Kelly L. Hawley

CD14 cooperates with complement receptor 3 to mediate MyD88-independent phagocytosis of Borrelia burgdorferi

Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is a poorly understood process, despite its importance during the host immune response to infection. B. burgdorferi has been shown to bind to different receptors on the surface of phagocytic cells, including the β2 integrin, complement receptor 3 (CR3). However, whether these receptors mediate the phagocytosis of the spirochete remains unknown. We now demonstrate that CR3 mediates the phagocytosis of the spirochete by murine macrophages and human monocytes. Interaction of B. burgdorferi with the integrin is not sufficient, however, to internalize the spirochete; phagocytosis requires the interaction of CR3 with the GPI-anchored protein, CD14, independently of TLR/MyD88-induced or inside-out signals. Interestingly, the absence of CR3 leads to marked increases in the production of TNF in vitro and in vivo, despite reduced spirochetal uptake. Furthermore, the absence of CR3 during infection with B. burgdorferi results in the inefficient control of bacterial burdens in the heart and increased Lyme carditis. Overall, our data identify CR3 as a MyD88-independent phagocytic receptor for B. burgdorferi that also participates in the modulation of the proinflammatory output of macrophages. These data also establish a unique mechanism of CR3-mediated phagocytosis that requires the direct cooperation of GPI-anchored proteins.

Phagosomal TLR signaling upon Borrelia burgdorferi infection

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.

CD14 Targets Complement Receptor 3 to Lipid Rafts during Phagocytosis of Borrelia burgdorferi

Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is mediated partly by the interaction of the spirochete with Complement Receptor (CR) 3. CR3 requires the GPI-anchored protein, CD14, in order to efficiently internalize CR3-B. burgdorferi complexes. GPI-anchored proteins reside in cholesterol-rich membrane microdomains, and through its interaction with partner proteins, help initiate signaling cascades. Here, we investigated the role of CD14 on the internalization of B. burgdorferi mediated by CR3. We show that CR3 partly colocalizes with CD14 in lipid rafts. The use of the cholesterol-sequestering compound methyl-β-cyclodextran completely prevents the internalization of the spirochete in CHO cells that co-express CD14 and CR3, while no effect was observed in CD11b-deficient macrophages. These results show that lipid rafts are required for CR3-dependent, but not independent, phagocytosis of B. burgdorferi. Our results also suggest that CD14 interacts with the C-lectin domain of CR3, favoring the formation of multi-complexes that allow their internalization, and the use of β-glucan, a known ligand for the C-lectin domain of CR3, can compensate for the lack of CD14 in CHO cells that express CR3. These results provide evidence to understand the mechanisms that govern the interaction between CR3 and CD14 during the phagocytosis of B. burgdorferi.

IFNγ Enhances CD64-Potentiated Phagocytosis of Treponema pallidum Opsonized with Human Syphilitic Serum by Human Macrophages

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.

Serum C3 Enhances Complement Receptor 3-Mediated Phagocytosis of Borrelia burgdorferi

Complement receptor (CR) 3 is a bona fide phagocytic receptor for Borrelia burgdorferi, the causative agent of Lyme borreliosis 1, 2. CR3 is also the receptor for the opsonin iC3b, a final degradation product of the complement component C3 3, 4. The transit of B. burgdorferi through the blood during the dissemination phase exposes the spirochete to serum components, including complement 5. B. burgdorferi contains proteins with anti-complement activities that trigger the inactivation of C3b through the binding of factor H (FH) on the surface of the bacterium 6. This could lead to the accumulation of the opsonin, iC3b, and the enhanced phagocytosis of the spirochete. To test whether the presence of serum would increase the phagocytosis of B. burgdorferi, we incubated bone marrow-derived macrophages (BMMs), generated as described 1, with heat-inactivated (HI; 56 oC, 30 min) or active normal mouse sera (NMS). Phagocytosis protocols were followed according to published reports 1, 2. The presence of 10% HI serum resulted in phagocytosis levels comparable to those in the absence of serum (Fig. ​(Fig.1A,B).1A,B). However, the presence of 10 % NMS enhanced the capacity of macrophages to internalize B. burgdorferi (Fig. ​(Fig.1A).1A). The same effect was observed on RAW264.7 (RAW) cells (Fig. ​(Fig.1C).1C). Thus, the contact of B. burgdorferi with serum components results in their enhanced phagocytic uptake. Next, we assessed whether the increased phagocytic activity associated with the presence of serum was due to C3-derived opsonin generation. We used sera from C3-deficient mice (C3 KO), in which B. burgdorferi dissemination is enhanced compared to WT mice 5. The presence of C3 KO serum abrogated the increased phagocytosis observed in the presence of NMS, both in BMMs (Fig. ​(Fig.1A,B)1A,B) and RAW cells (Fig. ​(Fig.11C).

Sequence Variation of Rare Outer Membrane Protein β-Barrel Domains in Clinical Strains Provides Insights into the Evolution of Treponema pallidum subsp. pallidum, the Syphilis Spirochete

ABSTRACT In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum. They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development. IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops. Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.

Inflammation and immune evasion coexist in Treponema pallidum–infected skin

IF: immunofluorescence SS: secondary syphilis INTRODUCTION Syphilis is a systemic, multistage, sexually transmitted infection caused by the highly invasive spirochetal bacterium, Treponema pallidum, subspecies pallidum. In the United States, the annual rate of primary and secondary syphilis (SS) between 2002 and 2016 has increased from 2.1 to 8.7 cases per 100,000. Gestational and congenital syphilis cases have also increased in the last few years. There is no evidence of a change in T pallidum susceptibility to penicillin as an explanation for the significant increase in the number of syphilis cases in the United States. It is more likely that changes in risk-taking behavior in the general population are responsible for this change. Although syphilis is easily treatable with penicillin, if left untreated up to one-third of syphilitic patients will go on to have the typical complications associated with tertiary syphilis. It is therefore critically important for clinicians to be well versed in the classic and not so classic dermatologic manifestations of the disease.

Controlled M1-to-M2 transition of aged macrophages by calcium phosphate coatings

Older adults suffer from weakened and delayed bone healing due to age-related alterations in bone cells and in the immune system. Given the interaction between the immune system and skeletal cells, therapies that address deficiencies in both the skeletal and the immune system are required to effectively treat bone injuries of older patients. The sequence of macrophage activation observed in healthy tissue repair involves a transition from a pro-inflammatory state followed by a pro-reparative state. In older patients, inflammation is slower to resolve and impedes healing. The goal of this study was to design a novel drug delivery system for temporal guidance of the polarization of macrophages using bone grafting materials. A biomimetic calcium phosphate coating (bCaP) physically and temporally separated the pro-inflammatory stimulus interferon-gamma (IFNγ) from the pro-reparative stimulus simvastatin (SIMV). Effective doses were identified using a human monocyte line (THP-1) and testing culminated with bone marrow macrophages obtained from old mice. Sequential M1-to-M2 activation was achieved with both cell types. These results suggest that this novel immunomodulatory drug delivery system holds potential for controlling macrophage activation in bones of older patients.

LB1.64 Cd64-mediated phagocytosis of human syphilitic serum opsonized treponema pallidum by human macrophages required ifn-gamma

Introduction Syphilis is a multi-stage, sexually transmitted disease caused by the spirochete Treponema pallidum (Tp). Clinical manifestations result from the treponeme’s ability to elicit a robust immune response while at the same time evading host defenses. Syphilitic lesions are comprised of a rich cellular infiltrate, which includes IFN-gamma (IFNg) producing T cells, NK cells and activated macrophages. We 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. The purpose of this study is to establish a potential role for macrophages and opsonic Ab in clearance of Tp and generation of tissue-based inflammation during human syphilis. Methods We used monocyte-derived macrophages to develop an ex vivo model for studying spirochete-macrophage interactions. We used macrophage-colony stimulating factor and IFNg for macrophage maturation and evaluated the immunophenotypic modulations by flow cytometry. We assessed Tp uptake, in the presence or absence of HSS by confocal microscopy. We also determined the cellular responses initiated by opsonophagocytosis of Tp using targeted transcriptional array analysis and cytokine bead array. Results IFNg polarisation of macrophages led to an increase in Fcg receptors (FcgRs) expression, phagocytosis of HSS opsonized Tp and cytokine production. Blockade of CD64 significantly diminished spirochetal uptake and pro-inflammatory cytokine secretion by the macrophages. Conclusion Our ex vivo studies provide a potential role for macrophages in clearance of Tp during human syphilis. These data are the first to demonstrate that CD64 in the primary FcR involved in opsonophagocytosis of Tp and IFNg plays a critical role in the macrophages responsiveness following uptake of the spirochete. Moreover, our study results also provide an ex vivo surrogate system for use in future syphilis vaccine studies.