The Formins FMNL1 and mDia1 Regulate Coiling Phagocytosis of Borrelia burgdorferi by Primary Human Macrophages

Abstract

Spirochetes of the Borrelia burgdorferi sensu lato complex are the causative agent of Lyme borreliosis, a tick-borne infectious disease primarily affecting the skin, nervous system, and joints. During infection, macrophages and dendritic cells are the first immune cells to encounter invading borreliae. Phagocytosis and intracellular processing of Borrelia by these cells is thus decisive for the eventual outcome of infection. Phagocytic uptake of Borrelia by macrophages proceeds preferentially through coiling phagocytosis, which is characterized by actin-rich unilateral pseudopods that capture and enwrap spirochetes. Actin-dependent growth of these pseudopods necessitates de novo nucleation of actin filaments, which is regulated by actin-nucleating factors such as Arp2/3 complex. Here, we demonstrate that, in addition, also actin-regulatory proteins of the formin family are important for uptake of borreliae by primary human macrophages. Using immunofluorescence, live-cell imaging, and ratiometric analysis, we find specific enrichment of the formins FMNL1 and mDia1 at macrophage pseudopods that are in contact with borreliae. Consistently, small interfering RNA (siRNA)-mediated knockdown of FMNL1 or mDia1 leads to decreased formation of Borrelia-induced pseudopods and to decreased internalization of borreliae by macrophages. Our results suggest that macrophage coiling phagocytosis is a complex process involving several actin nucleation/regulatory factors. They also point specifically to the formins mDia1 and FMNL1 as novel regulators of spirochete uptake by human immune cells.

Non-canonical activity of the podosomal formin FMNL1γ supports immune cell migration

“…2C), suggesting that FMNL1 may be important for directional movement -such as chemotaxis or in 3D environments -but that it is unnecessary for random motility. Surprisingly, we found no loss of phagocytic function in FMNL1 KO macrophages when non-specific, CR3-mediated or Fc-receptor-mediated routes of entry were examined, in contrast with other reports (Naj et al, 2013;Seth et al, 2006), which could be potentially explained by differences between FMNL1 KO and suppression using RNA interference (Fig. S2).…”

“…Two independent studies have suggested that FMNL1 is required for different types of macrophage phagocytosis (Naj et al, 2013;Seth et al, 2006). FMNL1 KO macrophages did not differ from WT in their ability to perform non-specific, CR3-mediated or FcγR-mediated phagocytosis.…”

ADiaphanous-related formin links Ras signaling directly to actin assembly in macropinocytosis and phagocytosis

“…This synergistic mode of action between different actin assembly factors has been proposed for actin polymerization in lamellipodia, in which Arp2/3 complexgenerated filaments are captured by the DRF FMNL2 to promote their elongation in a PFN/actin-dependent manner to drive efficient cell migration (60). Of note, the Arp2/3 complex and the DRFs mDia1 and FMNL1 also seem to synergize during a specialized form of internalization of Borrelia burgdorferi by macrophages, termed coiling phagocytosis (56), meaning that this type of cooperation between different actin assembly factors may be a general concept in various actin-based processes.…”

“…Consistently, the Arp2/3 complex was also shown to be required for large-scale endocytosis in higher eukaryotes (54). Formins, such as the DRFs mDia1, FMNL1/FRL1, and Daam1 (29,(55)(56)(57), have also been implicated in phagocytosis in immune cells, albeit the molecular function of these actin assembly factors is less clear.…”

Human Macrophages Utilize the Podosome Formin FMNL1 for Adhesion and Migration

“…However, little is known about most of the functions of these formins in macrophages. Dia1 and FMNL1 have been implicated in phagocytosis and we have shown FMNL1 is critical for adhesion, but the other formins have yet to be characterized for their function and localization [22] [37] [38]. Since we have shown FMNL1 to be vital to macrophage adhesion and podosome stability, it seems likely to also be important for migration.…”