Protocol for CUBIC Clearing and Whole Mount Imaging of Mouse Lung Lobes v1

This protocol is for Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC) of mouse lung tissue for whole lobe imaging using Zeiss Lightsheet Imaging. All experimental procedures were performed in the American Association for Accreditation of Laboratory Animal Care (AAALAC)-certified laboratory animal facility at the University of California San Diego, following protocols approved by the institutional animal care and use committee (IACUC). The procedures should incorporate all local requirements for standards of animal experimentation.

Dynamic Observation of Autophagy and Transcriptome Profiles in a Mouse Model of Bleomycin-Induced Pulmonary Fibrosis

Pulmonary fibrosis is a group of progressive, fibrotic, and fatal lung diseases, and the role of autophagy in pulmonary fibrosis is controversial. In the current research, we dynamically observed a bleomycin-induced pulmonary fibrosis mouse model after 3, 7, 14, 21, and 28 days and investigated the expression of autophagy markers. We found that autophagy markers were not significantly changed on the indicated days in the mouse lung tissue. Then, RNA-Seq was used to analyze the gene expression and associated functions and pathways in fibrotic lung tissue on different days post-bleomycin. In addition, short time series expression miner (STEM) analysis was performed to explore the temporal post-bleomycin gene expression. Through STEM, continually up- or downregulated profiles did not demonstrate the critical role of autophagy in the development of fibrosis. Furthermore, gene ontology (GO) annotations showed that continually upregulated profiles were mainly related to fibrosis synthesis, extracellular space, and inflammation, while enriched pathways were mainly related to the PI3K-Akt signaling pathway, ECM–receptor interactions, and focal adhesion signaling pathway. For continually downregulated profiles, GO annotations mainly involved sarcomere organization, muscle contraction, and muscle fiber development. The enriched KEGG signaling pathways were the cAMP signaling pathway, cGMP-PKG signaling pathway, calcium signaling pathway, and cardiac muscle contraction. Moreover, we analyzed autophagy-related genes’ expression in specific cells from a publicly available database of three human and one animal study of pulmonary fibrosis using single-cell sequencing technology. All results consistently demonstrated no critical role of autophagy in the pathogenesis of pulmonary fibrosis. In summary, autophagy may not critically and consistently change during the development of pulmonary fibrosis at different stages post-bleomycin in a mouse model. These continually up- or downregulated profiles, including gene profiles, and the corresponding functions and pathways may provide mechanistic insights into IPF therapy.

Vimentin-Rab7a Pathway Mediates the Migration of MSCs and Lead to Therapeutic Effects on ARDS

Acute respiratory distress syndrome (ARDS) is difficult to treat and has a high mortality rate. Mesenchymal stem cells (MSCs) have an important therapeutic effect in ARDS. While the mechanism of MSC migration to the lungs remains unclear, the role of MSCs is of great clinical significance. To this end, we constructed vimentin knockout mice, extracted bone MSCs from the mice, and used them for the treatment of LPS-induced ARDS. H&E staining and Masson staining of mouse lung tissue allowed us to assess the degree of damage and fibrosis of mouse lung tissue. By measuring serum TNF-α, TGF-β, and INF-γ, we were able to monitor the release of inflammatory factors. Finally, through immunoprecipitation and gene knockout experiments, we identified upstream molecules that regulate vimentin and elucidated the mechanism that mediates MSC migration. As a result, we found that MSCs from wild-type mice can significantly alleviate ARDS and reduce lung inflammation, while vimentin gene knockout reduced the therapeutic effect of MSCs in ARDS. Cytological experiments showed that vimentin gene knockout can significantly inhibit the migration of MSCs and showed that it changes the proliferation and differentiation status of MSCs. Further experiments found that vimentin’s regulation of MSC migration is mainly mediated by Rab7a. Rab7a knockout blocked the migration of MSCs and weakened the therapeutic effect of MSCs in ARDS. In conclusion, we have shown that the Vimentin-Rab7a pathway mediates migration of MSCs and leads to therapeutic effects in ARDS.

Immune-Associated Proteins Are Enriched in Lung Tissue-Derived Extracellular Vesicles during Allergen-Induced Eosinophilic Airway Inflammation

Studying the proteomes of tissue-derived extracellular vesicles (EVs) can lead to the identification of biomarkers of disease and can provide a better understanding of cell-to-cell communication in both healthy and diseased tissue. The aim of this study was to apply our previously established tissue-derived EV isolation protocol to mouse lungs in order to determine the changes in the proteomes of lung tissue-derived EVs during allergen-induced eosinophilic airway inflammation. A mouse model for allergic airway inflammation was used by sensitizing the mice intraperitoneal with ovalbumin (OVA), and one week after the final sensitization, the mice were challenged intranasal with OVA or PBS. The animals were sacrificed 24 h after the final challenge, and their lungs were removed and sliced into smaller pieces that were incubated in culture media with DNase I and Collagenase D for 30 min at 37 °C. Vesicles were isolated from the medium by ultracentrifugation and bottom-loaded iodixanol density cushions, and the proteomes were determined using quantitative mass spectrometry. More EVs were present in the lungs of the OVA-challenged mice compared to the PBS-challenged control mice. In total, 4510 proteins were quantified in all samples. Among them, over 1000 proteins were significantly altered (fold change >2), with 614 proteins being increased and 425 proteins being decreased in the EVs from OVA-challenged mice compared to EVs from PBS-challenged animals. The associated cellular components and biological processes were analyzed for the altered EV proteins, and the proteins enriched during allergen-induced airway inflammation were mainly associated with gene ontology (GO) terms related to immune responses. In conclusion, EVs can be isolated from mouse lung tissue, and the EVs’ proteomes undergo changes in response to allergen-induced airway inflammation. This suggests that the composition of lung-derived EVs is altered in diseases associated with inflammation of the lung, which may have implications in type-2 driven eosinophilic asthma pathogenesis.

Co-Modification With Mesenchymal Stem Cells Membrane and PDA Prevents Fe3O4-Induced Pulmonary Toxicity in Micevia AMPK-ULK1 Axis

Background: Fe3O4 nanoparticles are widely used in the diagnosis and treatment of diseases, but the toxicity should not be ignored. It has been reported that PDA modification can reduce the toxicity of Fe3O4 and increase the biocompatibility. However, a better modification method is still worth studying. We have developed a new method to coat Fe3O4@PDA nanoparticles with mesenchymal stem cells membrane (MSCM) and evaluated the lung toxicity of the modified particles to mice.Result: We found that MSCM modification significantly reduced the lung injury induced by Fe3O4 nanoparticles in mice. Compared with Fe3O4@PDA nanoparticles, co-modification with MSCM and PDA modification significantly reduced autophagy and apoptosis of mouse lung tissue, and reduced the activation of autophagy pathway AMPK-ULK1 axis. Thus, co-modification with MSCM and PDA prevents Fe3O4-induced pulmonary toxicity in mice by inhibiting the AMPK-ULK1 derived autophagy.Conclusion: MSCM coated Fe3O4@PDA nanoparticles were demonstrated to prevent lung damage from autophagy and reduce the toxicity of iron oxide nanomaterials. The co-modification of PDA and MSCM can improve the biocompatibility and facilitate their further bioapplication.

Identification and Functional Analysis of EPOR+ Tumor-Associated Macrophages in Human Osteosarcoma Lung Metastasis

Background. Tissue-resident macrophages can be educated to tumor-associated macrophages (TAMs) by the tumor microenvironment and many types of macrophages express erythropoietic receptor (EPOR); However, little is known about the expression of EPOR on TAMs and the identity of EPOR+ TAMs in osteosarcoma lung metastasis has thus far remained elusive. Methods. EPOR-eGFPcre mice were used to determine the expression of EPOR on lung tissue-resident macrophages. Flow cytometry, RT-PCR, and Western blot were examined to define the identity of EPOR+ TAMs in 106 osteosarcoma lung metastasis specimens. Moreover, the clinicopathologic factors and prognosis of patients with CD163+EPOR+ macrophages were compared. Results. We found that a subpopulation of mouse lung tissue-resident macrophages express EPOR and EPO enhances the proliferation of EPOR+ macrophages in mouse lung. A subpopulation of CD163+ macrophages expresses EPOR in human osteosarcoma lung metastasis specimens. CD163+EPOR+TAMs increase 2.5 times in human osteosarcoma lung metastasis tissues; CD206, CD163, and PD1, which are known to have a significant role in TAM function had high expression in CD163+EPOR+ TAMs compared with CD163+EPOR- TAMs. Furthermore, CD163+EPOR+ TAMs had higher M2 marker and cytokine expression in osteosarcoma tissues compared with para-osteosarcoma tissues. EPO enhanced the expression of M2 cytokines in primary CD163+EPOR+ TAMs. Importantly, the percentage of CD163+EPOR+ TAMs had a positive linear association with malignant phenotypes as well as poor disease-free survival and overall survival time. Conclusions. We have characterized TAMs expressing EPOR and CD163+EPOR+ macrophages as TAMs in osteosarcoma lung metastasis patients, which are highly associated with tumor aggressiveness.

4-phenylbutyric acid alleviates bleomycin-induced pulmonary fibrosis in mouse via inhibition of endoplasmic reticulum stress

Background: 4-phenylbutyric acid (4-PBA) is a chemical chaperone that may aid the folding of proteins and alleviate endoplasmic reticulum (ER) stress by inhibiting the unfolded protein response (UPR). This study explores the effects of 4-PBA on idiopathic pulmonary fibrosis (IPF) using a murine model of bleomycin (BLM)-induced pulmonary fibrosis.Methods: Pulmonary fibrosis was induced in C57BL/6 mice by intratracheal injection of BLM. Sixty mice were randomly allocated into three groups: BLM group (n=20), BLM+4-PBA group (n=20), and control group (n=20). Lung tissues and lung function were analyzed to evaluate the degree of pulmonary fibrosis and the survival of the mice was noted. The expression levels of the ER stress markers activating transcription factor 6 (ATF6) and C/EBP Homologous Protein (CHOP) were analyzed in lung tissues from IPF patients and healthy controls as well as the mice.Results: Lung tissues from IPF patients expressed significantly higher levels of ATF6 and CHOP compared to those from healthy controls. BLM induced significant collagen deposition in the lungs of the mice, which was prevented by 4-PBA. 4-PBA also dramatically improved pulmonary function and increased the survival rate in the BLM+4-PBA group compared to that in the BLM group. Both the protein and mRNA expression levels of ATF6 and CHOP were significantly reduced in mouse lung tissue after 2 weeks of 4-PBA treatment.Conclusions: This study demonstrated that 4-PBA treatment could alleviate BLM-induced pulmonary fibrosis in mice via the attenuation of ER stress.

Metabolomic Analysis Reveals Unique Biochemical Signatures Associated with Protection from Radiation Induced Lung Injury by Lack of cd47 Receptor Gene Expression

The goal of this study was to interrogate biochemical profiles manifested in mouse lung tissue originating from wild type (WT) and cd47 null mice with the aim of revealing the in vivo role of CD47 in the metabolic response to ionizing radiation, especially changes related to the known association of CD47 deficiency with increased tissue viability and survival. For this objective, we performed global metabolomic analysis in mouse lung tissue collected from (C57Bl/6 background) WT and cd47 null mice with and without exposure to 7.6 Gy whole body radiation. Principal component analysis and hierarchical clustering revealed a consistent separation between genotypes following radiation exposure. Random forest analysis also revealed a unique biochemical signature in WT and cd47 null mice following treatment. Our data show that cd47 null irradiated lung tissue activates a unique set of metabolic pathways that facilitate the handling of reactive oxygen species, lipid metabolism, nucleotide metabolism and nutrient metabolites which may be regulated by microbial processing. Given that cd47 has pleiotropic effects on responses to ionizing radiation, we not only propose this receptor as a therapeutic target but postulate that the biomarkers regulated in this study associated with radioprotection are potential mitigators of radiation-associated pathologies, including the onset of pulmonary disease.