Host defense mechanisms in secondary syphilitic lesions: a role for IFN-gamma-/IL-17-producing CD8+ T cells?

Abstract

Cell-mediated immunity is thought to be of critical importance in antisyphilitic host defense, but the exact mechanisms are still unknown. This fact is particularly true for HIV-infected persons with a deficit in CD4+ T-cell number. We therefore obtained lesional skin samples from HIV+ and HIV- patients with secondary syphilis at different time points of lesional age to search both for causative microorganisms and to characterize the inflammatory infiltrate. By doing so, we detected Treponema pallidum spirochetes with a much greater abundance in late lesions of HIV+ individuals compared with the HIV- cohort. The dominating inflammatory cells were T cells, macrophages, and neutrophils at all stages and plasma cells in older lesions. In HIV- persons, T cells consisted of equal numbers of CD4+ and CD8+ T-cells, whereas in HIV+ patients, the majority of T cells belonged to the CD8 lineage and produced both IFN-γ and IL-17. Regulatory T cells and Langerhans cells were reduced in these patients compared with their HIV- counterparts. Because of our observations, we propose that T cells of both the CD4 and CD8 lineage are needed for an at least partial protective antisyphilitic immunity. Compensation mechanisms in HIV+ individuals, such as an increase of Tc1/17 cells as well as a reduction in immunoregulatory Langerhans cells and T cells, apparently do not overcome the deficiencies in these patients to eliminate the spirochete.

Figure 1

Correlation of clinical symptoms and lesional age of secondary syphilitic eruptions. Patients were arbitrarily divided into four groups according to the length of persistent lesions [(A) 10 days or less, n = 6; (B) 11 to 20 days, n = 6; (C) 21 to 30 days, n = 7; (D) more than 30 days, n = 6]; type as well as severity of these lesions were recorded. The clinical pictures are representative of the indicated times.

Figure 2

Histopathological correlation with the lesional age of secondary syphilitic skin eruptions. Histopathological changes are displayed analogous to clinical alterations in four groups according to lesional persistence. Representative pictures with a magnification ×100 and ×400 are shown for lesional persistence of four different times: (A) 0–10 days (n = 6), (B) 11–20 days (n = 6), (C) 21–30 days (n = 7), and (D) more than 30 days (n = 6). Quantifications of epidermal and dermal alterations are shown in E–H.

Figure 3

Quantitative RT-PCR identifies IFN-γ as master cytokine for secondary syphilitic lesions. Quantitative RT-PCR was performed after trizol fixation of whole biopsies from secondary syphilitic lesions and analyzed for (A and D) so-called Th1 cytokines, (B and E) Th17 cytokines, and (C and F) Th2 and regulatory cytokines. Samples were subdivided according (A–C) the patients’ HIV status and (D–F) the time of persistence of the lesions. Data are normalized to β2-microglobulin of each specimen and represent the mean values of fold changes relative to healthy skin ± SEM. *P < 0.05.

Figure 4

Alterations of the lymphocytic infiltrate in syphilitic lesions of HIV⁺ and HIV⁻ patients. A: Detailed quantitative in situ analysis of lymphocytes within the epidermis and dermis of secondary syphilitic lesions and normal skin from healthy controls (HC). Single and double immunofluorescence staining was performed with the indicated markers from HIV⁺ and HIV⁻ patients. Data are given as absolute numbers of positive cells ± SEM. B: Triple immunofluorescence labeling of syphilitic lesions from HIV⁺ and HIV⁻ patients with anti-CD3 (FITC), anti-CD4 (TRITC), and anti-CD8 (APC) reveals an increase in CD8⁺ T-cells and total T-cells in biopsies from HIV-infected patients. Original magnification, ×400. C: Quantification of CD25⁺FoxP3⁺ T-cells by immunofluorescence double labeling. Data are given as absolute numbers of positive cells ± SEM. D: Triple immunofluorescence labeling of syphilitic lesions from HIV-infected and HIV⁻ patients with anti-CD3 (FITC), anti-CD25 (TRITC), and anti-FoxP3 (Cy5) reveals decreased numbers of regulatory T-cells in HIV⁺ patients. Arrows denote triple positive cells. Original magnification, ×400. E: Quantification of cell subsets was performed by single and double immunofluorescence staining with the indicated markers. Samples were subdivided according their lesional age. Data are given as the absolute numbers of positive cells ± SEM.

Figure 5

Dendritic cells and mononuclear phagocytes within secondary syphilitic lesions. A: Detailed quantitative in situ analysis of dendritic cells and mononuclear phagocytes within the epidermis and dermis of secondary syphilitic lesions divided according to their HIV status and normal skin from healthy controls (HC). Double immunofluorescence staining was performed with the indicated markers. Data are given as the absolute numbers of positive cells ± SEM. B: Double immunofluorescence labeling of syphilitic lesions from HIV⁺ and HIV⁻ patients with anti-CD1a (FITC) and anti-Langerin (TRITC) elucidates a deficit in epidermal Langerhans cells of HIV⁺ patients. C: Triple immunofluorescence labeling of syphilitic lesions from HIV-infected and HIV⁻ patients with anti-CD11b (FITC), anti-CD14 (TRITC), and anti-HLA-DR (APC) reveals an increase of triple-positive mononuclear phagocytes in biopsies from HIV-infected patients. B and C: Original magnification, ×400. D: Quantification of cell subsets with the indicated markers subdivided according to their lesional age. Data are given as the absolute numbers of positive cells ± SEM.

Figure 6

CD8⁺ T-cells as the main source of IFN-γ and IL-17. Triple immunofluorescence labeling of syphilitic lesions from HIV-infected and HIV⁻ patients with anti-CD3 (FITC), anti-CD8 (APC), and (A) anti–IFN-γ (TRITC), or (B) anti–IL-17 (TRITC) reveals that CD8-positive T-cells are IFN-γ– and IL-17–producing cells. C: Double labeling of IFN-γ and IL-17 showed an overlapping staining pattern. A–C: Arrows denote triple-positive cells. Original magnification, ×400. D: Data are given as the absolute numbers of positive cells ± SEM.

Figure 7

Increase of T. pallidum in late secondary syphilitic lesions. A and B: Immunohistochemistry of paraffin sections with an anti–T. pallidum antibody shows an increase of treponemas in HIV-infected patients. Original magnification, ×100 of the total pictures and ×400 of the insets. B: Quantification of treponemas in the epidermis and dermis of syphilitic patients by immunohistochemistry. C: Further splitting of the samples to early and late secondary syphilitic lesions reveals that the increase of treponemas in HIV⁺ patients accounts only for late lesions of HIV-infected individuals. B and C: Data are given as the absolute numbers of treponemas ± SEM. *P < 0.05. D: Quantification of T. pallidum transcripts by quantitative RT-PCR. Data represent the mean values of copies per ml eluate ± SEM. *P < 0.05.