Vigorous Response of Human Innate Functioning IgM Memory B Cells upon Infection by Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae, the cause of the sexually transmitted infection gonorrhea, elicits low levels of specific Ig that decline rapidly after the bacteria are cleared. Reinfection with the same serovar can occur, and prior gonococcal infection does not alter the Ig response upon subsequent exposure, suggesting that protective immunity is not induced. The mucosal Ig response apparent during gonorrhea does not correlate with that observed systemically, leading to a suggestion that it is locally generated. In considering whether N. gonorrhoeae directly influences B cells, we observed that gonococcal infection prolonged viability of primary human B cells in vitro and elicited robust activation and vigorous proliferative responses in the absence of T cells. Furthermore, we observed the specific expansion of IgD(+)CD27(+) B cells in response to gonococcal infection. These cells are innate in function, conferring protection against diverse microbes by producing low-affinity, broadly reactive IgM without inducing classical immunologic memory. Although gonococcal infection of B cells produced small amounts of gonococcal-specific IgM, IgM specific for irrelevant Ags were also produced, suggesting a broad, polyspecific Ig response. The gonococci were effectively bound and engulfed by B cells. TLR9-inhibitory CpGs blocked B cell responses, indicating that intracellular bacterial degradation allows for innate immune detection within the phagolysosome. To our knowledge, this is the first report of a bacterial pathogen having specific affinity for the human IgM memory B cells, driving their potent activation and polyclonal Ig response. This unfocused T-independent response explains the localized Ig response that occurs, despite an absence of immunologic memory elicited during gonorrhea.

Full text

The Journal of Immunology
Vigorous Response of Human Innate Functioning IgM
Memory B Cells upon Infection by Neisseria gonorrhoeae
Nancy S. Y. So,* Mario A. Ostrowski,
†
and Scott D. Gray-Owen*
Neisseria gonorrhoeae, the cause of the sexually transmitted infection gonorrhea, elicits low levels of specific Ig that decline rapidly
after the bacteria are cleared. Reinfection with the same serovar can occur, and prior gonococcal infection does not alter the Ig
response upon subsequent exposure, suggesting that protective immunity is not induced. The mucosal Ig response apparent during
gonorrhea does not correlate with that observed systemically, leading to a suggestion that it is locally generated. In considering
whether N. gonorrhoeae directly influences B cells, we observed that gonococcal infection prolonged viability of primary human
B cells in vitro and elicited robust activation and vigorous proliferative responses in the absence of T cells. Furthermore, we
observed the specific expansion of IgD
+
CD27
+
B cells in response to gonococcal infection. These cells are innate in function,
conferring protection against diverse microbes by producing low-affinity, broadly reactive IgM without inducing classical immu-
nologic memory. Although gonococcal infection of B cells produced small amounts of gonococcal-specific IgM, IgM specific for
irrelevant Ags were also produced, suggesting a broad, polyspecific Ig response. The gonococci were effectively bound and
engulfed by B cells. TLR9-inhibitory CpGs blocked B cell responses, indicating that intracellular bacterial degradation allows
for innate immune detection within the phagolysosome. To our knowledge, this is the first report of a bacterial pathogen having
specific affinity for the human IgM memory B cells, driving their potent activation and polyclonal Ig response. This unfocused T-
independent response explains the localized Ig response that occurs, despite an absence of immunologic memory elicited during
gonorrhea. The Journal of Immunology, 2012, 188: 4008–4022.
Neisseria gonorrhoeae (gonococci) is a human-specific
pathogen that causes the sexually transmitted infection
gonorrhea and is a major cause of morbidity. The rate of
gonorrhea infection is increasing worldwide, with a recent esti-
mate of 88 million new infections per year (1). Gonorrhea results
from overwhelming inflammation, producing purulent discharge
at the site of infection. The majority of women infected with N.
gonorrhoeae are clinically asymptomatic (2). Left untreated, this
can progress to serious complications including ectopic pregnan-
cies because of fallopian tube scarring, sterility, or acquired blind-
ness because of gonococcal conjunctivitis in children born to
infected mothers (3). Once infection is detected, gonorrhea can be
effectively treated with antibiotics; however, antibiotic resistance
is increasing at an alarming rate, and gonococcal infections may
soon become untreatable (4, 5). Gonococcal coinfection can also
increase viral shedding in HIV patients (6), further heightening the
urgency to control the spread of this important pathogen.
Despite the overzealous innate immune response that causes
symptomatic disease, adaptive immunity against uncomplicated
gonococcal infections is surprisingly poor. Although gonococcal-
specific Ig can be observed in infected individuals, Ab levels are
low compared with other mucosal infections, and there is no ev-
idence of a classical immunologic memory response upon re-expo-
sure, even during infection of rectal tissues containing immune-
inductive sites (7). As such, repeated gonococcal infections can
occur, sometimes with the same serotype of N. gonorrhoeae (8).
Although neisserial surface structures antigenically vary at an
impressive rate, this alone cannot explain the paucity of memory
response. Instead, the gonococci also appear capable of actively
suppressing the adaptive immune response. Neisseria sp. express
Opa protein adhesions, which, upon binding to human carci-
noembryonic Ag-related cellular adhesion molecule (CEACAM)
receptors, can trigger bacterial uptake and transcytosis through the
mucosal epithelial layer (9, 10). In addition to facilitating entry
into tissues, certain Opa variants trigger the coinhibitory function
of CEACAM1, which may suppress innate proinflammatory cy-
tokine production by epithelia (11) and inhibits the cellular acti-
vation and proliferation of CD4
+
T cells during infection (12, 13).
Although only a fraction of gonococcal Opa variants are specific
for CEACAM1 (14), there appears to be an in vivo selection for
this function because .95% of clinical Neisseria isolates bind to
CEACAM1 (15). The ability of N. gonorrhoeae to inhibit CD4
+
T cells during infection may explain the absence of classical
immune memory responses to this human-restricted pathogen.
Although the Opa-dependent inhibition of T cell function has
been well characterized (12, 13), there is not a complete absence
of Ig during gonorrhea. In fact, clinical evidence enticingly sug-
gests that gonococcal infection might directly influence the B cell
response. There is an increase in both B and T lymphocytes in the
endocervix of women with nonulcerative sexually transmitted
infections including N. gonorrhoeae (16), suggesting that the
bacteria may directly access the B cells. Ig production within the
*Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S
1A8, Canada; and
†
Clinical Sciences Division, University of Toronto, Toronto,
Ontario M5S 1A8, Canada
Received for publication March 11, 2011. Accepted for publication February 15,
2012.
This work was supported by Canadian Institute of Health Research Operating Grant
414775.
Address correspondence and reprint requests to Dr. Scott D. Gray-Owen, Department
of Molecular Genetics, University of Toronto, Room 4381, Medical Sciences Building,
1 Kings College Circle, Toronto, ON M5S 1A8, Canada. E-mail address: scott.gray.
owen@utoronto.ca
The online version of this article contains supplemental material.
Abbreviations used in this article: CEA, carcinoembryonicAg; CEACAM, carcinoem-
bryonic Ag-related cell adhesion molecule; HSPG, heparin sulphate proteoglycan;
iCpG, inhibitory CpG ODN TTAGGG; KLH, keyhole limpet hemocyanin; MAMP,
microbial-associated molecular pattern; MOI, multiplicity of infection; ODN, oligo-
deoxynucleotide; Opa
CEA
, carcinoembryonic Ag-related cell adhesion molecule-
specific Opa; Opa
HSPG
, heparan sulfate proteoglycan-specific Opa; TT, tetanus toxoid.
Copyright Ó2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100718

male and female genital tract is distinct from that of other mucosal
surfaces in that IgG predominates rather than IgA (17). Although
the levels of total mucosal IgG were slightly lower in gonococcal-
infected versus uninfected women, total mucosal IgM increased in
those with gonorrhea (7). Gonococcal-specific IgM was present in
59% of cervical mucus samples obtained from women with gon-
orrhea but were undetectable in samples obtained from individuals
who were uninfected (18). Curiously, the Ig response in serum did
not reflect that found in cervical mucus or vaginal washes, sug-
gesting that Ig production was localized rather than being a sys-
temic response (7). The production of IgM in local secretions did
require active gonococcal infection, because antibiotic therapy to
eliminate the infection correlated with a decrease in gonococcal-
specific IgM in cervical mucus samples in all women who were
treated (18). Puzzlingly, the antigonococcal response was not
greater against the infecting strain, because it frequently cross-
reacted equally or, in some cases, better with the prototypical
but antigenically distinct N. gonorrhoeae MS11 (7).
Such clinical data prompted us to consider that, in addition
to suppressing T cell responses (12), the gonococci may directly
affect B cell function. Previous work suggested that gonococcal
infection kills human B cells in a CEACAM1-dependent manner
(19); however, this does not explain the elevated Ig levels during
N. gonorrhoeae infection. Alternatively, because the production of
IgM by human B cells does not require supplemental signaling
from T cells (20, 21), it is enticing to consider that gonococcal
infection could directly induce the production of IgM, thereby
explaining the clinical observations.
N. gonorrhoeae is strictly a human pathogen, with several
virulence factors that show exquisite host specificity. As such, no
mouse model of infection exists that accurately mimics the pro-
gression of gonococcal disease in the human host. Furthermore,
taking into account the intrinsic differences between human and
mouse B cells [e.g., TLR4 signaling induces strong responses in
murine B cells but is not detected in human B cells (20, 22)], we
developed in vitro infection protocols using purified primary
human B cells. Unexpectedly, and in stark contrast to parallel
infections with the prototypical Gram-negative bacteria, Escher-
ichia coli, we observed that N. gonorrhoeae prolonged B cell
viability and triggered a robust cellular activation that led to the
polyclonal proliferation and differentiation of specifically the IgD
+
CD27
+
subset of human B cells, resulting in an innate, polyclonal
Ig response that reacted with both Neisseria-derived and unrelated
Ags. When combined with the ability of the gonococci to suppress
T cell activation (12, 13, 23), our observations explain the unfo-
cused and nonprotective Ig responses apparent in individuals with
uncomplicated gonococcal infection.
Materials and Methods
Cells
Primary human B cells were obtained by either standard venipuncture or,
when larger numbers of cells were required, through leukopheresis. All
participants gave informed consent in accordance with guidelines for the
conduct of clinical research at the University of Toronto and St. Michael’s
Hospital, respectively. The protocols used were approved by the University
of Toronto and St. Michael’s Hospital institutional review boards (Toronto,
ON, Canada).
Fresh peripheral mononuclear cells were obtained from Ficoll-Paque
Premium (GE Healthcare) gradients, according to the manufacturer’s
specifications. CD19
+
B cells were purified using either the EasySep
Human CD19 Positive Selection Kit (StemCell Technologies) or CD19
Microbeads (Miltenyi Biotec). B cell purity consistently exceeded 95%, as
measured by a FACSCalibur flow cytometer (BD Biosciences) with FlowJo
software (Tree Star) used for all flow data analysis.
Purified cells were maintained in RPMI 1640 medium (Invitrogen),
supplemented with 10% heat-inactivated FBS (CanSera), 4 mM GlutaMAX
(Invitrogen), and 50 mM HEPES (Bioshop, Burlington, ON, Canada) at pH
7.4. Cells were cultured at 37˚C in 5% CO
2
and humidified air.
Bacterial strains
Isogenic N. gonorrhoeae strains constitutively expressing no Opa (Opa
2
;
strain N302), the heparan sulfate proteoglycan-specific Opa (Opa
HSPG
)
(strain N303, expressing Opa
50
), or CEACAM-specific Opa (Opa
CEA
)
(strain N309, expressing Opa
52
) were derived from a pilus-deficient MS11
parent strain (24) and were graciously provided by Prof. T. F. Meyer (Max
Planck Institute for Infection Biology, Berlin, Germany). Clinical N.
gonorrhoeae strains N2061 and N2066 were isolated from male urethra.
Immunoblot analysis indicates that strain N2061 does not express any
Opa protein, N2066 expresses at least one Opa adhesin, and neither strain
expresses pili. These clinical strains were obtained from a sexually
transmitted disease clinic in the district of Pumwani in Nairobi, Kenya, and
were a gift from Dr. F.A. Plummer (University of Manitoba, Winnipeg,
MB, Canada). Neisseria species were grown from frozen stocks on GC
agar (Difco) supplemented with 1% (v/v) IsoVitalex (BBL Microbiology
Systems) at 37˚C in a 5% CO
2
atmosphere with humidity. A binocular
microscope was used for daily selection of gonococcal colony opacity
phenotypes for the MS11 strains. Opa protein expression was routinely
monitored by immunoblotting using the Opa cross-reactive mAb 4B12/
C11 (25), provided by Prof. M. Achtman (Environmental Research Insti-
tute, University College Cork, Cork, Ireland).
E. coli (DH5a) was grown from frozen stocks in aerated Luria–Burtani
broth overnight at 37˚C. Liquid cultures were subcultured onto Luria–
Burtani agar prior to infection assays.
Infection of lymphocytes
Freshly isolated human B cells were infected with a multiplicity of infection
(MOI) of 10 bacteria per cell, unless otherwise indicated. Gentamicin
(Bioshop) was routinely added to infected samples 3 h after bacterial
addition to prevent overgrowth. For E. coli, gentamicin was added im-
mediately upon infection to prevent bacterial overgrowth of the B cell
cultures. Although the gentamicin effectively inhibited bacterial growth,
it did not otherwise affect observed B cell responses.
Confocal microscopy
B cells were infected with the indicated N. gonorrhoeae and E. coli strains
at an MOI of 10 for durations between 1.5 and 44 h. Acid-washed glass
coverslips were coated with mouse mAb specific for human CD19 (clone
HIB19; eBioscience) to capture B cells for microscopy. Cells were fixed
with 4% paraformaldehyde (Sigma-Aldrich), and gonococci were detected
using a polyclonal rabbit antigonococcal serum (UTR01), followed by
Alexa 488-conjugated secondary Ab (Invitrogen-Molecular Probes) after
permeabilization of mammalian cell membranes using 0.4% Triton X-100
(Sigma-Aldrich). E. coli was detected using the purified polyclonal goat Ab
ab25823 (Abcam), followed by a goat-specific secondary Ab in a process
similar to the gonococcal staining protocol. Where indicated, bacteria were
prelabeled using Texas Red succinimidyl ester (Molecular Probes) rather
than using antisera. B cells were stained using F(ab9)
2
of biotinylated goat
anti-human IgM, IgG, and IgA (Jackson ImmunoResearch Laboratories),
followed by streptavidin-Alexa 488 (Molecular Probes).
To examine whether primary human B cells were able to engulf whole
bacteria, cells were infected with the indicated N. gonorrhoeae strains at an
MOI of 10 for 3 h. Acid-washed coverslips coated with the human CD19-
specific Ab clone HIB19 were used to capture B cells prior to fixation.
Extracellular gonococci were stained first, using a polyclonal rabbit anti-
gonococcal serum (UTR01), followed by anti-rabbit IgG Alexa 647
(Molecular Probes). Mammalian cell membranes were permeabilized us-
ing 0.4% Triton X-100 (Sigma-Aldrich), allowing for total gonococci to be
stained using the polyclonal rabbit antigonococcal serum (UTR01), fol-
lowed by anti-rabbit IgG Alexa 488 (Molecular Probes).
Bacteria and cells were visualized and recorded using a Plan-
Apochromat 3100/1.4 Oil differential interference contrast objective lens
on a Zeiss LSM 510 confocal microscope. All images were obtained at
room temperature. Zeiss LSM Image Browser version 4.2.0.121 was used
for subsequent image processing. Each infection condition was prepared on
duplicate or triplicate coverslips, and at least 60 N. gonorrhoeae-infected
cells and at least 100 cells for the E. coli infection condition were analyzed.
Analysis of B cell responses
All flow cytometric analysis was performed using a BD FACSCalibur. B cell
subsets were defined using the following Abs: IgD-PE or IgD-FITC (clone
IA6-2; BD Biosciences), CD27-FITC or CD27-allophycocyanin (clone
O323; eBioscience), and Cy5-labeled F(ab9)
2
fragments of goat and human
The Journal of Immunology 4009

IgG, IgM, or IgA (Jackson ImmunoResearch Laboratories). CD27 and IgD
immunostaining was performed on live cells, whereas all other immuno-
staining was performed on fixed cells. CompBeads (BD Biosciences) were
used for fluorescent compensation. All cells were fixed prior to flow
analysis.
To identify the B cell subpopulations associating with gonococci or E.
coli, live bacteria were labeled with Alexa 647 succinimidyl ester (Mo-
lecular Probes) and then used to infect purified B cells at an MOI of 2
bacteria per cell for 3 h before treatment with gentamicin. Bacterial as-
sociation to B cell subpopulations was visualized by flow cytometry.
For viability, activation, and proliferation assays, infected cells and
uninfected controls were fixed at time points indicated in the figure legends.
Where indicated, 16 mM staurosporine (Sigma-Aldrich) was added to
uninfected cells to induce B cell death as a control for viability experi-
ments. Forward- and side-scatter data and annexin V-PE (BD Biosciences)
staining were used to quantify cell death. Cellular activation was quantified
using CD86-PE (clone FUN-1; BD Biosciences) and forward- versus side-
scatter data, because activated cells increase in forward scatter compared
with cells at rest.
To examine proliferation, B cells were infected overnight with bacteria or
were left uninfected, at which point BrdU (Sigma-Aldrich) was added to the
culture media. BrdU incorporation was quantified using monoclonal mouse
anti-BrdU (clone IU-4; Invitrogen-Caltag Laboratories), which was directly
conjugated to biotin using Zenon Mouse IgG
1
Labeling Kit (Molecular
Probes). Streptavidin-allophycocyanin (Jackson ImmunoResearch Labora-
tories) was then used to visualize the Ab for detection by flow cytometry.
To identify and quantify populations of cells undergoing proliferation at the
time of fixation, Ki67-Alexa 647 (clone B56; BD Biosciences) was used.
B cell responses to TLR agonists were examined by culturing purified
B cells with the following reagents (all from InvivoGen): Pam
3
CSK
4
at 2.5
mg/ml, FSL1 at 1 mg/ml, E. coli K12 LPS at 1 mg/ml, and CpG ODN2006
at 5 mg/ml for 3 d, at which time they were stained for activation marker
CD86-PE (clone FUN-1; BD Biosciences) or nuclear proliferation Ag,
Ki67-Alexa 647 (clone B56, BD biosciences), and B cell subpopulation
markers CD27 and IgD as described above.
To examine the role of TLR9 signaling in N. gonorrhoeae infection of
B cells, inhibitory CpG oligodeoxynucleotide (ODN) TTAGGG (iCpG;
InvivoGen) was used, which functions as a dominant-negative TLR9 li-
gand. B cells were either left untreated/uninfected, treated with CpG
ODN2006 (InvivoGen) at 0.65 nmol/ml or infected with N. gonorrhoeae
strain N302 Opa
2
at a MOI of 10. Cell cultures were then treated with
either iCpG or ODN TTAGGG control (cCpG; InvivoGen), a control ODN
that possess similar sequence to the iCpG but without the ability to inhibit
TLR9 signaling, both at 10-fold molar concentrations of stimulating CpG
ODN2006 for 3 d, at which time cells were stained for Ki67-Alexa 647
(clone B56; BD Biosciences) and B cell subpopulation markers CD27 and
IgD as already described previously.
ELISA
All ELISAs were read at 450 nm using a 1420 Victor
3
(PerkinElmer) plate
reader, unless otherwise indicated. Total Ig in cell culture supernatant was
quantified by IgM, IgG, or IgA ELISA kits (Zeptometrix), used according
to manufacturer’s instructions.
To monitor Ag-specific Ig production, 96-well Maxisorp plates (Nunc)
were coated with either heat-killed Opa
2
gonococci (N302), keyhole
limpet hemocyanin (KLH; Sigma-Aldrich), or tetanus toxoid (TT; List
Biological Labs) resuspended in pH 7.4 PBS (Wisent). The plates were
dried, vacuum-sealed into airtight bags, and stored at 4˚C until use. Five-
percent BSA (Bioshop) in PBS was used to block wells, and 0.05% Tween
20 (Sigma-Aldrich) in PBS was used as wash buffer. Culture supernatants
from uninfected cells or those infected with either gonococci or E. coli were
diluted using 1% BSA in 0.05% Tween 20.
For KLH and TT ELISAs, biotinylated F(ab9)
2
fragments of goat anti-
human IgG, IgM, and IgA (Jackson ImmunoResearch Laboratories) were
used separately to determine the amount of each class of Ig produced in
response to bacterial infection. After incubation with streptavidin HRP
Ultra (Sigma-Aldrich), 3,39,5,59-Tetramethylbenzidine (KPL) was used as
the colorimetric reagent for HRP activity.
For N. gonorrhoeae plates, F(ab9)
2
fragments of goat anti-human IgG,
IgM, and IgA directly conjugated to alkaline phosphatase (Jackson
ImmunoResearch Laboratories) were used separately to determine the
amount of each class of Ig present in culture supernatants. BluePhos (KPL)
was used as the developing reagent. These ELISAs were read at 595 nm.
Gentamicin protection assay
To examine the viability of engulfed intracellular gonococci, B cells were
infected with an MOI of 50 with the indicated N. gonorrhoeae strains for
1.5 h. Infection cultures were then treated with and maintained in 100 mg/
ml gentamicin (Bioshop) to kill any extracellular (nonengulfed) gonococci.
At 2.5 and 6 h postinfection, cells were washed and concentrated in PBS
containing 1 mM MgCl
2
and 0.5 mM CaCl
2
using Transwells with a 3-mm
pore size to retain the cells (Corning). One-percent saponin (Sigma-
Aldrich) was added to lyse mammalian membranes. Serial dilutions of the
lysis mixture were plated on GC agar (Difco) supplemented with 1% (v/v)
IsoVitalex (BBL Microbiology Systems) and incubated at 37˚C in a 5%
CO
2
atmosphere with humidity for at least 48 h before numeration of
bacterial colonies.
Statistics
Differences between experimental conditions were determined to be sig-
nificant when p,0.05 by one-way ANOVA with a Dunnett’s posttest,
calculated by Prism 5.0 (GraphPad).
Results
N. gonorrhoeae associates with human peripheral blood
B cells
N. gonorrhoeae adheres directly to primary human CD4
+
T cells
in an Opa protein-dependent manner, allowing effective suppres-
sion of T cell activation (13). To establish whether N. gonorrhoeae
also interacts with primary human B cells, we exposed freshly
purified cells to isogenic strains of gonococci expressing either no
Opa adhesin, Opa
HSPG
, or Opa
CEA
, and then monitored their as-
sociation with the B cells over time using confocal microscopy.
Cellular association with the prototypical Gram-negative bacterium
E. coli was also examined to assess the general propensity of
B cells to bind bacteria. To confirm that bacteria were associating
with B cells and not a copurified cell type, the infected cells were
stained for BCR, as depicted in Fig. 1A. We then quantified the
percentage of B cells that were associated with at least one bac-
terium. As illustrated in Fig. 1B, substantially more B cells are
associated with gonococci than E. coli, regardless of which neis-
serial adhesin was expressed. However, unexpectedly, even after
44 h of infection, some B cells did not associate with gonococci,
suggesting that binding may be restricted to a specific subpopula-
tion of cells. Moreover, no recruitment of BCR to sites of bacterial
attachment was evident, indicating that this association is inde-
pendent of BCR clustering, a strategy used by other bacteria (26).
Although Fig. 1B quantifies the percentage of B cells with at
least one bacterium bound, this does not illustrate how many
bacteria associate with each cell. We quantified the number of
bacteria per cell (Fig. 1C) and observed that most infected B cells
are associated with one to five bacteria per cell. The ability of
gonococci to associate with cells is largely Opa-independent
(compare Opa
2
gonococci), although Opa expression did have
some influence on bacterial association with the lymphocytes in
some donors (e.g., compare donors 02L and 07L).
Gonococcal infection inhibits B cell death and promotes
prolonged B cell viability
Previous work suggested that in the presence of potent stimulation,
infection of primary human B cells with N. gonorrhoeae caused
B cell death (19). This could be explained either by a direct cy-
totoxic effect of the gonococci or by N. gonorrhoeae contributing
to activation-induced cell death. We explored whether N. gonor-
rhoeae infection affected the viability of freshly purified primary
human B cells by monitoring changes in cell morphology and
membrane integrity during infection experiments. Loss of mem-
brane integrity is an early event of cell death and results in a de-
crease in forward scatter and increase in side scatter, reflecting
corresponding changes in cell size and granularity, respectively
(Fig. 2A). Annexin V binding to phosphatidylserine exposed on
a depolarized cell membrane is also a well-established method to
measure cell viability (27). To confirm the positioning of the live
4010 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS

and dead populations within flow cytometry plots, B cell death
was induced by treating cells with staurosporine. Comparison
between gated populations in Fig. 2A with annexin V staining
profiles of these gates in Fig. 2B verify the positioning of live and
dead cells.
Infections using a range of MOIs for both N. gonorrhoeae and
E. coli were conducted in pilot experiments to assess their effect
on B cell viability. Infection with E. coli at an MOI of .10 resulted
in rapid B cell death (Supplemental Fig. 1A). Although a sim-
ilar effect was not apparent with N. gonorrhoeae (Supplemental
Fig. 1B), we selected an MOI of 10 for our standard assay system
because it allowed us to compare the effects of gonococcal infec-
tion with E. coli over time.
Primary human B cells are notoriously difficult to maintain in
viable culture. However, for each individual donor (as indicated by
different symbols within each plot), the percentage of viable cells
are significantly increased upon infection with 10 gonococci/cell
relative to those remaining uninfected, as indicated by forward
and side scatter (Fig. 2C). Cells exposed to E. coli at the same
MOI were morphologically indistinguishable from the uninfected
controls, whereas N. gonorrhoeae infection consistently boosted
B cell viability. Moreover, the percentage of gonococci-infected
B cells labeled with soluble annexin V was lower than cells either
left uninfected or exposed to E. coli (Fig. 2D). These results
confirm that the protective effect of N. gonorrhoeae is not a gen-
eral response to bacteria. It is pertinent to note that, although N.
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. gon-
orrhoeae expressing no Opa adhesin, Opa
HSPG
, or Opa
CEA
or Texas Red-labeled E. coli for 6 h and then visualized by confocal microscopy. Scale bar, 5 mm.
(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.
The Journal of Immunology 4011

gonorrhoeae clearly increased viability of the B cells, it did not
protect B cells from apoptotic death induced upon treatment with
staurosporine (data not shown). When considered together, these
data indicate that N. gonorrhoeae infection supports the survival
of primary human B cells and that this effect occurs regardless of
Opa protein expression, suggesting that it is not CEACAM1 de-
pendent.
N. gonorrhoeae elicits robust B cell activation
During flow cytometric analysis, we consistently observed a
striking increase in B cell size and granularity upon exposure to
N. gonorrhoeae, characteristic of a blasting phenotype that is in-
duced by BCR cross-linking (Fig. 3A, 3B). Flow cytometry for
one representative donor is illustrated in Fig. 3B, displaying robust
B cell blasting in response to either BCR cross-linking or infection
with N. gonorrhoeae while not occurring in cells left uninfected or
infected with E. coli. To compare multiple donors, the proportion
of blasting cells in each sample was normalized against the per-
cent of total live cells, the gating of which is reflected in Fig. 3B.
As evident in Fig. 3C, the proportion of blasting cells is signifi-
cantly higher in N. gonorrhoeae-infected but not E. coli-infected
cells compared with the uninfected samples. This effect is con-
sistent among all donors examined and occurs regardless of Opa
protein expression.
Because blasting is usually indicative of cellular activation, we
measured the expression of activation-induced costimulatory
molecule CD86 on cells infected with N. gonorrhoeae. Fig. 3D
illustrates that gonococcal infection, but not E. coli infection,
induces significantly more cells to express CD86 than when cells
were left uninfected. Combined, these results demonstrate a potent
activation response that is specific to the gonococci.
N. gonorrhoeae alone can provide the necessary signals to
induce proliferation of B cells in a T-independent manner
If cells receive the appropriate combination of signals, they will
not only become activated but will also proliferate. To ascertain
whether simple exposure to N. gonorrhoeae was sufficient to
promote B cell proliferation, BrdU incorporation was used to ob-
serve genome replication. Fig. 4A illustrates that infection with
gonococci promotes significantly increased BrdU incorporation by
B cells, compared with uninfected cells. Proliferation of B cells
infected with E. coli tended to reflect that of uninfected cells, al-
though the extent varied slightly between donors (Fig. 4B); this
again illustrates that B cell proliferation is not a general response to
microbial-associated molecular pattern (MAMP) molecules or other
effects of bacterial infection. However, kinetic analysis of infection
for all donors shows progressive B cell accumulation of BrdU in
response to infection with gonococci, consistent with proliferation
as a specific and reproducible response to N. gonorrhoeae (Fig. 4B).
Gonococcal interaction with B cell populations is specific and
favors binding to IgM memory B cells
Because it was clear that some but not all B cells associated with the
gonococci (Fig. 1B, 1C), we considered whether cellular responses
to infection correlated with the action of a specific B cell subset.
In human peripheral blood, naive, IgM memory, and switched
memory B cells can be differentiated by the combined expression
patterns of CD27 and IgD (28), as shown in Fig. 5A. In mature
cells in the periphery, CD27 expression typically indicates a
FIGURE 2. N. gonorrhoeae infection of primary human B cells pro-
motes 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 staur-
osporine, a protein kinase inhibitor that induces cell death at high con-
centrations, 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-posi-
tive and -negative populations within this culture. Cells cultured with
staurosporine produce only an annexin V-positive peak (unshaded). Rep-
resentative 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 indi-
vidual donor. Bars indicate mean. Asterisks indicate a comparison between
uninfected and infected cells. *p,0.05, **p,0.01. Data points corre-
sponding to the same individual donor are represented by the same symbol
within each plot.
4012 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS

memory cell lineage, whereas IgD is primarily expressed on naive
cells only.
Freshly purified IgD
+
CD27
2
(naive) B cells primarily express
surface IgM (Fig. 5B). IgD
+
CD27
+
(IgM memory) B cells also
consistently express surface IgM; however, IgG- and IgA-
expressing cells also exist within this population (Fig. 5B), sug-
gesting that these cells have recently switched Ig classes (29). Most
IgD
2
CD27
+
(switched memory) B cells have undergone Ig class
switching to express surface IgG or IgA (Fig. 5B). Although the
relative proportion of B cells that represent each subpopulation
differed slightly between donors, the average proportion of each
subpopulation in freshly purified uninfected PBMC preparations
was 57% naive (IgD
+
CD27
2
), 26% IgM memory (IgD
+
CD27
+
),
and 11% switched memory (IgD
2
CD27
+
) B cells (data not shown).
To identify which populations of B cells were associating with
N. gonorrhoeae, we prepared live fluorescently labeled N. gon-
orrhoeae or E. coli and used these to infect purified B cells for 3 h
with a low MOI of 2 bacteria per cell. Although E. coli binding to
all peripheral B cell subsets was very low, gonococci displayed
substantial binding to naive, IgM memory, and switched memory
populations of B cells (Fig. 5C). Comparable binding by the three
isogenic gonococcal strains suggests that this association is not
strictly dependent on receptors for either Opa variant, although the
ability of the B cell subpopulations to bind gonococci expressing
these different adhesins is not equal (Fig. 5D). Surprisingly, we
observed that the simple expression of Opa adhesin did not
translate into higher levels of bacterial binding, because Opa
HSPG
-
expressing N. gonorrhoeae associated less with the B cells than
did the strain that expresses no adhesin (Opa
2
; Fig. 5D). In
general, both IgM memory and switched memory subsets display
a greater affinity for gonococci than do naive cells, but the gon-
ococci had a particular affinity for the IgD
+
CD27
+
IgM memory
B cells (Fig. 5C, 5D). Strikingly, up to 80% of these IgM memory
B cells were associated with the gonococci. Although it is possible
that, in rare instances, some gonococcal recognition could be
mediated through the BCR, it is clear that this is not the primary
mechanism of binding because it is unreasonable to assume this
large proportion of B cells are N. gonorrhoeae specific in all
donors examined. The lack of BCR involvement is also consistent
with the fact that we observed no BCR recruitment to the bound
bacteria during our immunofluorescence-based studies (Fig. 1A).
There was little association of any B cell subpopulations with E.
coli, consistent with the interaction being unique to the gonococci.
IgM memory B cell population expands in response to infection
with N. gonorrhoeae
Considering that B cells proliferate in response to gonococcal
infection (Fig. 4), we examined whether one or more B cell subset
(s) were specifically induced. After 5 d of infection, the IgD
+
CD27
+
IgM memory cell population had clearly expanded in re-
sponse to gonococci, but not to E. coli, in all donors (Fig. 6). This
increase in the IgM memory cells was reflected by a tendency
toward a reduction in the naive and, less so, switched memory
B cell populations. However, in contrast to the significant increase
in IgM memory cells, the apparent changes in naive and switched
memory cells were not statistically significant (Fig. 6B). There-
fore, despite variability in the proportion of each of the B cell
subpopulations between individuals, there is a consistent expan-
sion of the IgM memory B cell subset in response to gonococci.
Moreover, the different levels of bacterial association conferred by
FIGURE 3. A robust activation response is observed
by primary human B cells infected with N. gonor-
rhoeae 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 mg/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 non-
blasting viable cells. Representative of two indepen-
dent experiments with different donors. (B) Blasting
cells were observed upon infection with N. gonor-
rhoeae 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. Repre-
sentative 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 per-
centage 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 com-
parison 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.
The Journal of Immunology 4013

Opa
CEA
and Opa
HSPG
(Fig. 5C, 5D) did not influence B cell ex-
pansion (Fig. 6B).
N. gonorrhoeae clinical isolates induce proliferation in both
IgM memory and switched memory B cell populations
N. gonorrhoeae undergo frequent antigenic and phase variation. To
confirm that the observed B cell responses were not unique to the
prototypical MS11 “laboratory” strain of N. gonorrhoeae, we also
infected B cells with two unrelated clinical isolates obtained from
male urethra. Flow cytometry was used to monitor the expression
of nuclear proliferation Ag Ki67 in all B cell subpopulations
3 d postinfection. All strains of N. gonorrhoeae tested induced
robust Ki67 expression by the IgD
+
CD27
+
IgM memory cells with
little or no staining in the other subsets (Fig. 7), confirming that the
observed increase in the IgM memory B cell population upon N.
gonorrhoeae infection (Fig. 6) is due to the selective proliferation
of the IgD
+
CD27
+
IgM memory cells. This effect is in clear
contrast to infection with E. coli, which induced no proliferation in
any B cell subpopulation (Fig. 7). The potent IgD
+
CD27
+
IgM
memory cell response thus appears to be an intrinsic feature of N.
gonorrhoeae rather than being a strain-specific effect.
Polyreactive IgM is produced by B cells infected with N.
gonorrhoeae
Although naive B cells require AgR signaling for the production of
Ig, IgM memory B cells have the potential to produce Ab in a
T-independent manner, both in the presence and absence of Ag
receptor signaling (30). For example, CpG DNA has been shown
to induce Ig production by the IgM memory B cells, even in
the absence of BCR signals (30). This prompted us to examine
whether Ig production could be elicited by N. gonorrhoeae in-
fection of purified primary human B cells. As depicted in Fig. 8A,
IgG, IgM, and IgA classes of Ab were all induced in significant
amounts upon infection with gonococci relative to uninfected
cells. This effect was not apparent upon infection with E. coli,
indicating that it is not a generalized response to bacteria or
bacterial-derived products such as endotoxin or other MAMPs.
IgM is the principal class of Ig elicited in response to N. gonor-
rhoeae, with ∼20-fold induction versus the uninfected controls
(Fig. 5A). Neither Opa expression nor the relative level of asso-
ciation by the isogenic neisserial strains with B cells (Fig. 5C, 5D)
correlated with total Ig titers induced or the relative proportion of
individual Ig classes (Fig. 8A). Considering that the study pop-
ulation was not believed to have high-risk sexual behaviors and
are therefore not uniformly exposed to N. gonorrhoeae, it is un-
likely that this is a result of conventional memory recall responses.
To examine whether gonococcal infection produced N. gonor-
rhoeae-specific Ab, we prepared ELISA plates coated with the
Opa
2
strain of gonococci. Using this strain to measure Ig
responses against all three isogenic N. gonorrhoeae strains elim-
inates potential skewing of results caused by responses to the
strongly immunogenic Opa proteins (31). N. gonorrhoeae infec-
tion elicited a small increase in gonococci-specific IgM (Fig. 8B),
whereas no detectable gonococci-specific IgG or IgA was evident
(data not shown). Importantly, the baseline amount of N. gonor-
rhoeae-reactive Ig in E. coli-infected samples closely reflected
that in uninfected samples, indicating that the observed increase in
Ig required exposure to the gonococci.
The relatively low increase in N. gonorrhoeae-specific Ig did
not reflect the significant responses apparent when total Ig was
measured (Fig. 8A). To assess the specificity of the Ig produced,
we performed ELISAs to monitor the production of Abs specific
for other, nonneisserial Ags upon B cell infection. For this, we
measured Ig specific for a recall Ag, TT, as well as an irrelevant
Ag, KLH. Although little TT-specific Ig was apparent in unin-
fected samples, significant increases in IgM specific for TT were
apparent in the culture supernatants of all N. gonorrhoeae-infected
cells (Fig. 8C) and the purified B cells from 5 of 10 donors pro-
duced TT-specific IgG upon exposure to the gonococci (data not
shown). Considering that a recall response to TT would be ex-
pected to primarily be IgG, this implies that the N. gonorrhoeae-
induced TT-specific IgM observed is not a conventional memory
response. TT-specific Abs were not detected in E. coli-infected
samples (Fig. 8C), again indicating that this is not a generalized
response to bacteria-derived products. B cells from the majority of
donors (90%) also produced IgM reactive with KLH following
infection with gonococci but not E. coli (Fig. 8D), even though we
would not expect any of the volunteer blood donors to have been
exposed to this Ag previously. When combined, these results in-
dicate that N. gonorrhoeae infection of primary B cells results in
broad, polyclonal activation and differentiation of IgM-bearing
B cells rather than a focused, clonal response to the gonococci.
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 corre-
sponding to the same individual donor are represented by the same symbol
within each plot.
4014 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS

B cell responses to N. gonorrhoeae infection involve TLR9
Because all strains of N. gonorrhoeae examined produced robust
proliferation in the IgM memory population of B cells, we ex-
plored the possibility that gonococci were being detected by in-
nate immune receptors, resulting in downstream proliferation and
differentiation of B cells. It has already been established that
TLR expression varies between human peripheral B cell sub-
populations, such that naive cells express few TLRs, each at very
low (to undetectable) levels when they are present, whereas both
IgM memory and switched memory subsets can express high
levels of TLR6, 7, 9, and 10 (20). Neisseria species are known to
constitutively express outer membrane porins that can be detected
by complexes of TLR2 and TLR1 (32, 33), in addition to pos-
sessing a lipo-oligosaccharide that is recognized by TLR4 (34). In
this study, we systematically examined primary human B cellular
responses to defined TLR agonists. To assess functional TLR re-
sponses in the B cell subsets, we examined a panel of specific TLR
agonists—Pam
3
CSK
4
(a synthetic triacylated lipoprotein that sig-
nals through TLR2/1), FSL1 (a synthetic diacylated lipoprotein that
signals through TLR2/6), E. coli K12 LPS (TLR4), and CpG
ODN2006 (TLR9)—for their ability to cause activation and pro-
liferation. Surprisingly, when considered in the context of TLR
expression-based studies (20), naive cell activation was significantly
increased in response to the TLR2/6 agonist FSL1 and TLR9 ago-
nist CpG ODN2006 (Fig. 9A). Naive cells were weakly activated in
response to treatment with TLR2/1 agonist Pam
3
CSK
4
; however,
this difference did not reach statistical significance (Fig. 9A).
CD27
+
memory B cells responded to Pam
3
CSK
4
, FSL1, and CpG
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
2
), IgM memory (IgD
+
,CD27
+
), and switched memory (IgD
2
,CD27
+
). Representative of four in-
dependent 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.
The Journal of Immunology 4015

ODN2006 with strong and significant increases in cellular activa-
tion (Fig. 9A). Treatment with LPS did not induce any response in
any of the B cell populations (Fig. 9A, 9B), confirming previously
published reports that TLR4 is not expressed in high enough levels
in any human B cell subset to facilitate signaling (20, 35).
Although naive cells expressed the CD86 activation marker
upon treatment with some TLR agonists, proliferation was not ob-
served in these cells in response to any of the TLR agonists tested
(Fig. 9B). Both IgM memory as well as switched memory B cells
underwent strong proliferation responses to CpG ODN2006. Al-
though both memory cell populations did increase proliferation
in response to treatment with Pam
3
CSK
4
and FSL1 compared
with untreated cells, the difference did not reach statistical sig-
nificance (Fig. 9B).
Treatment with all TLR agonists examined resulted in prolif-
eration by both IgM memory and switched memory subsets to
varying degrees; however, treatment with TLR9 induced by far the
strongest response within both populations (Fig. 9B). Others have
shown that treatment with CpG ODN2006, either alone or in
combination with other cytokines, induces only IgM class Ab from
IgM memory cells (suggesting that it cannot induce class switch-
ing), whereas switched memory cells predominately produce either
IgG or IgA Ig (20). Because we observed that N. gonorrhoeae
infection induces the specific expansion of IgM memory B cells
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 clin-
ical N. gonorrhoeae strains induces pro-
liferation 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 subpopula-
tions. 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 in-
dependent experiments with different
donors. (B) Quantification of the percent
of each B cell subpopulation postinfec-
tion under the same conditions outlined
in (A). Bars indicate mean. Asterisks in-
dicate 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.
4016 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS

(Fig. 6B) and that the majority of Ig induced is IgM (Fig. 8A), we
considered whether TLR9 signaling might also contribute to the
gonococcal-induced IgM memory B cell response.
A dominant-negative CpG-containing oligonucleotide that
inhibits TLR9 signaling (iCpG) has been shown to compete with
the stimulatory effects of other CpG-containing DNA in both
primary murine and primary human immune cells (36, 37). We
took advantage of this reagent to examine whether TLR9 con-
tributes to the gonococcal-induced induction of B cell prolifera-
tion. Although the iCpG had little effect on uninfected B cells, this
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
2
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. (Aand B) B cells were treated with 2.5 mg/ml
Pam
3
CSK
4
,1mg/ml FSL1, 1 mg/ml LPS, and 5 mg/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 mg/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 prolif-
eration 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.
The Journal of Immunology 4017

inhibitor effectively reduced the proportion of actively prolifer-
ating cells in response to the synthetic CpG-containing oligonu-
cleotide ODN2006 and to intact N. gonorrhoeae by mean values
of 54.9 and 35.6%, respectively (Fig. 9C); this effect was con-
sistent with cells from all donors tested. Thus, TLR9 contributes,
at least in part, to B cell responses induced by N. gonorrhoeae
infection.
N. gonorrhoeae can be engulfed and subsequently killed by
primary human B cells
TLR9, unlike other TLRs, is not expressed on the cell surface but
is found within endolysosomes (38). Although B cells will endo-
cytose soluble Ag (39), their ability to engulf intact bacteria is
largely unexplored. CD4
+
T cells do not engulf adherent N.
gonorrhoeae (13). However, we considered the possibility that
B cells might have the capacity to engulf and kill whole gon-
ococci, effectively delivering the CpG-containing bacterial DNA
directly to intracellular TLR9. As depicted in Fig. 10A, primary
human B cells are able to engulf whole gonococci, as determined
by differential staining of total and extracellular gonococci.
To determine the fate of the internalized bacteria, a gentamicin
protection assay was used to quantify viable intracellular gon-
ococci. This assay takes advantage of the fact that gentamicin does
not permeate mammalian membranes, so engulfed bacteria are
protected from the antibiotic. B cells were infected with the
specified strain of gonococci for 90 min before the addition of
gentamicin for 1 h. Viable intracellular gonococci are observed at
this point (2.5 h postinfection), supporting the microscopic evi-
dence that the bacteria were being internalized. However, when
the infection was allowed to persist foran additional 3.5 h (for a total
of 6 h), the vast majority of intracellular gonococci is dead (Fig.
10B). Taken together, these results suggest that the high-affinity
binding of N. gonorrhoeae to human B cells leads to engulfment of
whole bacteria. Once internalized, the bacteria die and lyse, de-
livering a large bolus of bacterial DNA directly to intracellular
lysosomal compartments where it can interact with TLR9 (Fig. 11).
Discussion
N. gonorrhoeae has a curious niche, such that the pathogen seems
to penetrate and persist within the subepithelial space (9); this site
is rich in nutrients but also patrolled by resident sentinel immune
cells (16, 40) that the bacteria must contend with. Leukocytes,
including B cells, accumulate in the endocervical mucosa during
both symptomatic and asymptomatic gonococcal infections (16),
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 mm. (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 co-
stimulatory 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 devel-
opment of a classical memory response upon re-exposure to the gonococci.
4018 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS

making it likely that infecting N. gonorrhoeae come into direct
contact with B cells at the site of infection as well as in the
draining lymph nodes. When considering this, it is surprising that
the Ab response elicited by N. gonorrhoeae is poor compared with
other mucosal infections and does not elicit a classical immuno-
logic memory response that would otherwise protect upon sub-
sequent exposure (7). B cells are APCs and express many pattern
recognition molecules capable of detecting bacterial-derived
products (20, 41–44). Although this suggests that B cells should
recognize all bacteria equally, our results unexpectedly reveal that
N. gonorrhoeae has a specific tropism for the IgM memory subset
of human B cells that elicits both their proliferation and a potent
but broadly reactive T cell-independent Ig response.
Past reports indicate that infection by a variety of bacteria can
cause B cell death (45, 46), yet we observed that N. gonorrhoeae
infection instead supports primary human B cell viability and
promotes their proliferation, even decreasing cell death that nor-
mally occurs during in vitro culture of freshly isolated human
B cells. In other cell types, both protection from (47, 48) and
promotion of (49) cell death have been observed upon infection
with N. gonorrhoeae. Although the majority of studies examining
this effect have focused on epithelial cells, it is becoming clear
that infection with N. gonorrhoeae, more often than not, inhibits
apoptosis (50). Previous work using preactivated human B cells
suggested that infection with CEACAM1-binding gonococci
results in B cell death (19), but we did not observe similar
responses using freshly purified primary human B cells. On the
basis of our findings described in this article, those previous
observations may reflect cell death attributable to the over-
whelming stimulation applied to the cells (gonococci as well as
cytokines), rather than a direct effect of the bacteria itself. Al-
though specific conclusions regarding the survival signals elicited
by the gonococci await further molecular studies, we propose
that the protection from cell death observed in our assays stem, in
part, from the innate nature of the lymphocytes that are responding.
The IgM memory B cells have a lower activation threshold than
that of naive cells (51), suggesting that they have evolved to re-
spond by rapid activation, proliferation, and differentiation in re-
sponse to microbes.
We observe that B cell infection with N. gonorrhoeae elicits
an impressive production of mainly IgM, which recognizes both
immunologically relevant (N. gonorrhoeae) and heterologous (TT
and KLH) Ags. The production of Ngo-specific IgM in response
to gonococcal infection in the absence of T cell help would be
expected if it was a specific BCR-dependent response, but our
observations indicate that N. gonorrhoeae recognition by B cells
is not due to the BCR. However, IgM specific for TT and KLH,
heterologous Ags consisting of single subunit proteins that contain
fewer potential epitopes than do the intact bacteria, was produced
in greater amounts upon infection with N. gonorrhoeae than was
IgM specific for the infecting pathogen. This both highlights that
the T-independent Ig response is broadly polyspecific and makes
it intriguing to consider that the gonococci may have a mecha-
nism by which to allow the selective expansion of heterologous,
nonneisserial Ig.
IgG and IgA production by B cells infected with N. gonorrhoeae
is significantly higher than the levels produced in response to
infection with E. coli (Fig. 7B). This suggests that either IgG and
IgA can arise from the IgM memory cells (via class switch re-
combination) or that switched memory cells, in addition to the
IgM memory cells, are differentiating into Ab-secreting cells in
response to the gonococci. We consider it plausible that the
switched memory cells may benefit from T-independent Neisseria-
induced “bystander” activation signals that originate from the IgM
memory B cells, which would at least partially explain the
switched memory response. Bernasconi et al. demonstrate by-
stander activation of switched memory B cells, wherein micro-
environments produced upon activation of classical Ag-specific
memory cells supports the activation of noncognate switched
memory cells in a T-dependent manner (30). Although previously
undescribed, the possibility that IgM memory B cells could play
a role in the bystander activation of switched memory cells is
enticing because it would suggest that these cells could poly-
clonally activate conventional memory B cells at first sign of in-
fection.
It is unreasonable to expect that the large proportion of B cells
that we observe respond to N. gonorrhoeae are specific for neis-
serial Ags, especially considering our observation that it is pri-
marily the IgM memory subset that vigorously responds to
infection by a pathogen that is uncommon in the study population.
Neisserial activation of B cells does not occur via BCR clustering,
such as that which occurs with the Moraxella catarrhalis super-
antigen Moraxella IgD-binding protein (26). To date, bacterial
products such as capsular polysaccharide vaccines (28, 52), and
purified CpG DNA (30) have been shown to expand the population
of human peripheral blood IgM memory B cells. Interestingly,
N. gonorrhoeae does not express a polysaccharide capsule, con-
firming that stimulation of this subset does not require antigenic
signaling by these structures. As such, to our knowledge, this is
the first report indicating the direct induction of polyclonal pro-
liferation and IgM production by the human IgM memory B cell
subpopulation in response to a bacterium.
N. gonorrhoeae is a complex organism, with many components
that have the potential to interact with host cell receptors and
cause immune activation, especially on APCs such as B cells.
Although it has been established that the IgM memory subset
expresses various TLRs (20), their TLR functionality had not, to
our knowledge, been systematically explored. Although neisserial
endotoxin might be considered an obvious candidate for eliciting
the B cell response when considering its stimulatory effect on
a variety of other immune cells (53–55), we did not observe
a TLR4 response in any of the human B cell subsets described in
this article (Fig. 9A, 9B).
Considering that the IgM memory B cells had a particular affin-
ity for N. gonorrhoeae but not E. coli, which was used as a pro-
totypical Gram-negative bacterial control to account for general-
ized MAMP responses, and that these cells also displayed a potent
response to a synthetic CpG oligonucleotide agonist, TLR9 acti-
vation following N. gonorrhoeae engulfment seemed a likely
explanation for the observed switched memory B cell response.
Consistent with this premise, gonococcal-induced B cell prolif-
eration was lower upon treatment with TLR9 antagonist iCpG
(Fig. 9C). Why the iCpG-mediated inhibition was incomplete
remains unclear. It may simply not be sufficient to fully compete
with a bolus of CpG-containing DNA released upon neisserial
lysis within the phagosome. Alternatively, B cell recognition of
other neisserial-specific factors may also contribute to this re-
sponse. Interesting to consider in this context is the fact that
neisserial outer membrane-expressed porins have been shown to
strongly induce both activation and proliferation by human and
murine B cells through interaction with surface-expressed TLR2
and TLR1 (33, 56), particularly because IgM memory express
sufficient TLR2 and TLR1 to respond to purified Pam
3
CSK
4
(Fig. 9A, 9B).
It is becoming increasingly clear that TLR9 is centrally im-
portant in the induction of immune cell responses to the pathogenic
Neisseria sp. (37, 57–59). In B cells, TLR9 is highly expressed
relative to other TLRs (20), but it is only found in intracellular
The Journal of Immunology 4019

compartments (38) rather than at the cell surface. Important in this
context has been the demonstration that the delivery of bacterial
DNA directly to TLR9-containing compartments is absolutely
required for B cell responses, because they are poor at engulfing
exogenous DNA (60). B cells are surprisingly effective at
engulfing whole gonococci (Fig. 10A, 10B), allowing for the de-
livery of gonococcal DNA to TLR9, whereas engulfment of E. coli
was rarely observed (data not shown). This difference may result
from the combination of the gonococci’s tropism for B cells, in-
creasing the time spent in contact with the cells, and/or the larger
size of E. coli hindering the engulfment of these bacteria.
IgM memory B cells represent the first line of defense by the
adaptive immune response because of their natural (not requiring
antigenic stimulation) production of low affinity but polyreactive
IgM, the majority of which express germline-encoded receptors
that can undergo somatic hypermutation (61). Some of this “nat-
ural” IgM presumably functions by opsonizing encapsulated
bacteria (62, 63) and neutralizing viruses (64, 65). An innate
population of B cells with similar characteristics to the human
IgM memory B cells exist in mice and are termed B-1 B cells (66).
Functionally, murine B-1 B cells are similar to human IgD
+
CD27
+
IgM memory cells because they are both considered to be the
source of natural Ab (67). However, the human IgM memory cell
population is found in the periphery and may represent a circu-
lating form of splenic marginal zone B cells (28), whereas similar
cells in mice are primarily associated with peritoneal and pleural
cavities (68). In the murine model of influenza lung infection, flu-
specific B-1 B cells have been shown to be present only in the
mediastinal lymph nodes and are not present in distal lymph nodes
or the surrounding lung tissue (65). However, there remains a
paucity of information regarding the development, regulation,
trafficking, and immune induction sites of the IgM memory B cells
during infection in humans. In fact, it remains unclear whether,
during infection, the activation of IgM memory B cells in humans
would be detectable systemically, or whether they primarily
function in local immune induction sites in a manner that reflects
that of the B-1 subset. Such shortcomings represent a particular
challenge to the field because they preclude the use of mice as a
model to understand human IgM memory cell function. Future
work in the field must still clarify if they are indeed similar cells.
Although the human IgM memory B cells have been conclu-
sively identified as a distinct B cell subpopulation relatively re-
cently (28, 66), it is becoming increasingly apparent that their
immune function is nonredundant in the production of an effective
immune response against invading microorganisms. In humans,
loss of IgM memory B cells correlates with recurrent bacterial
pneumonia in patients with common variable immunodeficiency
(52, 66). Furthermore, low numbers of IgM memory B cells found
in the periphery of HIV
+
patients have been shown to be a pre-
dictor of cryptococcosis (69). In a similar vein, B cell super-
antigen Staphylococcus aureus protein A targets the innate murine
B-1 B cells for deletion, leaving a void in the B cell repertoire that
remains unrestored 14 mo after the initial treatment (70). N.
gonorrhoeae appears to use an alternative approach to prevent the
production of a specific humoral response, suppressing T cell help
(12, 13) while simultaneously stimulating the IgM memory B cells
to produce polyclonal Ig responses; combined, this would effec-
tively lower the relative concentration of gonococcal-specific Ab.
However, considering that some of the Ig will bind the bacteria,
the relative benefit of a polyspecific Ig response to the infecting
pathogen versus the host remains a matter of ongoing debate (71).
Although the ability of IgM memory B cells to respond to
microbes in a T-independent manner is plainly apparent, it has been
recently suggested that murine innate B-1 B cells may possess
a form of “memory” that is distinct from the conventional T-
dependent response (72). In humans, an analysis of female sex
workers from Kenya showed the induction of an incomplete, but
serotype-specific, immunologic memory response following re-
peated N. gonorrhoeae infections over years of high-incidence
exposure (73). Although these women could be very gradually
acquiring immunity by traditional T-dependent mechanisms, it is
tempting to propose the possibility that this memory response
results from the repeated N. gonorrhoeae-dependent expansion of
human IgM memory B cells. Further in vivo studies will be re-
quired to test this postulate.
In this report, we establish that the human-specific pathogen N.
gonorrhoeae elicits a vigorous innate B cell response. Considering
that other bacteria, including E. coli used in this study and Listeria
monocytogenes (45) and Francisella tularensis (46) in previous
work, do not elicit a similar response, this appears to be a specific
effect of the gonococci rather than being a prototypical immune
response. Indeed, it is intriguing to speculate that N. gonorrhoeae
may promote the specific expansion of IgM memory B cells to
amplify the production of nonspecific Ig in a manner that nega-
tively affects specific immunity in infected individuals. We pro-
pose that, in the early stages of infection, circulating IgM memory
B cells may come into contact with the gonococci, either at the
site of infection or in the draining lymph nodes surrounding the
urogenital tract. This interaction promotes the engulfment of
whole gonococci by the B cells, which may contribute to the
clearance of the pathogen, but also allows for the direct detection
of bacterial products by innate receptors including TLR9. This
results in a vigorous T-independent proliferation, expanding the
IgM memory B cell population, and the induction of an acute,
localized, effector response in the form of low-affinity, polyclonal
IgM (see model, Fig. 11). As the infection progresses, the gon-
ococci may attach to CD4
+
T cells, an interaction mediated by
their Opa adhesins binding to the coinhibitory receptor CEA-
CAM1, because the vast majority of clinical neisserial isolates
have the ability to bind CEACAM1 (15). In contrast to B cells,
T cells do not engulf the gonococci; this facilitates the inhibitory
effect of CEACAM1-mediated phosphatase-dependent signaling
at the cell surface, thereby preventing T cell activation and clonal
expansion in response to TCR engagement (12, 13). Consequently,
the absence of effective T cell help precludes the production of
high-affinity class-switched Ab, but the T cell-independent pro-
duction of innate IgM production is unaffected. The result is
a broadly specific and thereby weakly effective humoral response
to gonococcal infection that may assist in clearing the current
infection but, in the context of an unguided B cell response,
provides no conventional immunologic memory responses upon
subsequent exposure to N. gonorrhoeae.
Such a model, involving the suppression of CD4
+
T cells while
activating the innate B cell response, is in agreement with clinical
observations indicating a modest increase in total IgM levels lo-
calized within the mucosa during natural gonococcal infection, the
unexpectedly rapid decline in N. gonorrhoeae-specific Ig once the
infection is cleared, and the absence of immunological memory
upon subsequent exposure to these bacteria (7, 18). In the context
of the remarkable antigenic variability of this pathogen, such di-
rect subversion of the protective immune response explains the
ongoing persistence of this highly evolved human pathogen.
Acknowledgments
We thank the volunteer blood donors, without whom our study would not
have been possible. We thank Dr. Chao Wang and Shannon McCaw for tech-
nical assistancein completing this work and Drs.Alberto Martin and Michael
J.H. Ratcliffe for insightful comments and critical reading of the manuscript.
4020 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS

Disclosures
The authors have no financial conflicts of interest.
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4022 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS