Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile
Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile
María Carolina Ortiz 0 1
Claudia Lefimil 0 1
Paula I. Rodas 0 1
Rolando Vernal 0 1
Mercedes Lopez 0 1
Claudio Acuña-Castillo 0 1
Mónica Imarai 0 1
Alejandro Escobar 0 1
0 1 Instituto de Investigación en Ciencias Odontológicas, Facultad de Odontología. Universidad de Chile , Santiago , Chile , 2 Center for Integrative Medicine and Innovative Science, Facultad de Medicina. Universidad Andrés Bello , Santiago , Chile , 3 Departamento de Odontología Conservadora, Facultad de Odontología. Universidad de Chile , Santiago , Chile , 4 Programa Disciplinario de Inmunología, Instituto de Ciencias Biomédicas, Facultad de Medicina. Universidad de Chile , Santiago , Chile , 5 Laboratorio de Inmunología, Departamento de Biología, Facultad de Química y Biología. Universidad de Santiago de Chile , Santiago , Chile
1 Editor: Michal A Olszewski, University of Michigan Health System, UNITED STATES
Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ) and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ) or alternative-M2 (M2-MФ) activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM) upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/ M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1), widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies.
Competing Interests: The authors have declared
that no competing interests exist.
Neisseria gonorrhoeae is the etiological agent of the sexually transmitted disease gonorrhea,
with a worldwide incidence and an estimate of over 100 million new infections per year . In
women, infection by N. gonorrhoeae is associated with several clinical manifestations such as
urethritis, cervicitis, pelvic inflammatory disease, ectopic pregnancy, chronic pelvic pain and
infertility . Moreover, gonococcus (GC) is often co-morbid with other STDs such as HIV,
which increases the risk of transmission of this disease [3, 4]. Due to its increasing
antimicrobial resistance and the absence of effective vaccines , gonorrhea remains as an important
public health issue.
The gonococcal infection is unable to induce a state of protective immunity. This is
supported by clinical data indicating that previous infections with N. gonorrhoeae do not improve
the immune response and gonorrhea can be repeatedly acquired [6, 7]. The mechanisms of
immune evasion exhibited by the pathogen are multiple and involve the innate and adaptive
immune response [8–11]. Studies in the murine model of gonococcal genital tract infection
show an increase of CD4+Foxp3+CD25+ regulatory T lymphocytes (Tregs) in the lymph
nodes draining of the genital tract. This increase correlates with an augmentation of
transforming growth factor (TGF)-β positive cells in the uterine stroma of infected animals . In
addition, N. gonorrhoeae enhances TGF-β production and thereby promotes a Th17-dependent
response with the concomitant suppression of Th1/Th2 protective responses . Recently, Liu
et al demonstrated that Th1/Th2 responses are suppressed by mechanisms dependent on
TGFβ and interleukin (IL)-10 as well as type 1 regulatory T (Tr1) cells . Moreover, the
interaction of gonococcal pili with CD4+T cells induces the activation and proliferation of
lymphocytes and stimulates the secretion of IL-10 . In contrast, Opa proteins mediate the binding
to CEACAM-1 expressed by CD4+ T cells and suppress activation and proliferation of naive
lymphocytes [12, 13].
Macrophages (MF) and dendritic cells (DCs) are critical cells in the innate immune
response, acting as sentinels in peripheral tissues and responding against pathogens sensed in
the environment. In this regard, it has been showed that N. gonorrhoeae potently inhibits the
ability of antigen-primed bone-marrow-derived DCs (BMDC) to trigger T-cell proliferation by
inducing expression of both immunosuppressive cytokines and tolerance-inducing cell surface
protein . Furthermore Escobar et al  recently demonstrated that GC modulates MF
and their functionality, producing a shift towards anti-inflammatory cytokine production,
inefficient upregulation in molecules involved in antigen presentation and T-cell activation and a
poor allogeneic T-cell stimulatory activity . These studies showed that N. gonorrhoeae also
suppresses adaptive immune responses through effects on antigen presenting cells (APCs).
Current view of MF considers them as a continuum of phenotypes with overlapping
expression of cell surface markers, secreted cytokines and chemokines, and transcriptional regulators.
In response to various signals, MF may undergo classical-M1 (M1-MF) or alternative-M2
(M2-MF) activation . The M1 phenotype promotes Th1 response and possesses strong
microbicidal and tumoricidal activity . In contrast, M2-MФ are involved in parasite
clearance, dampen inflammation, promotion of tissue remodeling, tumor progression and possess
immune-regulatory functions . M1 and M2 phenotype can be converted into each other in
specific microenvironments . During microbial infection, MF are polarized to M1 or M2
in response to microbial components and host immune mediators. Depending on the bacterial
species, M1 or M2 polarization can play either a beneficial or a detrimental role in disease
outcomes [19, 20]. The persistence of bacterial pathogens in tissues and the chronic evolution of
infectious diseases are linked to MF reprogramming towards heterogeneous M2 signatures.
For example, Coxiella burnetii elicits an atypical M2 profile in MF combining M1/M2
characteristics , while Yersinia enterocolitica stimulates a clear-cut M2 program in MF . The
presence of M2 is also critical for the chronic fate of mycobacterial infections, and high levels
of M2-derived IL-10 are found in early ulcerative lesions of Buruli disease . Although N.
gonorrhoeae has been reported to modulate MF [15, 23], GC influence on MF polarization
has not been yet explored. In order to address this issue we studied the effect of N. gonorrhoeae
using an in vitro model of human monocyte derived MФ (MDM). N. gonorrhoeae exposure
leads to the upregulation of IL-6 and IL-10, which are inflammatory and immunosuppressive
cytokines respectively. Interestingly, Programmed Death Ligand 1 (PD-L1) was also induced.
However, molecules necessary for an efficient adaptive immune response (CD86, MHCII)
were not affected. Consequently we showed that gonococci induce hyporesponsiveness of
interacting T cells, demonstrating for the first time that N. gonorrhoeae interferes with MФ
polarization favoring a shift towards a regulatory phenotype.
Donor buffy coats were obtained to generate macrophages. The study was approved by the
local Scientific ethic committee (Hospital Clínico Universidad de Chile, Act approval number
58). All donors provided written informed consent. After the samples had been collected,
each donor was allocated a trial number, demographic data were collected and the database
Bacteria and culture conditions
The Neisseria gonorrhoeae P9-17 strain used in this study was kindly provided by Dr. Myron
Christodoulides (University of Southampton, UK) . In particular, P9-17 (Pil+ Opab+)
variant of N. gonorrhoeae containing the red-shift mutant GFP (rs-GFP) plasmid was used.
Bacterial growth and analysis of colony morphology were handled as previously described .
Briefly, gonococcal variants were taken from frozen stocks, plated on GC agar plates (Difco,
Becton Dickinson) containing BBL Isovitalex (Becton Dickinson, Sparks, MD) and cultured at
37°C in 5% CO2 for 18 to 20 hours to obtain single colonies. Single colonies showing the proper
morphology were further grown for subsequent experiments.
Macrophages generation and polarization
Human monocytes were obtained from normal blood donor buffy coats by two-step gradient
centrifugation followed by an additional step using the RosetteSep™ Human Monocyte Enrich
ment Cocktail (STEMCELL Technologies). MF were obtained by culturing monocytes (84%
CD14+) for 7 days in RPMI 1640 (GIBCO, Invitrogen Corporation) supplemented with 10%
FBS (HyClone), 50 U/mL penicillin, 50 μg/mL streptomycin (Gibco Invitrogen) and 50 ng/mL
of M-CSF (MiltenyiBiotec) in 6-well plates at a density of 2 x 106 cells per well. Polarization
was induced by replacing the culture medium for RPMI 1640 supplemented with 5% FBS and
100 ng/mL LPS plus 20 ng/mL IFN-γ (for M1 polarization) or 1000 U/mL IL-4 (for M2
polarization) and culturing cells for an additional 24 hours. Three different cell types were generated:
resting fully differentiated 7 days MF (M0-MF), classically activated (M1-MF), and
alternatively activated (M2-MF).
Infection of primary macrophages
Gonococcal isolates were taken from frozen stocks and cultured on GC agar plates at 37°C in a
5% CO2 atmosphere. Bacteria were then scraped from confluent culture plates and
re-suspended in 1 mL of serum-free medium. Bacterial concentration was estimated by optical
density at 600 nm (1 O.D unit corresponding to 3.2 x 109 CFU/mL). M0-MF were infected with
GC at multiplicity of infection (MOI) of 10, 100 or 1000 for 4 hours. Then cultures were
supplemented with gentamicin (100 μg/mL) (Invitrogen Corp., Carlsbad, CA) to kill extracellular
bacteria. Cultures were returned to 37°C, 5% CO2 in humidified incubator and harvested 24
hours post infection for co-culture with T cells or down-stream assays.
Immunofluorescence microscopy analysis
M0-MF were grown on cover slips using antibiotic-free cell culture medium. Nearly confluent
cell monolayers were challenged with the rs-GFP GC strain at MOI of 100 and incubated for 4
hours at 37°C with 5% CO2. Then cell monolayers were washed five times with medium and
fixed for fluorescence microscopy in 1% paraformaldehyde in 1 x PBS (pH 7.4). DAPI and
rhodamine-phalloidin staining was carried out for visualizing the nucleus and F-actin respectively.
Association of GFP-fluorescent bacteria with stained MФ was determined using
Gentamicin protection assay
Assays were performed as described previously . Briefly, to quantify the total number
of MF-internalized gonococci, M0-MF were infected at MOI 100 for 4 or 8 hours. Next,
100 μg/mL of gentamicin (US Biological, Swampscott, MA) were added in order to kill the
extracellular bacteria. Cells were washed 3–5 times with 1 x PBS and lysed with 1% saponin
(Sigma, St Louis, MO) in 1 x PBS for 30 min. The lysates were collected, serially diluted and
aliquots were seeded onto supplemented GC agar plates and incubated 24 hours at 37°C and 5%
CO2. Finally, colony forming units (CFU) were counted. To confirm that gentamicin indeed
killed all non-internalized bacteria, 50 μL of the infection medium post gentamicin treatment
were seeded onto GC plates. No bacterial growth was observed.
Immunophenotyping and flow cytometry
The following directly conjugated anti-human monoclonal antibodies were used: CD4-FITC,
CD4-APC, CD8-PE, CD163-PE, CD206-FITC, CD86-PE-Cy5, CD64-APC, CD273-PE,
TLR4-PE-Cy7, CD40-FITC, HLA-DR-PECy5 and CD274-APC (eBioscience, San Diego).
Saturating amounts of antibody were used to stain approximately 3 x 105 cells in staining buffer (1 x
PBS, 2% FBS) at a final volume of 20 μl for 30 min at 4°C protected from the light. All samples
were washed with staining buffer and resuspended in 200 μl of FACS Buffer. Samples were
examined in a FACSCalibur (BD Biosciences) and analysis was performed using FlowJo
software (Tree Star, Inc., OR).
IL-10, IL-6, IL-1β, IL-23 and IL-12 levels were measured in supernatants 24 hours post
infection or after LPS-IFN-γ/IL-4 treatment (M1/M2 positive control) by enzyme-linked
immunosorbent assays using ELISA Ready-SET-Go! (eBioscience, USA), according to the
Mixed lymphocyte reaction (MLR) assay
Peripheral blood lymphocytes (PBL) were obtained from human peripheral blood cells
(PBMC) of a single donor. Briefly, PBMC were depleted from antigen-presenting cells by
adherence to T75 tissue culture flask supplied with 10% FBS RMPI 1640 medium without
agitation. Two hours later, non-adherent (NAD) cells were collected and incubated overnight into
another T75 tissue culture. The NAD cells containing PBL were collected and labeled with
CFSE (5 mM per 1 × 107 cells) (eBioscience, USA) for 10 min at 37°C. Cells were washed
extensively and 2 × 105 cells/well were cultured with 1 × 105 M1, M2 or GC-treated MФ from
another donor in round-bottomed 96-well plates in RPMI-1640 medium (Gibco Invitrogen)
with 10% fetal bovine serum (FBS, HyClone), 50 U/mL penicillin and 50 μg/mL streptomycin
(Gibco Invitrogen) at 37°C, in a 5% CO2 atmosphere for 7 days. As a positive control we used
PBL stimulated with 150 U/mL of IL-2 and 20 μg/mL of anti-CD3 (OKT-3). Medium was
changed at day 3. At day 7, co-cultures were collected and stained against CD4 using the
previously described conjugated antibodies. Proliferation analysis was performed using FlowJo
software (Tree Star, Inc., OR).
Quantitative Real Time-PCR (qRT-PCR)
The total RNA was extracted from macrophages as described previously . Reverse
transcription of RNA (5 μg) was performed using the Transcriptor First-Strand cDNA synthesis kit
following the manufacturer’s recommendations (Roche Applied Science, Mannheim,
Germany). To quantify the mRNA expression for the M1 and M2-associated cytokines, 50 ng of
cDNA were amplified by quantitative real-time PCR, using the appropriate primers and the
Sybr1Green Master Mix (Fermentas) in an ABI PRISM 7900 Sequence Detector System
(Applied Biosystems, Foster City, CA, USA). The cycle program used was: 95°C for 10 min,
followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. The fold change in
expression of the target gene relative to the 18S endogenous control was set at 2-ΔΔCt, where ΔΔCt =
(CtTarget – Ct18S)stimulated – (CtTarget – Ct18S)unstimulated.
Data was analyzed by two-way ANOVA with Tukey post-test and showed as mean ± standard
error (SEM), (Graphpad Prism V5.0). Statistical significance was considered at a p value less
than 0.05. The data presented are representative of at least three biological replicates.
Applying the widely used method to obtain fully differentiated macrophages (M0-MФ) from
M-CSF-treated human monocytes , we examined whether N. gonorrhoeae might
differentially activate M0-MФ towards a M1 or M2-associated profile. Due to the fact that N.
gonorrhoeae corresponds to facultative intracellular bacteria, we first tested whether M0-MF were
able to internalize the pathogen. M0-MФ were challenged with the rs-GFP GC variant and
then MF-internalized bacteria were evaluated by epifluorescence microscopy. Several bacteria
were observed in the MF cytoplasm 4 hours after infection (Fig 1A). In addition, gonococcus
internalization was also evaluated through a gentamicin protection assay, confirming that a
significant number of intracellular, viable bacteria could be recovered from the MF cytoplasm
(Fig 1B). Likewise, the bacterial counts increased at 8 hours post infection given that MF were
allowed to recognize and internalize bacteria during a longer period of time.
Once the M0-MF capacity to interact and internalize N. gonorrhoeae is confirmedwe
evaluated several M1 and M2-MФ-associated markers by flow cytometry 24 hours post
N. gonorrhoeae exposure. Stimulation with N. gonorrhoeae increased the expression of the
M2-MФ-associated marker CD163 at all the MOIs tested. CD206 M2-MФ-associated marker,
in contrast, was only increased at MOI 1000 (Fig 2).
Regarding M1-MФ-associated markers, CD64 and TLR-4 were significantly upregulated
upon treatment with LPS/IFN-γ (M1-MF) as expected. Yet these markers were not induced in
GC-treated MF, which exhibited similar levels to the non-stimulated M0-MF. Moreover N.
Fig 1. Gonococcus uptake by human MФ. (A) Epifluorescence micrographs of M0-MΦ incubated with rs-GFP GC variant for 4 hours. Right panel shows
merge of GFP (green)/ DAPI (blue)/ Rhodamine (red). Left panel is the merge of all three fluorescent channels overlaid on the phase contrast image to
denote cell boundaries. (B) Gonococcus internalization by human MФ was evaluated trough a gentamicin protection assay. M0-MΦ were infected at time 0
and after 4 and 8 hours of infection they were treated 1 hour with gentamicin to kill extracellular bacteria. M0-MΦ were then lysed after treatment with saponin
for 30 min. Cell lysates were serially diluted, plated in GC agar plates and incubated during 24 hours for CFU counting.
gonorrhoeae was not able to induce the co-stimulatory CD86 neither the major
histocompatibility complex (MHC class II) molecules. Finally CD40, another M1-MF–associated marker,
showed a tendency towards upregulation upon bacteria treatment, although no statistically
significant differences were observed between the different treatments.
N. gonorrhoeae induces a mixed cytokine profile in human macrophages
Considering that microorganisms can modulate the MФ phenotype , we aimed to
determine the effects of gonococcus on MF functionality by evaluating the cytokine profile induced
by N. gonorrhoeae. The pro-inflammatory M1-MФ-associated cytokines IL-6, IL-1β, and
IL23, as well as the anti-inflammatory cytokine IL-10-characteristic of the M2-MФ subtype-,
were measured in culture supernatants (Fig 3A–3D). Data obtained from 6 independent
experiments 24 hours after challenge revealed that GC at MOI of 100 and 1000 significantly induced
the production of the pro-inflammatory cytokine IL-6 in comparison to M0-MФ (Fig 3A). A
similar result was observed for IL-10 (Fig 3B) but at a lower dose of N. gonorrhoeae (MOI 10).
In other words, infected macrophages produced IL-10 rather than IL-6 and IL-1β. Even though
IL-1β and IL-23 levels did not reach statistically significant differences between GC-treated
MF and M0-MF, these cytokines exhibited a tendency to increase and decrease in a dose
dependent manner respectively (Fig 3C and 3D). Additionally, qRT-PCR analysis 4 hours post
infection confirmed the results described above in the sense that GC-infected macrophages
upregulated mRNA expression of IL-10 and IL-6. Remarkably TNF-α mRNA was also induced
(S1 Fig). It is important to mention that even though TNF-α and IL-6 are M1-MF-associated
cytokines, they are also characteristic of the M2b-MF subtype which also produces IL-10 .
Therefore, our results suggest that N. gonorrhoeae polarize human MF towards a M2 profile,
particularly a M2b-MF subtype.
Other molecule we thought interesting to study in the macrophage-GC context was PD-L1.
PD-L1 is a member of the co-stimulatory family of proteins and it is involved in the regulation
Fig 2. N. gonorrhoeae induced M1 and M2-MΦ associated markers. Expression of M1 and M2-MΦ distinctive surface markers were evaluated in
GCtreated MΦ by flow cytometry (monocytic cells gated). (A) Representative histograms for each evaluated marker from at least three independent
experiments. (B) Mean fluorescence intensity (MFI) average for each marker. Data represent at least 3 independent experiments; bars indicate SEM;
* p < 0.05. ** p < 0.01. *** p < 0.001 indicates significant induction compared to non-stimulated MΦ (M0-MΦ).
of the immune response [29–32]. Several reports indicate that PD-L1 participates in the
generation of Tregs and in maintaining self-tolerance [33–35] According to our flow cytometry
results, we found a significant upregulation of PD-L1 in M0-MF upon gonococcal infection
(Fig 4). These data along with secreted IL-10 levels suggest that N. gonorrhoeae polarize
M0-MF towards a more likely regulatory macrophage.
Fig 3. N. gonorrhoeae induced a mixed cytokine profile in human MФ. (A) IL-6, (B) IL-10, (C) IL-1β
and (D) IL-23 cytokines were evaluated 24 hours post infection with N. gonorrhoeae or M1/M2 polarization.
Data obtained are expressed as the mean ± SEM and represent at least three independent experiments.
** p < 0.01. *** p < 0.001 indicates significant induction compared to non-stimulated MΦ (M0-MΦ). ND. non
Fig 4. PD-L1 expression was upregulated in human MФ upon N. gonorrhoeae infection. Human MФ treated for 24 hours with medium only, or M1/M2
polarizing stimulus, or N. gonorrhoeae (MOI = 10, 100, 1000) were immunostained for flow cytometric analysis of PD-L1. (A) Representative overlay
histograms. (B) MFI average. Data obtained are expressed as the mean ± SEM and represent at least nine independent experiments. * p < 0.05. ** p < 0.01.
*** p < 0.001 indicates significant induction compared to non-stimulated MΦ (M0-MΦ).
Fig 5. Hyporesponsive alloantigen T-cell responses induced by MФ infected with N. gonorrhoeae. CFSE-labeled CD4+ cells proliferation after
non-adherent cells were co-cultured with human MФ treated for 24 hours with LPS-IFN-γ (M1-MΦ), IL-4 (M2-MΦ) or N. gonorrhoeae (MOI = 10, 100, 1000)
for 7 days at the ratio of 2:1. As a positive control of proliferation we used PBL stimulated with 150 U/mL of IL-2 and 20 μg/mL of anti-CD3 (OKT-3). (A)
Representative T CD4+ cell proliferation dot plots from one of the donors are shown. (B) CD4 + cells proliferation average under different conditions.
** p < 0.01 indicates that only M1-MФ profile is able to significantly induce proliferation of CD4+ cells in a mixed lymphocyte reaction.
Since the surface markers and cytokines profile induced by N. gonorrhoeae on infected MF are
well-matched with M2 profile, we addressed to study the capacity of GC-treated MФ to
stimulate T cells. PBL from a single donor were co-cultured with GC-treated MФ (or M1/M2-MФ
for the controls) from another donor in a mixed lymphocyte reaction (MLR) assay. After 7
days of co-culture with N. gonorrhoeae-treated MФ, CD4+ cells exhibited no significant
proliferation, evaluated trough CFSE dilution, as compared to M1-MF-exposed cells (Fig 5).
Although T cell proliferation was evaluated at three different MOIs (10, 100 and 1000), we did
not observe significant differences between them.
It has been previously demonstrated that N. gonorrhoeae is capable of inducing a tolerogenic
profile not only in RAW murine macrophage cell line but also in human dendritic cells [14,
15]. However, the effect of gonococcal infection in human MФ has not been yet reported. In
this study we first evaluated the effect of N. gonorrhoeae at different doses in the polarization of
MDM (referred to as M0-MФ). M1-MФ (or classically activated) and M2-MФ (or alternatively
activated) associated surface markers were measured by flow cytometry. We found N.
gonorrhoeae was indeed capable of inducing CD163 in M0-MФ at the lowest bacterial concentration
(MOI 10) whereas CD206 was only induced at MOI 1000. M2-MФ profile includes at least
three subsets: M2a, induced by IL-4 or IL-13; M2b, induced by immune complexes and
agonists of TLRs or IL-1 receptors; and M2c, induced by IL-10 and glucocorticoid hormones .
Although CD206 is the best characterized M2-MФ marker, as is present in all the M2-MФ
subtypes, there is controversy regarding CD163 expression on M2a phenotype (referred to as
M2-MФ in our study). Specifically, Zizzo et al  state that M1 and M2a-MФ (generated
upon stimulation with IL-4) exhibit low levels of CD163. Vogel et al  also determined
CD163 did not differ significantly in M2a-MФ compared to M0-MФ. This might explain why
we did not observe an upregulation of CD163 in our M2-MФ control.
Unlike M0-MФ treated with LPS/IFN-γ, N. gonorrhoeae was not able to induce the
M1-MФ-associated markers CD86, MHCII, TLR-4 nor CD64 at any of the three bacterial
doses tested (Fig 2). Indeed, the expression levels exhibited after GC-stimulation were similar
to those observed in M2 and M0-MФ controls. Although CD40 showed a tendency towards
upregulation upon infection, no significant differences were observed in comparison with the
M0-MФ control, neither between M0-MФ and the M1-MФ positive control. The latter
suggests that CD40 is not a suitable marker of the M1-MФ phenotype, which differs from other
studies that have established CD40 as the most distinctive M1-MФ profile marker .
Interestingly, the lack of induction of the cell surface co-stimulatory molecule CD86 upon infection
with N. gonorrhoeae seems to be an infrequent feature of pathogens in many studies using
transcriptional tools, which have indicated that CD86 along with other M1-MФ markers,
−including cytokines such as TNF, IL-6, IL-1β− are upregulated upon infection with several bacteria
for instance, Yersinia enterocolitica, Tropheryma whipplei , Salmonella enterica serovar
Typhimurium  and Mycobacterium tuberculosis . Since the CD86 and the MHC-II
molecules are extremely necessary to antigen presentation, it is likely that the MФ resulting
from the infection with N. gonorrhoeae have a poor proliferative capacity over T cells. These
data are supported by a previous report where N. gonorrhoeae was unable to induce significant
upregulation of neither CD86 nor MHC class II in the murine MФ cell line RAW .
Although N. gonorrhoeae is actually phagocytosed by MФ (Fig 1), our data suggest that the
bacteria might weaken antigen-presenting functions because the immune responses regulated
by the CD86/CD28 co-stimulatory pathway are impaired in the absence of CD28 signaling. As
is known, these immune responses are responsible of antibody production and induction of
cytotoxic T-cell activity . In addition, the low levels of TLR-4 exhibited by GC-infected
MФ might lead to deficient activation of APCs, thus resulting in chronic infection with
weakened bacterium elimination as previously reported in a mycobacterial model using TLR-4
mutant mice . CD64, also known as FcγR1, is another well-characterized M1-MФ-associate
marker that was not induced upon infection with N. gonorrhoeae (Fig 2). CD64 belongs to the
Fcγ family of receptors and binds IgG with high affinity . Importantly, upon Fc binding,
the CD64 receptor induces the association of the γ chain, triggering functional responses such
as phagocytosis. Binding of CD64 with IgG also mediates antibody-dependent cellular
cytotoxicity (ADCC) as well as induction of several cytokine genes transcription and release of
inflammatory mediators . Based on the above, we suggest that the low expression of this receptor
on MФ infected with N. gonorrhoeae might help gonococcus to evade some immune responses,
especially the ADCC-mediated response.
Once established the surface marker profile exhibited by GC-treated MФ, we evaluated
their functional polarization through the release of M1-MФ and M2-MФ-associated cytokines.
Interestingly, infection with N. gonorrhoeae significantly induced pro (IL-6 and TNF-α) and
anti-inflammatory (IL-10) cytokines in M0-MФ (Fig 3 and S1 Fig). IL-6 secretion triggered by
N. gonorrhoeae infection has been observed in vivo . In particular, it has been demonstrated
that GC not only induces the secretion of the pro-inflammatory cytokine IL-6 but also TNF-α
in APCs that are located in the stroma of the female mouse genital tract. This is supported by
the increased levels of TNF-α and IL-6 observed in vivo in vaginal secretions of Balb/c mice
after gonococcal infection . In vitro, Feinen et al  further demonstrated that BMDC
cultured with N. gonorrhoeae also produced IL-6 along with IL-23, but not IL-12. Moreover, upon
stimulation with N. gonorrhoeae, human THP-1-derived MФ also secreted IL-6 and IL-23, 1β
and TNF-α, but not IL-12, which suggest that human and mouse APCs behave similarly in
response to GC-stimulation. In our model, although IL-1β and IL-23 were also induced upon
infection, we did not obtain statistically significant differences between the
unstimulated/stimulated MФ. These data suggest that although response to N. gonorrhoeae might trigger some
inflammatory pathways (IL-6 production); this is not sufficient to activate the adaptive
immune system through co-stimulatory molecule induction. This would possibly result in a
chronic inflammatory condition without clearance of the pathogen as observed in infected
patients . Remarkably we did not detect IL-12 secretion in N. gonorrhoeae-infected MФ
neither in the positive control M1-MФ (data not shown), which is in accordance with previous
studies [5, 45]. An explanation for this is M1-MФ also released significant levels of IL-10,
which in turns might inhibit IL-12 secretion.
Interestingly, IL-10 was strongly induced in a dose-dependent manner upon infection with
N. gonorrhoeae. IL-10 induction by N. gonorrhoeae was recently demonstrated by a study of
Liu et al  which showed both in vitro and in vivo that N. gonorrhoeae strongly induced
IL10 and Tr1 cells.
IL-10 is one of the most important regulatory cytokines and it is induced following
stimulation with TLR ligands such as LPS . This fact explains the low levels of IL-10 secreted by
control M2-MФ (Fig 3). The role of IL-10 has also been observed upon infection with other
pathogens. Particularly, in lepromatous lesions caused by Mycobacterium ulcerans, in
infections with Coxiella burnetii and Mycobacterium tuberculosis [19, 22].
The cytokine profile (IL-10, IL-6, TNF-α) elicited upon infection with N. gonorrhoeae
correlates well with the M2b-MФ phenotype. Furthermore M2b-MФ as well as GC-infected
macrophages exhibit CD163 marker on their surface . Our findings lead us to suggest N.
gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the
M1-MФ-associated markers are induced.
Besides the assessment of IL-10 production, we evaluated other surface markers with
immunosuppressive properties, in particular Programmed Death Ligand 1 (PD-L1), on infected MФ.
PD-L1 is important in suppressing the immune system during specific events such as
autoimmune diseases and pregnancy [47–50]. In this work, we found PD-L1 was significantly induced
upon N. gonorrhoeae exposure in all the MOIs tested (Fig 4). It is important to mention that
PD-L1 was also upregulated in M1-MФ control. However, upregulation of CD86 expression in
M1-MФ was also observed (Fig 2B). Furthermore, the effect of N. gonorrhoeae in PD-L1 has
been documented by Zhu et al . Particularly, they observed PD-L1 upregulation upon N.
gonorrhoeae exposure in primary human DCs and murine bone marrow derived DCs
(BMDCs). Although little is known about the role of PD-L1 in MФ during bacterial infections,
several studies have reported this role in DCs [44, 51, 52]. Specifically, PD-L1-mediated DC
immunosuppression has been observed in response to commensally or pathogenic bacteria
that colonize the genital tract [53, 54].
In order to determine the functionality of N. gonorrhoeae-stimulated MФ and to evaluate
whether it might confer them a regulatory phenotype, N. gonorrhoeae-treated MФ were
co-cultured with allogenic CFSE labeled PBL. It was found that these MФ were not able to induce
CD4+ T cell proliferation, unlike our positive control M1-MФ did (Fig 5). These data correlate
with our previous study in which it was demonstrated that GC-treated RAW cells possess weak
allogeneic T-cell stimulatory activity [14, 15]. In addition, Zhu et al  showed that
GCexposed BMDCs and also human DCs failed to elicit antigen-induced CD4+ T lymphocyte
proliferation. Although further studies are needed to determine which is the exactly
mechanisms responsible of the hyporesponsive alloantigen responses exhibited by T cells upon
stimulation with N. gonorrhoeae-infected MФ, our work reports a novel strategy by which N.
gonorrhoeae modulates host innate immune response by polarizing M0-MF towards a
regulatory/M2-MF phenotype and provides new insights that might help to unravel the complexity
of the immune response against gonococcal infection.
S1 Fig. Transcriptional cytokine analysis of N. gonorrhoeae-stimulated human MФ.
Quantitative PCR analysis for cytokine mRNA expression on N. gonorrhoeae-stimulated MF. M1
and M2-MF were used as controls. Log2 expression levels for IL-10, IL-6, IL-1β, TNF-α and
IL-23. Results are expressed as the ratio of the expression level in stimulated vs. unstimulated
MF (M0-MF) and represent the mean ± SEM of three independent experiments.
We thank Michael Russell for critical review of the manuscript. Israel Guerrero for his help
with the microscopic analysis. Mr. Juan Fernández for the final proofreading and check of the
spelling and grammar.
Conceived and designed the experiments: AE MCO. Performed the experiments: AE MCO.
Analyzed the data: AE MCO MI. Contributed reagents/materials/analysis tools: CL CA-C ML
PIR RV. Wrote the paper: AE MCO.
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