Internalization and degradation of live Bb within phagosomal compartments of monocytes, macrophages and dendritic cells (DCs), allows for the release of lipoproteins, nucleic acids and other microbial products, triggering a broad and robust inflammatory response. Toll-like receptors (TLRs) are key players in the recognition of spirochetal ligands from whole viable organisms (i.e., vita-PAMPs). Herein we will review the role of endosomal TLRs in the response to the Lyme disease spirochete.

Syphilis is a multi-stage, sexually transmitted disease caused by the spirochete Treponema pallidum (Tp). Considered broadly, syphilis can be conceptualized as a dualistic process in which spirochete-driven inflammation, the cause of clinical manifestations, coexists to varying extents with bacterial persistence. Inflammation is elicited in the tissues, along with the persistence of spirochetes to keep driving a robust immune response while evading host defenses; this duality is best exemplified during the florid, disseminated stage called secondary syphilis (SS). SS lesions typically contain copious amounts of spirochetes along with a mixed cellular infiltrate consisting of CD4+ T cells, CD8+ T cells, NK cells, plasma cells, and macrophages. In the rabbit model, Tp are cleared by macrophages via antibody-mediated opsonophagocytosis. Previously, we demonstrated that human syphilitic serum (HSS) promotes efficient uptake of Tp by human monocytes and that opsonophagocytosis of Tp markedly enhances cytokine production. Herein, we used monocyte-derived macrophages to study Tp–macrophage interactions ex vivo. In the absence of HSS, monocyte-derived macrophages internalized low numbers of Tp and secreted little cytokine (e.g., TNF). By contrast, these same macrophages internalized large numbers of unopsonized Borrelia burgdorferi and secreted robust levels of cytokines. Maturation of macrophages with M-CSF and IFNγ resulted in a macrophage phenotype with increased expression of HLA-DR, CD14, inducible nitric oxide synthase, TLR2, TLR8, and the Fcγ receptors (FcγR) CD64 and CD16, even in the absence of LPS. Importantly, IFNγ-polarized macrophages resulted in a statistically significant increase in opsonophagocytosis of Tp accompanied by enhanced production of cytokines, macrophage activation markers (CD40, CD80), TLRs (TLR2, TLR7, TLR8), chemokines (CCL19, CXCL10, CXCL11), and TH1-promoting cytokines (IL-12, IL-15). Finally, the blockade of FcγRs, primarily CD64, significantly diminished spirochetal uptake and proinflammatory cytokine secretion by IFNγ-stimulated macrophages. Our ex vivo studies demonstrate the importance of CD64-potentiated uptake of opsonized Tp and suggest that IFNγ-activated macrophages have an important role in the context of early syphilis. Our study results also provide an ex vivo surrogate system for use in future syphilis vaccine studies.

Background Macrophages play prominent roles in bacteria recognition and clearance, including Borrelia burgdorferi (Bb), the Lyme disease spirochete. To elucidate mechanisms by which MyD88/TLR signaling enhances clearance of Bb by macrophages, we studied wildtype (WT) and MyD88−/−Bb-stimulated bone marrow-derived macrophages (BMDMs). Results MyD88−/− BMDMs exhibit impaired uptake of spirochetes but comparable maturation of phagosomes following internalization of spirochetes. RNA-sequencing of infected WT and MyD88−/− BMDMs identified a large cohort of differentially expressed MyD88-dependent genes associated with re-organization of actin and cytoskeleton during phagocytosis along with several MyD88-independent chemokines involved in inflammatory cell recruitment. We computationally generated networks which identified several MyD88-dependent intermediate proteins (Rhoq and Cyfip1) that are known to mediate inflammation and phagocytosis respectively. Conclusion Our findings show that MyD88 signaling enhances, but is not required, for bacterial uptake or phagosomal maturation and provide mechanistic insights into how MyD88-mediated phagosomal signaling enhances Bb uptake and clearance.

35Lyme disease is a tick-borne illness caused by the spirochete Borrelia burgdorferi (Bb). It 36 is believed that the robust inflammatory response induced by the host's innate immune 37 system is responsible for the clinical manifestations associated with Bb infection. The 38 macrophage plays a central role in the immune response to many bacterial infections and 39 is thought to play a central role in activation of the innate immune response to Bb. 40 Previous studies have shown that following phagocytosis of spirochetes by macrophages, 41 phagosome maturation results in degradation of Bb and liberation of bacterial lipoproteins 42 and nucleic acids, which are recognized by TLR2 and TLR8, respectively, and elicit 43MyD88-mediated phagosome signaling cascades. Bone marrow-derived macrophages 44 (BMDMs) from MyD88 -/mice show significantly reduced spirochete uptake and 45 inflammatory cytokine production when incubated with Bb ex vivo. Paradoxically, 46additional studies revealed that Bb-infected MyD88 -/mice exhibit inflammation in joint 47 and heart tissues. To determine the contribution of MyD88 to macrophage-mediated 48 spirochete clearance, we compared wildtype (WT) and MyD88 -/mice using a murine 49 model of Lyme disease. MyD88 -/mice showed increased Bb burdens in hearts 28 days 50 post infection, while H&E staining and immunohistochemistry showed significantly 51 increased inflammation and greater macrophage infiltrate in the hearts of MyD88 -/mice. 52This suggests that Bb triggers MyD88-independent inflammatory pathways in 53 macrophages to facilitate cell recruitment to tissues. Upon stimulation with Bb ex vivo, 54WT and MyD88 -/-BMDMs exhibit significant differences in bacteria uptake, suggesting 55 that MyD88 signaling mediates cytoskeleton remodeling and the formation of membrane 56 protrusions to enhance bacteria phagocytosis. A comprehensive transcriptome 57 comparison in Bb-infected WT and MyD88 -/-BMDMs identified a large cohort of MyD88-58 dependent genes that are differentially expressed in response to Bb, including genes 59 involved in actin and cytoskeleton organization (Daam1, Fmnl1). We also identified a 60 cohort of differentially-expressed MyD88-independent chemokines (Cxcl2, Ccl9) known 61 to recruit macrophages. We identified master regulators and generated networks which 62 model potential signaling pathways that mediate both phagocytosis and the inflammatory 63 AUTHOR SUMMARY 67Macrophages play prominent roles in bacteria recognition and clearance, including 68Borrelia burgdorferi (Bb), the Lyme disease spirochete. To elucidate mechanisms by 69 which MyD88/TLR signaling enhances clearance of Bb by macrophages, we studied Bb-70 infected wildtype (WT) and MyD88-/-mice and Bb-stimulated bone marrow-derived 71 macrophages (BMDMs). Bb-infected MyD88-/-mice show increased bacterial burdens, 72 macrophage infiltration and altered gene expression in inflamed heart tissue. MyD88-/-73 BMDMs exhibit impaired uptake of spirochetes but comparable maturation of 74 phagosomes following internaliza...