Article Figures & Data
Figures
- FIGURE 1.
N. gonorrhoeae association with primary human B cells. (A) B cells were infected with a MOI of 10 of either Texas Red-labeled N. gonorrhoeae expressing no Opa adhesin, OpaHSPG, or OpaCEA or Texas Red-labeled E. coli for 6 h and then visualized by confocal microscopy. Scale bar, 5 μm. (B) B cells were infected as described in (A) but using unlabeled bacteria that were stained postinfection, and bacterial association was assessed over a time course by confocal microscopy. (C) The total number of bacteria associated per cell was quantified 3 h postinfection, considering only cells that have at least one bacteria bound (excluding cells that did not associate with bacteria). Data for two representative donors are shown. DIC, differential interference contrast.
- FIGURE 2.
N. gonorrhoeae infection of primary human B cells promotes viability and does not induce cell death. (A) Viability was examined by comparing forward- and side-scatter flow cytometric parameters, which directly relate to cellular morphology. The percentage of viable cells was obtained by taking the percentage of live cells (live gate) divided by the percentage of total cells (all gate). B cells cultured alone include both live and dead cell populations, whereas B cell cultures treated with staurosporine, a protein kinase inhibitor that induces cell death at high concentrations, did not contain viable cells. Representative of four independent experiments with different donors. (B) The cell populations from (A) were analyzed for the ability to bind annexin V, a marker of cell death. Cells cultured alone (shaded) produce two peaks, indicative of annexin V-positive and -negative populations within this culture. Cells cultured with staurosporine produce only an annexin V-positive peak (unshaded). Representative of two independent experiments with different donors. B cells were infected with either N. gonorrhoeae or E. coli for 3 d and then assayed for culture viability by forward versus side scatter (C) or examined for cell death by annexin V staining (D). Each symbol in the scatter plots represents an experiment conducted with B cells obtained from an individual donor. Bars indicate mean. Asterisks indicate a comparison between uninfected and infected cells. *p < 0.05, **p < 0.01. Data points corresponding to the same individual donor are represented by the same symbol within each plot.
- FIGURE 3.
A robust activation response is observed by primary human B cells infected with N. gonorrhoeae but not with E. coli. All B cell cultures were infected for 3 d with either gonococci or E. coli at an MOI of 10 prior to cellular activation analysis. (A) B cells activated for 3 d with 10 μg/ml BCR cross-linker (anti-BCR) displayed a typical blasting phenotype, with an increase in cell size, as reflected in the shifting of the live cells along the forward-scatter axis. Note that the live gate encompasses both blasting and nonblasting viable cells. Representative of two independent experiments with different donors. (B) Blasting cells were observed upon infection with N. gonorrhoeae but not in uninfected or E. coli-infected cells. Compare the anti–BCR-activated cells in the live gate with N. gonorrhoeae or E. coli-infected cells. Representative of at least six independent experiments with different donors. (C) Quantification of the percent of cells that acquired a blasting phenotype after 3 d of infection with N. gonorrhoeae or E. coli. The percentage of blasting cells was obtained by taking the percentage of blasting cells (blasting gate) divided by percentage of total live cells (live gate). (D) Expression of costimulatory receptor and activation marker CD86 on B cells that were infected with either gonococci or E. coli. Bars indicate mean. Asterisks indicate a comparison between uninfected and infected cells. *p < 0.05, ***p < 0.001. Data points corresponding to the same individual donor are represented by the same symbol within each plot.
- FIGURE 4.
N. gonorrhoeae infection, but not E. coli infection, induces strong proliferative responses in human B cells. (A) BrdU incorporation was examined in B cells infected by either N. gonorrhoeae or E. coli for 3 d. (B) The kinetics of B cell proliferation was measured with purified cells from four different donors and shows that gonococci can effectively induce B cell-proliferative responses for up to 6 d (the longest time point we analyzed). Bars indicate mean. Asterisks indicate a comparison between uninfected and infected cells. *p < 0.05, **p < 0.01. Data points corresponding to the same individual donor are represented by the same symbol within each plot.
- FIGURE 5.
Three populations of B cells are found in human peripheral blood, and although gonococci are able to bind to all of them, N. gonorrhoeae association is strongest with IgM memory B cells. (A) The populations of B cells found in human peripheral blood can be differentiated by the combined expression profiles of CD27 and IgD. We show freshly isolated B cells from one representative donor can be separated into the three B cell populations found in human peripheral blood: naive (IgD+,CD27−), IgM memory (IgD+,CD27+), and switched memory (IgD−,CD27+). Representative of four independent experiments with different donors. (B) Expression of IgM, IgG, or IgA was examined in uninfected cells by gating on the B cell subpopulations. Solid line, IgG; dotted line, IgA; and dashed line, IgM. Representative of four independent experiments with different donors. (C) B cells were infected with a low MOI of 2 for 3 h to examine bacterial binding patterns. The percentage of bacteria bound to each population of cells was determined by flow cytometric analysis. (D) The results in (C) are regraphed to allow for ease of comparison, examining how the different cell populations associate with the different strains of N. gonorrhoeae or E. coli. Bars indicate mean. Asterisks indicate a comparison between gonococcal and E. coli-infected cells. **p < 0.01, ***p < 0.001. Data points corresponding to the same individual donor are represented by the same symbol within each plot.
- FIGURE 6.
N. gonorrhoeae infection, but not infection with E. coli, specifically expands the IgM memory B cell population. (A) B cells were infected for 5 d with either gonococci or E. coli at an MOI of 10 and then analyzed for changes in the populations of peripheral B cells. The percentage of IgM memory cells is indicated in each of the contour plots. Representative of three independent experiments with different donors. (B) Quantification of each B cell population postinfection under the same conditions outlined in (A). Bars indicate mean. Asterisks indicate a comparison between uninfected and infected cells. **p < 0.01. Data points corresponding to the same individual donor are represented by the same symbol within each plot.
- FIGURE 7.
B cell infection with clinical N. gonorrhoeae strains induces proliferation of both IgM and switched memory B cell populations. (A) B cells were infected at an MOI of 10 by each of the indicated bacterial strains for 3 d and then analyzed for nuclear proliferation Ag Ki67 within the B cell subpopulations. Gonococcal strains N2061 and N2066 were obtained from male urethra and were both pilin negative. Strain N2061 does not express Opa adhesin; however, N2066 expresses at least one Opa variant. Representative of three independent experiments with different donors. (B) Quantification of the percent of each B cell subpopulation postinfection under the same conditions outlined in (A). Bars indicate mean. Asterisks indicate a comparison between uninfected and infected cells. *p < 0.05, ***p < 0.001. Data points corresponding to the same individual donor are represented by the same symbol within each plot.
- FIGURE 8.
Primary B cells produce polyclonal IgM, including pathogen-specific Ab, in response to infection with N. gonorrhoeae, an effect that is not observed with E. coli infection. (A) Five days postinfection, cell-free supernatants taken from B cell cultures infected with MOI of 10 bacteria per cell were analyzed for total Ig production by ELISA. (B–D) ELISA plates were coated with Opa− gonococci, TT, or KLH to examine the Ag specificity of the Ig produced upon infection. After 5 d of infection, supernatants were applied to these plates to examine the production of Ig specific for gonococci (B), to determine whether pathogen-specific Ig is produced, TT (C), to examine whether polyclonal activation of B cells includes activation of switched memory cells, and keyhole limpet hemocyanin (D), to determine if Ig specific for irrelevant Ags is produced. All three classes of Ig were tested. All cells from donors examined produced IgM that recognized TT, whereas 9 of 10 donors produced IgM specific for KLH and 7 of 9 donors produced IgM specific for gonococci. All data points are graphed as a ratio of Ig produced relative to Ig levels present in cells left uninfected. Bars indicate mean. Asterisks indicate a comparison between uninfected and infected cells. *p < 0.05, **p < 0.01, ***p < 0.001. Data points corresponding to the same individual donor are represented by the same symbol within each plot.
- FIGURE 9.
TLR9 signaling is induced upon N. gonorrhoeae infection of primary human B cells. (A and B) B cells were treated with 2.5 μg/ml Pam3CSK4, 1 μg/ml FSL1, 1 μg/ml LPS, and 5 μg/ml CpG ODN2006, infected with MOI:10 of N. gonorrhoeae strain N302, or left untreated for 3 d before analysis of CD86 for cellular activation (A) and Ki67 for proliferation (B) was conducted by flow cytometry, differentiating between B cell subpopulations by staining for IgD and CD27. (C) B cells were either left untreated, infected with MOI:10 of N. gonorrhoeae strain N302, or treated with 5 μg/ml CpG ODN2006. iCpG was added to inhibit TLR9 signaling, and in parallel wells, a control for iCpG was added (cCpG). After 3 d of incubation, cells were stained for B cell subpopulations and proliferation by Ki67. The relative percent of Ki67-positive cells was obtained by taking the ratio between proliferation observed in iCpG-treated cells versus cCpG-treated cells. Bars indicate mean. Asterisks indicate a comparison between uninfected and infected cells. *p < 0.05, **p < 0.01, ***p < 0.001. Data points corresponding to the same individual donor are represented by the same symbol within each plot.
- FIGURE 10.
B cells are able to engulf and kill whole gonococci. (A) B cells were infected with MOI:50 of indicated strains of N. gonorrhoeae for 3 h, stained for extracellular gonococci first, and then permeabilized to stain for total gonococci. Intracellular N. gonorrhoeae are single-color stained. Scale bars, 5 μm. (B) B cells were infected with the indicated strain of gonococci for 90 min and then treated with gentamicin to kill any extracellular bacteria. At 2.5 and 6 h postinfection, cells were washed, lysed, and then plated for growth on GC agar. Bars indicate mean. Data points corresponding to the same individual donor are represented by the same symbol within each plot. DIC, differential interference contrast.
- FIGURE 11.
Proposed model describing how N. gonorrhoeae infection effectively prevents the production of a protective immune response in infected individuals. (A) Upon vaccination or infection with microbes that elicit T-dependent B cell responses, activated T cells can provide costimulatory signaling to B cells. This is required for clonal expansion of B cells and the generation of a highly specific Ig response against the Ag/microbe. (B) Activation and proliferation of CD4+ T cells is inhibited by infection with N. gonorrhoeae through the clinically relevant engagement of the coinhibitory receptor CEACAM1 by the neisserial Opa proteins. This prevents T cell and B cell collaboration, abrogating the production of a T cell-dependent memory response. However, B cells are able to respond to N. gonorrhoeae infection in a T-independent manner, an effect relying in part on N. gonorrhoeae engulfment allowing direct delivery of neisserial DNA to intracellular TLR9. This potently activates IgM memory cells to produce a polyclonal IgM response which includes Ig that may bind to the gonococci but also to irrelevant (nongonococcal) Ags. Cumulatively, this allows suboptimal control of the current infection and precludes development of a classical memory response upon re-exposure to the gonococci.
Additional Files
Data Supplement
Files in this Data Supplement:
- Supplemental Figure 1 (PDF, 39 Kb) - Supplemental Figure 1. Primary human B cell viability is affected upon infection with different MOIs of E. coli or N. gonorrhoeae.
In this issue
