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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.

SUBMITTER: Naj X

PROVIDER: S-EPMC3647995 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Interleukin-10 (IL-10) modulates inflammatory responses elicited in vitro and in vivo by Borrelia burgdorferi, the Lyme disease spirochete. How IL-10 modulates these inflammatory responses still remains elusive. We hypothesize that IL-10 inhibits effector functions of multiple genes induced by B. burgdorferi in macrophages to control concomitantly elicited inflammation. Because macrophages are essential in the initiation of inflammation, we used mouse J774 macrophages and live B. burgdorferi spirochetes as the model target cell and stimulant, respectively. First, we employed transcriptome profiling to identify genes that were induced by stimulation of cells with live spirochetes and that were perturbed by addition of IL-10 to spirochete cultures. Spirochetes significantly induced upregulation of 347 genes at both the 4-h and 24-h time points. IL-10 inhibited the expression levels, respectively, of 53 and 65 of the 4-h and 24-h genes, and potentiated, respectively, at 4 h and 24 h, 65 and 50 genes. Prominent among the novel identified IL-10-inhibited genes also validated by quantitative real-time PCR (qRT-PCR) were Toll-like receptor 1 (TLR1), TLR2, IRAK3, TRAF1, IRG1, PTGS2, MMP9, IFI44, IFIT1, and CD40. Proteome analysis using a multiplex enzyme-linked immunosorbent assay (ELISA) revealed the IL-10 modulation/and or potentiation of RANTES/CCL5, macrophage inflammatory protein 2 (MIP-2)/CXCL2, IP-10/CXCL10, MIP-1?/CCL3, granulocyte colony-stimulating factor (G-CSF)/CSF3, CXCL1, CXCL5, CCL2, CCL4, IL-6, tumor necrosis factor alpha (TNF-?), IL-1?, IL-1?, gamma interferon (IFN-?), and IL-9. Similar results were obtained using sonicated spirochetes or lipoprotein as stimulants. Our data show that IL-10 alters effectors induced by B. burgdorferi in macrophages to control concomitantly elicited inflammatory responses. Moreover, for the first time, this study provides global insight into potential mechanisms used by IL-10 to control Lyme disease inflammation.

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The formins FMNL1 and mDia1 regulate coiling phagocytosis of Borrelia burgdorferi by primary human macrophages.

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