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Generating large-scale, high-fidelity sequencing data is challenging and, furthermore, not much has been done to characterize adjuvants' effects at the repertoire level. Thus, we introduced an IgSeq pipeline that standardized library prep protocols and data analysis functions for accurate repertoire profiling. We then studied systemically effects of CpG and Alum on the Ig heavy chain repertoire using the ovalbumin (OVA) murine model. Ig repertoires of different tissues (spleen and bone marrow) and isotypes (IgG and IgM) were examined and compared in IGHV mutation, gene usage, CDR3 length, clonal diversity, and clonal selection. We found Ig repertoires of different compartments exhibited distinguishable profiles at the non-immunized steady state, and distinctions became more pronounced upon adjuvanted immunizations. Notably, Alum and CpG effects exhibited different tissue- and isotype-preferences. The former led to increased diversity of abundant clones in bone marrow, and the latter promoted the selection of IgG clones in both tissues., Competing Interests: The authors declare that there is no conflict of interest., (© 2023.)

Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae , poses a significant global public health threat. Infection in women can be asymptomatic and may result in severe reproductive complications. Escalating antibiotic resistance underscores the need for an effective vaccine. Approaches being explored include subunit vaccines and outer membrane vesicles (OMVs), but an ideal candidate remains elusive. Meningococcal OMV-based vaccines have been associated with reduced rates of gonorrhea in retrospective epidemiologic studies, and with accelerated gonococcal clearance in mouse vaginal colonization models. Cross-protection is attributed to shared antigens and possibly cross-reactive, bactericidal antibodies. Using a Candidate Antigen Selection Strategy (CASS) based on the gonococcal transcriptome during human mucosal infection, we identified new potential vaccine targets that, when used to immunize mice, induced the production of antibodies with bactericidal activity against N. gonorrhoeae strains. The current study determined antigen recognition by human sera from N. gonorrhoeae -infected subjects, evaluated their potential as a multi-antigen (combination) vaccine in mice and examined the impact of different adjuvants (Alum or Alum+MPLA) on functional antibody responses to N. gonorrhoeae . Our results indicated that a stronger Th1 immune response component induced by Alum+MPLA led to antibodies with improved bactericidal activity. In conclusion, a combination of CASS-derived antigens may be promising for developing effective gonococcal vaccines.

Vaccines have played a pivotal role in improving public health, however, many infectious diseases lack an effective vaccine. Controlling the spread of infectious diseases requires continuing studies to develop new and improved vaccines. Our laboratory has been investigating the immune enhancing mechanisms of Toll-like receptor (TLR) ligand-based adjuvants, including the TLR2 ligand Neisseria meningitidis outer membrane protein, PorB. Adjuvant use of PorB increases costimulatory factors on antigen presenting cells (APC), increases antigen specific antibody production, and cytokine producing T cells. We have demonstrated that macrophage expression of MyD88 (required for TLR2 signaling) is an absolute requirement for the improved antibody response induced by PorB. Here-in, we specifically investigated the role of subcapsular CD169+ marginal zone macrophages in antibody production induced by the use of TLR-ligand based adjuvants (PorB and CpG) and non-TLR-ligand adjuvants (aluminum salts). CD169 knockout mice and mice treated with low dose clodronate treated animals (which only remove marginal zone macrophages), were used to investigate the role of these macrophages in adjuvant-dependent antibody production. In both sets of mice, total antigen specific immunoglobulins (IgGs) were diminished regardless of adjuvant used. However, the greatest reduction was seen with the use of TLR ligands as adjuvants. In addition, the effect of the absence of CD169+ macrophages on adjuvant induced antigen and antigen presenting cell trafficking to the lymph nodes was examined using immunofluorescence by determining the relative extent of antigen loading on dendritic cells (DCs) and antigen deposition on follicular dendritic cells (FDC). Interestingly, only vaccine preparations containing PorB had significant decreases in antigen deposition in lymphoid follicles and germinal centers in CD169 knockout mice or mice treated with low dose clodronate as compared to wildtype controls. Mice immunized with CpG containing preparations demonstrated decreased FDC networks in the mice treated with low dose clodronate. Conversely, alum containing preparations only demonstrated significant decreases in IgG in CD169 knockout mice. These studies stress that importance of subcapsular macrophages and their unique role in adjuvant-mediated antibody production, potentially due to an effect of these adjuvants on antigen trafficking to the lymph node and deposition on follicular dendritic cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lisk, Yuen, Kuniholm, Antos, Reiser and Wetzler.)

Objective: The aim of this observational study was to determine the optimal timing of interleukin-6 receptor inhibitor (IL6ri) administration for coronavirus disease 2019 (COVID-19)., Methods: Patients with COVID-19 were given an IL6ri (sarilumab or tocilizumab) based on iteratively reviewed guidelines. IL6ri were initially reserved for critically ill patients, but after review, treatment was liberalized to patients with lower oxygen requirements. Patients were divided into two groups: those requiring ≤45% fraction of inspired oxygen (FiO 2 ) (termed stage IIB) and those requiring >45% FiO 2 (termed stage III) at the time of IL6ri administration. The main outcomes were all-cause mortality, discharge alive from hospital, and extubation., Results: A total of 255 COVID-19 patients were treated with IL6ri (149 stage IIB and 106 stage III). Patients treated in stage IIB had lower mortality than those treated in stage III (adjusted hazard ratio (aHR) 0.24, 95% confidence interval (CI) 0.08-0.74). Overall, 218 (85.5%) patients were discharged alive. Patients treated in stage IIB were more likely to be discharged (aHR 1.43, 95% CI 1.06-1.93) and were less likely to be intubated (aHR 0.43, 95% CI 0.24-0.79)., Conclusions: IL6ri administration prior to >45% FiO 2 requirement was associated with improved COVID-19 outcomes. This can guide clinical management pending results from randomized controlled trials., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Vaccines are arguably one of the greatest advancements in modern medicine. Subunit vaccines comprise the majority of current preparations and consist of two main components-antigen and adjuvant. The antigen is a small molecule against which the vaccine induces an immune response to provide protection via the immunostimulatory ability of the adjuvant. Our laboratory has investigated the adjuvant properties of Toll-like receptor (TLR) ligand-based adjuvants, especially the outer membrane protein from Neisseria mengingitidis , PorB. In this current study we used PorB, along with CpG, an intracellular TLR9 agonist, and a non-TLR adjuvant, aluminum salts (Alum), to further investigate cellular mechanisms of adjuvanticity, focusing on the fate of intact antigen in the germinal center and association with follicular dendritic cells (FDCs). FDCs are located in the B cell light zone of the germinal center and are imperative for affinity maturation. They are stromal cells that retain whole intact antigen allowing recognition by the B cell receptor of the germinal center B cells. Our studies demonstrate that TLR ligands, but not Alum, increase the FDC network, while PorB and Alum increased colocalization of FDC and the model soluble antigen, ovalbumin (OVA). As PorB is the only adjuvant tested that induces both a higher number of FDCs and increased deposition of antigen on FDCs, it has the greatest ability to increase FDC-antigen interaction, essential for induction of B cell affinity maturation. These studies demonstrate a further mechanism and potential superiority of PorB as an adjuvant and its influence on antibody production., (Copyright © 2020 Lisk, Yuen, Kuniholm, Antos, Reiser and Wetzler.)

Our laboratory has focused on Porin B (PorB), an outer membrane protein from Neisseria meningitidis and TLR2 ligand-based adjuvant, to characterize specific molecular and cellular pathways involved in improved immune responses induced by vaccine adjuvants. PorB's ability to form micellar nanoparticular multi-molecular organized structures and its interaction with Toll-like receptor 2/1 complexes likely accounts for its potent adjuvant activity. Downstream from this stimulation, we have observed enhanced antigen uptake in antigen presenting cells (APC), greater antigen deposition in secondary lymphoid organs, and promotion of germinal center reactions. In mice, antigen-specific IgGs were increased after PorB adjuvanted vaccination using the model antigen ovalbumin (OVA). Likewise, this formulation resulted in more IL-4 and IFN-γ positive T cells. Mice that received PorB adjuvanted vaccinations benefitted from lower bacterial burdens when challenged with recombinant Listeria monocytogenes expressing OVA. Mouse models lacking MyD88 signaling in various APC types helped identify macrophages as an essential cell type for the adjuvant activity of PorB. We believe the work presented here provides examples of the mechanistic studies required to understand how vaccine adjuvants are contributing to the establishment of protective immunity.

The emergence and spread of fully antimicrobial resistant Neisseria gonorrhoeae (GC) highlights a clear need for next-generation antigonococcal therapeutics. A broadly reactive anti-GC vaccine would best address this global public health threat. Polyantigenic outer membrane vesicles (OMVs) derived from GC can overcome the challenges posed by GC's high rate of phase and antigen variation. In fact, GC OMVs have already shown promise as a vaccine antigen; however, all previous studies have utilized vesicles contaminated by RMP, a bacterioprotective antigen known to entirely abrogate vaccine-induced bactericidal activity in vivo. Additionally, these studies primarily utilized vesicles isolated through techniques like membrane disruption with detergents, which are known to increase contamination of cytoplasmic components as compared to naturally released OMVs (nOMVs). This chapter describes the isolation and characterization of naturally released nOMVs through sequential size and weight restrictive filtration. nOMVs are characterized by morphology, proteomics, and bioactivity via various methods. Herein we also describe methods for further evaluation of the innate and induced immunogenicity of rmp-deficient GC nOMVs by cell stimulation and murine vaccination. Per these methods, nOMVs are found to be largely homogenous spherical structures approximately 70 nm in diameter containing a consistent subset of GC outer membrane proteins. The rmp-deficient vesicles demonstrate a morphology and, with the exception of RMP, antigenic profile consistent with that of nOMVs derived from wild time N. gonorrhoeae. Additionally, vesicles lacking RMP are able to engage and strongly activate a diverse array of pattern recognition receptors in vitro. These methods lay the groundwork for future experiments examining the in vivo protective efficacy of the anti-GC response induced by these nOMVs as well as studies examining the mechanism of vaccine induced female genital tract immunity.

Vaccines formulated with adjuvant have been effective against numerous infectious diseases, almost always due to induction of functional antibodies that recognizes the pathogen of interest. There is an unmet clinical need for vaccine adjuvants that induce T cells responses to potentially enhance protection against malignancies and intracellular pathogens, where a humoral response, alone, may not be adequate for protection. In this study, we demonstrate that a TLR2 ligand-based adjuvant, meningococcal PorB, has broad immunostimulatory activity with the ability to induce a robust and diverse vaccine antigen specific T cell response. We demonstrate that a vaccine formulated with PorB admixed with ovalbumin induces a wide variety of antigen specific antibody subclasses and effector molecules (MIG, MCP-1, IP-10, MIP-1α, KC & IL-2) with known roles for inducing T cell responses, along with elevated levels of Th1 and Th2 type cytokines upon antigen stimulation. We confirmed production of these cytokines by examining the antigen-specific T cells induced by PorB in vivo. After two immunizations with vaccine formulated with PorB/OVA, antigen-specific CD4 and CD8 T cells were significantly increased in numbers and produced IL-4 or IFN-γ upon ex vivo antigen re-stimulation. Finally, in a Listeria mouse infection model, vaccine formulated with PorB significantly reduced the bacterial burden upon a low dose infection and increased survival upon a high dose infection with recombinant Listeria monocytogenes engineered to express OVA (rLmOVA), a pathogen that requires OVA-antigen specific cytotoxic CD8 T cells for clearance. In summary, PorB is able to induce antigen specific broad B and T cell responses, illustrating its potential as a potent and new vaccine adjuvant., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: The emergence of fully antimicrobial resistant Neisseria gonorrhoeae has led global public health agencies to identify a critical need for next generation anti-gonococcal pharmaceuticals. The development and success of these compounds will rely upon valid pre-clinical models of gonorrhoeae infection. We recently developed and reported the first model of upper genital tract gonococcal infection. During initial characterization, we observed significant reproductive cycle-based variation in infection outcome. When uterine infection occurred in the diestrus phase, there was significantly greater pathology than during estrus phase. The aim of this study was to evaluate transcriptional profiles of infected uterine tissue from mice in either estrus or diestrus phase in order to elucidate possible mechanisms for these differences., Results: Genes and biological pathways with phase-independent induction during infection showed a chemokine dominant cytokine response to Neisseria gonorrhoeae. Despite general induction being phase-independent, this common anti-gonococcal response demonstrated greater induction during diestrus phase infection. Greater activity of granulocyte adhesion and diapedesis regulators during diestrus infection, particularly in chemokines and diapedesis regulators, was also shown. In addition to a greater induction of the common anti-gonococcal response, Gene Set Enrichment Analysis identified a diestrus-specific induction of type-1 interferon signaling pathways., Conclusions: This transcriptional analysis of murine uterine gonococcal infection during distinct points in the natural reproductive cycle provided evidence for a common anti-gonococcal response characterized by significant induction of granulocyte chemokine expression and high proinflammatory mediators. The basic biology of this host response to N. gonorrhoeae in estrus and diestrus is similar at the pathway level but varies drastically in magnitude. Overlaying this, we observed type-1 interferon induction specifically in diestrus infection where greater pathology is observed. This supports recent work suggesting this pathway has a significant, possibly host-detrimental, function in gonococcal infection. Together these findings lay the groundwork for further examination of the role of interferons in gonococcal infection. Additionally, this work enables the implementation of the diestrus uterine infection model using the newly characterized host response as a marker of pathology and its prevention as a correlate of candidate vaccine efficacy and ability to protect against the devastating consequences of N. gonorrhoeae-associated sequelae.

The gonococcal Opa proteins are an antigenically variable family of surface adhesins that bind human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CEACAM3, CEACAM5, and/or CEACAM6, cell surface glycoproteins that are differentially expressed on a broad spectrum of human cells and tissues. While they are presumed to be important for infection, the significance of various Opa-CEACAM-mediated cellular interactions in the context of the genital tract has remained unclear. Here, we observed that CEACAM1 and CEACAM5 are differentially expressed on epithelia lining the upper and lower portions of the human female genital tract, respectively. Using transgenic mouse lines expressing human CEACAMs in a manner that reflects this differential pattern, we considered the impact of Opa-CEACAM interactions during uncomplicated lower genital tract infections versus during pelvic inflammatory disease. Our results demonstrate that Opa-CEACAM5 binding on vaginal epithelia facilitates the long-term colonization of the lower genital tract, while Opa protein binding to CEACAM1 on uterine epithelia enhances gonococcal association and penetration into these tissues. While these Opa-dependent interactions with CEACAM-expressing epithelial surfaces promote infection, Opa binding by neutrophil-expressed CEACAMs counterbalances this by facilitating more effective gonococcal clearance. Furthermore, during uterine infections, CEACAM-dependent tissue invasion aggravates disease pathology by increasing the acute inflammatory response. Together, these findings demonstrate that the outcome of infection is determined by both the cell type-specific expression of human CEACAMs and the CEACAM specificity of the Opa variants expressed, which combine to determine the level of gonococcal association with the genital mucosa versus the extent of CEACAM-dependent inflammation and gonococcal clearance by neutrophils., (Copyright © 2018 American Society for Microbiology.)

TOLL-like receptor (TLR) ligands activate both innate and adaptive immune cells, while modulating the cellular immune response. The outer membrane protein (OMP) from Neisseria meninigitidis, PorB, is a naturally occurring TLR2 ligand and functions as an adjuvant. Here, we demonstrate that PorB increases the level of OVA in the endo-/lysosomal cellular compartment of BMDCs, increases antigen presenting cell (APC) trafficking to draining lymph nodes, and enhances antigen cross-presentation. PorB is capable of mounting an antigen specific T cell response by efficiently stimulating antigen cross-presentation in vivo and in vitro assessed by BMDC OT-I cocultivation assays. The enhanced antigen cross-presentation and the increased APC recruitment to secondary lymphoid tissues expand the scope of known adjuvant effects of PorB on the immune system. Our findings lead to a better understanding of how TLR-ligand based adjuvants can alter and modulate immune responses.

Vaccines are critical in the fight against infectious diseases, and immune-stimulating adjuvants are essential for enhancing vaccine efficacy. However, the precise mechanisms of action of most adjuvants are unknown. There is an urgent need for customized and adjuvant formulated vaccines against immune evading pathogens that remain a risk today. Understanding the specific role of various cell types in adjuvant-induced protective immune responses is vital for an effective vaccine design. We have investigated the role of cell-specific MyD88 signaling in vaccine adjuvant activity in vivo , using Neisserial porin B (PorB), a TLR2 ligand-based adjuvant, compared with an endosomal TLR9 ligand (CpG) and toll-like receptor (TLR)-independent (alum, MF59) adjuvants. We found that intact MyD88 signaling is essential, separately, in all three antigen-presenting cell types [B cells, macrophages, and dendritic cells (DCs)] for optimal TLR ligand-based adjuvant activity. The role of MyD88 signaling in B cell and DC in vaccine adjuvant has been previously investigated. In this study, we now demonstrate that the immune response was also reduced in mice with macrophage-specific MyD88 deletion (Mac-MyD88 -/- ). We demonstrate that TLR-dependent adjuvants are potent inducers of germinal center (GC) responses, but GCs are nearly absent in Mac-MyD88 -/- mice following immunization with TLR-dependent adjuvants PorB or CpG, but not with TLR-independent adjuvants MF59 or alum. Our findings reveal a unique and here-to-for unrecognized importance of intact MyD88 signaling in macrophages, to allow for a robust vaccine-induced immune responses when TLR ligand-based adjuvants are used.

Neisseria meningitidis (NM) is an opportunistic gram-negative human pathogen that colonizes the human nasopharyngeal epithelium. Asymptomatic carriage is common, but some meningococcal strains can invade nasopharyngeal epithelial cells and proceed to cause severe and often fatal infections. Invasion is predominantly driven by expression of bacterial virulence factors and host cell cognate receptors for bacterial recognition. Porins are among the Neisserial components involved in host cell activation and bacterial internalization processes. Similar to other virulence factors, porins present antigenic and structure variability among strains. Such sequence variability in the surface-exposed loop regions has been correlated to bacterial invasiveness and to variability in host cell responses via Toll-like receptor 2 (TLR2). Here, we examined whether TLR2 signaling by porins influences recovery of intracellular Neisseriae from epithelial cells in vitro. Our results show that TLR2 stimulation, either by the organism or exogenously, generally enhances Neisseriae internalization by epithelial cells. TLR2-driven intracellular signaling via ERK1/2, JNK and particularly NF-κB plays a role in this process. Based on these results, it is possible that expression of porin sequence variants that strongly induce TLR2 activation may be a mechanism to enhance the invasive features of pathogenic Neisseriae strains., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Unlabelled: There is an urgent need for the development of an antigonococcal vaccine due to the increasing drug resistance found in this pathogen. The U.S. Centers for Disease Control (CDC) have identified multidrug-resistant gonococci (GC) as among 3 "urgent" hazard-level threats to the U.S., Population: In light of this, on 29 to 30 June 2015, the National Institute for Allergy and Infectious Diseases (NIAID) sponsored a workshop entitled "Gonorrhea Vaccines: the Way Forward." The goal of the workshop was to gather leaders in the field to discuss several key questions on the current status of gonorrhea vaccine research and the path forward to a licensed gonorrhea vaccine. Representatives from academia, industry, U.S. Government agencies, and a state health department were in attendance. This review summarizes each of the 4 scientific sessions and a series of 4 breakout sessions that occurred during the one and a half days of the workshop. Topics raised as high priority for future development included (i) reinvigoration of basic research to understand gonococcal infection and immunity to allow intervention in processes essential for infection; (ii) clinical infection studies to establish parallels and distinctions between in vitro and animal infection models versus natural human genital and pharyngeal infection and to inform in silico modeling of vaccine impact; and (iii) development of an integrated pipeline for preclinical and early clinical evaluation and direct comparisons of potential vaccine antigens and adjuvants and routes of delivery., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Women with asymptomatic Neisseria gonorrhoeae infection are at risk of developing pelvic inflammatory disease (PID) if the bacteria ascend from the endocervix into the uterus and oviducts. Factors that affect disease severity, ranging from mild discomfort to severe inflammation, pain, and infertility, remain elusive. Herein we perform direct transcervical inoculation of N. gonorrhoeae into the uterus of mice to establish an infection that leads to PID. Profoundly different disease outcomes were apparent at different stages of the reproductive cycle. Mice that were infected during the diestrus stage of the reproductive cycle displayed extensive gonococcal penetration into the submucosa, severe inflammation, and clinical signs reflecting discomfort. Meanwhile, infection during the intervening estrus stage showed only modest effects. Furthermore, a gonococcal-specific humoral response was only elicited following the penetrative upper genital tract (UGT) infection during diestrus but not estrus. Strikingly, the potential for antibodies to contribute to protection during re-infection also depends upon the reproductive stage, as antigonococcal antibodies within the genital tract were markedly higher when mice were in diestrus. Combined, this work establishes a robust new model reflecting gonococcal PID in humans and reveals how the reproductive cycle determines the pathogenic outcome of gonococcal infections of the UGT.

Objective: To examine inappropriate antibiotic prescribing for acute respiratory tract infections (RTIs) in ambulatory care to help target antimicrobial stewardship interventions. Design and Setting Retrospective analysis of RTI visits within general internal medicine (GIM) and family medicine (FM) ambulatory practices at an inner-city academic medical center from 2008 to 2010., Methods: Patient, physician, and practice characteristics were analyzed using multivariable logistic regression to determine factors predictive of inappropriate prescribing; physicians in the highest and lowest antibiotic-prescribing quartiles were compared using χ2 analysis., Results: Visits with FM providers, female gender, and self-reported race/ethnicity as white or Hispanic were significantly associated with inappropriate antibiotic prescribing. Physicians in the lowest quartile prescribed antibiotics for 5%-28% (mean, 21%) of RTI visits; physicians in the highest quartile prescribed antibiotics for 54%-85% (mean, 65%) of RTI visits. High prescribers had fewer African-American patients and more patients who were younger and privately insured. High prescribers had more patients with chronic lung disease. A GIM practice pod with a low prescriber was 3.0 times more likely to have a second low prescriber than other practice pods, whereas pods with a high prescriber were 1.3 times more likely to have a second high prescriber., Conclusions: Medical specialty was the only physician factor predictive of inappropriate prescribing when patient gender, race, and comorbidities were taken into account. Possible disparities in care need further study. Stewardship education in medical school, enlisting low prescribers as physician leaders, and targeting interventions to the highest prescribers might be more effective approaches to antimicrobial stewardship.

Among all Neisseriae species, Neisseria meningitidis and Neisseria gonorrhoeae are the only human pathogens, causative agents of bacterial meningitis and gonorrhoea, respectively. PorB, a pan-Neisseriae trimeric porin that mediates diffusive transport of essential molecules across the bacterial outer membrane, is also known to activate host innate immunity via Toll-like receptor 2 (TLR2)-mediated signaling. The molecular mechanism of PorB binding to TLR2 is not known, but it has been hypothesized that electrostatic interactions contribute to ligand/receptor binding. Strain-specific sequence variability in the surface-exposed loops of PorB which are potentially implicated in TLR2 binding, may explain the difference in TLR2-mediated cell activation in vitro by PorB homologs from the commensal Neisseriae lactamica and the pathogen N. meningitidis. Here, we report a comparative structural analysis of PorB from N. meningitidis serogroup B strain 8765 (63% sequence homology with PorB from N. meningitidis serogroup W135) and a mutant in which amino acid substitutions in the extracellular loop 7 lead to significantly reduced TLR2-dependent activity in vitro. We observe that this mutation both alters the loop conformation and causes dramatic changes of electrostatic surface charge, both of which may affect TLR2 recognition and signaling., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Porphyromonas gingivalis is a primary etiological agent of chronic periodontal disease, an infection-driven chronic inflammatory disease that leads to the resorption of tooth-supporting alveolar bone. We previously reported that TLR2 is required for P. gingivalis-induced alveolar bone loss in vivo, and our in vitro work implicated TNF as a key downstream mediator. In this study, we show that TNF-deficient (Tnf(-/-)) mice are resistant to alveolar bone loss following oral infection with P. gingivalis, and thus establish a central role for TNF in experimental periodontal disease. Using bone marrow-derived macrophages (BMDM) from wild-type and gene-specific knockout mice, we demonstrate that the initial inflammatory response to P. gingivalis in naive macrophages is MyD88 dependent and requires cooperative signaling of TLR2 and TLR4. The ability of P. gingivalis to activate cells via TLR2 or TLR4 was confirmed in TLR2- or TLR4-transformed human embryonic kidney cells. Additional studies using bacterial mutants demonstrated a role for fimbriae in the modulation of TLR-mediated activation of NF-κB. Whereas both TLR2 and TLR4 contributed to TNF production in naive macrophages, P. gingivalis preferentially exploited TLR2 in endotoxin-tolerant BMDM to trigger excessive TNF production. We found that TNF induced surface TLR2 expression and augmented TLR-induced cytokine production in P. gingivalis-stimulated BMDM, establishing a previously unidentified TNF-dependent feedback loop. Adoptive transfer of TLR2-expressing macrophages to TLR2-deficient mice restored the ability of P. gingivalis to induce alveolar bone loss in vivo. Collectively, our results identify a TLR2- and TNF-dependent macrophage-specific mechanism underlying pathogen-induced inflammatory bone loss in vivo.

Toll-like receptors (TLRs) play a major role in host mucosal and systemic defense mechanisms by recognizing a diverse array of conserved pathogen-associated molecular patterns (PAMPs). TLR2, with TLR1 and TLR6, recognizes structurally diverse bacterial products such as lipidated factors (lipoproteins and peptidoglycans) and nonlipidated proteins, i.e., bacterial porins. PorB is a pan-neisserial porin expressed regardless of organisms' pathogenicity. However, commensal Neisseria lactamica organisms and purified N. lactamica PorB (published elsewhere as Nlac PorB) induce TLR2-dependent proinflammatory responses of lower magnitude than N. meningitidis organisms and N. meningitidis PorB (published elsewhere as Nme PorB). Both PorB types bind to TLR2 in vitro but with different apparent specificities. The structural and molecular details of PorB-TLR2 interaction are only beginning to be unraveled and may be due to electrostatic attraction. PorB molecules have significant strain-specific sequence variability within surface-exposed regions (loops) putatively involved in TLR2 interaction. By constructing chimeric recombinant PorB loop mutants in which surface-exposed loop residues have been switched between N. lactamica PorB and N. meningitidis PorB, we identified residues in loop 5 and loop 7 that influence TLR2-dependent cell activation using HEK cells and BEAS-2B cells. These loops are not uniquely responsible for PorB interaction with TLR2, but NF-κB and MAP kinases signaling downstream of TLR2 recognition are likely influenced by a hypothetical "TLR2-binding signature" within the sequence of PorB surface-exposed loops. Consistent with the effect of purified PorB in vitro, a chimeric N. meningitidis strain expressing N. lactamica PorB induces lower levels of interleukin 8 (IL-8) secretion than wild-type N. meningitidis, suggesting a role for PorB in induction of host cell activation by whole bacteria.

Toll-like receptors (TLRs) are recognition molecules for multiple pathogens, including bacteria, viruses, fungi, and parasites. TLR2 forms heterodimers with TLR1 and TLR6, which is the initial step in a cascade of events leading to significant innate immune responses, development of adaptive immunity to pathogens and protection from immune sequelae related to infection with these pathogens. This review will discuss the current status of TLR2 mediated immune responses by recognition of pathogen-associated molecular patterns (PAMPS) on these organisms. We will emphasize both canonical and non-canonical responses to TLR2 ligands with emphasis on whether the inflammation induced by these responses contributes to the disease state or to protection from diseases.

In vivo anti-polysaccharide Ig responses to isolated polysaccharide (PS) are T cell independent, rapid, and fail to generate memory. However, little is known regarding PS-specific Ig responses to intact gram-positive and gram-negative extracellular bacteria. We previously demonstrated that intact heat-killed Streptococcus pneumoniae, a gram-positive bacterium, elicited a rapid primary pneumococcal capsular PS (PPS) response in mice that was dependent on CD4(+) T cells, B7-dependent costimulation, and CD40-CD40L interactions. However, this response was ICOS independent and failed to generate a boosted PPS-specific secondary IgG response. In the current study, we analyzed the murine meningococcal type C PS (MCPS)-specific Ig response to i.p.-injected intact, heat-killed Neisseria meningitidis, serogroup C (MenC), a gram-negative bacterium. In contrast to S. pneumoniae, the IgG anti-MCPS response to MenC exhibited delayed primary kinetics and was highly boosted after secondary immunization, whereas the IgG anti-MCPS response to isolated MCPS was rapid, without secondary boosting, and consisted of only IgG1 and IgG3, as opposed to all four IgG isotypes in response to intact MenC. The secondary, but not primary, IgG anti-MCPS response to MenC was dependent on CD4(+) T cells, CD40L, CD28, and ICOS. The primary and secondary IgG anti-MCPS responses were lower in TLR4-defective (C3H/HeJ) but not TLR2(-/-) or MyD88(-/-) mice, but secondary boosting was still observed. Of interest, coimmunization of S. pneumoniae and MenC resulted in a boosted secondary IgG anti-PPS response to S. pneumoniae. Our data demonstrate that the nature of the in vivo anti-PS response is markedly influenced by the composition and/or architecture of the bacterial subcapsular domain.

The process of cellular apoptosis is mediated by a number of microbial pathogens to modulate host defense mechanisms. Inhibition of apoptosis is thought to favor microbial survival, replication or immune evasion, while induction of apoptosis is likely to promote escape of the organisms from host cells. Several studies have reported that infection with Neisseria spp. can inhibit or reduce apoptotic cell death, thus allowing adaptation, intracellular replication, and immune evasion, events that are likely to spread infection. In this chapter, various techniques are described for direct measurement of host cell responses to infection with Neisseria meningitidis and to treatment with pure Neisseria porins, the major proteins found in the outer membrane of the pathogen.

There is a need for developing vaccines that elicit mucosal immunity. Although oral or nasal vaccination methods would be ideal, current strategies have yielded mixed success. Toll-like receptor 2 (TLR2) ligands are effective adjuvants and are currently used in the Haemophilus influenzae type B vaccine. Induction of humoral immunity in the mucosa is critical for effective vaccination; thus, we sought to determine the effects of TLR2 ligands on human mucosal B cell differentiation. We demonstrate that TLR2 ligands induce CCR9 and CCR10 expression by circulating B cells and increased chemotaxis to cognate chemokines CCL25 and CCL28 suggesting that TLR2 induces B cell homing to the gastrointestinal tract. TLR2 stimulation of B cells also induced J chain and IgA production demonstrating the induction of mucosal-like antibody secreting cells. These observations suggest that vaccines containing TLR2-ligands as adjuvants could induce mucosal B cell immunity even when delivered in a non-mucosal manner., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin's surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.

Background: Francisella tularensis causes severe pulmonary disease, and nasal vaccination could be the ideal measure to effectively prevent it. Nevertheless, the efficacy of this type of vaccine is influenced by the lack of an effective mucosal adjuvant., Methodology/principal Findings: Mice were immunized via the nasal route with lipopolysaccharide isolated from F. tularensis and neisserial recombinant PorB as an adjuvant candidate. Then, mice were challenged via the same route with the F. tularensis attenuated live vaccine strain (LVS). Mouse survival and analysis of a number of immune parameters were conducted following intranasal challenge. Vaccination induced a systemic antibody response and 70% of mice were protected from challenge as showed by their improved survival and weight regain. Lungs from mice recovering from infection presented prominent lymphoid aggregates in peribronchial and perivascular areas, consistent with the location of bronchus-associated lymphoid tissue (BALT). BALT areas contained proliferating B and T cells, germinal centers, T cell infiltrates, dendritic cells (DCs). We also observed local production of antibody generating cells and homeostatic chemokines in BALT areas., Conclusions: These data indicate that PorB might be an optimal adjuvant candidate for improving the protective effect of F. tularensis antigens. The presence of BALT induced after intranasal challenge in vaccinated mice might play a role in regulation of local immunity and long-term protection, but more work is needed to elucidate mechanisms that lead to its formation.

Neisseria meningitidis is a Gram-negative pathogenic bacteria responsible for bacterial meningitis and septicemia. Porins are the most represented outer membrane proteins in the pathogenic Neisseria species, functioning as pores for the exchange of ions, and are characterized by a trimeric beta-barrel structure. Neisserial porins have been shown to act as adjuvants in the immune response via activation of B cells and other antigen-presenting cells. Their effect on the immune response is mediated by upregulation of the costimulatory molecule B7-2 (CD86) on the surface of antigen-presenting cells, an effect that is dependent on Toll-like receptor (TLR)2 and MyD88, through a cascade of signal transduction events mediated by direct binding of the porin to the TLR2-TLR1 heterodimer. This article summarizes work carried out investigating the mechanisms of the immune stimulating capacity of the neisserial porins (specifically meningococcal PorB), emphasizing cellular events involved in antigen-presenting cell activation and induction of expression of cell surface molecules involved in the immune response.

Meningococcal porin PorB is an inhibitor of apoptosis induced via the intrinsic pathway in various cell types. This effect is attributed to prevention of mitochondrial depolarization and of subsequent release of proapoptotic mitochondrial factors. To determine whether apoptosis is globally inhibited by PorB, we compared the intrinsic and extrinsic pathways in HeLa cells. Interestingly, PorB does not prevent extrinsic apoptosis induced by tumor necrosis factor alpha plus cycloheximide, suggesting a unique mitochondrial pathway specificity. Several intracellular factors regulated by NF-kappaB, including members of the Bcl-2 family and of the inhibitor of apoptosis (IAP) family, play major roles in controlling apoptosis, and some of them are thought to contribute to the antiapoptotic effect of the gonococcal porin, PIB. However, most of the members of the Bcl-2 family and the IAP family are not induced by meningococcal PorB in HeLa cells, with the exception of Bfl-1/A1. Interestingly, PorB does not induce NF-kappaB activation in HeLa cells, likely due to a lack of Toll-like receptor 2 (TLR2) expression in these cells. Bfl-1/A1 expression is also regulated by CBF1, a nuclear component of the Notch signaling pathway, independent of NF-kappaB activation. Since HeLa cells are protected by PorB from intrinsic apoptosis events, regardless of TLR2 and NF-kappaB expression, the possibility of a contribution of alternative signaling pathways to this effect cannot be excluded. In this paper, we describe an initial dissection of the cascade of cellular events involved in the antiapoptotic effect of PorB in the absence of TLR2.

Several microbial pathogens can modulate the host apoptotic response to infection, which may contribute to immune evasion. Various studies have reported that infection with the sexually transmitted disease pathogen Neisseria gonorrhoeae can either inhibit or induce apoptosis. N. gonorrhoeae infection initiates at the mucosal epithelium, and in women, cells from the ectocervix and endocervix are among the first host cells encountered by this pathogen. In this study, we defined the antiapoptotic effect of N. gonorrhoeae infection in human endocervical epithelial cells (End/E6E7 cells). We first established that N. gonorrhoeae strain FA1090B failed to induce cell death in End/E6E7 cells. Subsequently, we demonstrated that stimulation with N. gonorrhoeae protected these cells from staurosporine (STS)-induced apoptosis. Importantly, only End/E6E7 cells incubated with live bacteria and in direct association with N. gonorrhoeae were protected from STS-induced apoptosis, while heat-killed and antibiotic-killed bacteria failed to induce protection. Stimulation of End/E6E7 cells with live N. gonorrhoeae induced NF-kappaB activation and resulted in increased gene expression of the NF-kappaB-regulated antiapoptotic genes bfl-1, cIAP-2, and c-FLIP. Furthermore, cIAP-2 protein levels also increased in End/E6E7 cells incubated with gonococci. Collectively, our results indicate that the antiapoptotic effect of N. gonorrhoeae in human endocervical epithelial cells results from live infection via expression of host antiapoptotic proteins. Securing an intracellular niche through the inhibition of apoptosis may be an important mechanism utilized by N. gonorrhoeae for microbial survival and immune evasion in cervical epithelial cells.

Francisella tularensis causes severe pneumonia that can be fatal if it is left untreated. Due to its potential use as a biological weapon, research is being conducted to develop an effective vaccine and to select and study adjuvant molecules able to generate a better and long-lasting protective effect. PorB, a porin from Neisseria meningitidis, is a well-established Toll-like receptor 2 ligand and has been shown to be a promising vaccine adjuvant candidate due to its ability to enhance the T-cell costimulatory activity of antigen-presenting cells both in vitro and in vivo. BALB/c mice were immunized with lipopolysaccharide (LPS) isolated from the F. tularensis subsp. holarctica live vaccine strain (LVS), with or without PorB from N. meningitidis, and the antibody levels induced during the vaccination regimen and the level of protection against intranasal challenge with LVS were determined. Antigen administered alone induced a specific F. tularensis LPS immunoglobulin M (IgM) response that was not maintained over the weeks and that conferred protection to only 25% of the mice. In contrast, F. tularensis LPS given in combination with neisserial PorB induced consistent levels of specific IgM throughout the immunization and increased the proportion of surviving mice to 70%. Postchallenge cytokine analysis showed that interleukin-6 (IL-6), monocyte chemoattractant protein 1, and gamma interferon were markers of mortality and that IL-1beta was a correlate of survival, independent of the presence of PorB as an adjuvant. These data indicate that neisserial PorB might be an optimal candidate adjuvant for improving the protective effect of F. tularensis LPS and other subunit vaccines against tularemia, but there is still a need to test its efficacy against virulent type A and type B F. tularensis strains.

Neisserial porins are potent immune adjuvants and have been demonstrated to stimulate and induce the activation of human and murine B lymphocytes. Their immunopotentiating ability is due largely to the upregulation of the surface expression of the costimulatory ligand CD86 (B7-2) on B cells and other antigen-presenting cells. Porin-induced activation is dependent on the innate immune pattern recognition receptor Toll-like receptor 2 (TLR2). These data have led us to investigate the signal transduction events induced by PorB from Neisseria meningitidis and then, using inhibitors of these pathways, to establish the mechanism by which this bacterial major outer membrane protein induces CD86 upregulation and the proliferation of murine B cells. PorB was able to induce (i) protein tyrosine kinase (PTK) activity, (ii) the phosphorylation of Erk1 and Erk2, and (iii) IkappaB-alpha phosphorylation, leading to NF-kappaB nuclear translocation in B cells in a TLR2-dependent manner. PorB-induced NF-kappaB nuclear translocation was not dependent on either PTK or Erk1/2 activities. However, B-cell proliferation and the induction of increased surface expression of CD86 by PorB were dependent on PTK activity and not Erk1/2 activation. In conclusion, PorB acts through TLR2 as a B-cell mitogen, triggering tyrosine phosphorylation of various cellular proteins that are involved in proliferation and CD86 expression, as well as the phosphorylation of Erk1/2, which is not necessary for CD86 upregulation or the proliferation of B cells.

Porins from pathogenic Neisseriae are among several bacterial products with immune adjuvant activity. Neisseria meningitidis (Nme) PorB, has been shown to induce immune cells activation in a TLR2-dependent manner and acts as a vaccine immune adjuvant. The PorB porin from Neisseria lactamica (Nlac), a common nasopharyngeal commensal, shares significant structural and functional similarities with Nme PorB. In this work we ask whether the immune adjuvant ability of porins from pathogenic Neisserial strains is a characteristic shared with porins from non-pathogenic Neisserial species or whether it is unique for bacterial products derived from microorganisms capable of inducing inflammation and disease. We evaluated the potential immune adjuvant effect of Nlac PorB in mice using ovalbumin (OVA) as a prototype antigen. Immunization with Nlac PorB/OVA induced high OVA-specific IgG and IgM titers compared to OVA alone, similar to other adjuvants such as Nme PorB and alum. High titers of IgG1 and IgG2b were detected as well as production of IL-4, IL-10, IL-12 and INF-gamma in response to Nlac PorB, consistent with induction of both a Th1-type and a Th2-type immune response. OVA-specific proliferation was also determined in splenocytes from Nlac PorB/OVA-immunized mice. In addition, B cell activation in vitro and cytokine production in response to Nlac PorB was found to be mediated by TLR2, in a similar manner to Nme PorB.

Francisella tularensis can cause severe disseminated disease after respiratory infection. The identification of factors involved in mortality or recovery following induction of tularemia in the mouse will improve our understanding of the natural history of this disease and facilitate future evaluation of vaccine candidate preparations. BALB/c mice were infected intranasally with the live vaccine strain (LVS) of F. tularensis subsp. holarctica and euthanized at different stages of disease to analyze the induction of immune molecules, gross anatomical features of organs, bacterial burdens, and progression of the histopathological changes in lung and spleen. Tissue-specific interleukin-6 (IL-6), macrophage inflammatory protein 2, and monocyte chemotactic protein 1 were immune markers of mortality, while anti-LVS immunoglobulin M and IL-1beta were associated with survival. Moribund mice had enlarged spleens and lungs, while surviving mice had even more prominent splenomegaly and normal-appearing lungs. Histopathology of the spleens of severely ill mice was characterized by disrupted lymphoid follicles and fragmented nuclei, while the spleens of survivors appeared healthy but with increased numbers of megakaryocytes and erythrocytes. Histopathology of the lungs of severely ill mice indicated severe pneumonia. Lungs of survivors at early time points showed increased inflammation, while at late times they appeared healthy with peribronchial lymphoid aggregates. Our results suggest that host immune factors are able to affect bacterial dissemination after respiratory tularemia, provide new insights regarding the pathological characteristics of pulmonary tularemia leading to systemic disease, and potentially identify immune markers associated with recovery from the disease.

Toll-like receptor (TLR) activation is primarily thought to affect antigen-presenting cells (APCs) by inducing an innate immune response that can subsequently activate the adaptive immune system. However, there are increasing data that demonstrate expression and activation of TLRs on T cells, thus providing evidence for a direct role for TLRs in the activation of an adaptive immune response. A study recently demonstrated that Pam3CSK {N-palmitoyl-S-[2,3-bis(palmitoloxy)-(2RS)-propyl]-Cys-Ser-Lys(4)}, a TLR2 agonist lipopeptide, activates T helper 1 (T(H)1) cells and induces interferon-gamma (IFN-gamma) production, even in the absence of TLR1, which differs from its mechanism of activation of APCs. Moreover, whereas Pam3CSK-stimulated IFN-gamma production by T(H)1 cells is ablated in the absence of both myeloid differentiation marker 88 (MyD88), an adaptor protein in the TLR pathway, and interleukin-1 receptor (IL-1R)-associated kinase-4 (IRAK4), the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase (JNK) are still phosphorylated. These data suggest that TLR2 activation of T(H)1 cells occurs through a mechanism different from that described for APCs and provides further evidence of direct TLR activation of the adaptive immune system.

Efficacious adjuvants are important components of new vaccines. The neisserial outer membrane protein, PorB, is a TLR2 ligand with unique adjuvant activity. We demonstrate that PorB promotes Th2-skewed cellular immune response to the model Ag, OVA, in mice, including Ag-specific recall eosinophil recruitment to the peritoneum. PorB induces chemokine secretion by myeloid cells using both TLR2-dependent and -independent mechanisms, suggesting that anatomical distribution of TLR2(+) cells may not be a limiting factor for potential vaccine strategies. The results from this study suggest that PorB, and other TLR2 ligands, may be ideal for use against pathogens where eosinophilia may be protective, such as parasitic helminths.

Cholera toxin (CT) is one of the most effective and widely studied mucosal adjuvants. Although the ADP-ribosylating A subunit has been implicated in augmenting immune responses, the receptor-binding B subunit (CT-B) has greater immunogenicity and may be a repository of adjuvant activity without potential toxicity. In order to elucidate mechanisms of immune modulation by CT-B alone, primary B cells and macrophages were assessed for responses to CT-B in vitro, as measured by the expression of cell surface markers, cellular signaling events, and cytokine secretion. Increased phosphorylation of multiple signaling molecules, including Erk1/2 and p38, was detected. CT-B also induced transactivation of the transcription elements cyclic AMP-responsive element and NF-kappaB, the latter of which was inhibited by phosphotyrosine inhibition. While specific inhibition of MEK1/2 did not reduce CT-B induction of cell surface marker expression, it did attenuate CT-B-mediated interleukin-6 secretion. These data show that CT-B induces a set of signaling events related to cellular activation, surface molecule expression, and cytokine production that has potential implications for elucidating CT-B adjuvant activity in the absence of enzymatically active holotoxin.

Human B cells likely have a major role in the adjuvant activity of Toll-like receptor (TLR) 9 agonists by enhancing innate and adaptive immune responses. As several TLR2 ligands are promising vaccine adjuvant candidates, our aim was to characterize the effects of TLR2 stimulation on human B cell activation and differentiation using cells derived from healthy peripheral blood (PB), spleen, and diseased tonsils. We found a subset of partially differentiated TLR2+ PB and splenic B cells which responds to TLR2 agonists by mediating events involved in germinal center formation, such as upregulating CD77 and secreting chemokines. Furthermore, we show that TLR2-activated monocytes induce B cells to secrete significant quantities of IgM. Finally, activated TLR2+ B cells from tonsils are induced to secrete IgM directly by TLR2 ligands. Thus, TLR2 is likely involved in specific B cell-mediated functions and may be a viable vaccine adjuvant target in humans.

TLR2 plays a key role in the initiation of the cellular innate immune responses by a wide range of bacterial products. TLRs signaling, including TLR2 and its coreceptors TLR1 and TLR6, is mediated by a number of specific ligands. Although many of the TLR-mediated cell signaling pathways have been elucidated in the past few years, the molecular mechanisms that lead to cell activation are still poorly understood. In this study, we investigate the interaction of PorB from Neisseria meningitidis with TLR2 and describe the direct binding of a bacterial protein to TLR2 for the first time. Using labeled PorB, we demonstrate its binding to TLR2 both in its soluble form in vitro, and when it is over-expressed on the surface of human embryonic kidney 293 cells. We also show that TLR2-mediated binding of PorB is directly related to cellular activation. In addition, using 293 cells expressing the chimeric TLR2/TLR1 and TLR2/TLR6 complexes, we report the selectivity of PorB binding to the TLR2/TLR1 heterodimer, which is required for initiating signaling in transfected 293 cells and in murine B cells. Together, these data provide new evidence that TLR2 recognizes PorB through direct binding, and that PorB-induced cell activation is mediated by a TLR2/TLR1 complex.

Purified pili and porin from Neisseria quickly mobilize calcium (Ca(2+)) stores in monocytes and epithelial cells, ultimately influencing host cell viability as well as bacterial intracellular survival. Here, we examined the Ca(2+) transients induced in human epithelial cells during infection by live, piliated N. gonorrhoeae. Porin induced an influx of Ca(2+) from the extracellular medium less than 60 s post infection. The porin-induced transient is followed by a pilus-induced release of Ca(2+) from intracellular stores. The timing of these events is similar to that observed using purified proteins. Interestingly, the porin-induced Ca(2+) flux is required for the pilus-induced transient, indicating that the pilus-induced Ca(2+) release is, itself, Ca(2+) dependent. Several lines of evidence indicate that porin is present on pili. Moreover, pilus retraction strongly influences the porin- and pilus-induced Ca(2+) fluxes. These and other results strongly suggest that the pilus and porin cooperate to modulate calcium signalling in epithelial cells, and propose a model to explain how N. gonorrhoeae triggers Ca(2+) transients in the initial stages of pilus-mediated attachment.

The outer membrane protein PorB of Neisseria meningitidis is a pore-forming protein which has various effects on eukaryotic cells. It has been shown to (1) up-regulate the surface expression of the co-stimulatory molecule CD86 and of MHC class II (which are TLR2/MyD88 dependent and related to the porin's immune-potentiating ability), (2) be involved in prevention of apoptosis by modulating the mitochondrial membrane potential, and (3) form pores in eukaryotic cells. As an outer membrane protein, its native trimeric form isolation is complicated by its insoluble nature, requiring the presence of detergent throughout the whole procedure, and by its tight association with other outer membrane components, such as neisserial LOS or lipoproteins. In this study, an improved chromatographic purification method to obtain an homogeneous product free of endotoxin and lipoprotein is described, without loss of any of the above-mentioned properties of the porin. Furthermore, we have investigated the requirement of the native trimeric structure for the porin's activity. Inactivation of functional PorB trimers into non-functional monomers was achieved by incubation on ice. Thus, routine long- and medium-term storage at low temperature may be a cause of porin inactivation.

Iron is limiting in the human host, and bacterial pathogens respond to this environment by regulating gene expression through the ferric uptake regulator protein (Fur). In vitro studies have demonstrated that Neisseria gonorrhoeae controls the expression of several critical genes through an iron- and Fur-mediated mechanism. While most in vitro experiments are designed to determine the response of N. gonorrhoeae to an exogenous iron concentration of zero, these organisms are unlikely to be exposed to such severe limitations of iron in vivo. To determine if N. gonorrhoeae expresses iron- and Fur-regulated genes in vivo during uncomplicated gonococcal infection, we examined gene expression profiles of specimens obtained from male subjects with urethral infections. RNA was isolated from urethral swab specimens and used as a template to amplify, by reverse transcriptase PCR (RT-PCR), gonococcal genes known to be regulated by iron and Fur (tbpA, tbpB, and fur). The constitutively expressed gonococcal rmp gene was used as a positive control. RT-PCR analysis indicated that gonorrhea-positive specimens where rmp expression was seen were also 93% (51/55) fbpA positive, 87% (48/55) tbpA positive, and 86% (14 of 16 tested) tbpB positive. In addition, we detected a fur transcript in 79% (37 of 47 tested) of positive specimens. We also measured increases in levels of immunoglobulin G antibody against TbpA (91%) and TbpB (73%) antigens in sera from infected male subjects compared to those in uninfected controls. A positive trend between tbpA gene expression and TbpA antibody levels in sera indicated a relationship between levels of gene expression and immune response in male subjects infected with gonorrhea for the first time. These results indicate that gonococcal iron- and Fur-regulated tbpA and tbpB genes are expressed in gonococcal infection and that male subjects with mucosal gonococcal infections exhibit antibodies to these proteins.

Neisserial porins have been shown to act as B cell mitogens and immune adjuvants. PorA and PorB are the major outer membrane porin proteins of the human pathogen Neisseria meningitidis. We have shown that the mechanism of the immunopotentiating capability of porin involves up-regulation of the T cell costimulatory ligand, CD86. Due to neisserial porin's ability to activate B cells and potentiate immune responses, we hypothesized that porin also employs the potent immune stimulatory function of dendritic cells (DC). We examined the ability of purified N. meningitidis PorB to induce maturation of murine splenic and bone marrow-derived DC. PorB treatment induced DC maturation, as demonstrated by increased expression of CD86 and class I and II MHC molecules. In addition, PorB not only enhanced the allostimulatory activity of DC, but also augmented the ability of DC to stimulate T cells in an Ag-specific manner. PorB-matured DC secreted the inflammatory cytokine IL-6, which may have implications for the adjuvant property of porin. Induction of IL-6 by PorB is also significant because IL-6 is one of a number of cytokines produced during infection with N. meningitidis and may be involved in the inflammatory process observed during infection and disease. We previously demonstrated the requirement of MyD88 and TLR2 for PorB-induced B cell activation. In the present study, MyD88 and TLR2 were also essential for PorB-induced DC activation. This work is significant for elucidating the mechanism(s) of neisserial porin's immune stimulatory activity.

Cells expressing Toll-like receptor (TLR), TLR2 in association with TLR1, TLR6 or some other unknown co-receptor can respond upon interaction with a large variety of microbial ligands. The variety of TLR2 ligands is the greatest among all the TLRs and this is due to the heterodimerization needed for TLR2 mediated responses. Like other TLRs, TLR2 signaling induces antigen presenting cell activation, pro-inflammatory cytokine production and increased expression of co-stimulatory ligand expression. These events are important for induction of innate immune responses and improved acquired immunity. There is strong suggestive evidence that alteration or lack of TLR2 function in vivo may correlate to decreased immune protection from pathogens that contain TLR2 ligands, but more work needs to be performed to strengthen this correlation.

Neisseria meningitidis and Neisseria gonorrhoeae are Gram-negative pathogenic bacteria responsible for bacterial meningitis and septicemia, and the sexually transmitted disease gonorrhea, respectively. Porins are the most represented outer membrane proteins in the pathogenic Neisseria species, functioning as pores for the exchange of ions, and are characterized by a trimeric beta-barrel structure. Neisserial porins have been shown to act as adjuvants in the immune response via activation of B cells and other antigen-presenting cells (APCs). Their effect on the immune response is mediated by upregulation of the costimulatory molecule B7-2 (CD86) on the surface of APCs, an effect that is Toll-like receptor 2- and MyD88-dependent. The effect of neisserial porins on the immune system also involves interaction with components of the complement cascade. Furthermore, neisserial porins co-localize with mitochondria of target cells, where they appear to modulate apoptosis.

We have previously shown that purified meningococcal porin PorB associates with mitochondria and prevents apoptosis of B cells, Jurkat cells and HeLa cells (Massari et al., 2000, Proc Natl Acad Sci USA 97: 9070-9075). This work examines if intact meningococci have a similar effect as purified porins. It was first determined that intact live meningococci do not induce apoptosis of HeLa cells and do not perturb mitochondrial physiology. This latter consideration is important as Neisserial porins affect the susceptibility of cells to apoptosis by preventing mitochondrial depolarization and cytochrome c release, events involved in the apoptosis cascade. Purified PorB or PorB from live bacteria were found to translocate into and interact with mitochondria. It was then determined whether treatment of HeLa cells with meningococci could prevent staurosporine-mediated apoptosis due to an effect of PorB on the mitochondrial parameters. Incubation of HeLa cells with live meningococci prevented staurosporine-induced apoptosis, as ascertained by measurements of mitochondrial potential, translocation of mitochondrial cytochrome c to the cytosol, caspases activation, and nuclear DNA degradation. These data are consistent with our previous findings that purified PorB associates with mitochondria and prevents apoptosis, and demonstrates that the mechanism by which whole meningococci protects cells from apoptosis is a result of direct interaction of neisserial porin with mitochondria.

The immunopotentiating activity of neisserial porins, the major outer membrane protein of the pathogenic Neisseria, is mediated by its ability to stimulate B cells and up-regulate the surface expression of B7-2. This ability is dependent on MyD88 and Toll-like receptor (TLR)2 expression, as demonstrated by a lack of a response by B cells from MyD88 or TLR2 knockout mice to the porins. Using previously described TLR2-dependent reporter constructs, these results were confirmed and were shown to be due to induction of NF-kappaB nuclear translocation. This is the first demonstration of known vaccine adjuvant to stimulate immune cells via TLR2.

The role of lipopolysaccharide (LPS) in the specific humoral response to meningococcal porins was investigated by measuring anti-PorA or -PorB antibody levels in mice immunized with wild-type meningococcal strain H44/76 or with its recently described LPS-negative mutant. Two murine strains were used for these immunizations: C3H/HeJ, which is LPS hyporesponsive, or C3H/HeOuJ, which is LPS responsive. A high level of anti-PorB immunoglobulin G (IgG) response was induced in both strains of mice immunized with either organism. The response induced by the wild-type strain was greater in C3H/HeOuJ mice than in C3H/HeJ mice, while the response induced by the LPS-negative mutant was similar in the two murine strains. Additionally, the anti-PorB response was similar in C3H/HeJ mice immunized with either bacterial strain. In general, the anti-PorA IgG response was lower than the anti-PorB response. These findings indicate that the presence of LPS is not essential for the induction of an antineisserial porin humoral response but can augment such a response. To determine whether LPS has any effect on the B-cell-stimulatory effect of neisserial porins (essential for the adjuvant activity of neisserial porins), B cells from both murine strains were incubated with outer membrane complexes (OMCs) prepared from strain H44/76 and its LPS-negative mutant. OMCs from either meningococcal strain were able to increase the surface expression of the costimulatory ligand B7-2 on B cells from either murine strain. Consistent with previously reported findings, LPS does not significantly affect the ability of neisserial porins to induce the costimulatory ligand B7-2.

Microorganisms or microbial products have been shown to induce or protect cells from activation-induced cell death or apoptosis (1-3). Induction of apoptosis by some bacterial invaders, like shigella, might aid in spread of the organism (4), whereas inhibition of apoptosis by other microbes might aid in furthering their intracellular survival (2,3). Viral products have been shown to inhibit apoptosis by mimicking anti-apoptotic related proteins (e.g., Bcl2, FLIPS, etc.) (2,3,5). Thus far, most investigators have demonstrated that bacteria either have no effect or induce apoptosis of various cell types, mainly cells that they encounter upon invasion, e.g., epithelial cells, fibroblasts, and so on. Apoptotic cell death is also a key control mechanism of immune responses (6), but, to date, there have not been many investigations into the effect of microbes on apoptosis in immune cells. Dysregulation of immune cells associated with a lack of apoptosis and abnormal Fas-mediated cell death have been associated with immune dysfunction and hyperimmune states (7).

IgG antibody levels to the meningococcal PorA and PorB proteins in 56 acute and convalescent phase sera from 25 patients with meningococcal disease were compared by immunoblotting and ELISA. Heat-treated outer membrane vesicles from strain 44/76 (B:15:P1.7, 16) served as antigens for immunoblotting, whereas purified P1.7,16 PorA and P15 PorB from the same strain were used as antigens in the ELISA. In the blotting method, IgG binding to the porins was determined by digital scanning of the immunoreactive bands and calculated relative to the PorA binding of a reference serum on each blot. The coefficient of variation for the reference serum was 21.6% (a total of 144 strips) with smaller variations for each day's experiments. Blotting of all 56 sera at the standard 1/200 dilution measured anti-PorA and anti-PorB levels that correlated with those obtained by ELISA (Spearman rank-order correlation coefficient r(s)=0.48; P<0.001). At this dilution, the anti-PorA (r(s)=0.52; P<0. 004) and anti-PorB (r(s)=0.60; P<0.001) levels of the convalescent phase sera (n=29) corresponded with the ELISA measurements, whereas no correlation was found with the results for the acute phase sera, which mostly had low ELISA antibody levels (<2 microg/ml IgG). A corresponding blot analysis of convalescent sera from the seven patients, who had received the 44/76 outer membrane vesicle vaccine, demonstrated a high correlation coefficient for the anti-PorA levels (r(s)=0.95; P<0.001) vs. the ELISA results. No such correlation was observed for the PorB response in these sera, being nine-fold higher than the PorA response, because of a prozone effect on the blots at the standard dilution. However, blotting at a higher serum dilution (1/2000) resulted in anti-PorB levels that also correlated strongly (r(s)=0.93 P<0.001) with the ELISA measurements.

Neisserial porins are strong immune adjuvants and B cell activators. The effect of neisserial porin PorB on activation-induced cell death was investigated, as a potential additional mechanism of the porin's immunopotentiating ability. Neisserial porins interact with target cells to localize intracellularly in the mitochondrial compartment without negatively affecting cellular survival. Pretreatment with Neisseria meningitidis PorB porin decreased or abrogated the mitochondrial damage induced by apoptotic stimuli. In addition, end stage determinants of apoptosis, including DNA breakdown, were diminished by PorB. Immunoprecipitation experiments revealed that PorB interacts with the mitochondrial porin VDAC (voltage-dependent anion channel). The mechanism of the antiapoptotic effect of neisserial porins could be explained by the protein-protein interaction of PorB with VDAC, similar to the interaction of VDAC with antiapoptotic Bcl-2 proteins, resulting in an enhancement of cell survival and continued activation of B cells.