Critical role of Th17 responses in a murine model of Neisseria gonorrhoeae genital infection

Abstract

Host immune responses, including the characteristic influx of neutrophils, against Neisseria gonorrhoeae are poorly understood; adaptive immunity is minimal and non-protective. We hypothesize that N. gonorrhoeae selectively elicits Th17-dependent responses, which trigger innate defense mechanisms, including neutrophils and antimicrobial proteins, that it can resist. We found that N. gonorrhoeae induced the production of interleukin-17 (IL-17) in mouse T-cells and Th17-inducing cytokines in mouse and human APCs in vitro. IL-17 was induced in the iliac lymph nodes in vivo in a female mouse model of genital tract gonococcal infection. Antibody blockade of IL-17 or deletion of the major IL-17 receptor (IL-17R) in IL-17RA(KO) mice led to prolonged infection and diminished neutrophil influx. Genital tract tissue from IL-17RA(KO) mice showed reduced production of neutrophil-attractant chemokines in response to culture with N. gonorrhoeae. These results imply a crucial role for IL-17 and Th17 cells in the immune response to N. gonorrhoeae.

Figure 1

N. gonorrhoeae induces Th17-associated cytokines, but not Th1-associated cytokines. (A) Production of IL-17 from mouse splenic mononuclear cells, incubated in medium only (control) or with 2 μg/ml ConA or N. gonorrhoeae outer membrane vesicles (OMV) at various concentrations for either 1, 3, or 5 days. Supernatants were assayed for IL-17 by ELISA. (B) Production of IL-17 from mouse splenic mononuclear cells, incubated in medium only (control) or with 2 μg/ml ConA or N. gonorrhoeae at various multiplicities of infection (MOI) for either 1, 3, or 5 days. Supernatants were assayed for IL-17 by ELISA. (C) Production of IL-17 from C3H/HeJ (TLR4-deficient), C3H/FeJ (TLR4-normal), or TLR2-knockout mouse splenic mononuclear cells incubated for 3 days in medium only (control), or with N. gonorrhoeae PID2 at MOI 10:1, or with OMV (5μg/ml), or LOS (5μg/ml). Supernatants were assayed for IL-17 by ELISA. (D) Production of IL-6, IL-12, IL-17, IL-22, and IFN-γ from mouse splenic mononuclear cells incubated for 3 days in medium only (control) or with 2 μg/ml or 5 μg/ml of OMV, or N. gonorrhoeae at an MOI of 10:1. Supernatants were assayed for cytokines by ELISA. All experiments (A-D) were conducted in triplicate, and results are shown as mean ±SD; * indicates cytokine secretion significantly above control levels (P<0.01; Student’s t). (E) Flow cytometry profiles of murine spleen cells cultured for 3 days with gonococcal OMV (right panel), compared to control unstimulated cells (left panel). Cells were stained for intracellular IL-17 (PE) and surface γ^δ-T cell receptor (FITC).

Figure 2

BMDC produce IL-6 and IL-23 but not IL-12 in response to N. gonorrhoeae or its OMV. BMDCs were incubated for 24 hours in medium only (control) or with 2 μg/ml of ConA, N. gonorrhoeae at 25:1 MOI, or 5 μg/ml of gonococcal OMV. Supernatants were collected and assayed for cytokines by ELISA. All experiments were conducted in triplicate, and results are shown as mean ±SD; * indicates cytokine secretion significantly above control levels (P<0.05; Student’s t).

Figure 3

The human monocyte-like cell line THP-1 produces Th17-associated cytokines in response to N. gonorrhoeae or its OMV. Differentiated THP-1 cells were incubated for 24 hours in medium only (control) or with 5 μg/ml⁻¹ of LPS, 5 μg/ml of gonococcal OMV, or N. gonorrhoeae at an MOI of 25:1. Supernatants were assayed for cytokines by ELISA. All experiments were conducted in triplicate, and results are shown as mean ±SD; * indicates cytokine secretion significantly above control levels (P<0.01; Student’s t). LPS, lipopolysaccharide; MOI, multiplicity of infection; OMV, outer membrane vesicle.

Figure 4

Production of IL-17 from draining iliac lymph node cells from mice infected with N. gonorrhoeae, or sham infected, or unmanipulated controls. Lymph nodes were collected on days 1–5 after infection and supernatants were assayed for IL-17 by ELISA after culture for 24 hours. Results are shown as mean ±SD of triplicate cultures of lymph nodes pooled from 3 mice per time point. For days 2–5, the difference between infected and sham infected mice was statistically significant, ** P<0.005, * P<0.05 (Student’s t).

Figure 5

Blocking of IL-17A prolongs gonococcal genital tract infection and delays the recruitment of neutrophils in mice. BALB/c mice were treated with anti-IL-17 antibody (n=7) or rat IgG (n=8) and infected vaginally with N. gonorrhoeae. The course of infection was monitored by culture of vaginal mucus, and the neutrophil influx was determined by microscopic examination of vaginal smears. (A) Percentage of mice infected with N. gonorrhoeae; survival curves are significantly different (P<0.001; Kaplan-Meier). (B) Number of recoverable N. gonorrhoeae (CFU), mean ±SD; * indicates significant difference between treated and control groups (P<0.05; Student’s t). (C) Neutrophil influx, measured as the percentage of neutrophils relative to other cells, mean ±SD; * indicates significant difference between treated and control groups (P<0.05; 2-way ANOVA).

Figure 6

Prolongation of infection and suppression of neutrophil recruitment in IL-17RA^KO mice. IL-17RA^KO (n=7) or wild-type (n=8) C57BL/6 mice were infected vaginally with N. gonorrhoeae. The course of infection was monitored by culture of vaginal mucus, and the neutrophil influx was determined by microscopic examination of vaginal smears. (A) Percentage of mice infected with N. gonorrhoea e; survival curves are significantly different (P<0.001; Kaplan-Meier). (B) Number of recoverable N. gonorrhoeae (CFU), mean ±SD; * indicates significant difference between IL-17RA^KO and WT groups (P<0.05; Student’s t). (C) Neutrophil influx, measured as the percentage of neutrophils relative to other cells, mean ±SD; * indicates significant difference between IL-17RA^KO and WT groups (P<0.05; 2-way ANOVA).

Figure 7

Production of cytokines and chemokines from mouse vaginal explants cultured with N. gonorrhoeae. Genital tract tissue segments from wild type or IL-17RA^KO mice were incubated in vitro in medium only (control) or with N. gonorrhoeae at a concentration of 1 × 10⁷ CFU/ml. Supernatants were removed after 3 days and assayed for cytokines and chemokines by ELISA. Results are shown as mean ±SD of triplicate cultures. Cytokine or chemokine production in the presence of N. gonorrhoeae was significantly higher than in corresponding controls or in IL-17RA^KO cultures, ** P<0.01, * P<0.05 (Student’s t).