Discovery of intra-pulmonary adipocytes in pulmonary fibrosis

by MD. Kinan El Husseini

CCA-AHU, FHU APOLLO — Service de Pneumologie A, Hôpital Xavier Bichat, AP-HP, 75018 Paris, France

PhD candidate, Inserm UMR-S 1152, Physiopathologie et épidémiologie des maladies respiratoires, Université (PHERE), Paris Cité, F-75018 Paris, France

Idiopathic pulmonary fibrosis (IPF) is a lethal chronic lung disease mainly affecting male adults over 50 years old, with an overall median survival of 3 to 5 years at diagnosis. Two antifibrotic drugs (pirfenidone/nintedanib) are currently approved for the treatment of IPF, albeit with only a suspensive effect. In broad terms, lung fibrosis is characterized by extensive parenchymal remodeling and chaotic reactivation of developmental pathways in the lung, with disruption of cell growth, migration, and differentiation processes. For instance, abnormal mesenchymal differentiation can be observed, with an aberrant accumulation of smooth muscle tissue or, more rarely, ossification foci within the lung. In IPF, alveolar epithelial cells appear to play a major role in the initiation of lung fibrosis, while activated myofibroblasts are the main effectors of its progression.

In preliminary observations, our team discovered the extensive presence of intra-pulmonary adipose tissue located in the sub-pleural, remodeled regions of the lungs in patients with IPF. Adipocytes are mesenchymal cells whose existence in the distal parenchyma had not been reported before; they are usually found in the hilar region and along bronchovascular axes. In addition, these cells have been linked to fibrogenesis in other organs, such as the heart or the liver, through paracrine interactions with resident fibroblasts. Consequently, the discovery of intra-pulmonary adipocytes in lung fibrosis raises the question of their origin as well as potential involvement in lung fibrogenesis.

Thanks to the support from FHU APOLLO, I am currently undertaking a research project on this subject as part of my PhD, in Inserm Unit 1152 — PHERE (led by Pr. Bruno Crestani). This project is structured along three aims: 1) extensively characterize intra-pulmonary adipose tissue in patients with lung fibrosis; 2) demonstrate a paracrine interaction between pulmonary adipocytes and IPF fibroblasts in vitro and determine its mediators; 3) study the direct effect of adipocytes on fibrogenesis in vivo, in a murine model of pulmonary fibrosis.

Early results based on analyses on tissue samples from our cohort of lung fibrosis patients suggest that intra-pulmonary adipose tissue deposits are a frequent finding in lung fibrosis, with a prevalence of up to 86% in IPF (n=37/43). Individual deposits are generally located superficially under the pleura (0.13 ± 0.11 mm deep; 0.54 ± 0.44 mm thick; 1.48 ± 0.88 mm wide), in close contact with fibroblastic foci in the remodeled parenchyma. Pleural fat content (ratio between pleural surface with underlying adipose tissue and total pleural surface) varies widely between patients (on average, 36.2 ± 25.2%). Comparison of intra-pulmonary adipocytes to hilar adipocytes using immunohistochemistry techniques targeting common adipogenic biomarkers found that although both tissues exhibit typical white adipose tissue characteristics (large, uniloculated, PLIN1+/PLIN4+/UCP1- cells), lung adipocytes expressed FABP4 less frequently, albeit more intensely. Using a morphometric approach on tissue sections, I also found that lung adipocytes had a smaller size and more circular shape compared to hilar adipocytes. Although adipose tissue presence, as a dichotomous criterion, was not correlated to clinical parameters (body-mass index, age, tobacco exposure, antifibrotic treatment or pulmonary function), I am currently investigating a possible quantitative correlation between pulmonary function and pleural fat content. In addition, to gain a deeper understanding of the components of lung adipose tissue in IPF and how they compare to hilar adipose tissue, we will perform analyses using an in situ lipid cartography technique (MALDI-MSI), in collaboration with the research team led by Pr. Valérie Paradis (Inserm UMR 1149), and single nucleus RNA sequencing, on fresh and frozen lung samples from IPF patients.

Regarding the in vitro experiments, I am currently using primary cells, both lung adipocytes and lung fibroblasts, in coculture experiments, proliferation, survival and metabolism assays, and conditioned milieu cultures to determine the effect intra-pulmonary adipocytes have on lung fibroblasts. To obtain primary lung adipocytes, we developed an original method for extracting the pulmonary adipose tissue stromal vascular fraction from fresh lung samples of IPF patients, which contains adipocyte precursors. These cells are then differentiated into mature adipocytes of pulmonary origin. Preliminary results indicate an inhibitory effect of pulmonary adipocytes on myofibroblastic differentiation, linked to one or multiple secreted mediators which have not yet been identified. Building on our research team’s experience, I am also using fibroblast-derived three-dimensional matrices to determine if IPF lung fibroblasts may promote adipogenic differentiation in pulmonary preadipocytes, compared to control lung fibroblasts, through their secretion of extracellular matrix.

As part of preparations for murine experiments, I investigated the presence of adipose tissue in the lung of mice in 4 models of pulmonary fibrosis (intratracheal single/repeated instillation of bleomycin; lung irradiation; adenovirus TGFβ1-induced fibrosis), but no adipose deposit was found in these mice. Therefore, to study the effect of parenchymal adipocytes on fibrogenesis, we currently plan to incorporate an intratracheal/trans-thoracic injection of primed preadipocytes into a bleomycin-induced pulmonary fibrosis model.

Part of our research results will be presented at the American Thoracic Society International Conference in May 2022, and at the European Respiratory Society Congress in September 2022.

https://www.fhu-apollo.fr

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Fédération Hospitalo-Universitaire (FHU) APOLLO
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Notre objectif est d’apporter une thérapie personnalisée aux maladies cardiovasculaires, pulmonaires, rénales chroniques et aux rejets de greffes d’organes.