We developed a decellularized murine lung matrix bioreactor system that could be used to evaluate the potential of stem cells to regenerate lung tissue. Lungs from 2-3-month-old C57BL/6 female mice were excised en bloc with the trachea and heart, and decellularized with sequential solutions of distilled water, detergents, NaCl, and porcine pancreatic DNase. The remaining matrix was cannulated and suspended in small airway growth medium, attached to a ventilator to simulate normal, murine breathing-induced stretch. After 7 days in an incubator, lung matrices were analyzed histologically. Scanning electron microscopy and histochemical staining demonstrated that the pulmonary matrix was intact and that the geographic placement of the proximal and distal airways, alveoli and vessels, and the basement membrane of these structures all remained intact. Decellularization was confirmed by the absence of nuclear 4',6-diamidino-2-phenylindole staining and negative polymerase chain reaction for genomic DNA. Collagen content was maintained at normal levels. Elastin, laminin, and glycosaminglycans were also present, although at lower levels compared to nondecellularized lungs. The decellularized lung matrix bioreactor was capable of supporting growth of fetal alveolar type II cells. Analysis of day 7 cryosections of fetal-cell-injected lung matrices showed pro-Sp-C, cytokeratin 18, and 4',6-diamidino-2-phenylindole-positive cells lining alveolar areas that appeared to be attached to the matrix. These data illustrate the potential of using decellularized lungs as a natural three-dimensional bioengineering matrix as well as provide a model for the study of lung regeneration from pulmonary stem cells.

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Development of a Decellularized Lung Bioreactor System for Bioengineering the Lung: The Matrix Reloaded

When animals are infected with helminthic parasites, resistant hosts show type II helper T immune responses to expel worms. Recently, natural helper (NH) cells or nuocytes, newly identified type II innate lymphoid cells, are shown to express ST2 (IL-33 receptor) and produce IL-5 and IL-13 when stimulated with IL-33. Here we show the relevant roles of endogenous IL-33 for Strongyloides venezuelensis infection-induced lung eosinophilic inflammation by using Il33−/− mice. Alveolar epithelial type II cells (ATII) express IL-33 in their nucleus. Infection with S. venezuelensis or intranasal administration of chitin increases in the number of ATII cells and the level of IL-33. S. venezuelensis infection induces pulmonary accumulation of NH cells, which, after being stimulated with IL-33, proliferate and produce IL-5 and IL-13. Furthermore, S. venezuelensis infected Rag2−/− mice increase the number of ATII cells, NH cells, and eosinophils and the expression of IL-33 in their lungs. Finally, IL-33–stimulated NH cells induce lung eosinophilic inflammation and might aid to expel infected worms in the lungs.

https://www.pnas.org/content/pnas/109/9/3451.full.pdf

Contribution of IL-33–activated type II innate lymphoid cells to pulmonary eosinophilia in intestinal nematode-infected mice

Interleukin-33 (IL-33) is an IL-1 family cytokine that signals via its receptor T1/ST2, and is a key regulator of inflammation, notably the type-2 response implicated in allergic asthma. Critical to our understanding of the role of IL-33 is the identification of the cellular sources of IL-33. Although progress has been made in this area, the development of a robust live cell reporter of expression would allow the localisation of IL-33 during ongoing immune responses. We have generated a fluorescent reporter mouse line, Il33Cit/+, to define the expression profile of IL-33 in vivo and demonstrate its temporal and spatial expression during experimental allergic asthma responses. We found that type-2 pneumocytes constitute the major source of IL-33 upon allergic lung inflammation following exposure to OVA, fungal extract or ragweed pollen. Using Il33Cit/Cit mice (IL-33-deficient), we establish a role for IL-33 early in the initiation of type-2 responses and the induction of nuocytes (ILC2). We also demonstrate a potential mechanism of action by which IL-33 rapidly initiates type-2 immune responses. Il33Cit/+ mice have enabled new insights into the initiation of type-2 responses and will provide an important tool for further dissection of this important inflammatory pathway in vivo.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/eji.201242863

IL-33 citrine reporter mice reveal the temporal and spatial expression of IL-33 during allergic lung inflammation

Idiopathic pulmonary fibrosis (IPF) is a chronic fibroproliferative pulmonary disorder for which there are currently no treatments. Although the etiology of IPF is unknown, dysregulated TGF-β signaling has been implicated in its pathogenesis. Recent studies also suggest a central role for abnormal epithelial repair. In this study, we sought to elucidate the function of epithelial TGF-β signaling via TGF-β receptor II (TβRII) and its contribution to fibrosis by generating mice in which TβRII was specifically inactivated in mouse lung epithelium. These mice, which are referred to herein as TβRIINkx2.1-cre mice, were used to determine the impact of TβRII inactivation on (a) embryonic lung morphogenesis in vivo; and (b) the epithelial cell response to TGF-β signaling in vitro and in a bleomycin-induced, TGF-β–mediated mouse model of pulmonary fibrosis. Although postnatally viable with no discernible abnormalities in lung morphogenesis and epithelial cell differentiation, TβRIINkx2.1-cre mice developed emphysema, suggesting a requirement for epithelial TβRII in alveolar homeostasis. Absence of TβRII increased phosphorylation of Smad2 and decreased, but did not entirely block, phosphorylation of Smad3 in response to endogenous/physiologic TGF-β. However, TβRIINkx2.1-cre mice exhibited increased survival and resistance to bleomycin-induced pulmonary fibrosis. To our knowledge, these findings are the first to demonstrate a specific role for TGF-β signaling in the lung epithelium in the pathogenesis of pulmonary fibrosis.

/pdf/epithelium-specific-deletion-of-tgf-b-receptor-type-ii-1svlv5vym3.pdf

Epithelium-specific deletion of TGF-β receptor type II protects mice from bleomycin-induced pulmonary fibrosis

Building a good lung, capable of efficient gas exchange between air and blood, poses a number of conflicting problems for physiology and cell biology. One of these is the complex architecture that allows dynamic functional integration of the lung’s structural elements (1); another is the role of cells in making a very thin and yet robust air–blood barrier, the topic of this essay. From a physiological perspective, the air–blood barrier must not only be very thin, measuring no more than a few tenths of a micrometer, but also of very large expanse, over 100 m in the human lung (2, 3). However, it must be built of biologically active cells, capable of selfmaintenance and self-renewal, as well as serving several functions beyond being a passive diffusion barrier. This holds particularly for the alveolar epithelium that forms the external sheet of the barrier (4). In the age of molecular cell biology, fascinating discoveries have advanced our understanding of the biology of these cells, obtained in sophisticated studies partly in vivo, but for a large part in vitro (4–13). It is, however, important to also consider the exceptional conditions under which these cells develop and are sustained in the complex architecture of the integral functioning lung (1). In that view, the alveolar epithelium deserves particular attention, because it must form and secure the very large surface of the air spaces. The epithelial covering of the alveolar capillaries is so extraordinarily thin that it was long believed by many prominent histologists that the capillaries were “naked like a wound” (14, 15), as light microscopy could not reveal a continuous lining by epithelial cells. It was the pioneer in electron microscopy, Frank Low (1911–1998) who showed, in 1953, that the alveoli in mice and humans were lined by an uninterrupted epithelium made of two cell types: a “large” cuboidal cell with features of a secretory cell, and a “small” squamous cell that lined the surface of capillaries with very thin cytoplasmic leaflets (16). Low called the squamous cells the type I cells and the cuboidal cells the type II cells, designations still customary today. That settled the issue of a complete epithelial lining of alveoli, and it was soon accepted, even by the former proponents of a “naked” surface (17).

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On the Tricks Alveolar Epithelial Cells Play to Make a Good Lung

We investigated the influence of extracellular matrix on transport properties of mouse alveolar epithelial cell (AEC) monolayers (MAECM) and transdifferentiation of isolated mouse alveolar epitheli...

Characterization of mouse alveolar epithelial cell monolayers

The gas exchange surface of the lungs is lined by an epithelium consisting of alveolar type (AT) I and ATII cells. ATII cells function to produce surfactant, play a role in host defense and fluid and ion transport, and serve as progenitors. ATI cells are important for gas exchange and fluid and ion transport. Our understanding of the biology of these cells depends on the investigation of isolated cells. Here, we present methods for the isolation of mouse and rat ATII cells.

Juan R. Alvarez

Papers

Characterization of mouse alveolar epithelial cell monolayers

Isolation of Rat and Mouse Alveolar Type II Epithelial Cells.