Processing of pathogens by immune cells

GFP-expressing Borrelia burgdorferi-cell.
Immunofluorescence micrograph of a GFP-expressing B. burgdorferi cell (green). Scale bar: 2 µm. Click image to enlarge.

Spirochetes of the Borrelia burgdorferi sensu lato complex are the causative agent of Lyme borreliosis, a chronic multisystemic disorder affecting primarily the skin, nervous system, heart and joints. Borreliae are transmitted by a tick bite and disseminate through the blood stream to various organs.

In order to eliminate invading borreliae, professional phagocytes such as macrophages and dendritic cells are recruited to the site of infection. Uptake and subsequent degradation of the spirochetes is thus of critical importance for the prevention of borreliae dissemination and the development of Lyme disease.
Uptake of borreliae takes mostly place by a mechanism termed “coiling phagocytosis”. A characteristic feature of this mechanism is the formation of a unilateral F-actin rich pseudopod which enwraps the spirochete and pulls it inside the cell body.

Coiling phagocytosis.
Confocal micrograph of primary human macrophage expressing RFP-Lifeact (red) enwrapping several times (see arrowheads) a GFP-expressing B. burgdorferi-cell (green) with a pseudopod. Scale bar: 10 µm. Click image to enlarge.

Phagocytosis of borreliae by primary human macrophage.
Time-lapse movie of confocal z-stack showing a primary human macrophage expressing RFP-Lifeact (red) internalizing several GFP-expressing spirochetes (green) with actin-rich cell protrusions. To see movie, click on the image. (sequence 41 min; 4.4 MB)

We are interested in the molecular mechanisms regulating borrelia uptake and focus especially on the involvement of actin-regulating factors such as Arp2/3 complex and formins in this process. Earlier, we could show that coiling phagocytosis of borreliae is regulated by CDC42 and Rac1 and involves recruitment of Arp2/3 complex and WASP (Linder et al., 2001). Recent results show that also the the formins mDia1 and FMNL1 are enriched at macrophage filopodia that are induced by contact with borreliae.
Knockdown experiments underline the requirement of both formins for the formation of Borrelia-induced filopodia and for final internalization of the spirochetes (Naj et al., 2013; see model)

Borrelia induces filopodia formation in human macrophages. Z-stack of a primary macropage (stained for F-actin) without stimulation (A) or after 1h of coincubation with Borrelia burgdorferi (B). Scale bar: 5 µm. Click image to enlarge.
We could also identify the formin Daam1 as a further player in the regulation of phagocytosis of borreliae. Daam1 plays a dual role during Borrelia uptake, first as a regulator of Borrelia-capturing filopodia and second during the formation of the coiling pseudopod that enwraps the bacterial cell during internalization. (Hoffmann et al., 2014). Knockdown experiments underline the requirement of all three formins for successful uptake of borreliae by primary macrophages (Naj et al., 2013; Hoffmann et al., 2014; see model)

Enrichment of GFP-Daam1DC50 during phagocytosis of borreliae.
Confocal micrographs of primary human macrophage expressing GFP-Daam1DC50 (green; A) which is accumulated at the uptake structure of a Borrelia cell visualized by Hoechst 33342 staining of DNA (blue; B) with merge shown in (C).
Scale bar: 5 µm

GFP-Daam1C50 enrichment in uptake structure during internalization of Borrelia.
Time lapse movie shows primary macrophage expressing GFP-Daam1DC50 (green) capturing a Borrelia cell stained by Hoechst 33342 (blue). Note GFP-Daam1DC50 accumulations arising from the macrophage surface and closely following the sinuous morphology of the spirochete cell.
Time since the start of the experiment is indicated in minutes. Scale bar 5 µm.

Model of formin- and Arp2/3 complex dependent actin regulation in coiling phagocytosis of Borrelia.
((A) Upon stimulation with borreliae, macrophages form filopodial protrusions that arise from the cortical network. Filopodia are enriched in the formins mDia1 (localized at tips) and FMNL1 (localized at tips and shaft), which probably contribute to longitudinal growth of filopodia, and Daam1 (localized in filopodial shaft) which is probably involved through its actin-bundling activity. (B) Upon capturing of a Borrelia cell by filopodia, the spirochete is enwrapped by a coiling pseudopod. Until now, it was not clear whether coiling pseudopods develop from filopodia or if they constitute independent structures. Our live-cell experiments showed that Daam1-positive coiling pseudopods arise as a second independent structure from the macrophage surface and enwrap borreliae. The flexibility of coiling pseudopods that enables them to enwrap the spiral-shaped borreliae is probably due to dot-like accumulations of Arp2/3 complex, which lead to formation of small branched actin networks and probably act as “hinges” at coiling nodes. Click image to enlarge

Current studies are focused on the intracellular processing of borreliae by macrophages.

Hoffmann et al., 2014

Naj et al., 2013

Linder et al., 2001