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 this process 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 µm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (∼12 µm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent 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 suggest 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

“…Since PTFE plastic is not amenable to most imaging techniques, we took advantage of recently developed optical-quality glass surfaces prepared by adsorption of longchain hydrocarbons such as paraffin that promote high level of macrophage fusion (Faust et al, 2017;Faust et al, 2018). In this series of experiments, rather than macrophage cell lines, we used primary macrophages isolated from inflamed mouse peritonea (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. Using phase-contrast video microscopy, we determined that the kinetics of IL-4-induced macrophage fusion on paraffin-adsorbed glass (P-surface) were similar to that on PCTFE ( Figure 2A and B).…”

“…It is known that the Arp2/3 complex and its activators Cdc42 and WASp nucleate the core of actin filaments in podosomes (Linder et al, 2000). We have previously demonstrated that deficiency of WASp or Cdc42, or inhibition of Arp2/3 strongly reduces macrophage fusion and podosome formation (Faust et al, 2019). To examine the importance of these actin regulators in ZLS formation, we isolated macrophages form WASP -/or myeloid-cell-specific Cdc42 -/mice and tested their ability to assemble ZLSs.…”

“…The rabbit anti-paxillin (32084) Cdc42 -/mice were generated by crossing Cdc42 loxP/loxP mice with LysMcre mice, followed by screening the progeny for Cdc42 excision in myeloid leukocytes as previously described (Faust et al, 2019). All animals were given ad libitum access to food and water and maintained at 22 °C on a 12-hour light/dark cycle.…”

“…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). Furthermore, we have found that fusion-competent protrusions form at the sites enriched in podosomes.…”

“…Most, if not all, of the steps involved in macrophage fusion, appear to rely on the actin cytoskeleton. 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). The polymerization of actin filaments is known to be involved in the formation of diverse cellular protrusions, including lamellipodia, filopodia, and podosomes [for review see Ridley (2011) and Svitkina (2013)].…”