RAB5c controls the assembly of non-canonical autophagy machinery to promote phagosome maturation and microbicidal function of macrophages

Abstract

Non-canonical conjugation of ATG8 proteins, including LC3, to single membranes implicates the autophagy machinery in cell functions unrelated to metabolic stress. One such pathway is LC3-associated phagocytosis (LAP), which aids in phagosome maturation and subsequent signaling upon cargo uptake mediated by certain innate immunity-associated receptors. Here, we show that a specific isoform of RAB5 GTPases, the molecular switches controlling early endosome traffic, is necessary for LAP. We demonstrate that RAB5c regulates phagosome recruitment and function of complexes required for phosphatidylinositol-3-phosphate [PI(3)P] and reactive oxygen species (ROS) generation by macrophages. RAB5c facilitates phagosome translocation of the V-ATPase transmembrane core, which is needed for ATG16L1 binding and consequent LC3 conjugation. RAB5c depletion impaired macrophage elimination of the fungal pathogen Aspergillus fumigatus and disruption of the V-ATPase-ATG16L1 axis increased susceptibility in vivo. Therefore, early endosome-to-phagosome traffic is differentially regulated to promote LAP and ROS contributes to resistance against A. fumigatus by effecting LAP.

Keywords: Aspergillus fumigatus; LC3-associated phagocytosis; Non-canonical autophagy; RAB5c GTPase.

Figure 1.. Rab5c depletion impairs LC3-associated phagocytosis.

(A) Analysis of time-lapse confocal microscopy of mCherry-RAB5c (mCh-RAB5c) at phagosomes containing opsonized-zymosan (Op-Zym) in RAW264.7 cells. Fluorescence profile was normalized by the MFI at 0” for each phagosome. Squares are the mean and shaded area is ± S.E.M. for 12 phagosomes (relative to Video S1). (B) Representative confocal images of RAW264.7-mCh-RAB5c cells stimulated for 5 min with Op-Zym, Beads adsorbed with IgG (IgG-Beads), or albumin (BSA-Beads). Insets show mCh-RAB5c fluorescence intensity using the RAINBOW LUT. *, phagosomes. N: nuclei. Scale bar: 5 μm. (C-D) Representative confocal images (C) and MFI quantification (D) of mCh-LC3B at phagosomes in RAW246.7 cells transduced with scrambled shRNA (sh_Ctrl) or indicated shRNAs (#1; #2) against Rab5a, Rab5b, Rab5c, and fed Op-Zym for 25 min. Scale bar: 5 μm. (E-G) Representative confocal images (E) and MFI quantification of LC3 immunolabeling (AF-647) at phagosomes in RAW264.7 cells (F) and BMDM (G) transduced with sh_Ctrl or sh_Rab5c and fed Op-Zym for 25 min. Blue: nuclei (Hoechst). Scale bar: 5 μm. (H) Immunoblot analysis of LC3, RAB5a/b/c and EEA1 in IgG-Bead- or BSA-Bead-containing phagosomes purified from RAW264.7 cells expressing sh_Ctrl or sh_Rab5c 40 min after stimulation. UNC93B1 was used as loading control and GM130, constitutive of Golgi apparatus, as control of purification. Data are representative of 4–5 experiments performed independently. (I-J) Representative confocal images (I) and MFI quantification (J) of LAMP1 immunolabeling (AF-488) at phagosomes in RAW264.7 cells fed Op-Zym for 25 min. The fluorescence intensity is shown using the FIRE LUT. Green *: phagosome. Scale bar: 10 μm. (K-M) Flow cytometric analysis of Op-pHrodo Red-Zym in RAW264.7 cells 45 min post-stimulation. Histogram (K), quantification of MFI (L), and percentage of positive cells (M) for each group are shown. The black line on the histogram represents the fluorescence of non-stimulated sh_Ctrl cells. Bars are the mean value of biological replicates (n=4, each indicated as a circle), and error bars are ± S.E.M. Statistical significance was calculated by unpaired Student′s t-test. Data shown is representative of 3 experiments performed independently. (D, F, G, J) Gray objects are the MIF of each analyzed phagosome. Colored objects are the mean value for each independent experiment (n=3) and error bars are ± S.E.M. Statistical significance was calculated by unpaired Student′s t-test between the indicated groups.

Figure 2.. RAB5c depletion impairs PI(3)P production and stable assembly of NOX2 machinery on phagosomes but still increases phagosome ROS production.

RAW264.7 cells or BMDM were transduced with scrambled shRNA (sh_Ctrl) or Rab5c shRNA (sh_Rab5c). (A) Immunoblot analysis of VPS34-PI(3)K CIII complex on BSA-Bead- or IgG-Bead-containing phagosomes purified from RAW264.7 cells at 40 min after stimulation. Data shown is representative of 3–5 experiments performed independently. (B) Representative confocal images of RAW264.7-p40^phox-PX-Venus cells fed with Op-Zym for 25 min. The fluorescence intensity is displayed using the SMART LUT. *, phagosomes. Scale bar: 10 μm. (C) Quantification of time-lapse confocal microscopy analysis of p40^phox-PX-Venus at phagosomes containing Op-Zym in RAW264.7 cells. Fluorescence profile was normalized by the MFI at 0” for each phagosome. Squares are the mean and shaded areas are ± S.E.M. for 18 phagosomes. The area under the curve (AUC) was determined for each analyzed phagosome and statistical significance was calculated by unpaired Student′s t-test. Data shown is representative of 2 independent experiments (related to Video S3). (D-E) Immunoblot analysis (D) and densitometric quantification (E) of LC3A/B-II in RAW246.7 cells non-stimulated or incubated with Op-Zym for 25 min; C8 (2 μM) for 25 min; DMXAA (50 μg/mL) for 1 h; PP242 (1 μM) for 2 h, as indicated. GAPDH was used as loading control. Bars are the mean values of 3 independent experiments (each indicated as a circle), and error bars are ± S.E.M. Statistical significance was calculated by unpaired Student′s t-test. (F) Immunoblot analysis of NOX2 components on IgG-Bead or BSA-Bead containing phagosomes purified from RAW264.7 cells 40 min after stimulation. Data shown are representative of 4–5 experiments performed independently. (G) Representative DIC images of formazan deposits in phagosomes of RAW264.7 cells or BDMD fed Op-Zym for 15 min. Arrowheads indicate Op-Zym-containing phagosomes positive (Red) or negative (Yellow) for formazan deposition. (H-I) Percentage of NBT⁺ phagosomes containing Op-Zym in RAW264.7 cells (H) and BMDM (I). (J-L) Representative confocal images (J) and MIF quantification of p-p47^phox immunolabeling (AF-647) at phagosomes in RAW264.7 cells fed Op-Zym for 15 min (K) or 40 min (L).(M-N) Representative confocal images (M) and MIF quantification (N) of p-ERK1/2 immunolabeling (AF-647) in RAW264.7 cells fed with Op-Zym for 15 min. Blue: nuclei (DAPI). Scale bar: 10 μm. (K, L, N) Gray objects are the MIF of each analyzed phagosome. Colored objects are the mean value for each independent experiment (n=3) and error bars are ± S.E.M. Statistical significance was calculated unpaired Student′s t-test between the indicated groups. (O) Percentage of NBT⁺ phagosomes in RAW264.7 cells treated or not with ERK1/2 inhibitor (BVD-523; 100 μM) for 10 min and subsequently fed Op-Zym for 15 min. (H, I, O) Bars are the mean value of biological replicates (n=3–6, each indicated as a circle) and error bars are ± S.E.M. Statistical significance was calculated by unpaired Student′s t-test (H, I) or by one-way ANOVA and Tukey′s multiple comparisons (O) between the indicated groups. Data shown is representative of 3 (H, I) or 2 (O) experiments performed independently.

Figure 3.. RAB5c controls the assembly of the V-ATPase in the phagosome during LAP.

RAW264.7 cells were transduced with scrambled shRNA (sh_Ctrl) or Rab5c shRNA (sh_Rab5c). (A, B) Representative confocal images (A) and MFI quantification (B) of ATP6V1A immunolabeling (AF-647) at phagosomes in RAW264.7 cells fed Op-Zym for 25 min. Blue: nuclei (Hoechst). Scale bar: 10 μm. (C) Immunoblot analysis of components of the V-ATPase complex and ATG8 lipidation machinery on BSA-Bead- or IgG-Bead-containing phagosomes purified from RAW264.7 cells at 40 min after stimulation. Data are representative of 4–5 independent experiments. (D-E) MFI quantification of ATP6V1A (D) and LC3 (E) immunolabeling (AF-647) at phagosomes in RAW264.7 cells treated or not with DPI before feeding the cells with Op-Zym (lower concentration: 0.61 μM; higher concentration: 5 μM). (B, D, E) Gray objects are the MIF of each analyzed phagosome. Colored objects are the mean value for each independent experiment (n=3) and error bars are ± S.E.M. Statistical significance was calculated by unpaired Student′s t-test (B) or by one-way ANOVA and Tukey′s multiple comparisons (D, E) between the indicated groups.

Figure 4.. RAB5c-mediated V-ATPase assembly confers macrophage resistance to Aspergillus fumigatus.

(A-F) BMDM transduced with scrambled shRNA (sh_Ctrl) or Rab5c shRNA (sh_Rab5c) were stimulated with conidia of wild-type (ATCC46645; WT) or ΔpksP strains of A. fumigatus. (A, B) Representative confocal images (A) and percentage of LC3-decorated phagosomes at 25 min of stimulation. Insets show details of conidia, scale bar: 10 μm. (C) Percentage of phagocytes positive for NBT deposition at 25 min of stimulation. (D) Percentage of ATP6V1A-decorated phagosomes at 1 h of stimulation. (E, F) Viability of WT (E) or ΔpksP (F) conidia determined as the ratio CFU at 6 h/CFU at 1 h. (G-K) Atg16l1^FL and Atg16l1^ΔWD40 BMDM were stimulated with WT or ΔpksP conidia. (G) Percentage of LC3-decorated phagosomes at 25 min of stimulation. (H) Percentage of phagocytes positive for NBT deposition at 25 min of stimulation. (I, J) Viability of WT (I) or ΔpksP (J) conidia, as assessed by CFU counts as in (E). (K) Viability of WT conidia by BMDM pre-treated or not with DPI (5 μM), assessed by CFU counts as in (E). (L) Scheme of A. fumigatus lung infection. Atg16l1^FL and Atg16l1^ΔWD40 mice were intranasally instilled with vehicle (PBS) or infected with WT A. fumigatus for 72 h. (M) Fungal loads in the bronchoalveolar lavage fluid (BAL) samples of infected mice, assessed by CFU counts. (N) Colorimetric quantification of BAL levels of Albumin. (O) Representative brightfield microscopy images of Grocott-Gomori′s methenamine silver (upper panel) or Hematoxylin and Eosin-stained (bottom panel) lung mice sections. Scale bar: 500 μm. Insets show details of A. fumigatus hyphae. Scale bar: 50 μm. (P-S) ELISA quantification of BAL levels of IL-6 (P), CCL2 (Q), TNF-α (R) and IL-1β (S). (B-K) Bars are the mean value of biological replicates (n=3–5, each indicated as a circle). (M, N, P-S) Bars represent the mean of 10 mice (each indicated as a circle). (B-K, M, N, P-S) Error bars are ± S.E.M. Statistical significance was calculated by unpaired Student′s t-test (B-J; M, N, P-S) or by one-way ANOVA and Tukey′s multiple comparisons (K) between indicated groups. Data shown are representative of 2 (A-D; G, H, K, M--S) or 3 (E, F, I, J) experiments performed independently.