This study was carried out in accordance with the recommendations of the Institutional Review Boards at UConn Health, Farmington CT and Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM) in Cali, Colombia. All study participants were provided written informed consent. All animal experimentation was conducted following the Guide for the Care and Use of Laboratory Animals (8th Edition) and in accordance with protocols reviewed and approved by the UConn Health Institutional Animal Care and Use Committee under the auspices of Animal Welfare Assurance A347-01.

Four micrometer sections, cut from a 4-mm punch biopsy that were fixed in 10% buffered formalin and embedded in paraffin, were stained with hematoxylin and eosin (H&E) or labeled immunohistochemically with antibodies against CD4 (clone EP204, Epitomics, Cambridge, MA), CD8 (clone 4B11, Leica Biosystems Inc., Buffalo Grove, IL), CD56 (clone 56C04 Thermo-Scientific, Waltham, MA), CD68 (clone PG-M1, Dako, Carpinteria, CA) and CD138 (clone B-A38 Cell Marque, Rocklin, CA) using an automated immunohistochemistry staining platform (Bond Max, Leica-Microsystems, Buffalo Grove, IL). IHC detection of Tp was performed manually as previously described (10) using a rabbit polyclonal anti-Tp Ab (Biocare, Concord, CA, USA). Skin specimens from healthy volunteers of the same socioeconomic background and conditions were not available for analysis. Tissue known to contain Tp were used for the positive control of Tp IHCs. Additionally, secondary alone controls were used to assess any non-specific Ab binding for each Ab used.

Treponema pallidum (Nichols strain) was propagated by intratesticular inoculation of adult male New Zealand white rabbits and harvested in CMRL medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Atlantic Biologicals, Miami, FL, USA) at peak orchitis (12). Spirochetes were enumerated by dark-field microscopy on a Petroff-Hausser counting chamber (Hausser Scientific, Horsham, PA, USA). Virulent Borrelia burgdorferi (Bb) strain 297 encoding green fluorescent protein on a cp32-based shuttle vector (27) was propagated in BSK-H medium containing 6% rabbit serum (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) and 400 μg/ml of kanamycin (Sigma-Aldrich Chemical Co.). For macrophage incubation experiments, Bb temperature-shifted from 23 to 37°C were grown to late-logarithmic phase (~8 × 10⁷ spirochetes/ml), washed twice with RPMI, and resuspended in RPMI to a final density of ~3 × 10⁸ spirochetes/ml.

Peripheral blood was obtained from healthy donors determined to be seronegative for syphilis by Rapid Plasma Reagin test and/or Lyme disease by enzyme-linked immunosorbent assay (ELISA) performed in the clinical laboratory at John Dempsey Hospital. Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep and SepMate-50 tubes in accordance with the recommendations of the manufacturer (STEMCELL Technologies, Vancouver, BC, Canada). To generate all macrophage phenotypes, 3 × 10⁷ PBMCs were plated in 10 cm polystyrene Petri dishes (BD Falcon) and incubated for 2 h at 37°C with 5% CO₂. Adherent cells were washed thoroughly with ice-cold PBS to remove non-monocyte populations. Macrophage nomenclature used throughout follows the recommendations of Murray et al. (28). MΦ(−)s were generated by incubating the adherent monocytes in RPMI-1640 (RPMI) medium (Gibco, Thermo-Scientific) supplemented with 20% heat-inactivated (56°C for 30 min) normal human serum (NHS) (CORNING, Corning, NY, USA) and 1% penicillin–streptomycin (10,000 U/ml) (Gibco) for 10 days. MΦ(C)s were generated by incubating the adherent monocytes in RPMI, 20% heat-inactivated NHS, and 50 ng/ml of M-CSF (Peprotech, Rocky Hill, NJ, USA) (29) for 10 days. To generate MΦ(IFNγ)s, MΦ(C)s were divided on day 7 and 2.5 ng/ml of recombinant IFNγ (Roche Diagnostics, Mannheim, Germany) (30) was added to one portion for an additional three days of incubation. Media and cytokines were replenished every 3 days for a total of 10 days. Because Tp lacks endotoxin and would not be encountered by macrophages in syphilitic lesions, LPS, which is often used in macrophage differentiation protocols, was omitted from all culture media.

All Tp opsonization studies were performed using a pool of deidentified sera collected from five HIV-negative primary or SS patients seen at Parkland Memorial Hospital in Dallas, TX, USA (approved for use by the Institutional Review Board of the UConn Health) (31). Strong reactivity of the pool with Tp proteins was confirmed by immunoblot (Trinity Biotech, Carlsbad, CA, USA, Figure S2 in Supplementary Material).

Freshly harvested Tp, adjusted to a final concentration of 3 × 10⁸ treponemes/ml, were incubated with either 10% NHS or 10% pooled HSS for 2 h at RT prior to addition to macrophages. Macrophages, differentiated as described above, were plated at 1 × 10⁵ cells in 500 μl of RPMI supplemented with 10% heat-inactivated FBS (Hyclone Laboratories, Inc., Logan, UT, USA) in an eight-well chamber microscopy slide (Millipore, Billerica, MA, USA). Tp were added at a multiplicity of infection (MOI) of 30:1 for 8 h at 37°C with 5% CO₂. All culture media and reagents were confirmed to be free of LPS contamination (≤10 pg/ml) by Limulus amebocyte lysate assay quantification (Cambrex, MA, USA). Following incubation, supernatants were removed and macrophages were prepared for IFA to evaluate binding and uptake of treponemes. Cells were fixed and permeabilized with 2% paraformaldehyde and 0.01% Triton X-100 for 10 min at RT. They then were rinsed with PBS, blocked with PBS containing 10% normal goat serum (NGS) for 1 h at RT, and then incubated with rabbit polyclonal anti-Tp (1:100, Abcam, Cambridge, MA, USA) in PBS 1% NGS for 1 h at RT. After four successive washes with PBS, the cells were then incubated with goat anti-rabbit immunoglobulin G (IgG)-Texas Red (1:500) in PBS 1% NGS for 1 h at RT. After staining for Tp, actin cytoskeletons were stained with Phalloidin-AF488 (1:20) (Life Technologies, Carlsbad, CA, USA) for 20 min at RT. The cells were then washed thoroughly with PBS six times, rinsed with deionized water to remove salt and allowed to air dry. Finally, Vectashield containing DAPI (Vector Laboratories, Inc., Burlingame, CA, USA) was added and samples were sealed with a coverslip. To assess binding and internalization of Tp, images of 100–200 macrophages were acquired on an Olympus BX60 epifluorescence microscope equipped with a Retiga 2000R CCD camera (QImaging) or Ziess LSM 780 confocal microscope mounted on an inverted Axio Observer Z1. Similar numbers of macrophages were imaged for each experimental condition per biological replicate. Acquired images were processed with ImageJ (version 1.5.1 g) (NIH, USA) and uptake was quantitated in a blinded fashion. The percentage of macrophages with bound spirochetes was quantified by dividing the number of cells with surface bound spirochetes by the total number of macrophages imaged for each condition. The percentage of spirochete-positive macrophages were calculated by dividing the number of cells containing ≥1 internalized spirochetes by the total number of cells imaged. Phagocytic indices were calculated by dividing the number of degraded, internalized spirochetes by the total number of spirochete-positive macrophages for the same condition. To quantify the percentage of Tp remaining after the 8 h incubation period, 10 μl aliquots from Tp-stimulated-macrophage supernatants were enumerated, in triplicate, by dark-field microscopy. Percentage of Tp recovered was calculated using a “time zero” spirochetal count.

MΦ(−)s, after maturation as described above, were plated at 1 × 10⁵ cells in 500 μl of RPMI supplemented with 10% FBS in an eight-well chamber microscopy slide and incubated at 37°C with 5% CO₂ overnight to allow for attachment. Prior to incubation, medium was removed and replaced with fresh RPMI supplemented with 10% FBS. Bb were added at an MOI of 30:1 and incubated for 8 h. Following incubation, cells were fixed and permeablized and cytoskeletons were stained with Phalloidin-AF594 as described above. Binding and internalization of spirochetes was performed as described above.

TNF, IL-6, IL-1β, IL-10, IL-12, and IL-8 were measured in supernatants using a Human Inflammatory Cytokine Bead Array per the manufacturer’s (BD Biosciences, San Jose, CA, USA) protocol. Data were collected on a MACSQaunt Analyzer (Miltenyi Biotec, Germany) and analyzed with FCAP Array™ Software version 3.0 (BD Biosciences).

MΦ(C)s and MΦ(IFNγ)s were harvested from 10-cm polystyrene Petri dishes by replacing growth medium with PBS containing 1% FBS and gently lifting the cells with a scraper. The macrophages were washed once with PBS in 50 ml conical tubes by spinning at 300g for 10 min; the cells were decanted, resuspended in PBS 1% FBS, and dispensed into FACS tubes in preparation for staining. Cells were incubated for 10 min at 4°C with 10 μg/ml of purified human IgG (Sigma, St. Louis, MO, USA) for Fcγ receptor (FcR) blocking, followed by a 20 min incubation with fluorochrome-conjugated antibodies. Antibodies obtained from Biolegend (CD32-APC, CD64-PECy7, CD163-PerCP/Cy5.5, CD206-APC, anti-rabbit IgG-BV421), eBioscience (CD14-APC, CD16-PE, TLR2-FITC, TLR4-APC), Novus Biologicals (TLR7-PE, TLR8-AF647), BD Biosciences (HLA-DR-PE), Invitrogen (CD68-FITC), and Abcam [rabbit anti-human inducible nitric oxide synthase (iNOS)] were used at dilutions recommended by the manufacturers. For intracellular staining, the cells were surface-stained as described above, permeabilized in 250 μl of Cytofix/Cytoperm solution (BD Biosciences) for 20 min at 4°C, washed with PermWash buffer (BD Biosciences), stained with fluorochrome-conjugated antibodies (diluted in 50 μl of PermWash buffer) for 30 min at 4°C, and washed twice with PermWash and resuspended in FACS buffer. A minimum of 10,000 single cells events were acquired using a BD LSR-II flow cytometer and FACSDIVA™ software (BD Biosciences). Analysis of immunostaining was performed using FlowJo V10 for Mac (FlowJo LLC, Ashland, OR, USA). Mean fluorescence intensity (MFI) values were determined after subtracting background fluorescence (32).

1 × 10⁶ MΦ(C)s and MΦ(IFNγ)s were plated in 1 ml of RPMI containing 10% FBS in a 12-well tissue culture plate (Corning) and incubated with unopsonized or opsonized Tp for 8 h at an MOI 30:1. RNAs were extracted using a NucleoSpin RNA purification kit according to the manufacturer’s (Macherey-Nagel Inc., Bethlehem, PA, USA) instructions and their concentrations determined using a Nanodrop spectrophotometer (Thermo-Scientific). cDNA synthesis was performed using the High Capacity cDNA Archive Kit (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. Transcripts were amplified using TaqMan^® Human Immune Array (384-fluidics card) and TaqMan^® Human Phagocytosis Array (96-well plate) per manufacturer’s instructions (Applied Biosystems). Briefly, the Immune Array was performed in a 2 μ1 reaction volume containing 62.5 pg of cDNA, and 1 μ1 of gene expression master mix. The Phagocytosis Array was processed in a 20 μl reaction volume containing 5 ng of cDNA and 10 ml of gene expression master mix. Commercially available primers (Applied Biosystems) were used to amply transcripts for human IRF3 (Hs01547283_m1), IRF5 (Hs00158114_m1), IRF7 (Hs00185375_m1), and IFNB (Hs00277188_s1) as described previously (33). Amplification reactions were performed using a 7900HT Fast Real Time thermocycler (Applied Biosystems) using the following conditions: 95°C for 20 min, and 40 cycles of 95°C for 1 s and 60°C for 20 s. Expression levels for all transcripts were normalized to GAPDH and the relative changes in gene expression between experimental groups were calculated using the 2^−ΔΔCt method (34). To identify genes differentially expressed in the presence of IFNγ and absence of Tp, the average fold changes for unstimulated MΦ(IFNγ)s were calculated relative to the unstimulated MΦ(C)s. To identify genes differentially expressed in the presence of Tp, relative fold changes were calculated based on the unstimulated MΦ(IFNγ)s condition, with a two-fold change and p-value < 0.05 threshold (Student’s t-test) (35). Heat maps and graphs of the fold changes of transcript expression were generated using R statistical software, version 3.2.2 with package “ggplot2,” function “heatmap.2.” Fold change values displayed in the heat maps represent the average fold change compared to the corresponding unstimulated MΦ(IFNγ). No clustering analysis was performed.

1 × 10⁵ MΦ(IFNγ)s were plated in 500 μl of RPMI supplemented with 10% FBS in an eight-well chamber microscopy slide and incubated for 8 h with Tp at an MOI of 30:1. Following incubation, the cells, fixed and permeablized as above, were incubated with mouse anti-human CD64 (1:25, clone 10.1, Abcam) and rabbit polyclonal anti-Tp 1 h at RT, and then washed four times with PBS followed by incubation for 1 h at RT with goat anti-mouse IgG-OregonGreen488 (1:200, Invitrogen, Carlsbad, CA, USA) and goat anti-rabbit IgG-Texas Red (1:500, Life Technologies). Secondary Ab alone controls were evaluated for non-specific binding (Figure S6 in Supplementary Material). Images were acquired on a Zeiss LSM 780 confocal microscope mounted on an inverted Axio Observer Z1 and processed with ImageJ as described above. Colocalization of Tp and CD64 was visualized using the ImageJ plugin “Colocalization”; the coefficient of colocalization was determined using the plugin “JACoP.”

MΦ(IFNγ)s were pre-incubated with 10 μg/ml of mouse antihuman CD64 (clone 10.1, Abcam), mouse anti-human CD32 (clone 6C4, Affymetrix eBiosciences, San Diego, CA, USA), mouse anti-human CD16 (clone 3G8, Biolegend, San Diego, CA, USA), or 10 μg/ml each of all three antibodies for 1 h prior to adding spirochetes. Opsonophagocytosis assays were performed as described above.

General statistical analysis was conducted using GraphPad Prism 6.0 h (GraphPad Software, San Diego, CA, USA). Phagocytic uptake/index, Tp recovery counts, cytokine concentrations, cytometric MFI ratio, and fold increase or decrease for each gene transcript assayed were compared among the different stimuli. Either a paired or unpaired Student’s t-test (i.e., Mann–Whitney test or Wilcoxon test) was used for comparison across two groups. For the analysis of three or more conditions, we used a non-parametric statistical test for trend analysis (Friedman’s test with a Dunnett’s multiple comparisons post-test analysis). For each experiment, the standard error of the mean was calculated and a p-value < 0.05 was considered significant.

To set the stage for our ex vivo studies, we assessed the distribution of macrophages, lymphocytes, and plasma cells in spatial relationship to Tp within representative skin punch biopsy specimens obtained from three HIV-negative SS patients. In line with prior studies (6, 7, 10, 36–39), IHC analysis of SS skin biopsies revealed a rich dermal-epidermal infiltrate (Figure 1A), which in addition to CD4⁺, CD8⁺, and CD56⁺ lymphocytes (Figure S1 in Supplementary Material), had large numbers of CD68⁺ macrophages (40) (Figure 1B). Abundant CD68⁺ cells were present throughout the dermis, at the dermal–epidermal juncture, and, in some cases, surrounding vascular structures within the dermis (Figure 1B). Treponemal numbers and locations in the inflamed skin, as well as their spatial proximity with dermal macrophages, varied among samples (Figure 1C). In SS143 skin biopsy, a small cluster of CD68⁺ macrophages was visualized in close proximity to a spirochete surrounding a blood vessel wall. In SS167 sample, substantial numbers of spirochetes were seen within the interstitium of the papillary dermis (Figures 1B,C). In SS133, large groups of spirochetes also were observed traversing the dermal–epidermal junction and within the stratum basale and stratum spinosum of the epidermis (Figure 1C), whereas very few macrophages were found within the epidermis (Figure 1B). Of particular interest, CD138⁺ plasma cells were commonly observed in perivascular locations and throughout the papillary dermis (Figure 1D). In concert with previously published studies (2, 6, 7), our current results confirm the presence of macrophages in Tp infected tissue, and suggest that the cellular environment may be involved in shaping the phenotype of macrophages, in addition to providing a local source of opsonic antibodies needed for spirochetal recognition and clearance in Tp infected tissues.

Having confirmed that the macrophage is an important cellular element of the inflammatory response to the syphilis spirochete in vivo, we next performed ex vivo experiments to directly characterize macrophage-Tp interactions. We began by maturing adherent human monocytes into macrophages using 20% heat-inactivated NHS without additional exogenous growth factors (e.g., M-CSF). We then assessed the ability of these macrophages [MΦ(−)] to bind and phagocytose spirochetes. As a phagocytosis control, we incubated MΦ(−)s with Bb, a spirochetal pathogen that is readily bound and internalized by primary human phagocytes (41, 42). As shown in Figure 2A and quantified in Figure 2B, Bb binds to MΦ(−)s and is internalized into phagosomal vacuoles (Figures 2A,C). We then assessed if macrophages were able to similarly bind and internalize Tp in the presence or absence of NHS or with HSS. For these experiments, we first confirmed that our pooled HSS reacted with spirochetal antigens by using a Tp IgG Marblot strip test (Figure S2 in Supplementary Material). The pooled HSS was determined to be highly reactive with many of Tp’s proteins, likely including some opsonic targets. It is important to note that we previously demonstrated that the same pooled HSS recognizes the Nichols strain variant of L4, an immunodominant extracellular loop in the β-barrel of BamA and opsonic target (31).

As shown in Figure 2, Tp binds to macrophages with or without NHS. The observed binding does not cause a concomitant increase in bacterial uptake, which suggests that Tp attaches to a non-phagocytic receptor yet to be defined on the macrophage’s surface. On the other hand, addition of HSS caused a significant increase in spirochetal uptake (Figures 2A,C). In line with the increased uptake of opsonized Tp, fewer spirochetes were visualized bound to the macrophage surface (Figure 2B). Interestingly, while internalization of Bb resulted in copious secretion of TNF, opsonophagocytosis of Tp did not (Figure 2D). These findings confirm that HSS promotes opsonophagocytosis of Tp by human macrophages, but also raise the possibility that the milieu in which macrophages encounter spirochetes is an important determinant of macrophage activation and cytokine production following internalization of bacteria.

The observation that cytokine production in Tp stimulated MΦ(−)s was unexpectedly low prompted us to generate a macrophage phenotype that more closely resembles macrophages present within an IFNγ rich syphilitic immune microenvironment in vivo (10, 14). To do so, human monocytes were differentiated into macrophages by adding M-CSF alone [MΦ(C)] or M-CSF with IFNγ [MΦ(IFNγ)] and then characterized by flow cytometry, gene transcript analysis, and cytokine responsiveness. Given that Tp is completely devoid of LPS (43), we omitted this potent endotoxin from our maturation protocols, whereas LPS is often used in macrophage stimulation experiments (29, 44, 45). To more clearly define the effect of IFNγ, we first had to measure the expression of several markers associated with either classically and/or alternatively activated macrophages (29, 46, 47). Expression of CD68, a well characterized macrophage marker, was unaffected by IFNγ (Figure 3A), while expression of the antigen presentation molecule HLA-DR was appreciably increased (48) (Figure 3A). CD14, a glycosylphosphatidylinositol-linked membrane glycoprotein that interacts with several pattern recognition receptors (PRRs), including TLR2 and complement receptor-3 (CR3) (42, 49–51), was upregulated by IFNγ (Figure 3A). iNOS, notorious for its vital role in antimicrobial activity as part of the oxidative burst of macrophages, was also significantly increased by IFNγ (Figure 3B). We also assessed macrophage expression of the scavenger receptor CD163 (52) and the C-type lectin CD206 (29), which are associated with alternative macrophage activation. CD163 and CD206 were for the most part unaffected by IFNγ (Figure 3B). We then examined the expression of TLR2, 7, and 8, which we have shown to be upregulated in SS skin lesions (10). Expression of TLR7 was similar between the two macrophage phenotypes. On the other hand, TLR2, which is essential for spirochetal lipoprotein recognition, and TLR8, an endosomal TLR involved in single-stranded spirochetal RNA recognition (53), were increased in response to IFNγ (Figure 3C).

We further examined transcriptional differences between unstimulated MΦ(C)s and MΦ(IFNγ)s by using a commercially available microarray panel. As shown in Table 1, several genes that code for macrophage activation markers, microbial PRRs, cytokines and chemokines, were significantly upregulated by IFNγ. Among them, the activation markers CD40 and CD80, as well as CD38, a type II transmembrane glycoprotein involved in Ca²⁺ signaling during FcγR-mediated bacterial uptake (54) were transcriptionally increased. In line with the flow cytometry results shown in Figure 3, TLR2 and TLR8 were also upregulated in the arrays. PTGS2, the gene which codes for COX-2 and is associated with classically activated macrophages (44), and several inflammatory chemokines and cytokines (CXCL11, IL1B, IL1A, IL8, IL15) involved in cellular recruitment and activation were also transcriptionally upregulated. Of particular importance, all three FcγRs (CD16, CD32, and CD64) were upregulated in MΦ(IFNγ)s. In parallel stimulation experiments, we compared proinflammatory cytokine production in response to several TLR ligands (MMP, LPS, or R848) between MΦ(C)s and MΦ(IFNγ)s. We observed that, independently of the TLR ligand used, IFNγ primed macrophages produced significantly more cytokines than non-primed MΦ(C)s (Figure S3 in Supplementary Material). Our combined observations thus confirm that M-CSF cultivated macrophages in the presence of IFNγ exhibit a classically activated macrophage phenotype.

Having generated macrophages by closely considering the microenvironment of syphilitic skin lesions, we then asked how these cells would respond to opsonized Tp ex vivo. To address this question, we stimulated MΦ(C)s and MΦ(IFNγ)s with Tp, in the presence or absence of opsonic antibodies, and assessed their phagocytic potential and immune responsiveness by confocal microscopy and cytokine production, respectively. We observed binding of the spirochete to both macrophage phenotypes, irrespective of the presence of HSS (Figure S4A in Supplementary Material). Spirochetes were seen attached to the macrophages by their tips (Figure 4A, bottom left inset—yellow arrow) and in some cases across the entire length of the bacterial surface (Figure 4A, top right inset—blue arrow). The addition of HSS led to a marked increase in phagocytosis of Tp by both macrophage phenotypes (Figures 4A,B), although the phagocytic index was significantly greater in IFNγ stimulated cells (Figure 4C). In agreement with enhanced uptake of opsonized spirochetes, we recovered significantly fewer spirochetes when HSS was included in the stimulation experiments (Figure S4B in Supplementary Material). Tp stimulated MΦ(IFNγ)s secreted more TNF (Figure 4D) and IL-6 (Figure 4E) than their counterparts without IFNγ, but neither macrophage phenotype secreted IL-1β (Figure 4F). Overall, these findings confirm our hypothesis that elements present within the syphilitic tissue microenvironment, specifically IFNγ, alter macrophage uptake and responsiveness to opsonized Tp.

To further characterize the effect of opsonized Tp on the macrophage, we used targeted phagocytic and inflammatory transcriptional array analysis. For these experiments, the relative fold changes of gene transcripts between Tp stimulated and unstimulated MΦ(IFNγ)s were measured in three experimental conditions; Tp with no sera, with NHS and with HSS (Figure 5). We first compared genes induced in Tp stimulated MΦ(IFNγ)s with no sera or with NHS. Consistent with our findings above, where spirochetal uptake and cytokine secretion were similar between the two conditions in the absence of HSS, we observed that transcriptional profiles were also comparable, such that none of the genes met our differential expression threshold of two-fold change and p-value of <0.05 (Figure S5A in Supplementary Material). On the other hand, several genes were significantly upregulated in Tp stimulated MΦ(IFNγ)s in the presence of HSS when compared to similarly stimulated MΦ(IFNγ)s with NHS (Figure S5B in Supplementary Material). CD38, CD40 and CD80 were increased, whereas the activation marker CD86 varied greatly between the individual participants (Figure 5). TLR7 was markedly increased in 5/6 healthy volunteer macrophages, but this increase was not statistically significant. TLR2 and TLR8, which have been shown to be important in spirochetal recognition (33, 53, 55) were significantly upregulated as a result of FcγR-mediated uptake of Tp. Given the significant upregulation of TLR8 (Figure 5) and the importance of type I IFNs in response to spirochetes (33, 56), in parallel RT-PCR experiments, we also assessed the expression of IFNB, and three interferon regulatory factors (IRFs, IRF3, IRF5, and IRF7) by RT-PCR. We observed no change in expression of IRF3 and IRF5 (Figure S5C in Supplementary Material). IRF7 was significantly upregulated, but we did not detect a substantial change in IFNB expression (Figure S5D in Supplementary Material).

We observed that many cytokine and chemokines involved in cellular recruitment as well as induction of other physiological responses, including enhanced phagocytosis, were differentially regulated in Tp stimulated MΦ(IFNγ)s in the presence of HSS. Among them, granulocyte macrophage colony-stimulating factor (GM-CSF), which is encoded by CSF2 and has been described to trigger the differentiation and exiting of monocytes from the bone marrow was upregulated. There was a strong induction of chemotactic factors, including CXCL10 and CXCL11, which are important for recruitment of monocytes/macrophages to the site of infection. IL12B, which is known to stimulate T cells and NK cells to secrete IFNγ, was robustly increased in 5/6 healthy volunteer macrophages. The transcript for IL15 (Figure 5), a strong NK cell activating cytokine (57), was also elevated. Although IL-1β protein was not secreted by the macrophages following Tp stimulation (Figure 4D), IL1B was transcriptionally upregulated (Figure 5). The findings together suggest that neither Tp nor its PAMPs, escape the phagosome into the cytosol to induce caspase activation and cleavage of pro-IL-1β into the active cytokine in accord with the mechanism detailed by Netea et al. (58).

Fcγ receptors have been observed to be upregulated in syphilitic skin lesions by transcriptional analysis (10) and are important for binding of IgG with various receptor–ligand affinities (59). Based on our primary human macrophage data (Figures 2A and 4A) and published studies (10, 12, 19, 23) demonstrating the importance of HSS in spirochetal uptake, we hypothesized that FcγRs are the major phagocytic receptors for Tp. To study this premise, we first assessed the effect of IFNγ on CD16, CD32, and CD64 expression by flow cytometry. Expression of CD16 (FcγRIII), which weakly binds IgG (59), was only mildly affected by IFNγ, while expression of CD32 (FcγRII), another low affinity IgG binder, was not affected (Figure 6A). Conversely, expression of CD64 (FcγRI), a high affinity receptor which binds IgG, specifically IgG₁ and IgG₃ (59), was significantly upregulated by IFNγ (Figures 6A,B).

Due to the significant increase in CD64 expression induced by IFNγ (Figure 6A) and importance of syphilitic serum in spirochetal uptake (Figure 4C), we then assessed the localization of CD64 with both unopsonized and opsonized Tp by confocal microscopy. CD64 could be seen localizing with unopsonized Tp (Figure 7A, middle right) however when the spirochetes were opsonized with HSS, the intensity of the colocalization was more robust and is most likely a result of FcγRs clustering to the site of treponeme attachment (Figure 7A, lower right). To determine the role of FcγRs in spirochetal uptake, we used monoclonal antibodies against human CD16, CD32, and CD64 to block the interaction between opsonized spirochetes and each of the three Fc receptors and then compared bacterial uptake and inflammatory cytokine production between blocked and unblocked MΦ(IFNγ)s. As shown in Figure 7B, there were no significant reductions in uptake by blocking CD16 or CD32. However, blockade of CD64 did cause a significant decrease in phagocytosis of opsonized spirochetes (Figure 7B), as well as TNF production (Figure 7C). This finding was not surprising because IgG1 and IgG3, the prominent IgG subclasses in syphilitic serum (60), are high affinity IgG subclasses known to bind CD64. Importantly, the use of monoclonal antibodies against CD64 alone, or in combination with CD16 and CD32, did not result in complete abrogation of phagocytosis (Figure 7B), suggesting that additional phagocytic receptors could have a role in Tp uptake. Taken together, these data show for the first time that CD64 acts as the primary phagocytic receptor for FcγR-mediated uptake of opsonized Tp, and that IFNγ-mediated enhanced expression of CD64 promotes Fc-receptor–ligand interactions.