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.

Fig 1

Formation of F-actin-rich uptake structures during coiling phagocytosis of Borrelia burgdorferi by primary human macrophages. (A and B) Confocal laser scanning micrographs of primary macrophages expressing Lifeact-mRFP (for labeling F-actin; red), in contact with GFP-expressing B. burgdorferi spirochetes (green). (A) Fixed specimen. Note the alteration between F-actin-rich pseudopod coils (arrowheads) and a spirochete enwrapped by a pseudopod. Scale bar, 5 μm. (B) Live-cell microscopy of Borrelia uptake. Still frames from Video S1 in the supplemental material are shown. The white box indicates the area enlarged in the images shown in B′. Note the formation of several F-actin-rich coils of the pseudopodium enwrapping the spirochete (individual coils tracked by open and filled arrowheads, respectively) and eventual complete uptake of the spirochete into the macrophage cell. Time from the start of the experiment is indicated as min:sec. Scale bar, 2 μm.

Fig 2

(A to I) Localization of Arp2/3 complex and the formins FMNL1 and mDia1 at B. burgdorferi-containing uptake structures of macrophages. Confocal immunofluorescence micrographs of primary human macrophages coincubated with GFP-expressing B. burgdorferi spirochetes and stained for endogenous proteins using specific primary antibodies. Note the accumulation of the Arp2/3 complex subunit Arp2, of FMNL1, and of mDia1 in macrophage pseudopodia (arrowheads in first and third columns) which contact borreliae. Scale bar, 5 μm.

Fig 3

Borrelia-stimulated macrophage pseudopodia are enriched in EGFP-FMNL1β or EGFP-mDia1ΔDAD and are positive for the filopodium marker fascin. Confocal micrographs of macrophages expressing mCherry-fascin (red) and EGFP-FMNL1β (green; A) or EGFP-mDia1ΔDAD (green; B). (A and B) Still images from Videos S2 and S3 in the supplemental material. Dashed white boxes indicate the areas enlarged in the images shown in panels A′ and B′. Note localization of EGFP-FMNL1β along the length of filopodia, including the fascin-free tip (arrowhead), and its persistence over time (A′). Note the localization of EGFP-mDia1ΔDAD especially at the tips of filopodia (arrowheads) (B′). Scale bar, 2 μm. Time from the start of the experiments is indicated as min:sec. (C and D) Evaluation of number and length of filopodia in unstimulated macrophages and macrophages stimulated by addition of either live or heat-killed borreliae. To distinguish between microspikes and filopodia, only protrusions with a length of >3 μm were evaluated. Values are given as means ± SEM.

Fig 4

siRNA-induced depletion of FMNL1 or mDia1 impairs the internalization of B. burgdorferi by primary human macrophages. (A) Western blots of lysates from macrophages treated with luciferase-specific siRNA (as a control), FMNL1-specific siRNA (left blot) or mDia1-specific siRNA (right blot). Proteins were detected with specific antibodies, with β-actin used as a loading control. Molecular mass in kDa is indicated. (B) Quantification of Borrelia-induced pseudopodia. The number of macrophages with pseudopodia in control cells treated with luciferase siRNA was set to 100%. Note the pronounced reduction of pseudopodium formation in cells depleted of FMNL1 or mDia1. Values are shown as means ± SEM (*, P < 0.05). The proportions of macrophages with pseudopodia are 49.6% ± 7.6% for FMNL1 siRNA and 25.1% ± 4.2% for mDia1 siRNA. For each value, each time 30 cells from three different donors were evaluated in three independent experiments. (C to J) Principle of outside-inside staining. Confocal laser scanning micrographs of macrophages coincubated with borreliae. To distinguish between internalized and noninternalized spirochetes, specimens were fixed and not permeabilized (−perm), stained with Bss42 B. burgdorferi antibody and with Alexa Fluor 568-labeled secondary antibody (red; C and G), and subsequently permeabilized (+perm) and stained with the same primary antibody but with Alexa Fluor 488-labeled secondary antibody (green; D and H). Internalized borreliae are detected only by the second round of staining and appear green; borreliae on the outside of the macrophages are detected by both stainings (red and green) and appear yellow in the merged image (F and J). Macrophages were stained with Alexa Fluor 647-labeled phalloidin to detect F-actin (E and I). Scale bar, 5 μm. (K) Internalization of borreliae by macrophages treated with specific siRNAs, based on evaluation of respective outside-inside staining. The phagocytic index is indicated as the ratio of internalized to noninternalized spirochetes. Values are given as means ± SEM (*, P < 0.05). Note the pronounced reduction of the phagocytic index upon depletion of either FMNL1 or mDia1 (phagocytic index of 0.38 ± 0.1 for FMNL1 siRNA and 0.22 ± 0.02 for mDia1 siRNA). For each value, each time 30 cells from three different donors were evaluated in three independent experiments. (L) Coiling phagocytosis of borreliae is reduced upon knockdown of FMNL1 or mDia1. Fixed specimens of borreliae in contact with macrophages were evaluated for the presence of several, clearly recognizable whorls enwrapping spirochetes as an indicator for coiling phagocytosis. Values are given as means ± SEM (*, P < 0.05). The percentages of uptake by coiling phagocytosis were 31.2% ± 6.5% for control macrophages transfected with luciferase siRNA, 13.7% ± 3.7% for macrophages transfected with FMNL1-specific siRNA, and 13.3% ± 2.9% for macrophages transfected with mDia1-specific siRNA. For the numbers of attached spirochetes per macrophage cell, see Fig. S3 in the supplemental material.

Fig 5

Model of formin- and Arp2/3 complex-dependent actin regulation in coiling phagocytosis of Borrelia. (A) The cortical actin network of macrophages contains branched actin networks. (B) Upon stimulation with borreliae, macrophages form filopodial protrusions that arise from the cortical network. Filopodium tips are enriched in the formins FMNL1 and mDia1, which probably contributes to longitudinal growth of filopodia, and contain actin filaments bundled by fascin. (C to E) Upon capturing of a Borrelia cell, filopodia enwrap the spirochete. Enwrapping and lateral growth of filopodia into coiling pseudopods are probably enabled by dot-like accumulations of Arp2/3 complex, leading to small, branched actin networks at coiling nodes. Local accumulations of FMNL1 contribute to coiling pseudopod growth by modulating actin filament growth.