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

This information is current as Nancy S. Y. So, Mario A. Ostrowski and Scott D. of October 1, 2021. Gray-Owen J Immunol 2012; 188:4008-4022; Prepublished online 16 March 2012; doi: 10.4049/jimmunol.1100718 http://www.jimmunol.org/content/188/8/4008 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 Downloaded from 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. http://www.jimmunol.org/

eisseria gonorrhoeae (gonococci) is a human-specific Despite the overzealous innate immune response that causes pathogen that causes the sexually transmitted infection symptomatic disease, adaptive immunity against uncomplicated N gonorrhea and is a major cause of morbidity. The rate of gonococcal infections is surprisingly poor. Although gonococcal- gonorrhea infection is increasing worldwide, with a recent esti- specific Ig can be observed in infected individuals, Ab levels are mate of 88 million new infections per year (1). Gonorrhea results low compared with other mucosal infections, and there is no ev- from overwhelming inflammation, producing purulent discharge idence of a classical immunologic memory response upon re-expo- at the site of infection. The majority of women infected with N. sure, even during infection of rectal tissues containing immune- by guest on October 1, 2021 gonorrhoeae are clinically asymptomatic (2). Left untreated, this inductive sites (7). As such, repeated gonococcal infections can can progress to serious complications including ectopic pregnan- occur, sometimes with the same serotype of N. gonorrhoeae (8). cies because of fallopian tube scarring, sterility, or acquired blind- Although neisserial surface structures antigenically vary at an ness because of gonococcal conjunctivitis in children born to impressive rate, this alone cannot explain the paucity of memory infected mothers (3). Once infection is detected, gonorrhea can be response. Instead, the gonococci also appear capable of actively effectively treated with antibiotics; however, antibiotic resistance suppressing the adaptive immune response. Neisseria sp. express is increasing at an alarming rate, and gonococcal infections may Opa protein adhesions, which, upon binding to human carci- soon become untreatable (4, 5). Gonococcal coinfection can also noembryonic Ag-related cellular adhesion molecule (CEACAM) increase viral shedding in HIV patients (6), further heightening the receptors, can trigger bacterial uptake and transcytosis through the urgency to control the spread of this important pathogen. mucosal epithelial layer (9, 10). In addition to facilitating entry into tissues, certain Opa variants trigger the coinhibitory function *Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S of CEACAM1, which may suppress innate proinflammatory cy- 1A8, Canada; and †Clinical Sciences Division, University of Toronto, Toronto, tokine production by epithelia (11) and inhibits the cellular acti- Ontario M5S 1A8, Canada vation and proliferation of CD4+ T cells during infection (12, 13). Received for publication March 11, 2011. Accepted for publication February 15, Although only a fraction of gonococcal Opa variants are specific 2012. for CEACAM1 (14), there appears to be an in vivo selection for This work was supported by Canadian Institute of Health Research Operating Grant this function because .95% of clinical Neisseria isolates bind to 414775. CEACAM1 (15). The ability of N. gonorrhoeae to inhibit CD4+ Address correspondence and reprint requests to Dr. Scott D. Gray-Owen, Department of Molecular Genetics, University of Toronto, Room 4381, Medical Sciences Building, T cells during infection may explain the absence of classical 1 Kings College Circle, Toronto, ON M5S 1A8, Canada. E-mail address: scott.gray. immune memory responses to this human-restricted pathogen. [email protected] Although the Opa-dependent inhibition of T cell function has The online version of this article contains supplemental material. been well characterized (12, 13), there is not a complete absence Abbreviations used in this article: CEA, carcinoembryonic Ag; CEACAM, carcinoem- of Ig during gonorrhea. In fact, clinical evidence enticingly sug- bryonic Ag-related cell adhesion molecule; HSPG, heparin sulphate proteoglycan; iCpG, inhibitory CpG ODN TTAGGG; KLH, keyhole limpet hemocyanin; MAMP, gests that gonococcal infection might directly influence the B cell microbial-associated molecular pattern; MOI, multiplicity of infection; ODN, oligo- response. There is an increase in both B and T lymphocytes in the deoxynucleotide; OpaCEA, carcinoembryonic Ag-related cell adhesion molecule- endocervix of women with nonulcerative sexually transmitted specific Opa; Opa , heparan sulfate proteoglycan-specific Opa; TT, tetanus toxoid. HSPG infections including N. gonorrhoeae (16), suggesting that the Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 bacteria may directly access the B cells. Ig production within the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100718 The Journal of Immunology 4009 male and female genital tract is distinct from that of other mucosal (Invitrogen), and 50 mM HEPES (Bioshop, Burlington, ON, Canada) at pH surfaces in that IgG predominates rather than IgA (17). Although 7.4. Cells were cultured at 37˚C in 5% CO2 and humidified air. the levels of total mucosal IgG were slightly lower in gonococcal- Bacterial strains infected versus uninfected women, total mucosal IgM increased in 2 those with gonorrhea (7). Gonococcal-specific IgM was present in Isogenic N. gonorrhoeae strains constitutively expressing no Opa (Opa ; strain N302), the heparan sulfate proteoglycan-specific Opa (Opa ) 59% of cervical mucus samples obtained from women with gon- HSPG (strain N303, expressing Opa50), or CEACAM-specific Opa (OpaCEA) orrhea but were undetectable in samples obtained from individuals (strain N309, expressing Opa52) were derived from a pilus-deficient MS11 who were uninfected (18). Curiously, the Ig response in serum did parent strain (24) and were graciously provided by Prof. T. F. Meyer (Max not reflect that found in cervical mucus or vaginal washes, sug- Planck Institute for Infection Biology, Berlin, Germany). Clinical N. gonorrhoeae gesting that Ig production was localized rather than being a sys- strains N2061 and N2066 were isolated from male urethra. Immunoblot analysis indicates that strain N2061 does not express any temic response (7). The production of IgM in local secretions did Opa protein, N2066 expresses at least one Opa adhesin, and neither strain require active gonococcal infection, because antibiotic therapy to expresses pili. These clinical strains were obtained from a sexually eliminate the infection correlated with a decrease in gonococcal- transmitted disease clinic in the district of Pumwani in Nairobi, Kenya, and specific IgM in cervical mucus samples in all women who were were a gift from Dr. F.A. Plummer (University of Manitoba, Winnipeg, MB, Canada). Neisseria species were grown from frozen stocks on GC treated (18). Puzzlingly, the antigonococcal response was not agar (Difco) supplemented with 1% (v/v) IsoVitalex (BBL Microbiology greater against the infecting strain, because it frequently cross- Systems) at 37˚C in a 5% CO2 atmosphere with humidity. A binocular reacted equally or, in some cases, better with the prototypical microscope was used for daily selection of gonococcal colony opacity but antigenically distinct N. gonorrhoeae MS11 (7). phenotypes for the MS11 strains. Opa protein expression was routinely Such clinical data prompted us to consider that, in addition monitored by immunoblotting using the Opa cross-reactive mAb 4B12/

C11 (25), provided by Prof. M. Achtman (Environmental Research Insti- Downloaded from to suppressing T cell responses (12), the gonococci may directly tute, University College Cork, Cork, Ireland). affect B cell function. Previous work suggested that gonococcal E. coli (DH5a) was grown from frozen stocks in aerated Luria–Burtani infection kills human B cells in a CEACAM1-dependent manner broth overnight at 37˚C. Liquid cultures were subcultured onto Luria– (19); however, this does not explain the elevated Ig levels during Burtani agar prior to infection assays. N. gonorrhoeae infection. Alternatively, because the production of Infection of lymphocytes IgM by human B cells does not require supplemental signaling from T cells (20, 21), it is enticing to consider that gonococcal Freshly isolated human B cells were infected with a multiplicity of infection http://www.jimmunol.org/ (MOI) of 10 bacteria per cell, unless otherwise indicated. Gentamicin infection could directly induce the production of IgM, thereby (Bioshop) was routinely added to infected samples 3 h after bacterial explaining the clinical observations. addition to prevent overgrowth. For E. coli, gentamicin was added im- N. gonorrhoeae is strictly a human pathogen, with several mediately upon infection to prevent bacterial overgrowth of the B cell virulence factors that show exquisite host specificity. As such, no cultures. Although the gentamicin effectively inhibited bacterial growth, it did not otherwise affect observed B cell responses. mouse model of infection exists that accurately mimics the pro- gression of gonococcal disease in the human host. Furthermore, Confocal microscopy taking into account the intrinsic differences between human and B cells were infected with the indicated N. gonorrhoeae and E. coli strains mouse B cells [e.g., TLR4 signaling induces strong responses in

at an MOI of 10 for durations between 1.5 and 44 h. Acid-washed glass by guest on October 1, 2021 murine B cells but is not detected in human B cells (20, 22)], we coverslips were coated with mouse mAb specific for human CD19 (clone developed in vitro infection protocols using purified primary HIB19; eBioscience) to capture B cells for microscopy. Cells were fixed human B cells. Unexpectedly, and in stark contrast to parallel with 4% paraformaldehyde (Sigma-Aldrich), and gonococci were detected using a polyclonal rabbit antigonococcal serum (UTR01), followed by infections with the prototypical Gram-negative bacteria, Escher- Alexa 488-conjugated secondary Ab (Invitrogen-Molecular Probes) after ichia coli, we observed that N. gonorrhoeae prolonged B cell permeabilization of mammalian cell membranes using 0.4% Triton X-100 viability and triggered a robust cellular activation that led to the (Sigma-Aldrich). E. coli was detected using the purified polyclonal goat Ab polyclonal proliferation and differentiation of specifically the IgD+ ab25823 (Abcam), followed by a goat-specific secondary Ab in a process + similar to the gonococcal staining protocol. Where indicated, bacteria were CD27 subset of human B cells, resulting in an innate, polyclonal prelabeled using Texas Red succinimidyl ester (Molecular Probes) rather Ig response that reacted with both Neisseria-derived and unrelated than using antisera. B cells were stained using F(ab9)2 of biotinylated goat Ags. When combined with the ability of the gonococci to suppress anti-human IgM, IgG, and IgA (Jackson ImmunoResearch Laboratories), T cell activation (12, 13, 23), our observations explain the unfo- followed by streptavidin-Alexa 488 (Molecular Probes). cused and nonprotective Ig responses apparent in individuals with To examine whether primary human B cells were able to engulf whole bacteria, cells were infected with the indicated N. gonorrhoeae strains at an uncomplicated gonococcal infection. 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- Materials and Methods gonococcal serum (UTR01), followed by anti-rabbit IgG Alexa 647 Cells (Molecular Probes). Mammalian cell membranes were permeabilized us- ing 0.4% Triton X-100 (Sigma-Aldrich), allowing for total gonococci to be Primary human B cells were obtained by either standard venipuncture or, stained using the polyclonal rabbit antigonococcal serum (UTR01), fol- when larger numbers of cells were required, through leukopheresis. All lowed by anti-rabbit IgG Alexa 488 (Molecular Probes). participants gave informed consent in accordance with guidelines for the Bacteria and cells were visualized and recorded using a Plan- conduct of clinical research at the University of Toronto and St. Michael’s Apochromat 3100/1.4 Oil differential interference contrast objective lens Hospital, respectively. The protocols used were approved by the University on a Zeiss LSM 510 confocal microscope. All images were obtained at of Toronto and St. Michael’s Hospital institutional review boards (Toronto, room temperature. Zeiss LSM Image Browser version 4.2.0.121 was used ON, Canada). for subsequent image processing. Each infection condition was prepared on Fresh peripheral mononuclear cells were obtained from Ficoll-Paque duplicate or triplicate coverslips, and at least 60 N. gonorrhoeae-infected Premium (GE Healthcare) gradients, according to the manufacturer’s + cells and at least 100 cells for the E. coli infection condition were analyzed. specifications. CD19 B cells were purified using either the EasySep Human CD19 Positive Selection Kit (StemCell Technologies) or CD19 Analysis of B cell responses Microbeads (Miltenyi Biotec). B cell purity consistently exceeded 95%, as measured by a FACSCalibur flow cytometer (BD Biosciences) with FlowJo All flow cytometric analysis was performed using a BD FACSCalibur. B cell software (Tree Star) used for all flow data analysis. subsets were defined using the following Abs: IgD-PE or IgD-FITC (clone Purified cells were maintained in RPMI 1640 medium (Invitrogen), IA6-2; BD Biosciences), CD27-FITC or CD27-allophycocyanin (clone supplemented with 10% heat-inactivated FBS (CanSera), 4 mM GlutaMAX O323; eBioscience), and Cy5-labeled F(ab9)2 fragments of goat and human 4010 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS

IgG, IgM, or IgA (Jackson ImmunoResearch Laboratories). CD27 and IgD 1.5 h. Infection cultures were then treated with and maintained in 100 mg/ immunostaining was performed on live cells, whereas all other immuno- ml gentamicin (Bioshop) to kill any extracellular (nonengulfed) gonococci. staining was performed on fixed cells. CompBeads (BD Biosciences) were At 2.5 and 6 h postinfection, cells were washed and concentrated in PBS used for fluorescent compensation. All cells were fixed prior to flow containing 1 mM MgCl2 and 0.5 mM CaCl2 using Transwells with a 3-mm analysis. pore size to retain the cells (Corning). One-percent saponin (Sigma- To identify the B cell subpopulations associating with gonococci or E. Aldrich) was added to lyse mammalian membranes. Serial dilutions of the coli, live bacteria were labeled with Alexa 647 succinimidyl ester (Mo- lysis mixture were plated on GC agar (Difco) supplemented with 1% (v/v) lecular Probes) and then used to infect purified B cells at an MOI of 2 IsoVitalex (BBL Microbiology Systems) and incubated at 37˚C in a 5% bacteria per cell for 3 h before treatment with gentamicin. Bacterial as- CO2 atmosphere with humidity for at least 48 h before numeration of sociation to B cell subpopulations was visualized by flow cytometry. bacterial colonies. For viability, activation, and proliferation assays, infected cells and uninfected controls were fixed at time points indicated in the figure legends. Statistics m Where indicated, 16 M staurosporine (Sigma-Aldrich) was added to Differences between experimental conditions were determined to be sig- uninfected cells to induce B cell death as a control for viability experi- nificant when p , 0.05 by one-way ANOVA with a Dunnett’s posttest, ments. Forward- and side-scatter data and annexin V-PE (BD Biosciences) calculated by Prism 5.0 (GraphPad). 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 Results with cells at rest. N. gonorrhoeae associates with human peripheral blood To examine proliferation, B cells were infected overnight with bacteria or B cells were left uninfected, at which point BrdU (Sigma-Aldrich) was added to the culture media. BrdU incorporation was quantified using monoclonal mouse N. gonorrhoeae adheres directly to primary human CD4+ T cells anti-BrdU (clone IU-4; Invitrogen-Caltag Laboratories), which was directly in an Opa protein-dependent manner, allowing effective suppres- Downloaded from conjugated to biotin using Zenon Mouse IgG1 Labeling Kit (Molecular sion of T cell activation (13). To establish whether N. gonorrhoeae Probes). Streptavidin-allophycocyanin (Jackson ImmunoResearch Labora- tories) was then used to visualize the Ab for detection by flow cytometry. also interacts with primary human B cells, we exposed freshly To identify and quantify populations of cells undergoing proliferation at the purified cells to isogenic strains of gonococci expressing either no time of fixation, Ki67-Alexa 647 (clone B56; BD Biosciences) was used. Opa adhesin, OpaHSPG, or OpaCEA, and then monitored their as- B cell responses to TLR agonists were examined by culturing purified sociation with the B cells over time using confocal microscopy. B cells with the following reagents (all from InvivoGen): Pam3CSK4 at 2.5 m m m Cellular association with the prototypical Gram-negative bacterium

g/ml, FSL1 at 1 g/ml, E. coli K12 LPS at 1 g/ml, and CpG ODN2006 http://www.jimmunol.org/ at 5 mg/ml for 3 d, at which time they were stained for activation marker E. coli was also examined to assess the general propensity of CD86-PE (clone FUN-1; BD Biosciences) or nuclear proliferation Ag, B cells to bind bacteria. To confirm that bacteria were associating Ki67-Alexa 647 (clone B56, BD biosciences), and B cell subpopulation with B cells and not a copurified cell type, the infected cells were markers CD27 and IgD as described above. stained for BCR, as depicted in Fig. 1A. We then quantified the To examine the role of TLR9 signaling in N. gonorrhoeae infection of B cells, inhibitory CpG oligodeoxynucleotide (ODN) TTAGGG (iCpG; percentage of B cells that were associated with at least one bac- InvivoGen) was used, which functions as a dominant-negative TLR9 li- terium. As illustrated in Fig. 1B, substantially more B cells are gand. B cells were either left untreated/uninfected, treated with CpG associated with gonococci than E. coli, regardless of which neis- ODN2006 (InvivoGen) at 0.65 nmol/ml or infected with N. gonorrhoeae 2 serial adhesin was expressed. However, unexpectedly, even after strain N302 Opa at a MOI of 10. Cell cultures were then treated with either iCpG or ODN TTAGGG control (cCpG; InvivoGen), a control ODN 44 h of infection, some B cells did not associate with gonococci, by guest on October 1, 2021 that possess similar sequence to the iCpG but without the ability to inhibit suggesting that binding may be restricted to a specific subpopula- TLR9 signaling, both at 10-fold molar concentrations of stimulating CpG tion of cells. Moreover, no recruitment of BCR to sites of bacterial ODN2006 for 3 d, at which time cells were stained for Ki67-Alexa 647 attachment was evident, indicating that this association is inde- (clone B56; BD Biosciences) and B cell subpopulation markers CD27 and pendent of BCR clustering, a strategy used by other bacteria (26). IgD as already described previously. Although Fig. 1B quantifies the percentage of B cells with at ELISA least one bacterium bound, this does not illustrate how many All ELISAs were read at 450 nm using a 1420 Victor3 (PerkinElmer) plate bacteria associate with each cell. We quantified the number of reader, unless otherwise indicated. Total Ig in cell culture supernatant was bacteria per cell (Fig. 1C) and observed that most infected B cells quantified by IgM, IgG, or IgA ELISA kits (Zeptometrix), used according are associated with one to five bacteria per cell. The ability of to manufacturer’s instructions. gonococci to associate with cells is largely Opa-independent To monitor Ag-specific Ig production, 96-well Maxisorp plates (Nunc) 2 2 (compare Opa gonococci), although Opa expression did have were coated with either heat-killed Opa gonococci (N302), keyhole limpet hemocyanin (KLH; Sigma-Aldrich), or tetanus toxoid (TT; List some influence on bacterial association with the lymphocytes in Biological Labs) resuspended in pH 7.4 PBS (Wisent). The plates were some donors (e.g., compare donors 02L and 07L). 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 Gonococcal infection inhibits B cell death and promotes 20 (Sigma-Aldrich) in PBS was used as wash buffer. Culture supernatants prolonged B cell viability from uninfected cells or those infected with either gonococci or E. coli were diluted using 1% BSA in 0.05% Tween 20. Previous work suggested that in the presence of potent stimulation, For KLH and TT ELISAs, biotinylated F(ab9)2 fragments of goat anti- infection of primary human B cells with N. gonorrhoeae caused human IgG, IgM, and IgA (Jackson ImmunoResearch Laboratories) were B cell death (19). This could be explained either by a direct cy- used separately to determine the amount of each class of Ig produced in totoxic effect of the gonococci or by N. gonorrhoeae contributing response to bacterial infection. After incubation with streptavidin HRP to activation-induced cell death. We explored whether N. gonor- Ultra (Sigma-Aldrich), 3,39,5,59-Tetramethylbenzidine (KPL) was used as the colorimetric reagent for HRP activity. rhoeae infection affected the viability of freshly purified primary For N. gonorrhoeae plates, F(ab9)2 fragments of goat anti-human IgG, human B cells by monitoring changes in cell morphology and IgM, and IgA directly conjugated to alkaline phosphatase (Jackson membrane integrity during infection experiments. Loss of mem- ImmunoResearch Laboratories) were used separately to determine the brane integrity is an early event of cell death and results in a de- 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. crease in forward scatter and increase in side scatter, reflecting corresponding changes in cell size and granularity, respectively Gentamicin protection assay (Fig. 2A). Annexin V binding to phosphatidylserine exposed on To examine the viability of engulfed intracellular gonococci, B cells were a depolarized cell membrane is also a well-established method to infected with an MOI of 50 with the indicated N. gonorrhoeae strains for measure cell viability (27). To confirm the positioning of the live The Journal of Immunology 4011 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

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, OpaHSPG, or OpaCEA 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. and dead populations within flow cytometry plots, B cell death Primary human B cells are notoriously difficult to maintain in was induced by treating cells with staurosporine. Comparison viable culture. However, for each individual donor (as indicated by between gated populations in Fig. 2A with annexin V staining different symbols within each plot), the percentage of viable cells profiles of these gates in Fig. 2B verify the positioning of live and are significantly increased upon infection with 10 gonococci/cell dead cells. relative to those remaining uninfected, as indicated by forward Infections using a range of MOIs for both N. gonorrhoeae and and side scatter (Fig. 2C). Cells exposed to E. coli at the same E. coli were conducted in pilot experiments to assess their effect MOI were morphologically indistinguishable from the uninfected on B cell viability. Infection with E. coli at an MOI of .10 resulted controls, whereas N. gonorrhoeae infection consistently boosted in rapid B cell death (Supplemental Fig. 1A). Although a sim- B cell viability. Moreover, the percentage of gonococci-infected ilar effect was not apparent with N. gonorrhoeae (Supplemental B cells labeled with soluble annexin V was lower than cells either Fig. 1B), we selected an MOI of 10 for our standard assay system left uninfected or exposed to E. coli (Fig. 2D). These results because it allowed us to compare the effects of gonococcal infec- confirm that the protective effect of N. gonorrhoeae is not a gen- tion with E. coli over time. eral response to bacteria. It is pertinent to note that, although N. 4012 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS

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- Downloaded from 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 http://www.jimmunol.org/ 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 by guest on October 1, 2021 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 FIGURE 2. N. gonorrhoeae infection of primary human B cells pro- response to infection with gonococci, consistent with proliferation motes viability and does not induce cell death. (A) Viability was examined as a specific and reproducible response to N. gonorrhoeae (Fig. 4B). by comparing forward- and side-scatter flow cytometric parameters, which directly relate to cellular morphology. The percentage of viable cells was Gonococcal interaction with B cell populations is specific and obtained by taking the percentage of live cells (live gate) divided by the favors binding to IgM memory B cells percentage of total cells (all gate). B cells cultured alone include both live Because it was clear that some but not all B cells associated with the and dead cell populations, whereas B cell cultures treated with staur- gonococci (Fig. 1B, 1C), we considered whether cellular responses osporine, a protein kinase inhibitor that induces cell death at high con- to infection correlated with the action of a specific B cell subset. centrations, did not contain viable cells. Representative of four independent experiments with different donors. (B) The cell populations from (A) were In human peripheral blood, naive, IgM memory, and switched analyzed for the ability to bind annexin V, a marker of cell death. Cells memory B cells can be differentiated by the combined expression cultured alone (shaded) produce two peaks, indicative of annexin V-posi- patterns of CD27 and IgD (28), as shown in Fig. 5A. In mature tive and -negative populations within this culture. Cells cultured with cells in the periphery, CD27 expression typically indicates a 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 vidual donor. Bars indicate mean. Asterisks indicate a comparison between assayed for culture viability by forward versus side scatter (C) or examined uninfected and infected cells. *p , 0.05, **p , 0.01. Data points corre- for cell death by annexin V staining (D). Each symbol in the scatter plots sponding to the same individual donor are represented by the same symbol represents an experiment conducted with B cells obtained from an indi- within each plot. The Journal of Immunology 4013

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 Downloaded from 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 http://www.jimmunol.org/ 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. by guest on October 1, 2021 memory cell lineage, whereas IgD is primarily expressed on naive a greater affinity for gonococci than do naive cells, but the gon- cells only. ococci had a particular affinity for the IgD+CD27+ IgM memory Freshly purified IgD+CD272 (naive) B cells primarily express B cells (Fig. 5C, 5D). Strikingly, up to 80% of these IgM memory surface IgM (Fig. 5B). IgD+CD27+ (IgM memory) B cells also B cells were associated with the gonococci. Although it is possible consistently express surface IgM; however, IgG- and IgA- that, in rare instances, some gonococcal recognition could be expressing cells also exist within this population (Fig. 5B), sug- mediated through the BCR, it is clear that this is not the primary gesting that these cells have recently switched Ig classes (29). Most mechanism of binding because it is unreasonable to assume this IgD2CD27+ (switched memory) B cells have undergone Ig class large proportion of B cells are N. gonorrhoeae specific in all switching to express surface IgG or IgA (Fig. 5B). Although the donors examined. The lack of BCR involvement is also consistent relative proportion of B cells that represent each subpopulation with the fact that we observed no BCR recruitment to the bound differed slightly between donors, the average proportion of each bacteria during our immunofluorescence-based studies (Fig. 1A). subpopulation in freshly purified uninfected PBMC preparations There was little association of any B cell subpopulations with E. was 57% naive (IgD+CD272), 26% IgM memory (IgD+CD27+), coli, consistent with the interaction being unique to the gonococci. and 11% switched memory (IgD2CD27+) B cells (data not shown). To identify which populations of B cells were associating with IgM memory B cell population expands in response to infection N. gonorrhoeae, we prepared live fluorescently labeled N. gon- with N. gonorrhoeae orrhoeae or E. coli and used these to infect purified B cells for 3 h Considering that B cells proliferate in response to gonococcal with a low MOI of 2 bacteria per cell. Although E. coli binding to infection (Fig. 4), we examined whether one or more B cell subset all peripheral B cell subsets was very low, gonococci displayed (s) were specifically induced. After 5 d of infection, the IgD+ substantial binding to naive, IgM memory, and switched memory CD27+ IgM memory cell population had clearly expanded in re- populations of B cells (Fig. 5C). Comparable binding by the three sponse to gonococci, but not to E. coli, in all donors (Fig. 6). This isogenic gonococcal strains suggests that this association is not increase in the IgM memory cells was reflected by a tendency strictly dependent on receptors for either Opa variant, although the toward a reduction in the naive and, less so, switched memory ability of the B cell subpopulations to bind gonococci expressing B cell populations. However, in contrast to the significant increase these different adhesins is not equal (Fig. 5D). Surprisingly, we in IgM memory cells, the apparent changes in naive and switched observed that the simple expression of Opa adhesin did not memory cells were not statistically significant (Fig. 6B). There- translate into higher levels of bacterial binding, because OpaHSPG- fore, despite variability in the proportion of each of the B cell expressing N. gonorrhoeae associated less with the B cells than subpopulations between individuals, there is a consistent expan- did the strain that expresses no adhesin (Opa2; Fig. 5D). In sion of the IgM memory B cell subset in response to gonococci. general, both IgM memory and switched memory subsets display Moreover, the different levels of bacterial association conferred by 4014 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS

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- Downloaded from 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 http://www.jimmunol.org/ 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 Opa2 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- by guest on October 1, 2021 tion elicited a small increase in gonococci-specific IgM (Fig. 8B), whereas no detectable gonococci-specific IgG or IgA was evident FIGURE 4. N. gonorrhoeae infection, but not E. coli infection, induces (data not shown). Importantly, the baseline amount of N. gonor- strong proliferative responses in human B cells. (A) BrdU incorporation rhoeae-reactive Ig in E. coli-infected samples closely reflected was examined in B cells infected by either N. gonorrhoeae or E. coli for 3 d. that in uninfected samples, indicating that the observed increase in (B) The kinetics of B cell proliferation was measured with purified cells Ig required exposure to the gonococci. from four different donors and shows that gonococci can effectively induce The relatively low increase in N. gonorrhoeae-specific Ig did B cell-proliferative responses for up to 6 d (the longest time point we not reflect the significant responses apparent when total Ig was analyzed). Bars indicate mean. Asterisks indicate a comparison between measured (Fig. 8A). To assess the specificity of the Ig produced, , , uninfected and infected cells. *p 0.05, **p 0.01. Data points corre- we performed ELISAs to monitor the production of Abs specific sponding to the same individual donor are represented by the same symbol for other, nonneisserial Ags upon B cell infection. For this, we within each plot. measured Ig specific for a recall Ag, TT, as well as an irrelevant Ag, KLH. Although little TT-specific Ig was apparent in unin- OpaCEA and OpaHSPG (Fig. 5C, 5D) did not influence B cell ex- pansion (Fig. 6B). fected samples, significant increases in IgM specific for TT were apparent in the culture supernatants of all N. gonorrhoeae-infected N. gonorrhoeae clinical isolates induce proliferation in both cells (Fig. 8C) and the purified B cells from 5 of 10 donors pro- IgM memory and switched memory B cell populations duced TT-specific IgG upon exposure to the gonococci (data not N. gonorrhoeae undergo frequent antigenic and phase variation. To shown). Considering that a recall response to TT would be ex- confirm that the observed B cell responses were not unique to the pected to primarily be IgG, this implies that the N. gonorrhoeae- prototypical MS11 “laboratory” strain of N. gonorrhoeae, we also induced TT-specific IgM observed is not a conventional memory infected B cells with two unrelated clinical isolates obtained from response. TT-specific Abs were not detected in E. coli-infected male urethra. Flow cytometry was used to monitor the expression samples (Fig. 8C), again indicating that this is not a generalized of nuclear proliferation Ag Ki67 in all B cell subpopulations response to bacteria-derived products. B cells from the majority of 3 d postinfection. All strains of N. gonorrhoeae tested induced donors (90%) also produced IgM reactive with KLH following robust Ki67 expression by the IgD+CD27+ IgM memory cells with infection with gonococci but not E. coli (Fig. 8D), even though we little or no staining in the other subsets (Fig. 7), confirming that the would not expect any of the volunteer blood donors to have been observed increase in the IgM memory B cell population upon N. exposed to this Ag previously. When combined, these results in- gonorrhoeae infection (Fig. 6) is due to the selective proliferation dicate that N. gonorrhoeae infection of primary B cells results in of the IgD+CD27+ IgM memory cells. This effect is in clear broad, polyclonal activation and differentiation of IgM-bearing contrast to infection with E. coli, which induced no proliferation in B cells rather than a focused, clonal response to the gonococci. The Journal of Immunology 4015 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

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+,CD272), IgM memory (IgD+,CD27+), and switched memory (IgD2,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.

B cell responses to N. gonorrhoeae infection involve TLR9 this study, we systematically examined primary human B cellular Because all strains of N. gonorrhoeae examined produced robust responses to defined TLR agonists. To assess functional TLR re- proliferation in the IgM memory population of B cells, we ex- sponses in the B cell subsets, we examined a panel of specific TLR plored the possibility that gonococci were being detected by in- agonists—Pam3CSK4 (a synthetic triacylated lipoprotein that sig- nate immune receptors, resulting in downstream proliferation and nals through TLR2/1), FSL1 (a synthetic diacylated lipoprotein that differentiation of B cells. It has already been established that signals through TLR2/6), E. coli K12 LPS (TLR4), and CpG TLR expression varies between human peripheral B cell sub- ODN2006 (TLR9)—for their ability to cause activation and pro- populations, such that naive cells express few TLRs, each at very liferation. Surprisingly, when considered in the context of TLR low (to undetectable) levels when they are present, whereas both expression-based studies (20), naive cell activation was significantly IgM memory and switched memory subsets can express high increased in response to the TLR2/6 agonist FSL1 and TLR9 ago- levels of TLR6, 7, 9, and 10 (20). Neisseria species are known to nist CpG ODN2006 (Fig. 9A). Naive cells were weakly activated in constitutively express outer membrane porins that can be detected response to treatment with TLR2/1 agonist Pam3CSK4; however, by complexes of TLR2 and TLR1 (32, 33), in addition to pos- this difference did not reach statistical significance (Fig. 9A). + sessing a lipo-oligosaccharide that is recognized by TLR4 (34). In CD27 memory B cells responded to Pam3CSK4, FSL1, and CpG 4016 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS Downloaded from

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. http://www.jimmunol.org/

ODN2006 with strong and significant increases in cellular activa- with untreated cells, the difference did not reach statistical sig- tion (Fig. 9A). Treatment with LPS did not induce any response in nificance (Fig. 9B). any of the B cell populations (Fig. 9A, 9B), confirming previously Treatment with all TLR agonists examined resulted in prolif- published reports that TLR4 is not expressed in high enough levels eration by both IgM memory and switched memory subsets to in any human B cell subset to facilitate signaling (20, 35). varying degrees; however, treatment with TLR9 induced by far the Although naive cells expressed the CD86 activation marker strongest response within both populations (Fig. 9B). Others have upon treatment with some TLR agonists, proliferation was not ob- shown that treatment with CpG ODN2006, either alone or in by guest on October 1, 2021 served in these cells in response to any of the TLR agonists tested combination with other cytokines, induces only IgM class Ab from (Fig. 9B). Both IgM memory as well as switched memory B cells IgM memory cells (suggesting that it cannot induce class switch- underwent strong proliferation responses to CpG ODN2006. Al- ing), whereas switched memory cells predominately produce either though both memory cell populations did increase proliferation IgG or IgA Ig (20). Because we observed that N. gonorrhoeae in response to treatment with Pam3CSK4 and FSL1 compared infection induces the specific expansion of IgM memory B cells

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. The Journal of Immunology 4017 Downloaded from

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 Opa2 gonococci, TT, or KLH to examine the Ag specificity of the Ig B produced upon infection. After 5 d of infection, supernatants were applied to these plates to examine the production of Ig specific for gonococci ( ), to http://www.jimmunol.org/ 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.

(Fig. 6B) and that the majority of Ig induced is IgM (Fig. 8A), we the stimulatory effects of other CpG-containing DNA in both

considered whether TLR9 signaling might also contribute to the primary murine and primary human immune cells (36, 37). We by guest on October 1, 2021 gonococcal-induced IgM memory B cell response. took advantage of this reagent to examine whether TLR9 con- A dominant-negative CpG-containing oligonucleotide that tributes to the gonococcal-induced induction of B cell prolifera- inhibits TLR9 signaling (iCpG) has been shown to compete with tion. Although the iCpG had little effect on uninfected B cells, this

FIGURE 9. TLR9 signaling is induced upon N. gonorrhoeae infection of primary human B cells. (A and B) B cells were treated with 2.5 mg/ml

Pam3CSK4,1mg/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. 4018 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS inhibitor effectively reduced the proportion of actively prolifer- not permeate mammalian membranes, so engulfed bacteria are ating cells in response to the synthetic CpG-containing oligonu- protected from the antibiotic. B cells were infected with the cleotide ODN2006 and to intact N. gonorrhoeae by mean values specified strain of gonococci for 90 min before the addition of of 54.9 and 35.6%, respectively (Fig. 9C); this effect was con- gentamicin for 1 h. Viable intracellular gonococci are observed at sistent with cells from all donors tested. Thus, TLR9 contributes, this point (2.5 h postinfection), supporting the microscopic evi- at least in part, to B cell responses induced by N. gonorrhoeae dence that the bacteria were being internalized. However, when infection. the infection was allowed to persist for an additional 3.5 h (for a total of 6 h), the vast majority of intracellular gonococci is dead (Fig. N. gonorrhoeae can be engulfed and subsequently killed by 10B). Taken together, these results suggest that the high-affinity primary human B cells binding of N. gonorrhoeae to human B cells leads to engulfment of TLR9, unlike other TLRs, is not expressed on the cell surface but whole bacteria. Once internalized, the bacteria die and lyse, de- is found within endolysosomes (38). Although B cells will endo- livering a large bolus of bacterial DNA directly to intracellular cytose soluble Ag (39), their ability to engulf intact bacteria is lysosomal compartments where it can interact with TLR9 (Fig. 11). largely unexplored. CD4+ T cells do not engulf adherent N. gonorrhoeae (13). However, we considered the possibility that Discussion B cells might have the capacity to engulf and kill whole gon- N. gonorrhoeae has a curious niche, such that the pathogen seems ococci, effectively delivering the CpG-containing bacterial DNA to penetrate and persist within the subepithelial space (9); this site directly to intracellular TLR9. As depicted in Fig. 10A, primary is rich in nutrients but also patrolled by resident sentinel immune human B cells are able to engulf whole gonococci, as determined cells (16, 40) that the bacteria must contend with. Leukocytes, Downloaded from by differential staining of total and extracellular gonococci. including B cells, accumulate in the endocervical mucosa during To determine the fate of the internalized bacteria, a gentamicin both symptomatic and asymptomatic gonococcal infections (16), protection assay was used to quantify viable intracellular gon- ococci. This assay takes advantage of the fact that gentamicin does http://www.jimmunol.org/ by guest on October 1, 2021

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 FIGURE 10. B cells are able to engulf and kill whole gonococci. (A)B of the coinhibitory receptor CEACAM1 by the neisserial Opa proteins. cells were infected with MOI:50 of indicated strains of N. gonorrhoeae for This prevents T cell and B cell collaboration, abrogating the production of 3 h, stained for extracellular gonococci first, and then permeabilized to a T cell-dependent memory response. However, B cells are able to respond stain for total gonococci. Intracellular N. gonorrhoeae are single-color to N. gonorrhoeae infection in a T-independent manner, an effect relying in stained. Scale bars, 5 mm. (B) B cells were infected with the indicated part on N. gonorrhoeae engulfment allowing direct delivery of neisserial strain of gonococci for 90 min and then treated with gentamicin to kill any DNA to intracellular TLR9. This potently activates IgM memory cells to extracellular bacteria. At 2.5 and 6 h postinfection, cells were washed, produce a polyclonal IgM response which includes Ig that may bind to the lysed, and then plated for growth on GC agar. Bars indicate mean. Data gonococci but also to irrelevant (nongonococcal) Ags. Cumulatively, this points corresponding to the same individual donor are represented by the allows suboptimal control of the current infection and precludes devel- same symbol within each plot. DIC, differential interference contrast. opment of a classical memory response upon re-exposure to the gonococci. The Journal of Immunology 4019 making it likely that infecting N. gonorrhoeae come into direct memory B cells, which would at least partially explain the contact with B cells at the site of infection as well as in the switched memory response. Bernasconi et al. demonstrate by- draining lymph nodes. When considering this, it is surprising that stander activation of switched memory B cells, wherein micro- the Ab response elicited by N. gonorrhoeae is poor compared with environments produced upon activation of classical Ag-specific other mucosal infections and does not elicit a classical immuno- memory cells supports the activation of noncognate switched logic memory response that would otherwise protect upon sub- memory cells in a T-dependent manner (30). Although previously sequent exposure (7). B cells are APCs and express many pattern undescribed, the possibility that IgM memory B cells could play recognition molecules capable of detecting bacterial-derived a role in the bystander activation of switched memory cells is products (20, 41–44). Although this suggests that B cells should enticing because it would suggest that these cells could poly- recognize all bacteria equally, our results unexpectedly reveal that clonally activate conventional memory B cells at first sign of in- N. gonorrhoeae has a specific tropism for the IgM memory subset fection. of human B cells that elicits both their proliferation and a potent It is unreasonable to expect that the large proportion of B cells but broadly reactive T cell-independent Ig response. that we observe respond to N. gonorrhoeae are specific for neis- Past reports indicate that infection by a variety of bacteria can serial Ags, especially considering our observation that it is pri- cause B cell death (45, 46), yet we observed that N. gonorrhoeae marily the IgM memory subset that vigorously responds to infection instead supports primary human B cell viability and infection by a pathogen that is uncommon in the study population. promotes their proliferation, even decreasing cell death that nor- Neisserial activation of B cells does not occur via BCR clustering, mally occurs during in vitro culture of freshly isolated human such as that which occurs with the Moraxella catarrhalis super- B cells. In other cell types, both protection from (47, 48) and antigen Moraxella IgD-binding protein (26). To date, bacterial promotion of (49) cell death have been observed upon infection products such as capsular polysaccharide vaccines (28, 52), and Downloaded from with N. gonorrhoeae. Although the majority of studies examining purified CpG DNA (30) have been shown to expand the population this effect have focused on epithelial cells, it is becoming clear of human peripheral blood IgM memory B cells. Interestingly, that infection with N. gonorrhoeae, more often than not, inhibits N. gonorrhoeae does not express a polysaccharide capsule, con- apoptosis (50). Previous work using preactivated human B cells firming that stimulation of this subset does not require antigenic suggested that infection with CEACAM1-binding gonococci signaling by these structures. As such, to our knowledge, this is results in B cell death (19), but we did not observe similar the first report indicating the direct induction of polyclonal pro- http://www.jimmunol.org/ responses using freshly purified primary human B cells. On the liferation and IgM production by the human IgM memory B cell basis of our findings described in this article, those previous subpopulation in response to a bacterium. observations may reflect cell death attributable to the over- N. gonorrhoeae is a complex organism, with many components whelming stimulation applied to the cells (gonococci as well as that have the potential to interact with host cell receptors and cytokines), rather than a direct effect of the bacteria itself. Al- cause immune activation, especially on APCs such as B cells. though specific conclusions regarding the survival signals elicited Although it has been established that the IgM memory subset by the gonococci await further molecular studies, we propose expresses various TLRs (20), their TLR functionality had not, to that the protection from cell death observed in our assays stem, in our knowledge, been systematically explored. Although neisserial by guest on October 1, 2021 part, from the innate nature of the lymphocytes that are responding. endotoxin might be considered an obvious candidate for eliciting The IgM memory B cells have a lower activation threshold than the B cell response when considering its stimulatory effect on that of naive cells (51), suggesting that they have evolved to re- a variety of other immune cells (53–55), we did not observe spond by rapid activation, proliferation, and differentiation in re- a TLR4 response in any of the human B cell subsets described in sponse to microbes. this article (Fig. 9A, 9B). We observe that B cell infection with N. gonorrhoeae elicits Considering that the IgM memory B cells had a particular affin- an impressive production of mainly IgM, which recognizes both ity for N. gonorrhoeae but not E. coli, which was used as a pro- immunologically relevant (N. gonorrhoeae) and heterologous (TT totypical Gram-negative bacterial control to account for general- and KLH) Ags. The production of Ngo-specific IgM in response ized MAMP responses, and that these cells also displayed a potent to gonococcal infection in the absence of T cell help would be response to a synthetic CpG oligonucleotide agonist, TLR9 acti- expected if it was a specific BCR-dependent response, but our vation following N. gonorrhoeae engulfment seemed a likely observations indicate that N. gonorrhoeae recognition by B cells explanation for the observed switched memory B cell response. is not due to the BCR. However, IgM specific for TT and KLH, Consistent with this premise, gonococcal-induced B cell prolif- heterologous Ags consisting of single subunit proteins that contain eration was lower upon treatment with TLR9 antagonist iCpG fewer potential epitopes than do the intact bacteria, was produced (Fig. 9C). Why the iCpG-mediated inhibition was incomplete in greater amounts upon infection with N. gonorrhoeae than was remains unclear. It may simply not be sufficient to fully compete IgM specific for the infecting pathogen. This both highlights that with a bolus of CpG-containing DNA released upon neisserial the T-independent Ig response is broadly polyspecific and makes lysis within the phagosome. Alternatively, B cell recognition of it intriguing to consider that the gonococci may have a mecha- other neisserial-specific factors may also contribute to this re- nism by which to allow the selective expansion of heterologous, sponse. Interesting to consider in this context is the fact that nonneisserial Ig. neisserial outer membrane-expressed porins have been shown to IgG and IgA production by B cells infected with N. gonorrhoeae strongly induce both activation and proliferation by human and is significantly higher than the levels produced in response to murine B cells through interaction with surface-expressed TLR2 infection with E. coli (Fig. 7B). This suggests that either IgG and and TLR1 (33, 56), particularly because IgM memory express IgA can arise from the IgM memory cells (via class switch re- sufficient TLR2 and TLR1 to respond to purified Pam3CSK4 combination) or that switched memory cells, in addition to the (Fig. 9A, 9B). IgM memory cells, are differentiating into Ab-secreting cells in It is becoming increasingly clear that TLR9 is centrally im- response to the gonococci. We consider it plausible that the portant in the induction of immune cell responses to the pathogenic switched memory cells may benefit from T-independent Neisseria- Neisseria sp. (37, 57–59). In B cells, TLR9 is highly expressed induced “bystander” activation signals that originate from the IgM relative to other TLRs (20), but it is only found in intracellular 4020 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS compartments (38) rather than at the cell surface. Important in this a form of “memory” that is distinct from the conventional T- context has been the demonstration that the delivery of bacterial dependent response (72). In humans, an analysis of female sex DNA directly to TLR9-containing compartments is absolutely workers from Kenya showed the induction of an incomplete, but required for B cell responses, because they are poor at engulfing serotype-specific, immunologic memory response following re- exogenous DNA (60). B cells are surprisingly effective at peated N. gonorrhoeae infections over years of high-incidence engulfing whole gonococci (Fig. 10A, 10B), allowing for the de- exposure (73). Although these women could be very gradually livery of gonococcal DNA to TLR9, whereas engulfment of E. coli acquiring immunity by traditional T-dependent mechanisms, it is was rarely observed (data not shown). This difference may result tempting to propose the possibility that this memory response from the combination of the gonococci’s tropism for B cells, in- results from the repeated N. gonorrhoeae-dependent expansion of creasing the time spent in contact with the cells, and/or the larger human IgM memory B cells. Further in vivo studies will be re- size of E. coli hindering the engulfment of these bacteria. quired to test this postulate. IgM memory B cells represent the first line of defense by the In this report, we establish that the human-specific pathogen N. adaptive immune response because of their natural (not requiring gonorrhoeae elicits a vigorous innate B cell response. Considering antigenic stimulation) production of low affinity but polyreactive that other bacteria, including E. coli used in this study and Listeria IgM, the majority of which express germline-encoded receptors monocytogenes (45) and Francisella tularensis (46) in previous that can undergo somatic hypermutation (61). Some of this “nat- work, do not elicit a similar response, this appears to be a specific ural” IgM presumably functions by opsonizing encapsulated effect of the gonococci rather than being a prototypical immune bacteria (62, 63) and neutralizing viruses (64, 65). An innate response. Indeed, it is intriguing to speculate that N. gonorrhoeae population of B cells with similar characteristics to the human may promote the specific expansion of IgM memory B cells to IgM memory B cells exist in mice and are termed B-1 B cells (66). amplify the production of nonspecific Ig in a manner that nega- Downloaded from Functionally, murine B-1 B cells are similar to human IgD+CD27+ tively affects specific immunity in infected individuals. We pro- IgM memory cells because they are both considered to be the pose that, in the early stages of infection, circulating IgM memory source of natural Ab (67). However, the human IgM memory cell B cells may come into contact with the gonococci, either at the population is found in the periphery and may represent a circu- site of infection or in the draining lymph nodes surrounding the lating form of splenic marginal zone B cells (28), whereas similar urogenital tract. This interaction promotes the engulfment of cells in mice are primarily associated with peritoneal and pleural whole gonococci by the B cells, which may contribute to the http://www.jimmunol.org/ cavities (68). In the murine model of influenza lung infection, flu- clearance of the pathogen, but also allows for the direct detection specific B-1 B cells have been shown to be present only in the of bacterial products by innate receptors including TLR9. This mediastinal lymph nodes and are not present in distal lymph nodes results in a vigorous T-independent proliferation, expanding the or the surrounding lung tissue (65). However, there remains a IgM memory B cell population, and the induction of an acute, paucity of information regarding the development, regulation, localized, effector response in the form of low-affinity, polyclonal trafficking, and immune induction sites of the IgM memory B cells IgM (see model, Fig. 11). As the infection progresses, the gon- during infection in humans. In fact, it remains unclear whether, ococci may attach to CD4+ T cells, an interaction mediated by during infection, the activation of IgM memory B cells in humans their Opa adhesins binding to the coinhibitory receptor CEA- by guest on October 1, 2021 would be detectable systemically, or whether they primarily CAM1, because the vast majority of clinical neisserial isolates function in local immune induction sites in a manner that reflects have the ability to bind CEACAM1 (15). In contrast to B cells, that of the B-1 subset. Such shortcomings represent a particular T cells do not engulf the gonococci; this facilitates the inhibitory challenge to the field because they preclude the use of mice as a effect of CEACAM1-mediated phosphatase-dependent signaling model to understand human IgM memory cell function. Future at the cell surface, thereby preventing T cell activation and clonal work in the field must still clarify if they are indeed similar cells. expansion in response to TCR engagement (12, 13). Consequently, Although the human IgM memory B cells have been conclu- the absence of effective T cell help precludes the production of sively identified as a distinct B cell subpopulation relatively re- high-affinity class-switched Ab, but the T cell-independent pro- cently (28, 66), it is becoming increasingly apparent that their duction of innate IgM production is unaffected. The result is immune function is nonredundant in the production of an effective a broadly specific and thereby weakly effective humoral response immune response against invading microorganisms. In humans, to gonococcal infection that may assist in clearing the current loss of IgM memory B cells correlates with recurrent bacterial infection but, in the context of an unguided B cell response, pneumonia in patients with common variable immunodeficiency provides no conventional immunologic memory responses upon (52, 66). Furthermore, low numbers of IgM memory B cells found subsequent exposure to N. gonorrhoeae. in the periphery of HIV+ patients have been shown to be a pre- Such a model, involving the suppression of CD4+ T cells while dictor of cryptococcosis (69). In a similar vein, B cell super- activating the innate B cell response, is in agreement with clinical antigen Staphylococcus aureus protein A targets the innate murine observations indicating a modest increase in total IgM levels lo- B-1 B cells for deletion, leaving a void in the B cell repertoire that calized within the mucosa during natural gonococcal infection, the remains unrestored 14 mo after the initial treatment (70). N. unexpectedly rapid decline in N. gonorrhoeae-specific Ig once the gonorrhoeae appears to use an alternative approach to prevent the infection is cleared, and the absence of immunological memory production of a specific humoral response, suppressing T cell help upon subsequent exposure to these bacteria (7, 18). In the context (12, 13) while simultaneously stimulating the IgM memory B cells of the remarkable antigenic variability of this pathogen, such di- to produce polyclonal Ig responses; combined, this would effec- rect subversion of the protective immune response explains the tively lower the relative concentration of gonococcal-specific Ab. ongoing persistence of this highly evolved human pathogen. However, considering that some of the Ig will bind the bacteria, the relative benefit of a polyspecific Ig response to the infecting Acknowledgments pathogen versus the host remains a matter of ongoing debate (71). We thank the volunteer blood donors, without whom our study would not Although the ability of IgM memory B cells to respond to have been possible. We thank Dr. Chao Wang and Shannon McCaw for tech- microbes in a T-independent manner is plainly apparent, it has been nical assistance in completing this work and Drs. Alberto Martin and Michael recently suggested that murine innate B-1 B cells may possess J.H. Ratcliffe for insightful comments and critical reading of the manuscript. The Journal of Immunology 4021

Disclosures 26. Vidakovics, M. L., J. Jendholm, M. Mo¨rgelin, A. Ma˚nsson, C. Larsson, L. O. Cardell, and K. Riesbeck. 2010. B cell activation by outer membrane The authors have no financial conflicts of interest. vesicles—a novel virulence mechanism. PLoS Pathog. 6: e1000724. 27. Koopman, G., C. P. Reutelingsperger, G. A. Kuijten, R. M. Keehnen, S. T. Pals, and M. H. van Oers. 1994. Annexin V for flow cytometric detection of phos- References phatidylserine expression on B cells undergoing apoptosis. Blood 84: 1415–1420. 1. World Health Organization. 2011. Emergence of Multi-Drug Resistant Neisseria 28. Weller, S., M. C. Braun, B. K. Tan, A. Rosenwald, C. Cordier, M. E. Conley, gonorrhoeae—Threat of Global Rise in Untreatable Sexually Transmitted A. Plebani, D. S. Kumararatne, D. Bonnet, O. Tournilhac, et al. 2004. Human Infections. World Health Organization, Manila, Philippines. blood IgM “memory” B cells are circulating splenic marginal zone B cells 2. Handsfield, H. H. 1990. Neisseria gonorrhoeae in Principles and Practice of harboring a prediversified immunoglobulin repertoire. Blood 104: 3647–3654. Infectious Diseases. Churchill Livingstone, New York. 29. Werner-Favre, C., F. Bovia, P. Schneider, N. Holler, M. Barnet, V. Kindler, 3. Edwards, J. L., and M. A. Apicella. 2004. The molecular mechanisms used by J. Tschopp, and R. H. Zubler. 2001. IgG subclass switch capacity is low in Neisseria gonorrhoeae to initiate infection differ between men and women. Clin. switched and in IgM-only, but high in IgD+IgM+, post-germinal center (CD27+) Microbiol. Rev. 17: 965–981 (table of contents.). human B cells. Eur. J. Immunol. 31: 243–249. 4. Workowski, K. A., S. M. Berman, and J. M. Douglas, Jr. 2008. Emerging an- 30. Bernasconi, N. L., E. Traggiai, and A. Lanzavecchia. 2002. Maintenance of timicrobial resistance in Neisseria gonorrhoeae: urgent need to strengthen pre- serological memory by polyclonal activation of human memory B cells. Science vention strategies. Ann. Intern. Med. 148: 606–613. 298: 2199–2202. 5. World Health Organization. 2010. WHO Warns of Danger of Untreatable Gon- 31. Plummer, F. A., H. Chubb, J. N. Simonsen, M. Bosire, L. Slaney, N. J. Nagelkerke, orrhoea. World Health Organization, Manila, Philippines. I. Maclean, J. O. Ndinya-Achola, P. Waiyaki, and R. C. Brunham. 1994. Anti- 6. Mcclelland, R. S., C. C. Wang, K. Mandaliya, J. Overbaugh, M. T. Reiner, bodies to opacity proteins (Opa) correlate with a reduced risk of gonococcal D. D. Panteleeff, L. Lavreys, J. Ndinya-Achola, J. J. Bwayo, and J. K. Kreiss. salpingitis. J. Clin. Invest. 93: 1748–1755. 2001. Treatment of cervicitis is associated with decreased cervical shedding of 32. Massari, P., P. Henneke, Y. Ho, E. Latz, D. T. Golenbock, and L. M. Wetzler. HIV-1. AIDS 15: 105–110. 2002. Cutting edge: immune stimulation by neisserial porins is toll-like receptor 7. Hedges, S. R., M. S. Mayo, J. Mestecky, E. W. Hook, III, and M. W. Russell. 2 and MyD88 dependent. J. Immunol. 168: 1533–1537. 1999. Limited local and systemic antibody responses to Neisseria gonorrhoeae 33. Massari, P., A. Visintin, J. Gunawardana, K. A. Halmen, C. A. King, during uncomplicated genital infections. Infect. Immun. 67: 3937–3946. D. T. Golenbock, and L. M. Wetzler. 2006. Meningococcal porin PorB binds to 8. Fox, K. K., J. C. Thomas, D. H. Weiner, R. H. Davis, P. F. Sparling, and TLR2 and requires TLR1 for signaling. J. Immunol. 176: 2373–2380. Downloaded from M. S. Cohen. 1999. Longitudinal evaluation of serovar-specific immunity to 34. Pridmore, A. C., D. H. Wyllie, F. Abdillahi, L. Steeghs, P. van der Ley, Neisseria gonorrhoeae. Am. J. Epidemiol. 149: 353–358. S. K. Dower, and R. C. Read. 2001. A lipopolysaccharide-deficient mutant of 9. McGee, Z. A., D. S. Stephens, L. H. Hoffman, W. F. Schlech, III, and Neisseria meningitidis elicits attenuated cytokine release by human macrophages R. G. Horn. 1983. Mechanisms of mucosal invasion by pathogenic Neisseria. and signals via Toll-like receptor (TLR) 2 but not via TLR4/MD2. J. Infect. Dis. Rev. Infect. Dis. 5(Suppl. 4): S708–S714. 183: 89–96. 10. Wang, J., S. D. Gray-Owen, A. Knorre, T. F. Meyer, and C. Dehio. 1998. Opa 35. Bourke, E., D. Bosisio, J. Golay, N. Polentarutti, and A. Mantovani. 2003. The binding to cellular CD66 receptors mediates the transcellular traversal of Neis- Toll-like receptor repertoire of human B lymphocytes: inducible and selective

seria gonorrhoeae across polarized T84 epithelial cell monolayers. Mol. expression of TLR9 and TLR10 in normal and transformed cells. Blood 102: http://www.jimmunol.org/ Microbiol. 30: 657–671. 956–963. 11. Slevogt, H., S. Zabel, B. Opitz, A. Hocke, J. Eitel, P. D. N’guessan, L. Lucka, 36. Gursel, I., M. Gursel, H. Yamada, K. J. Ishii, F. Takeshita, and D. M. Klinman. K. Riesbeck, W. Zimmermann, J. Zweigner, et al. 2008. CEACAM1 inhibits 2003. Repetitive elements in mammalian telomeres suppress bacterial DNA- Toll-like receptor 2-triggered antibacterial responses of human pulmonary epi- induced immune activation. J. Immunol. 171: 1393–1400. thelial cells. Nat. Immunol. 9: 1270–1278. 37. Dobson-Belaire, W. N., A. Rebbapragada, R. J. Malott, F. Y. Yue, C. Kovacs, 12. Boulton, I. C., and S. D. Gray-Owen. 2002. Neisserial binding to CEACAM1 R. Kaul, M. A. Ostrowski, and S. D. Gray-Owen. 2010. Neisseria gonorrhoeae arrests the activation and proliferation of CD4+ T lymphocytes. Nat. Immunol. 3: effectively blocks HIV-1 replication by eliciting a potent TLR9-dependent in- 229–236. terferon-a response from plasmacytoid dendritic cells. Cell. Microbiol. 12: 13. Lee, H. S., M. A. Ostrowski, and S. D. Gray-Owen. 2008. CEACAM1 dynamics 1703–1717. during Neisseria gonorrhoeae suppression of CD4+ T lymphocyte activation. J. 38. Latz, E., A. Schoenemeyer, A. Visintin, K. A. Fitzgerald, B. G. Monks, Immunol. 180: 6827–6835. C. F. Knetter, E. Lien, N. J. Nilsen, T. Espevik, and D. T. Golenbock. 2004.

14. Gray-Owen, S. D., D. R. Lorenzen, A. Haude, T. F. Meyer, and C. Dehio. 1997. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. by guest on October 1, 2021 Differential Opa specificities for CD66 receptors influence tissue interactions Immunol. 5: 190–198. and cellular response to Neisseria gonorrhoeae. Mol. Microbiol. 26: 971–980. 39. Lanzavecchia, A. 1985. Antigen-specific interaction between T and B cells. 15. Virji, M., S. M. Watt, S. Barker, K. Makepeace, and R. Doyonnas. 1996. The N- Nature 314: 537–539. domain of the human CD66a adhesion molecule is a target for Opa proteins of 40. Prakash, M., S. Patterson, and M. S. Kapembwa. 2001. Evaluation of the cervical Neisseria meningitidis and Neisseria gonorrhoeae. Mol. Microbiol. 22: 929–939. cytobrush sampling technique for the preparation of CD45+ mononuclear cells 16. Levine, W. C., V. Pope, A. Bhoomkar, P. Tambe, J. S. Lewis, A. A. Zaidi, from the human cervix. J. Immunol. Methods 258: 37–46. C. E. Farshy, S. Mitchell, and D. F. Talkington. 1998. Increase in endocervical 41. Thornton, B. P., V. Vĕtvicka, and G. D. Ross. 1994. Natural antibody and CD4 lymphocytes among women with nonulcerative sexually transmitted dis- complement-mediated antigen processing and presentation by B lymphocytes. J. eases. J. Infect. Dis. 177: 167–174. Immunol. 152: 1727–1737. 17. Johansson, M., and N. Y. Lycke. 2003. Immunology of the human genital tract. 42. Kubagawa, H., S. Oka, Y. Kubagawa, I. Torii, E. Takayama, D. W. Kang, Curr. Opin. Infect. Dis. 16: 43–49. G. L. Gartland, L. F. Bertoli, H. Mori, H. Takatsu, et al. 2009. Identity of the 18. McMillan, A., G. McNeillage, H. Young, and S. S. Bain. 1979. Secretory anti- elusive IgM Fc receptor (FcmR) in humans. J. Exp. Med. 206: 2779–2793. body response of the cervix to infection with Neisseria gonorrhoeae. Br. J. 43. Shibuya, A., N. Sakamoto, Y. Shimizu, K. Shibuya, M. Osawa, T. Hiroyama, Vener. Dis. 55: 265–270. H. J. Eyre, G. R. Sutherland, Y. Endo, T. Fujita, et al. 2000. Fc a/m receptor 19. Pantelic, M., Y. J. Kim, S. Bolland, I. Chen, J. Shively, and T. Chen. 2005. mediates endocytosis of IgM-coated microbes. Nat. Immunol. 1: 441–446. Neisseria gonorrhoeae kills carcinoembryonic antigen-related cellular adhesion 44. Wing, J. B., D. L. Jack, M. E. Lee, A. A. Pacey, G. R. Kinghorn, and R. C. Read. molecule 1 (CD66a)-expressing human B cells and inhibits antibody production. 2009. Mannose-binding lectin is present in human semen and modulates cellular Infect. Immun. 73: 4171–4179. adhesion of Neisseria gonorrhoeae in vitro. Clin. Exp. Immunol. 157: 408–414. 20. Bernasconi, N. L., N. Onai, and A. Lanzavecchia. 2003. A role for Toll-like 45. Menon, A., M. L. Shroyer, J. L. Wampler, C. B. Chawan, and A. K. Bhunia. 2003. receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in na- In vitro study of Listeria monocytogenes infection to murine primary and human ive B cells and constitutive expression in memory B cells. Blood 101: 4500–4504. transformed B cells. Comp. Immunol. Microbiol. Infect. Dis. 26: 157–174. 21. Capolunghi, F., S. Cascioli, E. Giorda, M. M. Rosado, A. Plebani, C. Auriti, 46. Krocova, Z., A. Ha¨rtlova, D. Souckova, L. Zivna, M. Kroca, E. Rudolf, G. Seganti, R. Zuntini, S. Ferrari, M. Cagliuso, et al. 2008. CpG drives human A. Macela, and J. Stulik. 2008. Interaction of B cells with intracellular pathogen transitional B cells to terminal differentiation and production of natural anti- Francisella tularensis. Microb. Pathog. 45: 79–85. bodies. J. Immunol. 180: 800–808. 47. Follows, S. A., J. Murlidharan, P. Massari, L. M. Wetzler, and C. A. Genco. 22. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda, K. Takeda, 2009. Neisseria gonorrhoeae infection protects human endocervical epithelial and S. Akira. 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are cells from apoptosis via expression of host antiapoptotic proteins. Infect. Immun. hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene 77: 3602–3610. product. J. Immunol. 162: 3749–3752. 48. Binnicker, M. J., R. D. Williams, and M. A. Apicella. 2003. Infection of human 23. Gray-Owen, S. D., and R. S. Blumberg. 2006. CEACAM1: contact-dependent urethral epithelium with Neisseria gonorrhoeae elicits an upregulation of host control of immunity. Nat. Rev. Immunol. 6: 433–446. anti-apoptotic factors and protects cells from staurosporine-induced apoptosis. 24. Kupsch, E. M., B. Knepper, T. Kuroki, I. Heuer, and T. F. Meyer. 1993. Variable Cell. Microbiol. 5: 549–560. opacity (Opa) outer membrane proteins account for the cell tropisms displayed 49. Mu¨ller, A., D. Gu¨nther, F. Du¨x, M. Naumann, T. F. Meyer, and T. Rudel. 1999. by Neisseria gonorrhoeae for human leukocytes and epithelial cells. EMBO J. Neisserial porin (PorB) causes rapid calcium influx in target cells and induces 12: 641–650. apoptosis by the activation of cysteine proteases. EMBO J. 18: 339–352. 25. Achtman, M., M. Neibert, B. A. Crowe, W. Strittmatter, B. Kusecek, E. Weyse, 50. Follows, S. A., and P. Massari. 2010. Consequences of pathogenic Neisseria M. J. Walsh, B. Slawig, G. Morelli, A. Moll, et al. 1988. Purification and infection on cellular apoptosis. In Neisseria: Molecular Mechanisms of characterization of eight class 5 outer membrane protein variants from a clone of Pathogenesis. C. A. Genco, and L. Wetzler, eds. Caister Academic Press, Nor- Neisseria meningitidis serogroup A. J. Exp. Med. 168: 507–525. folk, U.K., p. 145–162. 4022 N. GONORRHOEAE STIMULATION OF HUMAN IgM MEMORY B CELLS

51. Good, K. L., D. T. Avery, and S. G. Tangye. 2009. Resting human memory absence of antigen-driven responses is the hallmark of the IgM+IgD+CD27+ B cells are intrinsically programmed for enhanced survival and responsiveness to B cell repertoire in infants. J. Exp. Med. 205: 1331–1342. diverse stimuli compared to naive B cells. J. Immunol. 182: 890–901. 62. Shi, Y., T. Yamazaki, Y. Okubo, Y. Uehara, K. Sugane, and K. Agematsu. 2005. 52. Carsetti, R., M. M. Rosado, S. Donnanno, V. Guazzi, A. Soresina, A. Meini, Regulation of aged humoral immune defense against pneumococcal bacteria by A. Plebani, F. Aiuti, and I. Quinti. 2005. The loss of IgM memory B cells IgM memory B cell. J. Immunol. 175: 3262–3267. correlates with clinical disease in common variable immunodeficiency. J. Al- 63. Zhou, Z. H., Y. Zhang, Y. F. Hu, L. M. Wahl, J. O. Cisar, and A. L. Notkins. lergy Clin. Immunol. 115: 412–417. 2007. The broad antibacterial activity of the natural antibody repertoire is due to 53. Liu, M., C. M. John, and G. A. Jarvis. 2010. Phosphoryl moieties of lipid A from polyreactive antibodies. Cell Host Microbe 1: 51–61. Neisseria meningitidis and N. gonorrhoeae lipooligosaccharides play an im- 64. Ochsenbein, A. F., T. Fehr, C. Lutz, M. Suter, F. Brombacher, H. Hengartner, and portant role in activation of both MyD88- and TRIF-dependent TLR4-MD-2 R. M. Zinkernagel. 1999. Control of early viral and bacterial distribution and signaling pathways. J. Immunol. 185: 6974–6984. disease by natural antibodies. Science 286: 2156–2159. 54. Pridmore, A. C., G. A. Jarvis, C. M. John, D. L. Jack, S. K. Dower, and 65. Choi, Y. S., and N. Baumgarth. 2008. Dual role for B-1a cells in immunity to R. C. Read. 2003. Activation of toll-like receptor 2 (TLR2) and TLR4/MD2 by influenza virus infection. J. Exp. Med. 205: 3053–3064. Neisseria is independent of capsule and lipooligosaccharide (LOS) sialylation but 66. Kruetzmann, S., M. M. Rosado, H. Weber, U. Germing, O. Tournilhac, varies widely among LOS from different strains. Infect. Immun. 71: 3901–3908. H. H. Peter, R. Berner, A. Peters, T. Boehm, A. Plebani, et al. 2003. Human 55. Uronen-Hansson, H., L. Steeghs, J. Allen, G. L. Dixon, M. Osman, P. van der immunoglobulin M memory B cells controlling Streptococcus pneumoniae Ley, S. Y. Wong, R. Callard, and N. Klein. 2004. Human dendritic cell activation infections are generated in the spleen. J. Exp. Med. 197: 939–945. by Neisseria meningitidis: phagocytosis depends on expression of lip- 67. Baumgarth, N., O. C. Herman, G. C. Jager, L. Brown, L. A. Herzenberg, and ooligosaccharide (LOS) by the bacteria and is required for optimal cytokine L. A. Herzenberg. 1999. Innate and acquired humoral immunities to influenza production. Cell. Microbiol. 6: 625–637. virus are mediated by distinct arms of the immune system. Proc. Natl. Acad. Sci. 56. Wetzler, L. M., Y. Ho, and H. Reiser. 1996. Neisserial porins induce USA 96: 2250–2255. B lymphocytes to express costimulatory B7-2 molecules and to proliferate. J. 68. Herzenberg, L. A., and A. B. Kantor. 1993. B-cell lineages exist in the mouse. Exp. Med. 183: 1151–1159. Immunol. Today 14: 79–83, discussion 88–90. 57. Magnusson, M., R. Tobes, J. Sancho, and E. Pareja. 2007. Cutting edge: natural 69. Subramaniam, K., B. Metzger, L. H. Hanau, A. Guh, L. Rucker, S. Badri, and DNA repetitive extragenic sequences from gram-negative pathogens strongly L. A. Pirofski. 2009. IgM+ memory B cell expression predicts HIV-associated stimulate TLR9. J. Immunol. 179: 31–35. cryptococcosis status. J. Infect. Dis. 200: 244–251. 58. Mogensen, T. H., S. R. Paludan, M. Kilian, and L. Ostergaard. 2006. Live 70. Silverman, G. J., S. P. Cary, D. C. Dwyer, L. Luo, R. Wagenknecht, and Downloaded from Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis V. E. Curtiss. 2000. A B cell superantigen-induced persistent “Hole” in the B-1 activate the inflammatory response through Toll-like receptors 2, 4, and 9 in repertoire. J. Exp. Med. 192: 87–98. species-specific patterns. J. Leukoc. Biol. 80: 267–277. 71. Montes, C. L., E. V. Acosta-Rodrı´guez, M. C. Merino, D. A. Bermejo, and 59. Sjo¨linder, H., T. H. Mogensen, M. Kilian, A. B. Jonsson, and S. R. Paludan. A. Gruppi. 2007. Polyclonal B cell activation in infections: infectious agents’ 2008. Important role for Toll-like receptor 9 in host defense against meningo- devilry or defense mechanism of the host? J. Leukoc. Biol. 82: 1027–1032. coccal sepsis. Infect. Immun. 76: 5421–5428. 72. Alugupalli, K. R., J. M. Leong, R. T. Woodland, M. Muramatsu, T. Honjo, and 60. Roberts, T. L., M. L. Turner, J. A. Dunn, P. Lenert, I. L. Ross, M. J. Sweet, and R. M. Gerstein. 2004. B1b lymphocytes confer T cell-independent long-lasting

K. J. Stacey. 2011. B cells do not take up bacterial DNA: an essential role for immunity. Immunity 21: 379–390. http://www.jimmunol.org/ antigen in exposure of DNA to Toll-like receptor-9. Immunol. Cell Biol. 89: 517– 73. Plummer, F. A., J. N. Simonsen, H. Chubb, L. Slaney, J. Kimata, M. Bosire, 525. J. O. Ndinya-Achola, and E. N. Ngugi. 1989. Epidemiologic evidence for the 61. Weller, S., M. Mamani-Matsuda, C. Picard, C. Cordier, D. Lecoeuche, development of serovar-specific immunity after gonococcal infection. J. Clin. F. Gauthier, J. C. Weill, and C. A. Reynaud. 2008. Somatic diversification in the Invest. 83: 1472–1476. by guest on October 1, 2021