MSCs alleviate LPS-induced acute lung injury by inhibiting the proinflammatory function of macrophages in mouse lung organoid-macrophage model

Abstract

Acute lung injury (ALI) is an inflammatory disease associated with alveolar injury, subsequent macrophage activation, inflammatory cell infiltration, and cytokine production. Mesenchymal stem cells (MSCs) are beneficial for application in the treatment of inflammatory diseases due to their immunomodulatory effects. However, the mechanisms of regulatory effects by MSCs on macrophages in ALI need more in-depth study. Lung tissues were collected from mice for mouse lung organoid construction. Alveolar macrophages (AMs) derived from bronchoalveolar lavage and interstitial macrophages (IMs) derived from lung tissue were co-cultured, with novel matrigel-spreading lung organoids to construct an in vitro model of lung organoids-immune cells. Mouse compact bone-derived MSCs were co-cultured with organoids-macrophages to confirm their therapeutic effect on acute lung injury. Changes in transcriptome expression profile were analyzed by RNA sequencing. Well-established lung organoids expressed various lung cell type-specific markers. Lung organoids grown on spreading matrigel had the property of functional cells growing outside the lumen. Lipopolysaccharide (LPS)-induced injury promoted macrophage chemotaxis toward lung organoids and enhanced the expression of inflammation-associated genes in inflammation-injured lung organoids-macrophages compared with controls. Treatment with MSCs inhibited the injury progress and reduced the levels of inflammatory components. Furthermore, through the nuclear factor-κB pathway, MSC treatment inhibited inflammatory and phenotypic transformation of AMs and modulated the antigen-presenting function of IMs, thereby affecting the inflammatory phenotype of lung organoids. Lung organoids grown by spreading matrigel facilitate the reception of external stimuli and the construction of in vitro models containing immune cells, which is a potential novel model for disease research. MSCs exert protective effects against lung injury by regulating different functions of AMs and IMs in the lung, indicating a potential mechanism for therapeutic intervention.

Keywords: Acute lung injury; Immunoregulation; Mesenchymal stem cell; Organoid–macrophage co-culture.

Fig. 1

Construction and characterization of mouse lung organoids. A Schematic of the experimental strategy for isolation and culture of mouse lung organoids. B Progressive growth over time of primary (P0) lung organoids of mouse lung tissue origin (scale bar: 100 μm). C Representative images of mouse lung tissue-derived lung organoids taken under a scanning electron microscope. The lung organoids present intact 3D spherical structures. The white arrows indicate the ciliated structures of the lung epithelial cells observable on the ruptured inner surface (scale bar: 10 μm). D Representative images of lung organoids of mouse lung tissue origin taken under transmission electron microscopy, with white arrows indicating the presence of typical ciliated cells in the cultured lung organoids, and microtubule structures visible inside the ciliated cells. E Representative images of lung organoids of mouse lung tissue origin taken under a laser confocal microscope, where lung organoids were observed to contain multiple cell types expressing the ciliated cell-specific marker α-tubulin (red), the goblet cell-specific marker MUC5AC (green), the AT2 cell-specific marker SFTPC (red), the club cell-specific marker CC10 (red), and the basal cell-specific marker KRT5 (red) (scale bar: 50 μm)

Fig. 2

Matrigel-spreading growth alters the polarization properties of lung organoids. A Schematic of comparison growth pattern between mouse lung organoids wrapped in and laid on matrigel. B Representative image of hematoxylin and eosin (HE) staining of mouse lung organoids grown wrapped in matrigel, showing simple spherical structures (scale bar: 100 μm). C Representative image of HE staining of mouse lung organoids grown by matrigel spreading with thicker cell layer structure (scale bar: 100 μm). D Scanning electron microscope representative image of mouse lung organoids grown by matrigel spreading, with ciliated cluster structures with long cilia observed on the surface of the organoids (scale bar: 10 μm). E Representative image of immunohistochemical staining of mouse lung organoids grown by matrigel spreading, with ciliated cells growing outward toward the lumen and basal cells not wrapping around the periphery of the organoid alone, but showing polarized flip-flop growth features (scale bar: 50 μm)

Fig. 3

Lung organoids–immune cells model of mice grown by matrigel-spreading better simulates lung tissue structure. A Flow cytometry analysis to identify AMs isolated from bronchoalveolar lavage fluid. B Schematic diagram of lung organoids and AMs co-culture wrapped in matrigel. Representative photomicrographs of lung organoids and AMs co-culture wrapped in matrigel (scale bar: 100 μm). C Schematic diagram of lung organoids and AMs co-culture laid on matrigel spreading. Representative photomicrographs of lung organoids and AMs co-cultured under matrigel-spreading conditions (scale bar: 100 μm). D Representative image of immunohistochemical staining of lung organoids co-cultured with AMs, where F4/80-positive AMs could be present in the lumen of the organoid to better mimic the in vivo tissue structure (scale bar: 50 μm)

Fig. 4

MSCs suppress lung organoids’ inflammatory responses by modulating AMs. A Representative photomicrographs of lung organoids–AMs, lung organoids–AMs subjected to LPS stimulation for 72 h, and lung organoids–AMs subjected to lipopolysaccharide (LPS) stimulation for 72 h while co-cultured with MSCs under the condition of matrigel spreading (scale bar: 100 μm). The black arrows indicate AMs aggregating toward lung organoids under LPS stimulation. B Representative image of transmission electron microscopy of lung organoids–AMs after 72 h of LPS stimulation (scale bar: 5 μm). The white arrows indicate AMs. C Representative scanning electron microscopy images of lung organoids–AMs after 72 h of LPS stimulation (scale bar: 50 μm). The white arrows indicate AMs. D Statistical plots of mRNA levels of CCL3, CCL4, CCL5, CXCL1, CXCL2, IL-1β, IL-6, and TNF-α expression in lung organoids–AMs with LPS stimulation and co-culture with MSCs (n = 5, *P < 0.05, **P < 0.01, ***P < 0.001). E Representative graphs of ROS detection in lung organoids–AMs subjected to LPS stimulation for 72 h while co-cultured with MSCs (scale bar: 50 μm). The white arrows indicate AMs showing green fluorescence, with high expression of ROS. F Representative images of western blotting of NLRP3 in lung organoids–AMs subjected to LPS stimulation for 72 h while co-cultured with MSCs

Fig. 5

MSCs suppress lung organoids’ inflammatory responses by modulating IMs. A Representative photomicrographs of lung organoids–IMs, lung organoids–IMs subjected to LPS stimulation for 72 h, and lung organoids–IMs subjected to LPS stimulation for 72 h while co-cultured with MSCs under the condition of matrigel spreading (scale bar: 100 μm). The black arrows indicate IMs aggregating toward lung organoids under LPS stimulation. B Representative image of transmission electron microscopy of lung organoids–IMs after 72 h of LPS stimulation (scale bar: 5 μm). The white arrows indicate IMs. C Representative scanning electron microscopy images of lung organoids–IMs after 72 h of LPS stimulation (scale bar: 25 μm). The white arrows indicate IMs. D Statistical plots of mRNA levels of CCL3, CCL4, CCL5, CXCL1, CXCL2, IL-1β, IL-6, and TNF-α expression in lung organoids–IMs with LPS stimulation and co-culture with MSCs (n = 5, *P < 0.05, **P < 0.01, ***P < 0.001). E Representative graphs of ROS detection in lung organoids–IMs subjected to LPS stimulation for 72 h while co-cultured with MSCs (scale bar: 50 μm). The white arrows indicate IMs showing green fluorescence, with high expression of ROS. F Representative images of western blotting of NLRP3 in lung organoids–IMs subjected to LPS stimulation for 72 h while co-cultured with MSCs

Fig. 6

MSCs inhibit LPS stimulation-induced inflammatory responses in AMs through the NF-κB pathway. A PCA plots of transcriptome expression in the lung organoid–AM group, LPS lung organoid–AM group, and LPS/MSC lung organoid–AM group. B Volcano plot for DEGs between lung organoid–AM group and LPS lung organoid–AM group. C Heatmap for DEGs between lung organoid–AM group and LPS lung organoid–AM group. D KEGG pathway enrichment analysis of the upregulated genes of lung organoid–AM group after LPS stimulation. E Volcano plot for DEGs between LPS lung organoid–AM group and LPS/MSC lung organoid–AM group. F Heatmap for DEGs between LPS lung organoid–AM group and LPS/MSC lung organoid–AM group. G KEGG pathway enrichment analysis of the downregulated genes of lung organoid–AM group after LPS stimulation by MSC treatment. H GSEA of DEGs of the downregulated genes of lung organoid–AM group after LPS stimulation by MSC treatment. I Representative images of western blotting of NF-kB P65, p-NF-kB P65, and TRIM15 in lung organoids-AMs subjected to LPS stimulation for 72 h while co-cultured with MSCs. Quantitative analysis of the relative expression level of p-NF-κB P65 and NF-κB P65 (n = 3). J Expression of intracellular iNOS (M1 macrophage marker) and Arg-1 (M2 macrophage marker) in AMs after being treated with various formulations analyzed by flow cytometry (n = 4). Data are expressed as mean ± SD. *P < 0.05, ***P < 0.001

Fig. 7

MSCs inhibit LPS stimulation-induced inflammatory responses and MHC II in IMs via the NF-κB pathway. A PCA plots of transcriptome expression in the lung organoid–IM group, LPS lung organoid–IM group, and LPS/MSC lung organoid–IM group. B Volcano plot for DEGs between lung organoid–IM group and LPS lung organoid–IM group. C Heatmap for DEGs between lung organoid–IM group and LPS lung organoid–IM group. D KEGG pathway enrichment analysis of the upregulated genes of lung organoid–IM group after LPS stimulation. E Volcano plot for DEGs between LPS lung organoid–IM group and LPS/MSC lung organoid–IM group. F Heatmap for DEGs between LPS lung organoid–IM group and LPS/MSC lung organoid–IM group. G KEGG pathway enrichment analysis of the downregulated genes of lung organoid–IM group after LPS stimulation by MSC treatment. H GSEA of DEGs of the downregulated genes of lung organoid–IM group after LPS stimulation by MSC treatment. I Representative images of western blotting of NF-kB P65 and p-NF-kB P65 in lung organoids-IMs subjected to LPS stimulation for 72 h while co-cultured with MSCs. Quantitative analysis of the relative expression level of p-NF-κB P65 and NF-κB P65 (n = 3). J Expression of MHC II in IMs after being treated with various formulations analyzed by flow cytometry (n = 3–4). Data are expressed as mean ± SD. **P < 0.01, ***P< 0.001