Microphysiological Models of Lung Epithelium-Alveolar Macrophage Co-Cultures to Study Chronic Lung Disease

Abstract

The interactions between immune cells and epithelial cells influence the progression of many respiratory diseases, such as chronic obstructive pulmonary disease (COPD). In vitro models allow for the examination of cells in controlled environments. However, these models lack the complex 3D architecture and vast multicellular interactions between the lung resident cells and infiltrating immune cells that can mediate cellular response to insults. In this study, three complementary microphysiological systems are presented to delineate the effects of cigarette smoke and respiratory disease on the lung epithelium. First, the Transwell system allows the co-culture of pulmonary immune and epithelial cells to evaluate cellular and monolayer phenotypic changes in response to cigarette smoke exposure. Next, the human and mouse precision-cut lung slices system provides a physiologically relevant model to study the effects of chronic insults like cigarette smoke with the dissection of specific interaction of immune cell subtypes within the structurally complex tissue environment. Finally, the lung-on-a-chip model provides an adaptable system for live imaging of polarized epithelial tissues that mimic the in vivo environment of the airways. Using a combination of these models, a complementary approach is provided to better address the intricate mechanisms of lung disease.

Keywords: COPD; PCLS; alveolar macrophages; cigarette smoke; immune cells co‐cultures; lung epithelium; microphysiological systems.

Figure 1.

Microphysiological models of coculture and cigarette smoke of the lung epithelium. (A) Diagram of Transwell-based airway epithelial and alveolar macrophage coculture. (B) Diagram of the 3-compartment lung-on-a-chip model, with a cell-laden gel in the central channel and human bronchial airway epithelial cells growing on the wall between the epithelial and gel compartments to form a barrier. (D) PCLS explant cultures exposed to direct cigarette smoke using our in-house developed smoking chamber. Diagram created using BioRender.com.

Figure 2.

Immunostaining of murine cocultures of epithelial and immune cells. (A) Murine airway epithelial cells cocultured with alveolar macrophages (AMs), scale bar: 70 μm. (B) Murine airway epithelial cells cocultured with PBMCs, scale bar: 25 μm. (C) Control staining of monocultured alveolar macrophages, scale bar: 70 μm. (D) Control staining of PBMCs, which lack Siglec F expression, scale bar: 25 μm. Fluorescently labeled surface markers identify macrophages after 7 days of coculture. Alveolar macrophage markers CD11c (green, left) and Siglec F (red, center) are present in both cocultures, indicating an altered phenotype in the cocultured PBMCs. Merged images (right) are shown with nuclear Hoechst stain (blue).

Figure 3.

Graphical representation of the cigarette smoke exposure model optimized using our in-house developed smoking chamber.16 (A) Diagram of the Transwell holding inside the chamber. (B) Image of our device with a PCLS holding Transwell as well as a cell monolayer Transwell. Diagram created using BioRender.com.

Figure 4.

Identification of murine alveolar macrophage polarization after coculture and exposure to cigarette smoke. (A) Graphical representation of macrophage polarization after coculture and exposure to cigarette smoke as determined by FACS. All numbers of reported M1 and M2 cells are represented by fold change as compared to sorted M1/M2 macrophages in unexposed cocultures. (B) CD11c vs. CD206 expression profiles of unexposed cocultured murine alveolar macrophages. (C) CD11c vs. CD206 expression profiles of 1-day CS and (D) air-exposed cocultured murine alveolar macrophages. (E) CD11c vs. CD206 expression profiles of 10-day CS and (F) air-exposed cocultured murine alveolar macrophages. (G) CD11c vs. CD206 expression profiles of 10-day CS and (H) air-exposed cocultured murine alveolar macrophages after an additional 10 days of recovery.

Figure 5.

Fluorescent labeling of F-actin in Transwell models of human and murine airway epithelial cells and macrophage coculture. (A) Fluorescence recovery of murine airway epithelial cells and alveolar macrophages exposed to 10 days of air or cigarette smoke. (B) Fluorescence recovery of murine epithelial cells with added alveolar macrophages. (C) Fluorescence recovery of human airway epithelial cells with added differentiated THP-1 macrophages had a similar increase in recovery in response to coculture. (D) Fluorescence recovery of human airway epithelial cells coculture with differentiated THP-1 macrophages. (E) Human airway bronchial epithelial cells and differentiated THP-1 macrophages exposed to 1 day of CS. (F) Human airway bronchial epithelial cells and differentiated THP-1 macrophages were exposed to 10 days of CS 10-day of CS. Error bars = SE, n=5.

Figure 6.

Human precision-cut lung slices cigarette smoke exposure model. Three different normal human donors and seven replicate slices per experimental condition. P values are shown at the top of each graph in bold. (A) H&E of normal human lung exposed to humidified air. (B) H&E of normal human lungs exposed to cigarette smoke (CS). (C) LDH release of each replicate slice, shown as averages of triplicate values per slice. (D) MLI analysis of H&E slides shows the average width and height of vertical and horizontal chords measured per image. (E) TMRM fluorescence of human PCLS slices is shown as averages of triplicate values per slice. (F) Metabolic activity results obtained using the Alamar blue assay are shown as the average of the duplicate values of each slice. Scale bar 2 mm.

Figure 7.

Fluorescence confocal live imaging of coculture murine PCLS. Images were taken with a 63x oil objective using the Zeiss LSM 880 Confocal with Airyscan FAST Module. Each experimental condition was performed in replicates of 6. Alveolar Macrophages (AMs) (green), F-actin (red), DAPI (blue). (A) CFMDA stained isolated alveolar macrophages. (B) control PCLS pre-stained with Cell Mask deep red Actin tracking stain plus added CFMDA labeled macrophages. (C) CS exposure experimental setup. (Air + AMs) PCLS with added alveolar macrophages exposed to air. (Second Air + AMs) different z-plane of PCLS exposed to air showing area with more infiltrating alveolar macrophages and less polymerized actin. (CS + No AMs) PCLS without added alveolar macrophages exposed to Cigarette smoke (CS). (CS + AMs) PCLS with added alveolar macrophages exposed to CS. (Second CS + AMs) different z-plane of PCLS exposed to CS showing area with more infiltrating alveolar macrophages and less polymerized actin. Scale bar: 10 μm.

Figure 8.

H&E histology Images of murine PCLS. Images were taken digitally using the Concentriq digital pathology website at 20x magnification. (A) H&E of PCLS with no added alveolar macrophages (AMs). (B) H&E of PCLS with added AMs (WT + Air-Airway) Airways of Wild Type (B6) PCLS exposed to air. (WT + Air-Alveoli) Alveoli of Wild Type (B6) PCLS exposed to air. (WT + CS-Airway) Airway of Wild Type (B6) PCLS exposed to cigarette smoke (CS). (WT + CS-Alveoli) Alveoli of Wild Type (B6) PCLS exposed to CS. Scale bar: 200 mm.

Figure 9.

Culture of human airway epithelial cells on a lung-on-a-chip. (A) Schematic of the 3-channel lung chip showing the placement of epithelial cells and macrophages in each channel. The central chamber is filled with suspended macrophages in Matrigel. The epithelial cells grow on the interface between the epithelial and gel compartments at a 90-degree angle to form a barrier between the two compartments. The third channel provides media to the chip when the epithelial compartment is filled with air. (B) Image of the PDMS chip prior to seeding cells, scale bar: 3 mm. (C) Brightfield image of the interface between the epithelial and acellular gel compartment after epithelial cells have grown to confluence on the epithelial monoculture chip, scale bar: 300 μm. (D) Brightfield image of the interface between the epithelial and THP-1-seeded gel channel after epithelial cells have grown to confluence on the epithelial-immune coculture chip. Differentiated THP-1 macrophages are suspended in Matrigel in the gel channel, and have moved to the epithelial channel, scale bar: 300 μm. (E) Brightfield image of the endothelial chip, at the interface between the cellular channel and acellular gel compartment after human endothelial cells have grown to confluence, scale bar: 300 μm. (F) Confocal immunofluorescence image of the monoculture epithelial chip at air-liquid interface. Human bronchial epithelial cells stained for nuclei (blue) and E-cadherin (green) grow on the epithelial channel-gel compartment after 1 week of air-liquid interface culture, scale bar: 25 μm. (G) Confocal immunofluorescence image of endothelial chip in submerged culture. Human primary microvascular endothelial cells stained for nuclei (blue) and CD31 (red) grown in the endothelial channel, scale bar: 50 μm.