Immune cells fold and damage fungal hyphae

Significance Macrophages protect against microbial infection, in part by engulfing and killing invading microbes. Fungal pathogens such as Candida albicans are known to evade phagocytic killing by forming hyphae that are physically challenging to engulf because of their length. We now find that macrophages can respond by folding the hyphae of C. albicans (and other fungal species). Hyphal folding implies that immune cells can continue to apply mechanical force after their cargo has been internalized. The involvement of Dectin-1, β2-integrin, and actin–myosin polymerization provides initial mechanistic insight. Folding damages hyphae, inhibits their growth, and facilitates their complete engulfment. Therefore, hyphal folding represents an additional weapon in the immune cell armory that presumably contributes to fungal clearance.

. Secondary antibody controls for localisation experiments. Live C. albicans SC5314 or fixed tup1Δ cells were mixed with murine BMDMs, fixed after 3 h, stained and subjected to phase differential interference contrast (DIC) and fluorescence microscopy. These same cell populations were used to examine vinculin, paxillin or talin localisation. The same secondary antibody was used to analyse dectin-1, vinculin, paxillin and talin localisation, and no significant background staining was observed for this antibody. Interactions between live C. albicans SC5314 and murine BMDMs were monitored. After 4 h, cells were fixed, stained and subjected to phase differential interference contrast (DIC) and fluorescence microscopy: fungal chitin (Calcofluor White; CFW, blue); dectin-1 (anti-dectin-1, red); talin (anti-talin antibody, green (A)); paxillin (anti-paxillin antibody, green (B)). dectin-1 did not appear to co-localize with talin (A) or paxillin (B).
Movie S1 (separate file). BMDM engulfs and folds a live C. albicans SC5314 hypha. This video represents approximately 360 minutes of live imaging of macrophage-C. albicans interactions (Materials and Methods). The movie shows multiple folding events and fracture of the hypha that ends up centre stage.
Movie S2 (separate file). Thio-mac containing a live C. albicans SC5314 hypha, which is folded. This video represents approximately 240 minutes of live imaging of macrophage-C. albicans interactions (Materials and Methods), during which the hypha is folded and damaged.
Movie S3 (separate file). J774.1 cell engulfs a live C. albicans SC5314 yeast with germ tube. This video, which represents approximately 200 minutes from a 4 h movie (Materials and Methods), shows the engulfment of a mother cell and short germ tube by a macrophage, the growth of the germ tube in the macrophage, and then the resultant filament being folded by the macrophage.
Movie S4 (separate file). RAW264.7 cell engulfs and folds a live C. albicans SC5314. This video represents approximately 360 minutes (Materials and Methods). It shows a macrophage trapping and engulfing a yeast cell that is undergoing morphogenesis. The germ tube continues to extend, and the filament is then folded by the macrophage.

Movie S5 (separate file).
Human monocyte-derived macrophages phagocytosing and folding live C. albicans SC5314. In this video, which represents 360 minutes in real time (Materials and Methods), the macrophage centre stage phagocytoses two C. albicans yeast cells, which undergo morphogenesis. The germ tubes continue to extend, and then both hyphae are folded. Meanwhile, the macrophage below it (and to the left) folds another hypha twice.
Movie S6 (separate file). BMDM engulfs a long fixed C. albicans tup1Δ hypha. In this video, which represents about 93 minutes in real time (Materials and Methods), a macrophage starts to phagocytose a long tup1Δ filament and, as it does so, the macrophage folds the filament allowing it to fully engulf the fungus.
Movie S7 (separate file). BMDMs engulf and fold fixed C. albicans tup1Δ hyphae. This video represents 200 minutes in real time (Materials and Methods). It shows two macrophages (middle left) that trap and phagocytose tup1Δ hyphae. Then a third macrophage is shown folding another hypha multiple times (bottom).
Movie S8 (separate file). BMDM engulfs and folds a hyphal filament of the fungal species, M. sterilia. This video, which represents 60 minutes in real time (Materials and Methods), shows a macrophages folding a M. sterilia hypha, revealing that macrophages can fold the hyphae of an evolutionarily divergent, non-pathogenic environmental fungus.
Movie S9 (separate file). BMDM engulfs and fragments a short C. albicans tup1Δ hypha. This video represents 200 minutes in real time (Materials and Methods). The macrophage (starting centre stage) engulfs a relatively short fixed C. albicans tup1Δ filament. The macrophage then folds the filament at the yeast-hypha junction, and severs the yeast part from the remainder of the filament.
Movie S10 (separate file). Live 3D imaging of macrophage actin dynamics over a C. albicans filament. This video represents 107 minutes in real time (Materials and Methods). A fixed tup1Δ hypha stained with CFW (blue) is phagocytosed by a BMDM, and the actin patterns are shown to change dynamically over the dead fungal filament using silicon rhodamine live actin staining (orange).
Movie S11 (separate file). Live 3D imaging of macrophage actin dynamics over an emerging C. albicans germ tube. This video represents 103 minutes in real time (Materials and Methods). A RAW264.7 macrophage expressing Life-ACT (green) displays intense and dynamic actin patterns around phagosomes as live C. albicans SC5314 cells are engulfed. Dynamic actin persists around phagosomes formed for some time, around a hypha emerging from a mother yeast cell.