Table 1.
Clinical and laboratory characteristics of secondary syphilis patients.
Table 2.
Antibody staining panels used for flow cytometric analysis.
Figure 1.
Secondary syphilis induces immunophenotypic alterations in human monocytes.
Peripheral blood mononuclear cells (PBMCs) were isolated from secondary syphilis (SS) patients before and around 60 days after penicillin treatment and compared to healthy controls. The flow cytometry dot-plot reveals forward and side scatter features of gated monocytes from three different and representative patient samples. The dot plots reveal a population of monocytes with increased size and granularity in SS patients, which normalized at the follow-up visit.
Figure 2.
Cell surface activation markers in secondary syphilis patient monocytes.
Cell surface activation markers were examined in monocytes from secondary syphilis (SS) patients before (Pre-Tx) and around 60 days after penicillin treatment (Post-Tx) and compared to healthy controls. (A) A modest but significant decrease in CD40 MFI was evident between paired acute and convalescent samples obtained from SS patients before and after treatment. (B) Significant increases in CD14 MFI expression were observed between syphilis patients and healthy volunteers prior to antibiotic treatment and between pre- and post-penicillin treatment (p values are shown in the figure).
Figure 3.
Immunophenotypic alterations in dendritic cell (DC) populations.
Circulating DCs were analyzed by flow cytometry in secondary syphilis (SS) patients before (Pre-Tx) and after penicillin treatment (Post-Tx). DCs were characterized by flow cytometry parameters as being HLA-DR+ and Lineage cocktail negative (not shown) and the expression of CD11c into monocytoid (CD11c+) and plasmacytoid (CD11c−) and expression of the co-stimulatory molecule CD83. A marked decrease was observed in the CD11c+ population in the blood of 7/12 SS patients and at the follow-up visit this population recovered in these same SS patients.
Figure 4.
Secondary syphilis (SS) patients exhibit a significant decrease in total NK-cell populations.
(A) Gating procedure to determine NK-cells subsets according to CD56 and CD16 expression by flow cytometry are shown. (B and C) A significantly larger percentage of SS subjects exhibit NK-cell values below the 5th percentile of established published normal values (line depicts the cutoff), when compared to healthy controls (* indicates p<0.05).
Figure 5.
NK-cell subsets distribution in secondary syphilis (SS) patients.
SS patients exhibit significant decreases in (A) cytokine-producing NK-cells (* p = 0.02) and (B) cytotoxic NK-cells (* p<0.001). Values are shown at enrollment (Pre-Tx) and after penicillin treatment (Post-Tx). Neither cell subset appears to recover following treatment.
Figure 6.
Emergence of a CD56-negative NK-cell population in secondary syphilis (SS) patients.
(A) The CD56negative CD16+ NK-cells subset in untreated SS patients are shown by the black arrows. (B) Significant increases (* p = 0.003) in CD56 negative CD16+ NK-cell population was seen in SS patients. This anomaly was not present in healthy controls.
Figure 7.
Immunophenotypic cellular composition of the inflammatory infiltrate in secondary syphilis patient skin lesions.
IHC staining depicts CD68+ macrophages (A and B), CD4+ T-cells (C and D), CD8+ T-cells (E and F) and CD56+ NK-cells (G and H). Panels B, D, E and H are high magnification images of the red boxed areas in A, C, E and G.
Figure 8.
Spirochetal clusters are present in secondary syphilis skin lesions.
Representative skin biopsy from a posterior neck secondary syphilis (SS) lesion was processed for IHC. (A/B) H&E stain of SS lesions. (C/D) IHC staining reveals abundant spirochetes embedded within a mixed cellular inflammatory infiltrate (shown in the red box) in the papillary dermis. The blue arrow points to a tissue histiocyte and the read arrows to two dermal lymphocytes.
Table 3.
Transcriptional Profile - Secondary syphilis skin biopsies (n = 12) vs healthy control skin (n = 3).
Figure 9.
T. pallidum (Tp) uptake and recovery by IFA.
Purified human monocytes obtained from healthy controls were stimulated with fresh Tp Nichols strain (MOI 1∶1, 10∶1 and 30∶1) were incubated for 8-hours alone or where indicated in the presence of 10% heat inactivated normal human serum (NHS) or human syphilitic serum (HSS). (A) Percentage of phagocytosed Tp was greater when HSS was present. (B) Percentage of non-phagocytosed Tp which were recovered in supernatants at the end of incubation time is shown in each of the two graphs. Spirochetal recovery was substantially higher in the absence of HSS; nonetheless more than half of the bacteria avoided recognition and uptake despite the presence of HSS (p values shown correspond to statistical comparisons between groups by ANOVA). (C) Percentage of spirochetes recovered was similar in the presence of HSS at three different MOIs.
Figure 10.
Cytokine output in response to opsonized Tp.
Isolated human monocytes were stimulated for 8-hours with live Tp at three different spirochete to monocyte ratios (MOIs) (1∶1, 10∶1 and 30∶1) in the presence or absence of 10% heat inactivated human syphilitic serum (HSS). LPS was used as a positive control. Cytokines in supernatants were quantitated in picograms (pg)/ml by cytokine bead array as described in Methods. A, C: Higher cytokine production (TNFα and IL-1β) was elicited when spirochete-stimulated monocytes were immersed in 10% heat inactivated HSS. B, D: Cytokine output was greater with higher MOIs (* indicates where p values are <0.05 between different conditions studied by paired or unpaired student's T-test analysis as described in the Methods, NS = non-significant differences).