Figure 1.
N. gonorrhoeae survives in association with monocytes.
A. Survival of gonococci with human THP-1 monocytes at 2 and 5 hr post infection. Percent monocyte-associated gonococcal survival is calculated in reference to the number of viable gonococci cultured from infected THP-1 cells after 1 hr of phagocytosis. Error bars are ± standard deviation from the mean of 4 independent experiments. B: Viability of adherent and intracellular and extracellular GC-FA19 in differentiated THP-1 cells, i.e. adherent macrophages, were visualized with BacLight staining where red is dead and green is live gonococci. Adherent THP-1 cell membranes were permeabilized with 0.1% saponin to stain phagocytosed gonococci. i: 1 hr post infection stained without saponin. ii: 1 hr post infection stained in the presence of 0.1% saponin. iii: 5 hr post infection stained without saponin. iv: 5 hr post infection stained in the presence of 0.1% saponin. These data are representative of two independent experiments. C: Quantitative RT-PCR of iron-responsive and -unresponsive genes in monocyte-associated GC-FA19 at 5 hrs post infection compared to 1 hr post infection. Error bars represent SD from the mean of three independent experiments. * P values (<0.005) were calculated in comparison to rmpM gene expression. N.S.: not significant.
Figure 2.
Cytokine release from THP-1 cells infected with N. gonorrhoeae strain FA19.
A. Human macrophage-like monocytic THP-1 cells were infected with GC-FA19 at an MOI of 10 and incubated overnight. Released cytokines TNFα, IL-6, IL-1β and chemokines CXCL10 (IP-10) and MCP-1 were quantified in the supernatants of infected THP-1 cells using ELISA. Cytokine release was not detectable in the supernatants of uninfected THP-1 cells. Error bars represent the SD from the mean of triplicate readings and data are representative of three independent experiments. B. Intracellular gonococci (black arrows) were visualized by differential staining of infected THP-1 (i) and MM6 (ii) cells showing cytoplasmic vacuoles (yellow arrow) (iii) indicating monocyte activation and differentiation into macrophage-like cells, shown in the upper panel. Uninfected THP-1 (iv) and MM6 (v, vi) cells are shown in the lower panel.
Figure 3.
Infection with live gonococci induces more cytokine release than infection with heat-killed gonococci.
Human macrophage-like monocytic MM6 (A) and THP-1 (B) cells were infected with live or heat-killed (H-K) GC-FA19 at an MOI of 10 and incubated overnight. Released cytokines TNFα and IL-1β were quantified in the supernatants of infected cells by ELISA. Cytokine release was not detectable in the supernatants of uninfected cells. Error bars represent the SD from the mean of triplicate readings and data are representative of two independent experiments. P values (<0.001) were calculated for values of infections with live GC-FA19 compared to heat-killed and * TNFα, ** IL-1β in reference to no infection values.
Figure 4.
Hepcidin expression in monocytes is highly upregulated upon infection with live gonococci.
A: Human macrophage-like monocytic MM6 cells were infected with live or heat-killed (H-K) GC-FA19 and incubated for 5 hr or 18 hr. B: THP-1 cells were infected with live GC-FA19 in the presence or absence of 300 µM of the iron chelator deferiprone (DFP) and incubated for 5 hr or 18 hr. RNA was extracted from infected cells and from controls (uninfected cells) and hepcidin gene expression was assessed by quantitative RT-PCR and normalized to that of β-actin. The fold change in hepcidin gene expression was calculated in reference to uninfected controls using the ΔΔCT method. Error bars represent the SD from the mean of quadruplicate wells and data are representative of three independent experiments. **P values (<0.001) were calculated for values of hepcidin expression at 5 hr or 18 hr post infection. *P and *#P values (<0.001) were calculated for values of hepcidin expression upon infection with live GC-FA19 compared to heat-killed (H-K) at 5 hr and 18 hr post infection, respectively. N.S.: not significant. C: Western blot analysis of hepcidin protein expression in THP-1 cell extracts detected with polyclonal anti-hepcidin antibody. Lane 1: molecular weight marker; Lane 2: Untreated THP-1 cells; Lane 3: THP-1 cells treated with exogenous hepcidin (1 ng/ml); Lane 4: Uninfected THP-1 cells; Lane 5: THP-1 infected with GC-FA19. *Anti-hepcidin immunoreactive bands might be dimers of prohepcidin protein (∼16–18 kDa) or processed hepcidin (∼12 kDa). D: Activity of synthetic hepcidin-25 antimicrobial peptide against N. gonorrhoeae FA19 WT and the FA19 lptA::spc isogenic mutant as determined by minimum bactericidal concentration (MBC) assay. The model antimicrobial peptide, polymyxin B, is active against FA19 and highly active against the FA19 lptA::spc mutant, and was used as a control. Data are representative of four independent experiments. For the FA19 lptA::spc mutant, experiments were performed twice.
Figure 5.
Infection with gonococci induces iron retention in monocytes.
THP-1 macrophage-like monocytic cells were infected with GC-FA19 at an MOI of 10 overnight and iron retention in monocytes was determined using the Calcein-AM fluorescent probe method. Uninfected cells were incubated simultaneously and used as controls. Calcein-AM fluorescence was measured by excitation at 488 nm and emission at 528 nm wavelength (see methods). Calcein-AM fluorescence is quenched upon binding iron and is therefore inversely correlated with intracellular iron accumulation. Error bars represent the SD from the mean of quadruplicate readings and data are representative of three independent experiments. **P values (<0.001) were calculated in reference to no infection values.
Figure 6.
Gonococcal infection downregulates ferroportin gene expression in human THP-1 monocytes.
THP-1 cells were infected at an MOI of 10 with live GC-FA19 and incubated for 5 or 18 hr. RNA was extracted from infected and uninfected cells. Ferroportin gene expression was assessed by quantitative RT-PCR and normalized to that of β-actin. Ferroportin gene expression fold change was calculated in reference to uninfected controls. Error bars represent the SD from the mean of at least quadruplicate wells and data are representative of two independent experiments.
Figure 7.
Gonococcal infection upregulates expression of the cytosolic iron transporter NRAMP1 gene and the antibacterial iron carrier NGAL gene in monocytes.
Human macrophage-like monocytic MM6 cells were infected with live GC-FA19 at an MOI of 10 and incubated for 18 hr. RNA was extracted from infected cells and from controls (uninfected cells). NRAMP1 and NGAL gene expression was assessed by quantitative RT-PCR and normalized to that of β-actin. NRAMP1 and NGAL gene expression fold change was calculated in reference to uninfected controls. Error bars represent the SD from the mean of at least quadruplicate wells and data are representative of three independent experiments. P values (<0.001) were calculated in reference to no infection values.
Figure 8.
Gonococcal infection downregulates expression of the labile iron-detoxifying enzyme BDH2-encoding gene in monocytes.
Human macrophage-like monocytic THP-1 cells were infected with GC-FA19 at an MOI of 10 in the presence or absence of 300 µM of deferiprone (DFP) (an iron chelator) and incubated for 18 hr. RNA was extracted from infected cells and from controls (uninfected cells) and BDH2 gene expression was assessed by quantitative RT-PCR and normalized to that of β-actin. BDH2 gene expression fold change was calculated in reference to uninfected controls. Error bars represent the SD from the mean of at least quadruplicate wells and data are representative of three independent experiments. P values (<0.01) were calculated in reference to no infection values.
Figure 9.
Gonococcal infection of peripheral monocytes from healthy human donors induces cytokine release, upregulates hepcidin and downregulates ferroportin and BDH2 gene expression.
A: Peripheral monocytes derived from healthy donors were infected with GC-FA19 at an MOI of 10 and incubated overnight. Released cytokines IL-6 and TNFα and the antibacterial protein NGAL were quantified in the supernatants of infected monocytes by ELISA. Error bars represent the SD from the mean of four different healthy donors, each assayed in duplicate readings. B, C and D: Hepcidin, ferroportin, and BDH2 gene expression in healthy donor monocytes infected with GC-FA19 used in panel A above was determined by quantitative RT-PCR and normalized to uninfected monocytes. Error bars represent the SD from the mean fold change in gene expression from different healthy donors, each assayed in triplicate. *P value <0.01.
Figure 10.
Gonococcal infection modulates iron-limiting innate immune defenses in murine RAW264 macrophages.
A: Nitric oxide release from RAW264 cells infected with GC-FA19 was measured after 18 hr of incubation. Iron-regulated gene expression was determined by qRT-PCR from RNA extracted from the RAW264 infected cells used in the experiment shown in panel A: Hepcidin (B); LCN2 (C); BDH2 (D); NRAMP1 (E). Error bars represent the SD from the mean fold change of qRT-PCR, each assayed in triplicate. This result is representative of two independent experiments.
Figure 11.
N. gonorrhoeae survives in association with murine RAW264 macrophages.
Viability of GC-FA19 in RAW264 macrophages was visualized with BacLight staining. i: GC-FA19 in medium alone incubated for 1 hr at room temperature prior to staining where red is dead and green is live GC-FA19. ii: Uninfected RAW264 macrophages incubated at 37°C for 1 hr prior to staining in the presence of 0.1% saponin. iii: RAW264 macrophages infected with GC-FA19 for 1 hr and stained without saponin. iv: RAW264 macrophages infected with GC-FA19 for 1 hr and stained in the presence of 0.1% saponin. v: RAW264 macrophages infected with GC-FA19 for 5 hr and stained in the presence of 0.1% saponin. These data are representative of three independent experiments.
Figure 12.
Model for how gonococcal infection in macrophages alters cellular iron homeostasis to facilitate iron acquisition.
Macrophages play an essential role in iron homeostasis by engulfing senescent red blood cells (RBC) and recycling iron. Macrophages also play a very important role in host defense. GC infection in macrophages induces the expression of hepcidin (which then degrades the iron exporter ferroportin) and downregulates ferroportin gene expression, causing iron retention in macrophages. GC infection also induces expression of NRAMP1 (the cytosolic iron transporter) and NGAL (an iron carrier protein), and downregulates expression of BDH2, the enzyme that catalyzes the synthesis of the mammalian siderophore 2,5-DHBA which helps detoxify the labile iron pool (LIP). Collectively, these alterations in cellular iron homeostasis lead to increased iron bioavailability that facilitates iron acquisition and promotes gonococcal intracellular survival.