Neisseria gonorrhoeae induces a tolerogenic phenotype in macrophages to modulate host immunity

Abstract

Neisseria gonorrhoeae is the etiological agent of gonorrhoea, which is a sexually transmitted disease widespread throughout the world. N. gonorrhoeae does not improve immune response in patients with reinfection, suggesting that gonococcus displays several mechanisms to evade immune response and survive in the host. N. gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and dendritic cells. In this study, we determined whether N. gonorrhoeae directly conditions the phenotype of RAW 264.7 murine macrophage cell line and its response. We established that gonococcus was effectively phagocytosed by the RAW 264.7 cells and upregulates production of immunoregulatory cytokines (IL-10 and TGF- β 1) but not the production of proinflammatory cytokine TNF- α , indicating that gonococcus induces a shift towards anti-inflammatory cytokine production. Moreover, N. gonorrhoeae did not induce significant upregulation of costimulatory CD86 and MHC class II molecules. We also showed that N. gonorrhoeae infected macrophage cell line fails to elicit proliferative CD4+ response. This implies that macrophage that can phagocytose gonococcus do not display proper antigen-presenting functions. These results indicate that N. gonorrhoeae induces a tolerogenic phenotype in antigen-presenting cells, which seems to be one of the mechanisms to induce evasion of immune response.

Figure 1

Gonococcus uptake by murine macrophage cell line: fluorescence micrograph of murine macrophage cell line RAW 264.7 incubated with GFP-expressing Neisseria gonorrhoeae (green) variant P9-17 for (a) 1 h, (b) 2 h, (c) 3 h, and (d) orthogonal view of the intracellular gonococci (green spots); a midplane Z-section of height 1.25 μm is shown. Phase contrast denotes cell boundaries. Nuclei are in red. (e) Gentamicin protection assay for infection of RAW cells with N. gonorrhoeae variant P9-17 (n = 3).

Figure 2

Cytokines induced in murine macrophage cell line infected with N. gonorrhoeae P9-17 LPS is the positive control. A statistically significant difference is found when data is compared to the level of cytokines found in the medium from cultures without infection. (a) Secretion of TNF-α by RAW cells measured 24 h after challenge. Data represent the levels of cytokine relative to levels induced by LPS. Bars are the mean ± SEM of 6 independent experiments. (b) Secretion of IL-10 after 24 h treatment. n = 5  ** indicates that P9-17 induces higher levels of IL-10 than LPS and other treatments P < 0.01. (c) TGF-β1 levels on the surface of treated macrophages. Values correspond to the mean fluorescence intensity (G-mean) values relative to the levels found in untreated cells. Data are presented as the mean ± SEM of n = 7 challenge experiments. * indicates that P9-17 induces higher levels of TGF-β1 than LPS and other treatments P < 0.05.

Figure 3

Quantification of MHC II and CD86 expression on the surface of gonococcus-infected RAW cells using flow cytometry. Macrophages were stained with anti-CD11b specific antibodies. (a) MHC class II in RAW cells, n = 6. (b) CD86 in RAW cells, n = 8. Bars represent mean ± SEM of independent experiments. Representative histogram plot are shown in the right panel. * indicates that P9-17 induced lower levels of MHC II compared to levels induced by LPS, P < 0.05; ** indicates that P9-17 induced lower levels of CD86 compared to levels induced by LPS, P < 0.05.

Figure 4

Hyporesponsive alloantigen T-cell responses induced by RAW cells infected with N. gonorrhoeae P9-17. Allogeneic H-2^b splenocytes were cultured with RAW cells treated with gonococcus, medium and LPS. At day 5, CD4+ T-cell proliferation was determined by CFSE dilution analysis. Representative histogram plots are shown in the upper panel. Percentages of proliferation are indicated in the upper left quadrant. Bars represent mean ± SEM of 5 independent experiments performed in triplicate. ** indicates that P9-17 induces lower percentages of alloantigen proliferation than LPS and other treatments P < 0.01.