Date Published: March 18, 2019
Publisher: Public Library of Science
Author(s): Astrid Skjesol, Mariia Yurchenko, Korbinian Bösl, Caroline Gravastrand, Kaja Elisabeth Nilsen, Lene Melsæther Grøvdal, Federica Agliano, Francesco Patane, Germana Lentini, Hera Kim, Giuseppe Teti, Aditya Kumar Sharma, Richard K. Kandasamy, Bjørnar Sporsheim, Kristian K. Starheim, Douglas T. Golenbock, Harald Stenmark, Mary McCaffrey, Terje Espevik, Harald Husebye, Dana J. Philpott.
Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.
Phagocytosis is a complex and versatile process that eliminates pathogens and is performed by specialized cells such as macrophages . Phagocytosis requires cell surface receptors recognizing the pathogen  and Rho GTPases controlling local actin dynamics that drive engulfment [2–5]. Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS) present on Gram-negative bacteria , and data from mouse macrophages show that TLR4 is required for the phagocytosis of E. coli [7, 8]. Moreover, LPS-stimulated phagocytosis of E. coli occurs through actin polymerization controlled by Rho GTPases, Rac1 and Cdc42, although the mechanisms are unclear .
In the present study, we show that TLR4 mediates phagocytosis of E. coli in macrophages via its adaptor TRAM. TRAM performs this function by interacting with FIP2 which subsequently activates the Rac1 and Cdc42 Rho GTPases for controlling actin-dynamics. A consequence of this is that FIP2 strongly regulates phagosomal signalling that involves IRF3 activation. Receptor recognition during phagocytosis launches signalling pathways that induce remodelling of the actin cytoskeleton and extension of membrane protrusions that surround the particle to form a phagocytic cup . In early phases of E. coli phagosome formation, TLR4 is recruited to the phagocytic cup to provide a platform for subsequent TRAM-TRIF signalling . Our findings demonstrate that TRAM recruitment to this platform requires FIP2 and that TLR4-TRAM-TRIF signalling is needed for phagocytosis.