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

Abstract

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 Trpml3^{IRES-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.

Fig. 1. Characterization of TRPML3 expression in the lungs using single-cell transcriptomics and a Trpml3^{IRES-Cre/eR26-τGFP} reporter mouse model.

a Cartoon showing the breeding strategy to obtain Trpml3^{IRES-Cre/eR26-τGFP} mice. b Immunofluorescence images using antibodies against different cell markers (red) in 10 µm lung cryosections from transcardially perfused (4% PFA) Trpml3^{IRES-Cre/eR26-τGFP} mice. TRPML3 expression were visually detected in MΦ, T-cells, B-cells, AT2-cells, and Killer T-cells by colocalization analysis with the respective marker. c Quantification of data as shown in b. Percentage of cell type expressing TRPML3 was determined in five randomly chosen zoom-in sections, each (mean ± SEM). d, f FACS analysis of lung tissue and BAL of Trpml3^{IRES-Cre/eR26-τGFP} mice. Shown in d is the gating strategy used to identify TRPML3 + immune cells in the lungs. Further details are provided in the Methods section. Gating strategy and dot plots revealed TRPML3 being expressed mostly in AMΦ in the lung. e Quantitative analysis based on dot plots shown in d. Bar and pie charts show that the highest percentage of GFP + ( = TRPML3+) cells in the lung tissue corresponds to MΦ (71,58%; mean ± SEM, collected from 5 Trpml3^{IRES-Cre/eR26-τGFP} mice). f Gating strategy used to identify TRPML3+ cells in BAL isolated from Trpml3^{IRES-Cre/eR26-τGFP} mice. g Quantitative analysis based on dot plots as shown in f. Bar and pie charts show that the highest percentage of GFP + ( = TRPML3+) cells in the BAL corresponds to MΦ (97.5%; mean ± SEM, collected from 4 Trpml3^{IRES-Cre/eR26-τGFP} mice). h Transcriptomics data of single-cell suspensions from whole WT mouse lungs. Dot plot shows the percentage of cells expressing Mcoln3 using dot size and the average expression level of Mcoln3 based on the unique molecular identifier (UMI) counts. Mcoln3 expression was determined in 32 different cell types. Source data are provided as a Source Data file.

Fig. 2. Lung function parameters in WT and Trpml3^−/− mice (Mcoln3^tm1.2Hels and Mcoln3^tm1.1Jga).

Lung function measurements were performed using the SCIREQs FlexiVent System (see Methods). Different manoeuvres were applied. Single Frequency Forced Oscillation Technique (FOT) allows to study the subject’s response to a sinusoidal waveform, obtaining parameters such as Elastance (E) and Compliance (C). Broadband FOT measures the subject’s response to a signal, including a broad range of frequencies, below and above the subject’s breathing frequency. Outcomes are, e.g., Tissue Elasticity (H). Deep Inflation inflates the lungs to a total lung capacity state. Initial and end volumes are used to calculate Inspiratory Capacity (IC). Pressure-volume (PV) loops capture the quasistatic mechanical properties of the respiratory system such as Quasi-Static Compliance (Cst) and Total Lung Capacity (A). a, b In two different 4–5 months old Trpml3^−/− mouse models on different background, each (BL6 and FVB), a significant reduction of Elastance (E) of the whole respiratory system was observed, whereas the Compliance (C) was significantly increased (basal, untreated). *p < 0.05, **p < 0.01; Student’s t test, unpaired, two-tailed. c Differences of E, C, H, Cst, IC, and A in PBS versus elastase-treated 4–5 months old Trpml3^−/− and WT mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Two-way ANOVA followed by Tukey’s post hoc test. One single dot corresponds to one mouse, each in a–c. Average values are mean values ± SEM, each. d Pressure-volume (PV) loops of experiments as shown in c. Data are mean ± SEM calculated for each group. e Representative images of H&E-stained lung tissue sections from mouse lungs (BL6 WT and Trpml3^−/−) exposed to Elastase or PBS showing the respective extent of airspace enlargements. Scale bar 100 µm. f Quantification of airspace enlargement as mean linear chord length. Lung tissue sections from 6–8 mice per group were analysed. Each dot corresponds to one biologically independent lung tissue sample. Average values are mean values ± SEM, each. *p < 0.05, ****p < 0.0001; One-way ANOVA followed by Tukey’s post hoc test. Source data are provided as a Source Data file.

Fig. 3. Effect of isoform-selective TRPML3 agonist ML3-SA1 on mouse TRPML1, 2, 3 in HEK293 cells and functional characterization of endogenous TRPML3 currents in murine AMΦ organelles.

a Chemical structures of SN-2 and ML3-SA1 (= EVP-77). b Fura-2 calcium imaging experiments using HEK293 cells expressing human or murine TRPML1(NC), TRPML2, or TRPML3, respectively, indicating the specific levels of activation. Channels were stimulated with either SN-2, ML3-SA1, or ML-SA1 (10 µM, each). Shown are average values (mean ± SEM). Each dot represents one biologically independent experiment with 10–20 cells, each. ****p < 0.0001; Two-way ANOVA followed by Tukey’s post hoc test. c Dose-response curves obtained from experiments as described in b using ML3-SA1 on murine TRPML1-3 expressing HEK293 cells. d–g Representative currents from YM201636-enlarged LE/LY or Wort./Lat.B-enlarged EE isolated from murine (WT or Trpml3^−/−) primary AMΦ, elicited by an application of 10 µM ML3-SA1, respectively. h–i Statistical summary of data shown in d–g. Each dot corresponds to one biologically independent experiment. Average values are mean values ± SEM, each. In all experiments, conditions were set to evoke maximal TRPML3 current activity (neutral pH, low sodium). **p < 0.01, ***p < 0.001; One-way ANOVA followed by Tukey’s post hoc test. j Representative currents from vacuolin-enlarged Tf+ RE isolated from murine (WT or Trpml3^−/−) primary AMΦ, elicited by an application of 10 µM ML3-SA1, respectively. k qRT-PCR results for Trpml1, Trpml2, and Trpml3 in AMΦ normalized to HPRT (n = 3 biologically independent experiments, each. Average values are mean values ± SEM, each). Source data are provided as a Source Data file.

Fig. 4. Increased MMP-12 levels in WT and Trpml3^−/−.

a Quantification of the levels of different chemokines/cytokines and MMPs in BALF isolated from 4-month old WT and Trpml3^−/− mice using Multiplex analysis. b Repeated Multiplex analysis of MMP-12 levels in BALF. c, d MMP-12 quantification in BALF isolated from 4-month old WT and Trpml3^−/− mice using ELISA. One single dot corresponds to BALF from one mouse, each in c, d. e, f qRT-PCR data showing mRNA expression levels of Mmp-12 in AMΦ (WT and Trpml3^−/−). g Quantification of total cell numbers in BALF using the CASY1 cell counter. h Quantification of cell numbers in BALF using morphological criteria on May-Grünwald-Giemsa-stained cytospins. i MMP-12 quantification in the supernatant of cultured AMΦ isolated from 4-month old WT and Trpml3^−/− mice using ELISA. One single dot corresponds to the AMΦ SN from one well, each. Statistical analysis of datasets a-i was performed by using Student’s t test, unpaired, two-tailed (**p < 0.01, ****p < 0.0001). j, k MMP quantification in the supernatant of cultured AMΦ isolated from 4-month old WT and Trpml3^−/− mice using Multiplex and ELISA. One single dot corresponds to the AMΦ SN from one well, each. Two-way ANOVA followed by Tukey’s post hoc test; ***p < 0.001 (j) or Student’s t test, unpaired, two-tailed; **p < 0.01 (k). l Desmosine ELISA of BALF isolated from WT and Trpml3^−/− mice. One single dot corresponds to BALF from one mouse. Student’s t test, unpaired, two-tailed; **p < 0.01. m Verhoeff-Van Gieson (VVG) staining of formalin-fixed, paraffin-embedded lung sections of female, 4-month old WT or Trpml3^−/− mice (Mcoln3^tm.1.1Jga) treated either with PBS or porcine pancreatic elastase. Elastic fibers are stained blue-black, collagen appears red, and other tissue elements yellow. Scale bar 100 µm. n Quantification of elastin fibers as counts per field in VVG stained lung tissue sections from 6–8 mice per group. One dot corresponds to the mean count of elastin fibers in 8-10 fields of view per mouse lung. *p < 0.05, ****p < 0.0001; One-way ANOVA followed by Tukey’s post hoc test. In all figures, each single dot corresponds to one biologically independent sample. Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 5. Early endosomal trafficking in WT and Trpml3^−/− AMΦ.

a, b Transferrin (Tf) trafficking assay showing the decrease of Tf fluorescence in AMΦ (WT and Trpml3^−/−) within 20 min after the pulse with Tf-AlexaFluor488 (Tf accumulation). Mean ± SEM, 4 biologically independent experiments, each. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Two-way ANOVA followed by Bonferroni’s post hoc test. c Tf fluorescence in AMΦ (WT and Trpml3^−/−) after 20 min pulse with Tf-AlexaFluor488 (0 min timepoint, measures Tf uptake). Mean ± SEM, four biologically independent experiments. *p < 0.05, **p < 0.01, Student’s t test, unpaired, two-tailed. d Representative confocal images and quantification of the colocalization of EEA1 and Tf in AMΦ (WT and Trpml3^−/−). Statistical analysis was performed using Student’s t test, unpaired, two-tailed. Mean ± SEM, three biologically independent experiments. *p < 0.05, ***p < 0.001. e, f TfR expression analysis using Western blot. e Shown are two independent WB blots for TfR (90 kDa) and ß-Actin (45 kDa; loading control) using 5 WT and 5 Trpml3^−/− AMΦ lysates on each blot. f Quantification of WB data as shown in e. TfR protein was normalized to ß-Actin and values from Trpml3^−/− AMΦ were normalized to WT AMΦ. One single dot corresponds to one mouse, each (mean ± SEM). g, h Whole-cell patch-clamp experiments to determine membrane capacitance (measure of cell surface area). GTPγS induces an increase in surface area. Co-application of ML3-SA1 significantly reduces the effect of GTPγS in WT AMΦ, but not in Trpml3^−/− AMΦ ( = loss of membrane surface). Significance: GTPγS vs. GTPγS + ML3-SA1 from 140 to 150 sec *, from 152 to 156 sec **, from 158 to 172 sec ***, from 174 to 194 **, then till 200 sec *** (yellow dots). *p < 0.05, **p < 0.01, ***p < 0.001; two-way ANOVA followed by Tukey’s multiple comparisons test. Shown are mean values ± SEM, n (in parentheses) = biologically independent experiments. i, j Bar diagrams (mean ± SEM) showing the parameters Tau (= time until 2/3 of the maximum amplitude is reached) and Delay (= time until capacitance changes). *p < 0.05, Student’s t test, unpaired, two-tailed. Source data are provided as a Source Data file.

Fig. 6. Endocytosis in WT and Trpml3^−/− AMΦ.

a Shown are representative confocal images obtained from endocytosis experiments using dextran coupled to Alexa Fluor 568. Images show AMΦ (WT vs. Trpml3^−/−) that have been pulsed with fluorescently labelled dextran for different time periods. Scale bar 5 µm. b Quantification of dextran uptake showing significantly decreased rates of endocytosis in Trpml3^−/− AMΦ compared to WT AMΦ at various time points. A sum of at least 130 cells were analysed per timepoint and genotype deriving from five biologically independent experiments for both Trpml3^−/− lines (Mcoln3^tm1.2Hels and Mcoln3^tm1.1Jga), respectively. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Two-way ANOVA followed by Bonferroni’s post hoc test. c Effect of different endocytosis inhibitors on MMP-12 levels in WT and Trpml3^−/− AMΦ supernatants (SN). *p < 0.05, **p < 0.01, ****p < 0.0001; One-way ANOVA followed by Dunnett’s post hoc test. One single dot corresponds to the AMΦ SN from one well, each. 11 WT and 11 Trpml3^−/− mice were lavaged to obtain the number of cells for all wells. Data are mean ± SEM. d Cartoon showing endocytosis of MMP-12 via three different endocytosis pathways (CME, CIE, MP) and the effect of endocytosis inhibitors on MMP-12 uptake: According to the results shown in (c) the MMP-12 uptake in AMΦ corresponds to CIE and MP, resulting in higher concentrations of MMP-12 in the extracellular fluid after inhibition of these pathways. CME seems to be not involved. e Effect of the selective TRPML3 agonist ML3-SA1 (incubation o.n., 30 µM) on MMP-12 levels in WT and Trpml3^−/− AMΦ supernatants (SN). *p < 0.05, Student’s t test, unpaired, two-tailed. One single dot corresponds to the AMΦ SN from one well, each. 5 WT and 5 Trpml3^−/− mice were lavaged to obtain the appropriate number of cells for all wells. Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 7. Lysosomal exocytosis, pH and TRPML1 activity in WT and Trpml3^−/− AMΦ.

a Lysosomal exocytosis experiments measuring hexosaminidase release from WT and Trpml3^−/− AMΦ. Maximum effects were obtained with ionomycin (4 µM). TRPML3 activator ML3-SA1 elicited no significant effects in both WT and Trpml3^−/− AMΦ. Each dot corresponds to one biologically independent experiment. Average values are mean values ± SEM, each. b Cartoon illustrating LAMP1 translocation assay shown in c, d. Upon lysosomal exocytosis the lysosomal protein LAMP1 is detected on the plasma membrane (PM) by anti-LAMP1 followed by Alexa Fluor 488-conjugated secondary antibody. c Representative images of LAMP1 translocation assay using WT and Trpml3^−/− AMΦ. Shown are results obtained after 120 min treatment with DMSO, ML3-SA1 (30 µM), or 10 min treatment with ionomycin (4 µM). Scale bar 10 µm. d Quantification of experiments as shown in c (mean ± SEM from 3 biologically independent experiments, each). e Fura-2 calcium imaging experiments using HEK293 cells expressing human or murine TRPML1(NC), TRPML2 or TRPML3, respectively, indicating the specific levels of activation. Channels were stimulated with either ML1-SA1 ( = EVP-169) or ML-SA1 (10 µM, each). Shown are average values (mean ± SEM). Each dot represents one biologically independent experiment with 10–20 cells, each. ****p < 0.0001; Two-way ANOVA followed by Tukey’s multiple comparisons test. f Chemical structures of ML-SA1 and its derivative ML1-SA1 ( = EVP-169). g, h Quantification (g) and representative currents (h) from YM201636-enlarged LE/LY isolated from WT or Trpml3^−/− AMΦ, elicited by an application of 10 µM ML1-SA1. Each dot corresponds to one biologically independent experiment. Average values are mean values ± SEM, each. *p < 0.05, ***p < 0.001; One-way ANOVA followed by Tukey’s post hoc test. i Results obtained from endolysosomal pH measurements using WT or Trpml3^−/− AMΦ. Measurements were performed by ratiometric fluorescence imaging with Oregon Green^,. Data are mean ± SD. j Mean endolysosomal pH values (mean ± SD) in WT and Trpml3^−/− AMΦ were calculated using the calibration curves presented in i (n = 4, each). Source data are provided as a Source Data file.

Fig. 8. TIMPs in WT and Trpml3^−/− BALF and schematic of emphysema development in Trpml3^−/− lungs.

a, b TIMP-1 and TIMP-2 levels in BALF obtained from WT and Trpml3^−/− mice measured by ELISA. Data are mean ± SEM collected from up to 8 mice per genotype per mouse line. Statistical analysis was performed using Student’s t test, unpaired, two-tailed. One single dot corresponds to one mouse, each in a, b. c Scheme showing the mechanism of emphysema development in Trpml3^−/− mouse lungs. In WT lungs the amount of MMP-12 outside the AMΦ is regulated by TIMP-1/2, as well as endocytosis of MMP-12 and lysosomal degradation. We observed increased MMP-12 levels in BALF and lower endocytosis rates in Trpml3^−/− AMΦ. Vice versa selective activation of TRPML3 resulted in reduced MMP-12 levels in BALF. Therefore, it is postulated that loss of TRPML3 results in extracellular matrix (ECM) remodeling and emphysema characterized by destruction of the alveolar walls as depicted. Source data are provided as a Source Data file.

Fig. 9. Effects of tobacco smoke exposure in WT and Trpml3^−/− mice (Mcoln3^tm1.1Jga).

a Lung function measurements were performed using the SCIREQs FlexiVent System in analogy to experiments shown in Fig. 2. Elastance and Compliance in Trpml3^−/− mice are changed in the direction of an emphysematous lung, both under filtered air (FA) and under cigarette smoke (CS). b Quantification of airspace enlargement as mean linear chord length. Lung tissue sections from 6–8 mice per group were analysed. Each dot corresponds to one biologically independent lung tissue sample. Average values are mean values ± SEM, each. *p < 0.05, **p < 0.01, ***p < 0.001; One-way ANOVA followed by Tukey’s post hoc test. c Representative images of H&E-stained lung tissue sections (as quantified in b) from mouse lungs (BL6 WT and Trpml3^−/−) exposed to CS or FA showing the respective extent of airspace enlargements. Scale bar 100 µm. d Transcriptomics data of single-cell suspensions from female and WT whole mouse lungs that were exposed to FA or CS for 2 or 6 months. Dot plot shows the percentage of cells expressing Mcoln3 using dot size and the average expression level of Mcoln3 coded by color grading. e mRNA expression level of MCOLN3 in publicly available transcriptomics datasets obtained from the lungs of COPD patients with smoking history compared to healthy smokers (GSE27597), and in macrophages (MΦ) isolated from the bronchoalveolar lavage (BAL) of smokers compared to nonsmokers (GSE8823 and GSE2125), one single dot per person. Expression levels were normalized to the representative control groups. FC fold change. **p < 0.01, ***p < 0.001, ****p < 0.0001; two-tailed Mann-Whitney test. Source data are provided as a Source Data file.