Understanding Lung Cell Homeostasis to Reverse Lung Cell Remodeling

Background

On October 21 and 22, 2021, experts met to highlight recent research findings and remaining questions about lung homeostasis, repair, and remodeling. Presenters and discussants covered basic research, clinical investigation, and the latest ’omics technologies used to understand mechanisms of airway remodeling and ways to reverse it. Sessions focused on (1) realizing the promise of ’omics and other new technologies; (2) embracing cellular diversity; and (3) triggers and paths to remodeling. All presentations addressed health disparities and sex and gender influences. The format consisted of short talks in each session followed by moderated Q&A with panelists. Each session held a breakout to discuss the gaps and opportunities in each session’s topic areas. The virtual meeting had over 330 participants with a wide range of expertise, clinical and research focus. The workshop was organized by the NHLBI Division of Lung Diseases.

Workshop Goals

The workshop discussed challenges related to lung cell homeostasis and pathways driving remodeling and repair. This workshop was an opportunity to share new research, discuss gaps in the field, and suggest the next steps with respect to gaining a better understanding of the current issues and how to promote translation to the clinic. This workshop brought together experts from various fields of research to share their insights, begin partnerships, foster collaborations, and provide ideas for the future research needs in these topics.

Discussions

The workshop presentations and discussions centered around the following key topic areas:

Session 1: Realizing the Promise of ’omics and Other New Technologies

This session introduced the use of new technologies such as single-cell genomics which enable a granular look at molecular changes and can reveal the heterogeneity and subpopulations of cells in the lung. Single-cell RNA sequencing (scRNAseq) technology enables researchers to identify associations between mRNA and corresponding cells. Additionally, single cell multi-omics can reveal novel cell types and cell changes to provide a deeper understanding of homeostasis and remodeling. Combined with other technologies such as CRISPR and other ’omics, an increased granularity can be achieved at the molecular level. These technologies can delineate single-cell gene expression and changes across thousands of cells in parallel, using high multiplex CRISPR screens and Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq), a method for mapping chromatin accessibility genome-wide. Spatial genomics technologies allow 2D visualization of gene expression for each cell with spatial coordinates within tissues, capturing biologically relevant information in a spatial context. Through this microscopy-based approach, thousands of genes can be detected in tens of thousands of cells in parallel at very high resolution. Further, novel lineage tracing with single-cell genomics can provide additional insight into cell fate through remodeling and homeostasis. In parallel, great advances have been made with organoid models, which continue to be improved as models of human lung disease.

Session 2: Embracing Cellular Diversity

In this session, speakers continued with themes presented in Session 1 and described how new ’omics and technologies can reveal cellular diversity in lung cell types and function, and how lung structure changes during remodeling and disease progression. New cell types and fates were described, along with their implications for disease progression and therapeutic targeting. Researchers noted the importance of locating and understanding regional differences in the airways, in order to better understand what is normal biology versus regions of abnormal tissue composition. A case was made for continuing to characterize the cell types present across different airway regions in order to understand the initiation sites of lung disease and how these cells have different characteristics in lung diseases. (Interstitial lung disease [ILD], fibrosis, COVID-related lung disease and pneumonia, and chronic obstructive pulmonary disease [COPD] were given as examples.) Throughout Sessions 1 and 2, speakers addressed how they have used these technologies to identify the roles of specific cell types in lung development, homeostasis, and disease, including AT2 cells, fibroblasts, vascular smooth muscle cells, endothelial cells, epithelial cells, resident macrophages, and neuroendocrine cells.

Session 3: Triggers and Paths to Remodeling

In this session, researchers explored how new technologies impact the study of immune cell function in the normal and damaged lung and reveal how these cells contribute to inflammation or repair; how early life exposures can set up the lung for abnormal remodeling in later life; and how the effects of early exposures may be reversed. This session extended the conversations of Sessions 1 and 2 on the crosstalk between structural lung cells and immune cells throughout the lung, including how chemokines and cytokines play a role in lung reprogramming. Under the umbrella of understanding lung cellular homeostasis, researchers emphasized the role of circadian rhythms in lung inflammation and repair of injury, particularly noting interactions between lung cellular development and circadian biology. Finally, speakers noted the need for additional research to understand the impact of early life exposure on later lung injury and permanent structural deficits.

Session 4: Taking Cells to Cures

During Session 4, cell- and CRISPR-based gene therapy were discussed in relation to respiratory disease. Topics included strategies for therapeutic cell transplantation into the lung and the use of human organoids as models for testing gene function and for drug discovery. Speakers noted that considerable progress has been made in applying induced pluripotent stem cell (iPSC) technology in lung diseases. In particular, cystic fibrosis researchers have been able to derive airway stem cells (basal cells, tissue-specific stem cells) from iPSCs and to demonstrate multi-lineage differentiation capacity of these cells with properties of self-renewal. CRISPR-based gene therapy was presented as a means to manipulate and correct cells, as well as to understand the role of specific genes in homeostasis or disease. Other engineered models also show promise for studies of lung repair and regeneration, including synthetic scaffolds, lung cell chips, and organoids. In summary, engineered systems with controlled complexity provide unprecedented opportunities for mechanistic studies and tools for identification of therapeutic candidates.

Research Opportunities and Critical Gaps:

The workshop participants identified a number of knowledge gaps and research opportunities:

Specific Research Gaps:

• There is need for the community to reach consensus on lung cell nomenclature and signatures from healthy and diseased samples in single-cell datasets.
• Aligned with heterogeneity of cell populations in the lung, researchers noted that the submucosa (glands) and the connective tissue of the airways remain underexplored.
• Research is needed on the cellular bases of organ-level circadian behavior and inter-organ clocks.
• There are potential benefits in creating an integrated cross-disciplinary platform for curation of large ‘omics datasets and for other spatial transcriptomics, for human, mice, and other species data generated in the lung.
• There is need to utilize 4D imaging over times of lung development and lung regeneration.
• Better knowledge of transcription factors is required for initiating human lung progenitor cell self-renewal.
• An understanding of how healthy cells transition into a disease state is needed to identify therapeutic targets and biomarkers of response.
• There are critical gaps in knowledge regarding specific cell-types in particular lung diseases:
  • How does fibroblast heterogeneity impact interstitial lung disease and other lung diseases?
  • How can we prevent the development of activated myofibroblasts?
  • How do fibroblasts interact with macrophages, a key part of fibrosis?
  • In pulmonary hypertension, what specific inflammatory lineages interact with vascular cell types at different stages of disease? What are their molecular intermediates? Which cells within open and occluded arteries are modulated by T cells and T cell-derived signals?
  • How is human endothelium regulated by lung-specific microenvironmental cues and therapies?
• There is need to build better animal models to recapitulate human lung diseases because many human lung cells or cell trajectories are not represented in mice; ferrets, humanized mice, and large animal models are promising alternatives.

Research Opportunities:

• An infrastructure or resource for obtaining additional samples from healthy and diseased lungs would enable advanced ‘omics and spatial assays.
• Systematic lung cell profiling at different time points across the human lifespan, in different genders, and in more diverse populations would provide invaluable knowledge.
• Integration of circadian biology into research studies to understand clock control and its contribution to lung health and disease would add an important dimension to lung research.
• Validate cellular functions from single-cell and in vitro data, in in vivo models to understand the role of individual cell types in lung homeostasis.
• Greater collaboration and data-sharing within the lung community would enhance research productivity.
• Greater sharing and annotation of data from LungMAP can aid investigators not directly involved with that program.
• Building cellular models to study cell types and cell trajectories and use of perturbation techniques (ex vivo perfused lung, drug-seq) would be of particular benefit.
• Sophisticated iPSC-derived organotypic lung cultures analogous to primary lung organotypic cultures which also utilize bioengineering approaches and genetic manipulation tools show great promise as an in vitro model of the human lung.
  • Genetic manipulations in these systems could clarify mechanisms of differentiation, disease development, and therapeutic responses.
  • Testing scaffolds and biomaterials such as fibrinogen and Matrigel in these systems could accelerate the development of functional implants.