Proinflammatory Microenvironment During Kingella kingae Infection Modulates Osteoclastogenesis.

Kingella kingae is an emerging pathogen that causes septic arthritis, osteomyelitis, and bacteremia in children from 6 to 48 months of age. The presence of bacteria within or near the bone is associated with an inflammatory process that results in osteolysis, but the underlying pathogenic mechanisms involved are largely unknown. To determine the link between K. kingae and bone loss, we have assessed whether infection per se or through the genesis of a pro-inflammatory microenvironment can promote osteoclastogenesis. For that purpose, we examined both the direct effect of K. kingae and the immune-mediated mechanism involved in K. kingae-infected macrophage-induced osteoclastogenesis. Our results indicate that osteoclastogenesis is stimulated by K. kingae infection directly and indirectly by fueling a potent pro-inflammatory response that drives macrophages to undergo functional osteoclasts via TNF-α and IL-1ß induction. Such osteoclastogenic capability of K. kingae is counteracted by their outer membrane vesicles (OMV) in a concentration-dependent manner. In conclusion, this model allowed elucidating the interplay between the K. kingae and their OMV to modulate osteoclastogenesis from exposed macrophages, thus contributing to the modulation in joint and bone damage.

Virulence determinants of the emerging pathogen Kingella kingae.

Kingella kingae is a gram-negative coccobacillus that is a fastidious commensal organism in the oropharynx and is being recognized increasingly as a common cause of osteoarticular infections and other invasive diseases in young children. The pathogenesis of K. kingae disease begins with bacterial adherence to respiratory epithelium, followed by translocation across the epithelial barrier, survival in the bloodstream, and dissemination to distant sites, including bones, joints, and the endocardium, among others. Characterization of the determinants of K. kingae pathogenicity has revealed a novel model of adherence that involves the interplay of type IV pili, a non-pilus adhesin, and a polysaccharide capsule and a novel model of resistance to serum killing and neutrophil killing that involves complementary functions of a polysaccharide capsule and an exopolysaccharide. These models likely apply to other bacterial pathogens as well.

Prevalence and serogroup changes of Neisseria meningitidis in South Korea, 2010-2016.

Determination of the major serogroups is an important step for establishing a vaccine programme and management strategy targeting Neisseria meningitidis. From April 2010 to November 2016, a total of 25 N. meningitidis isolates were collected in South Korea, in collaboration with the Korean Society of Clinical Microbiology. Among isolates, 19 isolates were recovered from blood and/or cerebrospinal fluid (CSF) in 46 patients who suffered from invasive meningococcal disease (IMD), and six isolates were found in sputum or the throat. The most common serogroup was serogroup B (overall, 36%, n = 9/25; IMD, 37%, n = 7/19), which was isolated in every year of the research period except for 2011. There were five serogroup W isolates recovered from patients in military service. W was no longer isolated after initiation of a vaccine programme for military trainees, but serogroup B caused meningitis in an army recruit training centre in 2015. In MLST analysis, 14 sequence types were found, and all isolates belonging to W showed the same molecular epidemiologic characteristics (W:P1.5-1, 2-2:F3-9:ST-8912). All isolates showed susceptibility to ceftriaxone, meropenem, ciprofloxacin, minocycline, and rifampin; however, the susceptibility rates to penicillin and ampicillin for isolates with W and C capsules were 22% and 30%, respectively.

Neisseria meningitidis, pathogenetic mechanisms to overcome the human immune defences.

Neisseria meningitidis is hosted only by humans and colonizes the nasopharynx; it survives in the human body by reaching an equilibrium with its exclusive host. Indeed, while cases of invasive disease are rare, the number of asymptomatic Neisseria meningitides carriers is far higher. The aim of this paper is to summarize the current knowledge of survival strategies of Neisseria meningitides against the human immune defences. Neisseria meningitidis possesses a variety of adaptive characteristics which enable it to avoid being killed by the immune system, such as the capsule, the lipopolysaccharide, groups of proteins that block the action of the antimicrobial proteins (AMP), proteins that inhibit the complement system, and components that prevent both the maturation and the perfect functioning of phagocytes. The main means of adhesion of Neisseria meningitides to the host cells are Pili, constituted by several proteins of whom the most important is Pilin E. Opacity-associated proteins (Opa) and (Opc) are two proteins that make an important contribution to the process of adhesion to the cell. Porins A and B contribute to neisserial adhesion and penetration into the cells, and also inhibit the complement system. Factor H binding protein (fhbp) binds factor H, allowing the bacteria to survive in the blood. Neisserial adhesin A (NadA) is a minor adhesin that is expressed by 50% of the pathogenic strains. NadA is known to be involved in cell adhesion and invasion and in the induction of proinflammatory cytokines. Neisserial heparin binding antigen (NHBA) binds heparin, thus increasing the resistance of the bacterium in the serum.

CEACAM1 negatively regulates IL-1ß production in LPS activated neutrophils by recruiting SHP-1 to a SYK-TLR4-CEACAM1 complex.

LPS-activated neutrophils secrete IL-1ß by activation of TLR-4. Based on studies in macrophages, it is likely that ROS and lysosomal destabilization regulated by Syk activation may also be involved. Since neutrophils have abundant expression of the ITIM-containing co-receptor CEACAM1 and Gram-negative bacteria such as Neisseria utilize CEACAM1 as a receptor that inhibits inflammation, we hypothesized that the overall production of IL-1ß in LPS treated neutrophils may be negatively regulated by CEACAM1. We found that LPS treated neutrophils induced phosphorylation of Syk resulting in the formation of a complex including TLR4, p-Syk, and p-CEACAM1, which in turn, recruited the inhibitory phosphatase SHP-1. LPS treatment leads to ROS production, lysosomal damage, caspase-1 activation and IL-1ß secretion in neutrophils. The absence of this regulation in Ceacam1⁻/⁻ neutrophils led to hyper production of IL-1ß in response to LPS. The hyper production of IL-1ß was abrogated by in vivo reconstitution of wild type but not ITIM-mutated CEACAM1 bone marrow stem cells. Blocking Syk activation by kinase inhibitors or RNAi reduced Syk phosphorylation, lysosomal destabilization, ROS production, and caspase-1 activation in Ceacam1⁻/⁻ neutrophils. We conclude that LPS treatment of neutrophils triggers formation of a complex of TLR4 with pSyk and pCEACAM1, which upon recruitment of SHP-1 to the ITIMs of pCEACAM1, inhibits IL-1ß production by the inflammasome. Thus, CEACAM1 fine-tunes IL-1ß production in LPS treated neutrophils, explaining why the additional utilization of CEACAM1 as a pathogen receptor would further inhibit inflammation.

Retropharyngeal abscess from an unusual organism-Kingella denitrificans-in a patient on low-dose methotrexate.

Retropharyngeal abscess can be a life-threatening emergency with potential for airway compromise. We report a case of retropharyngeal abscess caused by Kingella denitrificans in a 66-year-old Caucasian man taking low-dose methotrexate for psoriatic arthritis. K denitrificans has not been previously reported as a cause of retropharyngeal abscess. K denitrificans, along with other Kingella species, is often found to be part of the normal upper respiratory tract flora. Potentially life-threatening complications of retropharyngeal abscess can occur in immunocompromised patients. Although complications of immunosuppression are thought to be rare with the use of low-dose pulse methotrexate for non-neoplastic conditions, such complications with prolonged use of low-dose pulse methotrexate may be seen.

Complement deficiency states and associated infections.

A major function of the immune system is to protect the host from microbial infections. The complement system plays important roles in both the innate and the adaptive immune defense and also acts as a bridge between these arms of immunity. This is obvious from complement deficiencies which in varying degree, depending on which factor is missing, are associated with increased infection susceptibility and also increased risk for other, mainly autoimmune diseases. Genetically determined deficiencies are described for almost all complement proteins but the consequences show a wide variation. Here the genetic defects and molecular abnormalities in complement deficient persons, related clinically relevant infections and the options for prevention and therapy are reviewed. The roles of complement in host defense against common infections are also discussed.

Nasopharyngeal colonization by Neisseria lactamica and induction of protective immunity against Neisseria meningitidis.

BACKGROUND: Natural immunity to Neisseria meningitidis may result from nasopharyngeal carriage of closely related commensals, such as Neisseria lactamica. METHODS: We enrolled 61 students with no current carriage of Neisseria species and inoculated them intranasally with 10,000 colony-forming units of Neisseria lactamica or sham control. Colonization was monitored in oropharyngeal samples over 6 months. We measured specific mucosal and systemic antibody responses to N. lactamica and serum bactericidal antibody (SBA) and opsonophagocytic antibodies to a panel of N. meningitidis serogroup B strains. We also inoculated an additional cohort following vaccination with N. lactamica outer-membrane vesicles (OMV) produced from the same strain. RESULTS: Twenty-six (63.4%) of 41 inoculated individuals became colonized with N. lactamica; 85% remained colonized at 12 weeks. Noncarriers were resistant to rechallenge, and carriers who terminated carriage were relatively resistant to rechallenge. No carriers acquired N. meningitidis carriage over 24 weeks, compared with 3 control subjects (15%). Carriers developed serum IgG and salivary IgA antibodies to the inoculated N. lactamica strain by 4 weeks; noncarriers and control subjects did not. Cross-reactive serum bactericidal antibody responses to N.meningitidis were negligible in carriers, but they developed broad opsonophagocytic antimeningococcal antibodies. OMV vaccinees developed systemic and mucosal anti-N. lactamica antibodies and were relatively resistant to N. lactamica carriage but not to natural acquisition of N. meningitidis. CONCLUSIONS: Carriers of N. lactamica develop mucosal and systemic humoral immunity to N. lactamica together with cross-reacting systemic opsonophagocytic but not bactericidal antibodies to N. meningitidis. Possession of humoral immunity to N. lactamica inhibits acquisition of N. lactamica but not of N. meningitidis. Some individuals are intrinsically resistant to N. lactamica carriage, independent of humoral immunity.

Human airway epithelial cell responses to Neisseria lactamica and purified porin via Toll-like receptor 2-dependent signaling.

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.

Distinct cell death programs in monocytes regulate innate responses following challenge with common causes of invasive bacterial disease.

Peripheral blood monocytes represent the rapid response component of mononuclear phagocyte host defense, generating vigorous but finite antibacterial responses. We investigated the fate of highly purified primary human monocytes following phagocytosis of different bacteria. Exposure to high bacterial loads resulted in rapid loss of cell viability and decreased functional competence. Cell death typically involved classical apoptosis. Exposure to high numbers of Escherichia coli and Klebsiella pneumoniae induced nonapoptotic death with loss of cell membrane integrity, marked disruption of phagolysosomes, and caspase-1 activation, while a subset of cells also released caspase-1-regulated extracellular traps. Classical apoptosis increased if extracellular bacterial replication was reduced and decreased if intracellular ATP levels were reduced during these infections. Both classical apoptosis and the alternative forms of cell death allowed monocytes, whose functional competence was exhausted, to downregulate reactive oxygen species and proinflammatory cytokine responses. In contrast, sustained stimulation of glycolytic metabolism and mitochondrial oxidative phosphorylation, with associated hypoxia inducible factor-1alpha upregulation, maintained intracellular ATP levels and prolonged monocyte functional longevity, as assessed by maintenance of phagocytosis, reactive oxygen species production, and proinflammatory cytokine generation. Monocyte innate responses to bacteria are short-lived and are limited by an intrinsic program of apoptosis, a response that is subverted by overwhelming infection with E. coli and K. pneumoniae or bacterial stimulation of cell metabolism. In this regard, the fate of monocytes following bacterial challenge more closely resembles neutrophils than macrophages.

Absence of mucosal immunity in the human upper respiratory tract to the commensal bacteria Neisseria lactamica but not pathogenic Neisseria meningitidis during the peak age of nasopharyngeal carriage.

The normal flora that colonizes the mucosal epithelia has evolved diverse strategies to evade, modulate, or suppress the immune system and avoid clearance. Neisseria lactamica and Neisseria meningitidis are closely related obligate inhabitants of the human upper respiratory tract. N. lactamica is a commensal but N. meningitidis is an opportunistic pathogen that occasionally causes invasive disease such as meningitis and septicemia. We demonstrate that unlike N. meningitidis, N. lactamica does not prime the development of mucosal T or B cell memory during the peak period of colonization. This cannot be explained by the induction of peripheral tolerance or regulatory CD4(+)CD25(+) T cell activity. Instead, N. lactamica mediates a B cell-dependent mitogenic proliferative response that is absent to N. meningitidis. This mitogenic response is associated with the production of T cell-independent polyclonal IgM that we propose functions by shielding colonizing N. lactamica from the adaptive immune system, maintaining immunological ignorance in the host. We conclude that, in contrast to N. meningitidis, N. lactamica maintains a commensal relationship with the host in the absence of an adaptive immune response. This may prolong the period of susceptibility to colonization by both pathogenic and nonpathogenic Neisseria species.

Neisseria meningitidis escape from the bactericidal activity of a monoclonal antibody is mediated by phase variation of lgtG and enhanced by a mutator phenotype.

Bacteria adapt to environmental changes through high-frequency switches in expression of specific phenotypes. Localized hypermutation mediated by simple sequence repeats is an important mechanism of such phase variation (PV) in Neisseria meningitidis. Loss or gain of nucleotides in a poly(C) tract located in the reading frame results in switches in expression of lgtG and determines whether a glucose or a phosphoethanolamine (PEtn) is added at a specific position in the inner core lipopolysaccharide (LPS). Monoclonal antibody (MAb) B5 is bactericidal for N. meningitidis strain 8047 when PEtn is present in the inner core LPS and lgtG is switched "off." Escape from the bactericidal activity of this antibody was examined by subjecting strain 8047 to multiple cycles of growth in the presence of MAb B5 and human serum. Escape variants with alterations in the lgtG repeat tract rapidly accumulated in bacterial populations during selection with this antibody. Strain 8047 was outcompeted in this assay by the 8047 Delta mutS strain due to the elevated PV rate of this mismatch repair mutant and hence the greater proportion of preexisting phase variants of lgtG in the inoculum. This mutS mutant was also more virulent than strain 8047 during escape from passive protection by MAb B5 in an in vivo infant rat model of bacteremia. These results provide an example of how PV rates can modulate the occurrence and severity of infection and have important implications for understanding the evolution of bacterial fitness in species subject to environmental variations that occur during persistence within and transmission between hosts.

Murine glia express the immunosuppressive cytokine, interleukin-10, following exposure to Borrelia burgdorferi or Neisseria meningitidis.

There is growing appreciation that resident glial cells can initiate and/or regulate inflammation following trauma or infection in the central nervous system (CNS). We have previously demonstrated the ability of microglia and astrocytes, resident glial cells of the CNS, to respond to bacterial pathogens by rapid production of inflammatory mediators. However, inflammation within the brain parenchyma is notably absent during some chronic bacterial infections in humans and nonhuman primates. In the present study, we demonstrate the ability of the immunosuppressive cytokine, interleukin-10 (IL-10), to inhibit inflammatory immune responses of primary microglia and astrocytes to B. burgdorferi and N. meningitidis, two disparate gram negative bacterial species that can cross the blood-brain barrier in humans. Importantly, we demonstrate that these organisms induce the delayed production of significant quantities of IL-10 by both microglia and astrocytes. Furthermore, we demonstrate that such production occurs independent of the actions of bacterial lipopolysaccharide and is secondary to the autocrine or paracrine actions of other glia-derived soluble mediators. The late onset of IL-10 production by resident glia following activation, the previously documented expression of specific receptors for this cytokine on microglia and astrocytes, and the ability of IL-10 to inhibit bacterially induced immune responses by these cells, suggest a mechanism by which resident glial cells can limit potentially damaging inflammation within the CNS in response to invading pathogens, and could explain the suppression of inflammation seen within the brain parenchyma during chronic bacterial infections.

Characterization and immunogenicity of Kingella kingae outer-membrane proteins.

In recent years, Kingella kingae has emerged as an important pediatric pathogen but the antigenicity of the organism and the host immune response have not been studied. Outer membrane proteins (OMPs) of 57 K. kingae isolates were characterized and the immune response of 19 children with invasive infections was studied by immunoblotting. Kingella kingae OMPs were remarkably similar disregarding place and time of isolation and associated clinical condition (asymptomatic carriage, bacteremia, endocarditis, septic arthritis or osteomyelitis). Most OMPs were immunogenic but the specific bands that reacted in each strain and the intensity of the reactions varied substantially. When convalescent sera were reacted with heterologous strains, bands that either were not recognized by the homologous serum or were not present in the homologous strain were visualized. These results demonstrate that OMPs of K. kingae are highly conserved but suggest that some epitopes are polymorphic, resulting in a variable pattern of immune response.

Immune response to invasive Kingella kingae infections, age-related incidence of disease, and levels of antibody to outer-membrane proteins.

The immune response to Kingella kingae was determined by enzyme-linked immunosorbent assay, using outer-membrane proteins as coating antigen, in 19 children with invasive infection. The age-related incidence of K. kingae disease in southern Israel during 1988-2002 was calculated and correlated with serum antibody levels in healthy children. Significant increases in immunoglobulin G (IgG) levels were found in children convalescing after invasive infections. The incidence was 1.3, 40.3, 23.9, 5.7, and 1.9 cases/100,000 children among those aged 0-5, 6-11, 12-23, 24-35, and 36-47 months, respectively. A low attack rate and undetectable serum IgA and high IgG levels were found during the first 6 months of life, which indicates that protection was conferred by maternally derived immunity. The high attack rate found among 6-24-month-old children coincides with the age at which antibody levels were lowest. Low incidence of disease and increasing antibody levels were found among older children, which probably represents cumulative experience with K. kingae antigens via colonization or infection.

Mucosal vaccination against encapsulated respiratory bacteria--new potentials for conjugate vaccines?

Polysaccharide (PS)-encapsulated bacteria such as Haemophilus influenzae type b (Hib), Streptococcus pneumoniae (pneumococcus), Neisseria meningitides (meningococcus) and group B streptococcus (GBS), cause a major proportion of disease in early childhood. Native PS vaccines are immunogenic and provide protection against disease in healthy adults but do not induce immunological memory. PSs are T-cell-independent antigens and do not elicit antibodies in infants and young children, but by conjugating PS to proteins they become T-cell dependent and immunogenic at an early age. Despite excellent efficacy of PS-protein conjugate vaccines against invasive disease, protection against mucosal infections such as pneumococcal otitis media has been less efficacious. Circulating PS-specific antibodies may protect against infections at mucosal sites, but mucosal immunoglobulin A antibodies may also contribute significantly to protection against mucosal infections. Mucosal immunization of experimental animals with conjugate vaccines against Hib, pneumococcus, meningococcus and GBS induces systemic and mucosal immune responses, which provide protection against carriage, otitis media and invasive disease in a variety of challenge models, providing new means for protection against encapsulated bacteria. In addition, mucosal immunization of neonatal mice with a pneumococcal conjugate and the nontoxic adjuvant LT-K63 has been superior to parenteral immunization in eliciting protective antibodies and PS-specific memory, and thus circumventing the limitations of antibody responses to PS that are responsible for enhanced susceptibility of neonates and infants to infections caused by encapsulated bacteria. Through T-cell dependent enhanced immunogenicity of PS-protein conjugate vaccines, mucosal immunization could be an attractive approach for early life immunization against encapsulated bacteria.

The role of porins in neisserial pathogenesis and immunity.

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.