Cell-cell and cell-matrix interactions driving progressive fibrosis in interstitial lung diseases

Project Summary Interstitial lung diseases (ILD) are a large group of lung diseases that are characterized by chronic, progressive pulmonary fibrosis. We hypothesize that in all ILDs the progressive fibrosis is driven by ongoing injury/stress in the airway epithelial cells that set up crosstalk with their neighboring mesenchymal cells resulting in a chronic

wound healing process that alters the matrix and changes the cellular niche. The goal of the proposed research is to address how interactions between the epithelial, mesenchymal and matrix compartments drive progression of fibrosis in all forms of ILD. We are proposing to study these interactions between the epithelial, mesenchymal

and matrix compartments, which is now feasible with spatial transcriptomics, single cell secretion protein analysis, and small region proteomics. Most studies have looked at end stage disease and fibrotic lesions where most of the epithelial cells are lost and consequently the initiating and propagating factors are not well

understood. We propose to study Interstitial Lung Abnormality (ILA) lesions, which represent very early ILD lesions, early stage ILD lesions when patients start to become symptomatic, and late stage ILD regions that still contain epithelial cells so we can study their interactions with mesenchymal cells and matrix. To test our

hypothesis, we have developed a 3D scaffolded lung cell co-culture model system with progressive fibrosis that closely models these cellular compartments in ILD tissue. We propose the following aims: Aim 1: To understand epithelial-mesenchymal interactions in the airway that drive fibrosis using spatial

transcriptomics. We will use our biobank of ILD and ILA samples and prospectively collect ILD and ILA patient samples to profile the airway epithelium and underlying fibroblasts from bronchioles to distal airspaces with spatial small region transcriptomics (GeoMx) and single cell spatial genome-wide transcriptomics (Stereoseq).

Aim 2: To understand epithelial, mesenchymal and matrix interactions in the airway that drive fibrosis. Aim 2a: We will use the secretory-single cell profiling (Sec-seq) technology to capture single airway epithelial cells, from fresh ILA and ILD tissue and trap the secretions from each cell to identify paracrine factors.

Aim 2b: We will use nanoscale small region proteomics to identify proteins in the adjacent epithelial, mesenchymal and basement membrane matrix compartments from ILA and ILD samples. Aim 3: To identify mechanisms by which persistent airway injury induces progressive fibrosis by using cell co- culture models of iPSC derived AT2 cells with specific mutations associated with familial IPF, in conjunction with

primary healthy or IPF lung fibroblasts. These powerful reductionist models allow the study of specific epithelial- mesenchymal-matrix interactions that drive progressive fibrosis. We have assembled a team with expertise in spatial transcriptomics and ILDs (Gomperts), novel bioinformatics pipelines and integration of genomics data (Plath), regional small-scale proteomics in ILDs (Clair), iPSC-derived

AT2 disease modeling and ILDs (Kotton) and bioengineered models of ILD (Gomperts).