Fig 2: Grn KO mice show enlarged lysosomes and an accumulation of myelin debris in microglial lysosomes(A) Representative images of the pons of 21-month-old wild-type (WT) and Grn KO mice co-stained for lysosome (Lamp1) and microglia (Iba1) markers. Arrows indicate enlarged lysosomes. Boxes indicate the region enlarged in the enlarged panel.(B) Representative images of myelin (Mbp), lysosome (Lamp1) and microglia marker (CD68) in the pons of 21-month-old Grn KO mice.(C) EM image of myelin debris engulfed by microglia in pons of 12-month-old Grn KO mice. Box indicates the region enlarged in the subsequent panel.(D) Representative images of Mbp staining in different brain regions of 21-month-old WT and Grn KO mice. Arrows indicate myelin debris. Scale bars, 10 μm (A–C) and 50 μm (D).(E) Quantitative analyses of Mbp staining in different brain regions of 21-month-old WT and Grn KO mice (n = 10 per group). Data are presented as mean ± SEM. ***p < 0.001, ****p < 0.0001, two-way ANOVA, Tukey’s multiple-comparison test. n.s., not significant.

Fig 3: Characterisation of GAA-KO Sol8 cellular models. (A) mRNA relative expression of mGAA in the different Sol8 clones (? ATG_14, E2_1, E5_6 and E7_1) compared to WT cells. (B) GAA activity in Sol8 WT cells and mutated clones (? ATG, E2_1, E5_6 and E7_1). (C) Glycogen content in Sol8 myoblasts differentiated into myotubes (see M&M) in WT and clones (? ATG, E2_1, E5_6 and E7_1). (D) Western blot analysis of Sol8 cells lysates using anti-p62/sequestosome-1 (SQSTM1) and anti-LAMP-1 antibodies. Anti-ERK1,2 was used as a loading control. (E) Quantification of p62 levels normalised to ERK1,2. (F) Quantification of LAMP-1 levels normalised to ERK1,2. (G) mRNA relative expression of CI-MPRs in clones (? ATG, E2_1, E5_6 and E7_1) and Sol8 WT cells. (H) Surface CI-MPRs relative expression (mean fluorescence of intensity-MFI) in Sol8 WT compared to E2_1, E2_bulk, E5_bulk and E7_bulk. (I) Representative dot-plot of CI-MPRs expression (MFI) in Sol8 WT, E2_1, E2_bulk, E5_bulk and E7_bulk. Statistical analyses: unpaired t-test, two-tails (A–C), unpaired t-test one-tail (E–H) (* p < 0.05, ** p < 0.01, *** p < 0.001. Values represent means +/- SEM of at least 3 separate experiments.

Fig 4: Phagocytosis- and STING-dependent induction of type I interferon by SYNB1891.a–c WT, TLR4-/- and STING-/- BMDCs were treated with Control EcN (MOI: 25), pre-induced SYN1891 (MOI: 25), LPS (100 ng/mL), or smSTING agonist (5 µg/mL) for 4 h (n = 3 biological replicates per group per genotype). BMDCs from each genotype incubated alone served as negative controls. Cells were analyzed for the upregulation of Ifnb1 (a), Il6 (b) and Il12a (c) mRNA (****P < 0.0001 two-way ANOVA with Tukey’s multiple comparisons tests, see Supplementary Fig. 3a for IFN-ß1 protein quantification). d–g Representative fluorescent images and phagocytosis quantification. WT BMDCs were incubated with pre-induced SYNB1891-gfp (MOI: 25) for 1 h in control media (d, e) or pre-treated for 1 h with Cytochalasin D (10 µM) before bacterial incubation (f). Non-internalized bacteria were washed out and cells were stained for microscopy. Cell nuclei were labeled with Hoechst (Blue), F-actin was stained with ActinRed 555 probe (Red), SYNB1891-gfp were labeled with anti-GFP (Green), and phagosomal transmembrane protein LAMP-1 was labeled with anti-LAMP1 (Purple). White arrows point to bacteria co-localized within DC actin cytoskeleton (d–f). These bacteria are surrounded by phagosomal membrane stained with LAMP-1 (e). The number of bacteria per dendritic cell per field of view (FOV) was quantified in (g). 12 FOVs were evaluated in the experiment (7 for control and 5 for CytoD group) resulting in >200 cells per treatment quantified (**P < 0.0001, two-tailed unpaired Student’s t test). Images and data are representative of 2 independent experiments. h, i RAW 264.7 macrophages or (j, k) WT BMDCs were treated as described above in (a) (n = 3 biological replicates per group). In the indicated groups cells were pre-treated for 1 h with Cytochalasin D (10 µM). Macrophages or BMDCs incubated in media alone served as a negative control. Cells were analyzed for the upregulation of Ifnb1 (g, i) and Il6 (h, j) mRNA (*P = 0.0182, **P = 0.0017–0.006, ***P = 0.0005, ****P < 0.0001 two-way ANOVA with Tukey’s multiple comparisons tests, see Supplementary Fig. 3h–k for protein quantification,). a–c, g–k Data are representative of two or more independent experiments per cell type with mean and s.d. shown. Each circle represents an independent experimental replicate.

Fig 5: Nilotinib treatment of Mtb-infected mice activates lysosome functions of lung myeloid subsets and reduces lung bacterial burdens.(A) Experimental protocol for treatment of Mtb infected C57BL/6 mice. Mice infected with Mtb H37Rv-ZsGreen were treated with vehicle or nilotinib (20 mg/kg/day) intraperitoneally, 5 days/week, for a total of 15 doses beginning at 7 dpi and ending at 27 dpi. At 28 dpi, mice were euthanized, and lungs were harvested for the assays shown.(B) Nilotinib treatment of mice decreases overall Mtb lung bacterial burdens (28 dpi).(C) Nilotinib treatment of Mtb-infected mice increases expression of lysosome genes in lungs (qPCR analysis). Gapdh was used as a control.(D) Nilotinib treatment of Mtb-infected mice increases LAMP1 expression in recruited lung phagocyte subsets; flow cytometry quantitation of intracellular LAMP1 MFI in lung subsets from mice infected with H37Rv-ZsGreen treated with vehicle or nilotinib (28 dpi).(E) Nilotinib treatment of Mtb-infected mice increases lysosomal V-ATPase subunit ATP6V1B2 expression in recruited lung phagocyte subsets; flow cytometry quantitation of intracellular ATP6V1B2 MFI in lung subsets from mice infected with H37Rv-ZsGreen treated with vehicle or nilotinib (28 dpi).(F) Nilotinib treatment of Mtb-infected mice increases lysosomal acidification. Cresyl violet MFI quantitated by flow cytometry for lung subsets from H37Rv-ZsGreen-infected mice treated with vehicle or nilotinib (28 dpi).(G) Nilotinib treatment of Mtb-infected mice increases lysosomal cathepsin B activity in recruited phagocytes, but not resident (alveolar) macrophages. MFI of fluorogenic MagicRed® CTSB product in lung subsets from mice treated with vehicle or nilotinib (28 dpi) quantitated by flow cytometry.Results are presented as mean ± SD, n=4–8, representative of 2 independent experiments. *p<0.05, **p<0.01, ***p<0.001, by unpaired Student’s t test (B) or multiple unpaired Student’s t test (C-G).