An actin-based protrusion originating from a podosome-enriched region initiates macrophage fusion

Abstract

Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating macrophage fusion is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase contrast video microscopy demonstrated that in the majority of events, short protrusions (3 ± 1 µm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (16 ± 7 µm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent actinbased protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP-and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data indicate that an actin-based protrusion formed at the leading edge initiates macrophage fusion.

Transition of podosomes into zipper-like structures in macrophage-derived multinucleated giant cells

“…Podosomes were often assembled into clusters of two or three podosomes, which were also surrounded by vinculin and talin. The size of individual podosomes (∼0.7 µm), as well as those in clusters, was in agreement with that determined previously in living MGCs and in fixed samples ( Faust et al. , 2019 ).…”

“…, 2017 , 2018 ).In this series of experiments, rather than macrophage cell lines, we used primary macrophages isolated from the inflamed mouse peritoneum ( Helming and Gordon, 2007a ; Podolnikova et al. , 2016 ; Faust et al. , 2019 ) to avoid the robust proliferation observed in the cultures of macrophage cell lines.…”

“…, 2000 ). We previously demonstrated that deficiency of WASp or Cdc42 or inhibition of Arp2/3 strongly reduces macrophage fusion and podosome formation ( Faust et al. , 2019 ).…”

“…It has long been known that cytochalasins B and D that prevent actin polymerization also inhibit macrophage fusion ( DeFife et al. , 1999 ; Faust et al. , 2019 ).…”

“…However, the precise targets of actin-disrupting agents that inhibit macrophage fusion have not been identified. Recently, we have characterized the early steps of IL-4–mediated macrophage fusion and showed that an actin-based protrusion at the leading edge initiates macrophage fusion ( Faust et al. , 2019 ).…”