Syphilis Infection Differentially Regulates the Phenotype and Function of γδ T Cells in HIV-1-Infected Patients Depends on the HIV-1 Disease Stage

Abstract

A rapidly escalating outbreak of syphilis infection has been affected men who have sex with men, particularly those with HIV-1 infection. γδ T cells are unconventional immune cells with two main subsets, Vδ1 T cells and Vδ2 T cells, which possess a combination of innate and adaptive immune features allowing them against HIV-1. However, whether syphilis infection affects the phenotype and function of γδ T cells in HIV-1-infected patients remains unclear, especially in acute HIV-1 infection (AHI). In this study, we enrolled 57 HIV-1-infected patients (24 with HIV-1 infection only and 33 coinfected with syphilis) from an acute HIV-1-infected cohort in Beijing (PRIMO). A comprehensive analysis of γδ T-cell phenotype and function was performed by flow cytometry. We found syphilis coinfection could reverse the imbalance of Vδ1/Vδ2 ratio in AHI. Syphilis infection results in decreased γδ T-cell activation in AHI, but increased γδ T-cell activation in chronic HIV-1 infection (CHI). Moreover, patients with CHI had larger numbers of IL-17-producing γδ T cells than those with AHI, regardless of syphilis status. Thus, syphilis affected the γδ T-cell immune response differently in patients depending on the stages of HIV-1 disease. In addition, the percentage of IL-17-producing γδ T cells was positively correlated with the percentage of neutrophils. These results suggest that the γδ T-cell/IL-17/neutrophil axis is involved in HIV-1 pathogenesis and disease progression. Taken together, our observations provide new insight into the roles of γδ T cells in immunopathogenesis of syphilis and HIV-1 coinfection, particularly during AHI, and our findings may be helpful for the prevention of syphilis and other sexually transmitted infections and highlight the great significance on the remedy of patients coinfected with HIV-1.

Keywords: IL-17; acute/chronic HIV-1 infection; innate immune response; syphilis; γδ T cells.

Figure 1

The flow chart summarizing the enrollment of subjects in the study. HIV-1-negative men who have sex with men (MSM) were enrolled in a prospective cohort study to identify acute HIV-1 infection. At enrollment, participants were screened for HIV-1 infection and syphilis by detecting HIV-1 RNA, HIV-1 antibody, rapid plasma reagin (RPR), and T. pallidum particle agglutination assay (TPPA) respectively. Then they were followed up every 2 months and HIV-1RNA, HIV-1 antibody, RPR, and TPPA were tested at each visit. Once acute HIV-1-infected individuals were captured, they continued to be followed up to 2–3 years to observe the natural progression of HIV-1 infection. According to the Fiebig stage, when the estimate date of infection was longer than 180 days, it is considered as a chronic HIV-1 infection. Acute or chronic HIV-1-infected patients with RPR⁺ were separated into HIV⁺RPR⁺ group in acute or chronic infection. Otherwise, they were enrolled into HIV⁺RPR⁻ group in acute or chronic infection.

Figure 2

The gating strategy for flow cytometric analysis of γδ T cells. Among all events, forward angle and side scatter light gating were gated on lymphocytes and were used to exclude cell debris from the analysis. Forward height and forward area were used to exclude doublet cells, and cells were labeled with LIVE/DEAD fixable viability stain 510, and dead cells were excluded. Then, CD3⁺γδTCR⁺cells, Vδ1 and Vδ2 subsets, functional subsets (T_Naive, T_CM, T_EM, and T_EMRA), activation (CD38 and HLA-DR), and cytokines secretion (IL-17 and IFN-γ) were gated, analyzed, and compared between healthy controls (HC) and HIV-1-infected individuals. The final analysis was performed with FlowJo software, which generated a graphical output. FSC, forward scatter; SSC, side scatter.

Figure 3

Comparison of the frequencies of γδ T cells, Vδ₁ and Vδ₂ T cells among healthy controls, HIV-1-infected and HIV-1/syphilis coinfected patients. Based on the results of rapid plasma reagin (RPR), HIV-1-infected patients were divided into HIV⁺RPR⁻ and HIV⁺RPR⁺ groups. The percentages of γδ T cells (A), Vδ₁ T cells (B), and Vδ₂ T cells (C) were compared among HC (●), HIV⁺RPR⁻ (), and HIV⁺RPR⁺ () groups. Next, HIV⁺RPR⁻ and HIV⁺RPR⁺ groups were subdivided according to the acute or chronic nature of their HIV-1 infection. The percentages of γδ T cells (D), Vδ₁ T cells (E), and Vδ₂ T cells (F) were compared among HIV⁺RPR⁻ () and HIV⁺RPR⁺ () groups in acute HIV-1 infection and HIV⁺RPR⁻ () and HIV⁺RPR⁺ () groups in chronic HIV-1 infection. The significance of differences was determined by calculating P values in Mann–Whitney tests and one-way ANOVA test. *P < 0.05, **P < 0.01, ***P < 0.001. HC, healthy controls; HIV⁺RPR⁺, patients coinfected with HIV-1 and syphilis; HIV⁺RPR⁻, patients infected with HIV-1 without syphilis.

Figure 4

Syphilis coinfection has effects on the functional subsets of γδ T cells in HIV-1-infected patients. γδ T cells were classified into four different functional subsets according to their CD27 and CD45RA expression. The percentages of T_Naiveγδ T cells (A), T_CM γδ T cells (B), T_EM γδ T cells (C), and T_EMRA γδ T cells (D) were compared among HC, HIV⁺RPR⁻ (), and HIV⁺RPR⁺ () groups in acute HIV-1-infected patients and HIV⁺RPR⁻ () and HIV⁺RPR⁺ () groups in chronic HIV-1-infected patients. The significance of differences was assessed by calculating P values in Mann–Whitney tests. *P < 0.05, **P < 0.01, ***P < 0.001. RPR, rapid plasma reagin.

Figure 5

Syphilis coinfection affects differently γδ T-cell activation in patients with acute and chronic HIV-1 infection. γδ T-cell activation was assessed by evaluating the expression of CD38 and HLA-DR. The frequencies of CD38⁺ γδ T cells (A), HLA-DR⁺ γδ T cells (B), and CD38⁺HLA-DR⁺ γδ T cells (C) were compared among HC, HIV⁺RPR⁻ (), and HIV⁺RPR⁺ () groups in acute HIV-1-infected patients and HIV⁺RPR⁻ () and HIV⁺RPR⁺ () groups in chronic HIV-1-infected patients. The significance of differences was determined by calculating P values in Mann–Whitney tests. *P < 0.05, **P < 0.01, ***P < 0.001. RPR, rapid plasma reagin.

Figure 6

Syphilis coinfection promotes the production of IL-17 by γδ T cells in patients with chronic HIV-1 infection. Peripheral blood mononuclear cells (1 × 10⁶ cells/ml) were used to seed 24-well plates, and they were incubated with PMA (50 ng/ml)/ionomycin (1 µg/ml) for 6 h and BFA (10 µg/ml) was added 2 h before cell harvests. Intracellular staining for IL-17 and IFN-γ was assessed by flow cytometry. Comparisons of the frequencies of IL-17-producing γδ T cells (A) and IFN-γ-producing γδ T cells (B) among HIV⁺RPR⁻ () and HIV⁺RPR⁺ () groups in acute HIV-1-infected patients and HIV⁺RPR⁻ () and HIV⁺RPR⁺ () groups in chronic HIV-1-infected patients. Correlations of the proportions of IL-17-producing γδ T cells (C) and IFN-γ-producing γδ T cells (D) with the percentage of neutrophils in HIV⁺RPR⁺ () and HIV⁺RPR⁻ () groups were analyzed. The significance of differences was assessed by calculating P values in Mann–Whitney tests. Spearman’s rank correlation analysis was used to assess correlations. *P < 0.05, **P < 0.01, ***P < 0.001. RPR, rapid plasma reagin.