Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3−/− mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

Histology data was analysed using the computer-assisted stereological toolbox software Visopharm Integrator System (VIS) v6.0.0.1765 (newCAST, Visiopharm). The Seurat R package v3.2.2 was used for analysis of the RNA sequencing data. Immunofluorescence images were processed using ZEN 2.6 (blue edition). Immunofluorescence intensities were analysed using ImageJ (v1.52p). Histology images were processed using CaseViewer 2.4 and Slide Converter 2.3.2 (3DHISTECH). FACS data were analysed with FlowJo v10.0.7r2 software (FlowJo LLC, BD). Western blot bands were quantified using ImageJ (v1.52p). Data collected from Whole-cell patch-clamp experiments were analysed using the software IGOR Pro v6 (WaveMetrics). Data collected from whole-EE, whole-RE and whole-LE/LY patch clamp experiments were analysed using the softwares OriginPro v6.  Fig. 1g): one sample needed to be excluded due to mistakes during sample processing MMP-12 ELISA of BALF (as shown in Fig. 4c): one extremeley high outlier for each group, respectively, was identified and excluded Counts of E-fibers/field (as shown in Fig. 4n): one outlier was identified and excluded from the group "PBS, BL6 WT" TIMP-2 ELISA of BALF (as shown in Fig. 8b): one outlier was identified and excluded from the group "WT (BL6)" MCL measurements (as shown in Fig. 9b): one outlier was identified and excluded from the group "Mcoln3tm1.1Jga, CS" All findings were reproducible with group sizes and number of independent repeats as mentioned in the figure legends or methods.
For quantification of airspace enlargements the computer-assisted stereological toolbox software Visopharm Integrator System randomly Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging chose 30 fields of view per lung/replica to be analysed. For lung function measurements grouping of mice was not performed randomly to ensure that the mice within groups have the same gender, age and correct genotype. For all other experiments based on cells or samples isolated from mice, WT and TRPML3 KO mice were processed in parallel and mice were specifically chosen to match age and gender between the genotypes. For all in vitro cell culture experiments wells were randomly allocated to experimental groups.

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Only well established and commercially avaible antibodies were used. Validation of commercial antibodies was carried out by the manufacturer performing regular quality control of each lot as stated in the data sheet or on the manufacturers website. E.g. Biolegend: "Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis." E.g. Cell Signaling: "When an antibody is recommend for a particular application, it indicates that the antibody has passed rigorous application-specific testing standards." E.g. ThermoFisher: Antibodies undergo a "rigorous 2-part testing approach" including "Target specifity verification" and "Functional application validation". E.g. Miltenyi Biotec: Antibodies are tested for "Lot-to-lot consistent performance". Antibody specifity is validated using "Knockout validation via target genome editing". Antibody sensitivity is evaluated through "Functional testing of every product prior to release". E.g. Merck (Sigma): "Each of the thousands of antibodies in our portfolio are certified through our standard validation process to ensure quality and reproducibility." E.g. Abcam: "To confirm antibody specificity, we have introduced knockout validation as a standard level of validation, with over 800 knockout-validated antibodies and counting." All antibodies were used for applications that were tested by the manufacturer, as specified on the respective data sheet. We validated dilutions of antibodies based on recommendations of the manufacturer or on their use in previous publications.
The cell line was routinely tested, and tested negative for Mycoplasma.

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Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.