblasts at day 12 (α-SMA: red, DAPI: cyan). Scale bar 50 µm. (C) Representative maximum intensity projections from high-content images of different co-culture conditions showing iAT2s growing with human fibroblasts for 12 days. Scale bar 500 µm. Zoom-ins show a 3x optical magnification. (D) Scatter plots (dashed black lines: median) indicate size (µm2) of organoids in co-cultures at day 12 across three independent biological replicates. (E) Number of formed organoids in co-cultures at day 12, N = 3. (F) Metabolic activity of co-cultured organoids at 5, 8 and 12 days of co-culture. Results show the increase in percentage across two biological replicates in comparison to d5 iAT2 organoids alone, representing the baseline of 100% metabolic activity (dashed grey line across dataset). Statistics: unpaired t-Test, *p<0.05, **p<0.01.

Figure 2

Relative gene expression at day 12 measured in AT2s co-cultures with ILD or IMR90 control fibroblasts in two seeding densities (high or low) compared to reference gene expression (HK; average of ß-Actin (ACTB) and hypoxanthine guanine phosphoribosyl transferase (HRPT)). (A) Epithelial and stem cell markers and (B) Genes associated with aberrant differentiation of epithelium. N = 3, unpaired t-Test, **p<0.005, ***p<0.0005.

Figure 3

Differential protein expression comparing ILD fibroblasts (F_ILD) vs. non-chronic lung disease fibroblasts (F_control). (A) Volcano plot visualizing significantly regulated proteins (47 up, 55 down) detected by MS secretome analysis of ILD or non-CLD control fibroblasts. Data showing log₂ fold change against the adjusted P value [log10]. Significantly upregulated proteins are depicted in red and significantly downregulated proteins in blue. (Total: 102 significantly regulated proteins with 5% FDR < 0.05, adj. p_value < 0.05). Pathway enrichment and protein interaction network of proteins with (B) increased and (C) decreased abundance using the Cytoscape plugin ClueGo. The following ontologies were used: KEGG, molecular functions and biological processes. The connectivity of the pathways is described by functional nodes and edges that are shared between proteins with a kappa score of 0.4. Only enriched pathways are visualized and the node size indicates the p-value (p-value ≤ 0.05). Proteins from the same pathway share the same node color and the bold fonts indicate the most important functional pathways that define the names of each group. Enriched Pathways: 1. rheumatoid arthritis, 2. chemokine-mediated signaling pathway, 3. neutrophil chemotaxis, 4. chemokine activity, 5. TNF signaling pathway, 6. IL17 signaling pathway, 7. cellular response to chemokine, 8. response to chemokine.

Figure 4

(A) Immunofluorescence of untreated alveolar organoids at day 14 of culture (SFTPC: red, NKX2.1: green, DAPI: cyan). (B) Representative maximum intensity projections from high-content images of (i) untreated alveolar organoids, (ii) 0.5 ng/mL or (iii) 5 ng/mL IL11 treatment. Treatment started at day 7 of culture every 48h. Scale bar, 500 µm. (C) Organoid formation capacity of alveolar organoids treated with 0.5 or 5ng/mL IL11. (D) Metabolic activity of alveolar organoids treated with 0.5 or 5ng/mL IL11. (C, D) Each value is graphed as percentage of the respective starting culture at day 7 set to 100%.