We examined the interactions of live and lysed spirochetes with innate immune cells. THP-1 monocytoid cells were activated to comparable extents by live Borrelia burgdorferi and by B. burgdorferi and Treponema pallidum lysates but were poorly activated by live T. pallidum. Because THP-1 cells poorly internalized live spirochetes, we turned to an ex vivo peripheral blood mononuclear cell system that would more closely reflect spirochetemononuclear phagocyte interactions that occur during actual infection. In this system, B. burgdorferi induced significantly greater monocyte activation and inflammatory cytokine production than did borrelial lysates or T. pallidum, and only B. burgdorferi elicited gamma interferon (IFN-␥) from NK cells. B. burgdorferi was phagocytosed avidly by monocytes, while T. pallidum was not, suggesting that the enhanced response to live B. burgdorferi was due to phagocytosis of the organism. When cytochalasin D was used to block phagocytosis of live B. burgdorferi, cytokine production decreased to levels comparable to those induced by B. burgdorferi lysates, while the IFN-␥ response was abrogated altogether. In the presence of human syphilitic serum, T. pallidum was efficiently internalized and initiated responses resembling those observed with live B. burgdorferi, including the production of IFN-␥ by NK cells. Depletion of monocytes revealed that they were the primary source of inflammatory cytokines, while dendritic cells (DCs) directed IFN-␥ production from innate lymphocytes. Thus, phagocytosis of live spirochetes initiates cell activation programs in monocytes and DCs that differ qualitatively and quantitatively from those induced at the cell surface by lipoprotein-enriched lysates. The greater stimulatory capacity of B. burgdorferi versus T. pallidum appears to be explained by the successful recognition and phagocytosis of B. burgdorferi by host cells and the ability of T. pallidum to avoid detection and uptake by virtue of its denuded outer membrane rather than by differences in surface lipoprotein expression.

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...

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