Mitofusins regulate lipid metabolism to mediate the development of lung fibrosis

Accumulating evidence illustrates a fundamental role for mitochondria in lung alveolar type 2 epithelial cell (AEC2) dysfunction in the pathogenesis of idiopathic pulmonary fibrosis. However, the role of mitochondrial fusion in AEC2 function and lung fibrosis development remains unknown. Here we report that the absence of the mitochondrial fusion proteins mitofusin1 (MFN1) and mitofusin2 (MFN2) in murine AEC2 cells leads to morbidity and mortality associated with spontaneous lung fibrosis. We uncover a crucial role for MFN1 and MFN2 in the production of surfactant lipids with MFN1 and MFN2 regulating the synthesis of phospholipids and cholesterol in AEC2 cells. Loss of MFN1, MFN2 or inhibiting lipid synthesis via fatty acid synthase deficiency in AEC2 cells exacerbates bleomycin-induced lung fibrosis. We propose a tenet that mitochondrial fusion and lipid metabolism are tightly linked to regulate AEC2 cell injury and subsequent fibrotic remodeling in the lung.

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Software and code
Policy information about availability of computer code Data collection n/a Data analysis 1. Analyses of TEM Images, immunoblots, and immunofluorescent images were performed using FIJI running Image J software (version 1.52b). 2. FlowJo analytical software (version 10) was used for analyses of flow cytometric data. 3. For RNA-seq analysis: The raw sequencing reads in binary base call (BCL) format were processed through bcl2fastq 2.19 (Illumina) for FASTQ format conversion and demultiplexing. RNA reads were aligned and mapped to the mm9 mouse reference genome by TopHAEC2 (version 2.0.11), and transcriptome reconstruction was performed by Cufflinks (version 2.1.1). The abundance of transcripts was measured with Cufflinks in Fragments Per Kilobase of exon model per Million mapped reads (FPKM). Differentially expressed genes were identified using the limma package (http://bioconductor.org/packages/release/bioc/html/limma.html). To assess the differential expression, p-values were derived from linear modelling and empirical Bayes moderation and adjusted for multiple testing by the Benjamini-Hochberg method. Gene ontology (GO) over-representation analysis was performed using the clusterProfiler package (http:// bioconductor.org/packages/release/bioc/html/clusterProfiler.html). The enrichment maps were visualized by Cytoscape (version 3.6.1), and the functional clusters were highlighted and labeled manually. Heat maps were plotted using Heatmap Illustrator software (Heml 1.0) (hemi.biocuckoo.org) and the pheatmap package (https://github.com/raivokolde/pheatmap), based on the z scores calculated using the gene expressions by FPKM. 4. For heatmap generation using lipidomic data, Heatmap Illustrator software (Heml 1.0) (hemi.biocuckoo.org) was used. 5. All statistical analyses were performed using SPSS version 17.0 (IBM Corporation) or GraphPad Prism version 5.0 (GraphPad Software) For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

October 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Sample sizes for all experiments were made as large as possible to ensure the robustness of the results. For bleomycin-induced lung fibrosis, many experiments include at least 9 mice, and at least 2-3 individual experiments were performed. Statistics were calculated across all biological and technical replicates. For RNA sequencing analysis, AEC2 cells from at least 3-4 mice per group were isolated. For lipidomic analysis, AEC2 cells from 3-8 mice were isolated to form 3-4 samples. For TEM image analysis, AEC2 cells from 2-3 mice were randomly selected for quantification. All the details about the sample size for each experiment were described in the respective figure legends.
Data exclusions No data were excluded.

Replication
All the reported findings are based on multiple biological and technical repeats, and can be reproduced without difficulties.
Randomization For bleomycin-induced lung fibrosis model, mice in different experimental groups were matched for age, gender and weight, and were selected randomly among mice with specific genotypes. Both males and females were used in the study. For image analysis, AEC2 or MLE 12 cells were randomly selected for quantification.

Blinding
The genotyping and experimental conditions, such as bleomycin treatment, of the animals was known to the investigators. The blinding to the experimental conditions is difficult since bleomycin treatment frequently leads to wasting and respiratory distress of the mice, particularly in the knockout mice. For image analysis, such as quantification of the metrics related to mitochondrial morphology, blinding to the condition was attempted as possible to ensure unbiased observations.

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Field-collected samples
No field-collected samples were used in this study.

Ethics oversight
All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee at Weill Cornell Medicine Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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Methodology
Sample preparation All samples are whole lung single cell suspensions, with or without CD45 negative selection and EpCAM positive selection. In brief, whole lung cell suspension was obtained after dispase digestion, followed by homogenization. DAPI was added for cell viability detection during flow cytometric sorting. Please refer to Online methods for details.

Instrument
Flow cytometric analysis was performed using a LSRFortessa cell analyzer, while flow cytometric cell sorting was performed by an Influx cell sorter (BD Biosciences).