Autophagy proteins are not universally required for phagosome maturation

Abstract

Phagocytosis plays a central role in immunity and tissue homeostasis. After internalization of cargo into single-membrane phagosomes, these compartments undergo a maturation sequences that terminates in lysosome fusion and cargo degradation. Components of the autophagy pathway have recently been linked to phagosome maturation in a process called LC3-associated phagocytosis (LAP). In this process, autophagy machinery is thought to conjugate LC3 directly onto the phagosomal membrane to promote lysosome fusion. However, a recent study has suggested that ATG proteins may in fact impair phagosome maturation to promote antigen presentation. Here, we examined the impact of ATG proteins on phagosome maturation in murine cells using FCGR2A/FcγR-dependent phagocytosis as a model. We show that phagosome maturation is not affected in Atg5-deficient mouse embryonic fibroblasts, or in Atg5- or Atg7-deficient bone marrow-derived macrophages using standard assays of phagosome maturation. We propose that ATG proteins may be required for phagosome maturation under some conditions, but are not universally required for this process.

Keywords: ATG5; ATG7; ATG8/LC3; LC3-associated phagocytosis; autophagy; phagosome maturation.

Figure 1.

Phagosome maturation of IgG-coated particles is unaffected in atg5^−/− MEFs. WT and atg5^−/− MEFs were transfected FCGR2A-GFP (green) to render them phagocytic. (A, B) MEFs were challenged with OpZ and fixed at 30, 60, 90, or 120 min post challenge, then stained for LAMP1 (red) and OpZ (blue) and imaged (A). A representative image is shown at 60 min post challenge with a LAMP⁺ particle marked by an arrow. (B) The percentage of LAMP1⁺ OpZ was enumerated in WT and atg5^−/− MEFs. At least 200 OpZ were counted per condition. (C, D) MEFs were incubated with LysoBrite (red) for 30 min and then challenged with SRBC (blue) for 30, 60 or 90 min and imaged live (C). A representative image is shown at 60 min post challenge with a LysoBrite⁺ particle marked by an arrow. (D) The percentage of LysoBrite⁺ SRBC was enumerated in WT and atg5^−/− MEFs. At least 50 SRBC were counted per condition. Scale bar: 8 μm.

Figure 2.

Phagosome maturation of IgG-coated particles is unaffected in autophagy-deficient CSF1-differentiated BMDMs. (A, B, C) CSF1-derived BMDMs were challenged with OpZ and fixed at 30, 60, 90, or 120 min, then stained for LAMP1 (green) and OpZ (blue) and imaged (A). A representative image is shown at 60 min post challenge with a LAMP⁺ particle marked by an arrow. The percentage of LAMP1⁺ OpZ was enumerated in WT and atg5^−/− (B) and WT and atg7^−/− (C) BMDMs. At least 300 OpZ were counted per condition. (D, E, F) CSF1-derived BMDMs were incubated with LysoBrite (red) for 30 min and then challenged with SRBC (blue) for 30, 60 or 90 min and imaged live (D). A representative image is shown at 60 min post challenge with a LysoBrite⁺ particle marked by an arrow. The percentage of LysoBrite⁺ SRBC was enumerated in WT and atg5^−/− (E) and WT and atg7^−/− (F) BMDMs. At least 50 SRBC were counted per condition. (G, H, I) CSF1-derived BMDMs were pulsed with DQ-BSA (red) for 1 h, chased for 1 h in regular medium, then challenged with SRBC (blue) for 30, 60 or 90 min and imaged live (G). A representative image is shown at 60 min post challenge with a DQ-BSA⁺ particle marked by an arrow. The percentage of DQ-BSA⁺ SRBC was enumerated in WT and atg5^−/− (H) and WT and atg7^−/− (I) BMDMs. At least 50 SRBC were counted per condition. Scale bar: 8 μm.