Pneumolysin promotes host cell necroptosis and bacterial competence during pneumococcal meningitis as shown by whole-animal dual RNA-seq.

Pneumolysin is a major virulence factor of Streptococcus pneumoniae that plays a key role in interaction with the host during invasive disease. How pneumolysin influences these dynamics between host and pathogen interaction during early phase of central nervous system infection in pneumococcal meningitis remains unclear. Using a whole-animal in vivo dual RNA sequencing (RNA-seq) approach, we identify pneumolysin-specific transcriptional responses in both S. pneumoniae and zebrafish (Danio rerio) during early pneumococcal meningitis. By functional enrichment analysis, we identify host pathways known to be activated by pneumolysin and discover the importance of necroptosis for host survival. Inhibition of this pathway using the drug GSK'872 increases host mortality during pneumococcal meningitis. On the pathogen's side, we show that pneumolysin-dependent competence activation is crucial for intra-host replication and virulence. Altogether, this study provides new insights into pneumolysin-specific transcriptional responses and identifies key pathways involved in pneumococcal meningitis.

The role of plasminogen activator inhibitor-2 in pneumococcal meningitis.

Pneumococcal meningitis is associated with dysregulation of the coagulation cascade. Previously, we detected upregulation of cerebral plasminogen activator inhibitor-2 (PAI-2) mRNA expression during pneumococcal meningitis. Diverse functions have been ascribed to PAI-2, but its role remains unclear. We analyzed the function of SERPINB2 (coding for PAI-2) in patients with bacterial meningitis, in a well-established pneumococcal meningitis mouse model, using Serpinb2 knockout mice, and in vitro in wt and PAI-2-deficient bone marrow-derived macrophages (BMDMs). We measured PAI-2 in cerebrospinal fluid of patients, and performed functional, histopathological, protein and mRNA expression analyses in vivo and in vitro. We found a substantial increase of PAI-2 concentration in CSF of patients with pneumococcal meningitis, and up-regulation and increased release of PAI-2 in mice. PAI-2 deficiency was associated with increased mortality in murine pneumococcal meningitis and cerebral hemorrhages. Serpinb2-/- mice exhibited increased C5a levels, but decreased IL-10 levels in the brain during pneumococcal infection. Our in vitro experiments confirmed increased expression and release of PAI-2 by wt BMDM and decreased IL-10 liberation by PAI-2-deficient BMDM upon pneumococcal challenge. Our data show that PAI-2 is elevated during in pneumococcal meningitis in humans and mice. PAI-2 deficiency causes an inflammatory imbalance, resulting in increased brain pathology and mortality.

Gene polymorphisms of TLR10: effects on bacterial meningitis outcomes in Angolan children.

This study examined whether gene polymorphisms for toll-like receptor 10 (TLR10) associated with the susceptibility to and outcomes of bacterial meningitis (BM) in Angolan children. The study cohort consisted of 190 BM patients and the determination of ten single-nucleotide polymorphisms (SNPs) by Sanger sequencing. Patients with BM caused by Streptococcus pneumoniae who carried the following variants of TLR10 SNPs exhibited an increased risk of coexisting pneumonia: rs10004195 (T > A) (p = 0.025), rs10856837 (G > A) (p = 0.018) or rs11096956 (G > T) (p = 0.010). Yet, TLR10 SNPs rs11466652 (A > G), rs10856837 (G > A) and rs11096956 (G > T) influenced the protein levels in the cerebrospinal fluid (CSF). Moreover, compared with the wild type, patients with pneumococcal meningitis carrying a variant genotype of TLR10 SNP rs11466648 (A > G) exhibited an increased risk of developing blindness (p = 0.025), whereas patients with TLR10 SNP rs10004195 (T > A) exhibited a lower risk of convulsions at admission (p = 0.039) and a lower risk of altered consciousness (p = 0.029). This study suggests a relationship exists between coexisting pneumonia, protein levels in CSF, blindness, convulsions and an altered consciousness with genetic variations of TLR10 in BM in Angolan children.

Association of toll-like receptor 10 polymorphisms with pediatric pneumococcal meningitis.

We aimed to investigate whether the gene polymorphisms of TLR10 were associated with risk and severity of pneumococcal meningitis (PM) and serum cytokine levels in children. Three single nucleotide polymorphisms (SNPs) of TLR10 rs4129009 (2676A > G), rs10004195 (1018T > A) and rs11466617 (40735A > G) were studied in 95 laboratory-confirmed PM pediatric patients and 330 healthy controls by PCR-based sequencing. Ten serum cytokines were determined by multiplex immunoassay. The frequency of variant haplotype GAG of TLR10 was significantly lower in patients than controls (11.3% vs 33.3%, p < 0.001), although frequencies of the genotypes and alleles of the three SNPs did not differ between patients and controls. Frequency of variant haplotype GAG was significantly lower in patients who had CSF protein >1000 mg/L than those who had CSF protein ≤1000 mg/L (3.50% vs 32.4%, p < 0.001). Moreover, higher frequency of the haplotype GAG was found in patients who had higher levels of inflammatory cytokines such as IFN-γ, TNF-α and IL-1ß. Our finding suggested that the variant haplotype GAG in TLR10 is associated with decreased risk of PM in Chinese children.

Double deficiency of toll-like receptors 2 and 4 alters long-term neurological sequelae in mice cured of pneumococcal meningitis.

Toll-like receptor (TLR) 2 and 4 signalling pathways are central to the body's defence against invading pathogens during pneumococcal meningitis. Whereas several studies support their importance in innate immunity, thereby preventing host mortality, any role in protecting neurological function during meningeal infection is ill-understood. Here we investigated both the acute immunological reaction and the long-term neurobehavioural consequences of experimental pneumococcal meningitis in mice lacking both TLR2 and TLR4. The absence of these TLRs significantly impaired survival in mice inoculated intracerebroventricularly with Streptococcus pneumoniae. During the acute phase of infection, TLR2/4-deficient mice had lower cerebrospinal fluid concentrations of interleukin-1ß, and higher interferon-γ, than their wild-type counterparts. After antibiotic cure, TLR2/4 double deficiency was associated with aggravation of behavioural impairment in mice, as shown by diurnal hypolocomotion throughout the adaptation phases in the Intellicage of TLR-deficient mice compared to their wild-type counterparts. While TLR2/4 double deficiency did not affect the cognitive ability of mice in a patrolling task, it aggravated the impairment of cognitive flexibility. We conclude that TLR2 and TLR4 are central to regulating the host inflammatory response in pneumococcal meningitis, which may mediate diverse compensatory mechanisms that protect the host not only against mortality but also long-term neurological complications.

Joint sequencing of human and pathogen genomes reveals the genetics of pneumococcal meningitis.

Streptococcus pneumoniae is a common nasopharyngeal colonizer, but can also cause life-threatening invasive diseases such as empyema, bacteremia and meningitis. Genetic variation of host and pathogen is known to play a role in invasive pneumococcal disease, though to what extent is unknown. In a genome-wide association study of human and pathogen we show that human variation explains almost half of variation in susceptibility to pneumococcal meningitis and one-third of variation in severity, identifying variants in CCDC33 associated with susceptibility. Pneumococcal genetic variation explains a large amount of invasive potential (70%), but has no effect on severity. Serotype alone is insufficient to explain invasiveness, suggesting other pneumococcal factors are involved in progression to invasive disease. We identify pneumococcal genes involved in invasiveness including pspC and zmpD, and perform a human-bacteria interaction analysis. These genes are potential candidates for the development of more broadly-acting pneumococcal vaccines.

Resveratrol Acts Anti-Inflammatory and Neuroprotective in an Infant Rat Model of Pneumococcal Meningitis by Modulating the Hippocampal miRNome.

Resveratrol (RSV) is anti-inflammatory and neuroprotective, cross the blood-brain barrier (BBB) and has a safe profile. Besides, RSV modulates the expression of some miRNAs related to neurological disorders. Thus, we hypothesized that RSV can be neuroprotective in pneumococcal meningitis by modulating the global microRNA expression profile (miRNome). Eleven-day old rats were intracysternally infected with S. pneumoniae (~ 2 × 106 c.f.u.) and were orally administered with RSV (50 mg/kg) or vehicle in pre-treatment (before infection) or post-treatment schedules (3 and 18 h p.i.). At 24 h p.i., animals were euthanized and apoptotic cells were counted in the hippocampal dentate gyrus of the right brain hemispheres. The hippocampi from left hemispheres were used for cytokines and chemokines multiplex assay and miRNome profiling with TaqMan OpenArray Rodent MicroRNA. Infected rats treated with RSV had lower apoptotic scores and IL-1ß, CCL2, and CCL3 levels when compared to the infected group receiving placebo. Seven miRNAs were down regulated, and 18 were up regulated by pneumococcal acute meningitis. Thirty-seven miRNAs were down regulated, and three were up regulated (hsa-miR-15b-5p, hsa-miR-25-3p, hsa-miR-125b-5p) by the interaction between meningitis and RSV. Pathway enriched analysis revealed that meningitis and RSV modulate the expression of miRNAs targeting critical pathways related to the pathophysiology of bacterial meningitis. Nevertheless, hsa-miR-25-3p and hsa-miR-125b-5p target the transcription factor TEF-1, for which there are binding sites in Il-1ß, Ccl2, and Ccl3 genes. RSV is anti-inflammatory and neuroprotective in an infant rat model of pneumococcal meningitis and these positive effects involve the modulation of the hippocampal miRNome.

Association between TLR2 + 2477G/A polymorphism and bacterial meningitis: a meta-analysis.

Toll-like receptor 2 (TLR2) is a key member of TLRs, which is crucial in the initial inflammatory response against bacteria. TLR2, is also the initial barrier against bacterial infection and plays an important role in recognising a variety of bacterial lipoproteins. Several studies have been performed to investigate the TLR2 + 2477G/A polymorphism and bacterial meningitis susceptibility. Unfortunately, the results of previous studies were controversial. Therefore, we performed a meta-analysis to derive a more precise estimation of the association. The association between the TLR2 + 2477G/A polymorphism and bacterial meningitis susceptibility was assessed by odds ratios together with their 95% confidence intervals (CI). Six studies were enrolled in the present meta-analysis. Overall, no significant association between TLR2 + 2477G/A polymorphism and bacterial meningitis risk were found under allele contrast (A vs. G: OR = 1.15, 95% CI = 0.93-1.43, P = 0.202), recessive genetic model (AA vs. AG/GG: OR = 1.12, 95% CI = 0.90-1.41, P = 0.313). The significant association was found between TLR2 + 2477G/A polymorphism and pneumococcal meningitis risk under allele contrast (A vs. G: OR = 1.54, 95% CI = 1.01-2.36, P = 0.046), recessive genetic model (AA vs. AG/GG: OR = 1.63, 95% CI = 1.03-2.57, P = 0.035). We conclude that TLR2 + 2477G/A polymorphism is not associated with meningococcal meningitis risk but contributes an increased risk of pneumococcal meningitis.

Pneumococcal meningitis and endocarditis in an infant: possible improved survival with factor V Leiden mutation.

Streptococcus pneumoniae infections continue to remain associated with high morbidity and mortality. Although the incidence of invasive meningeal and/or lung disease are not uncommon, Streptococcus pneumoniae endocarditis is rare especially in healthy pediatric population. New studies have suggested a strong association between factor V leiden (FVL) mutation and favorable outcomes in critically ill children. A healthy 10 month old presented with sepsis and meningeal signs, was later confirmed to have Streptococcus pneumoniae meningitis and endocarditis. She was found to have factor V leiden mutation and made a complete recovery despite initial complications. CONCLUSION: Presence of factor V leiden mutation in critically ill children with severe septicaemia possibly contributes to better outcomes. What is known: • Mortality and morbidity remain high with invasive pneumococcal disease. • Pneumococcal endocarditis is rare in healthy pediatric population and results in significant morbidity and mortality What is new: • New studies have suggested a strong association between factor V leiden (FVL) mutation and favorable outcomes in critically ill children. • The presence of factor V mutation in children with extensive invasive pneumococcal disease possibly contributes to a better outcome.

Mannose-binding lectin-deficient genotypes as a risk factor of pneumococcal meningitis in infants.

OBJECTIVES: The objective of this study was to evaluate to evaluate the role of mannose-binding-lectin deficient genotypes in pneumococcal meningitis (PM) in children. METHODS: We performed a 16-year retrospective study (January 2001 to March 2016) including patients ≤ 18 years with PM. Variables including attack rate of pneumococcal serotype (high or low invasive capacity) and MBL2 genotypes associated with low serum MBL levels were recorded. RESULTS: Forty-eight patients were included in the study. Median age was 18.5 months and 17/48 episodes (35.4%) occurred in children ≤ 12 months old. Serotypes with high-invasive disease potential were identified in 15/48 episodes (31.2%). MBL2 deficient genotypes accounted for 18.8% (9/48). Children ≤ 12 months old had a 7-fold risk (95% CI: 1.6-29.9; p < 0.01) of having a MBL2 deficient genotype in comparison to those > 12 months old. A sub-analysis of patients by age group revealed significant proportions of carriers of MBL2 deficient genotypes among those ≤ 12 months old with PM caused by opportunistic serotypes (54.5%), admitted to the PICU (Pediatric Intensive Care Unit) (46.7%) and of White ethnicity (35.7%). These proportions were significantly higher than in older children (all p<0.05). CONCLUSIONS: Our results suggest that differences in MBL2 genotype in children ≤12 months old affects susceptibility to PM, and it may have an important role in the episodes caused by non-high invasive disease potential serotypes.

Analysis of TLR2, TLR4, and TLR9 single nucleotide polymorphisms in children with bacterial meningitis and their healthy family members.

BACKGROUND: The aim was to analyse TLR2 rs5743708, TLR2 rs4696480, TLR4 rs4986790, TLR9 rs5743836, and TLR9 rs352140 single nucleotide polymorphisms (SNPs) in children with pneumococcal and meningococcal meningitis and their family members. METHODS: The study group consisted of 39 children with bacterial meningitis (25 with meningococcal meningitis and 14 with pneumococcal meningitis) and 49 family members. Laboratory test results and the course of the diseases were analyzed. Genomic DNA was extracted from 1.2ml of peripheral blood in order to analyze the five SNPs. RESULTS: Patients with pneumococcal and meningococcal meningitis showed a similar male/female ratio, mean age, and duration of symptoms. There were no statistically significant differences in biochemical markers between the two groups. All patients possessed at least one polymorphic variant of the analyzed SNPs. The most common SNP was TLR9 rs352140, detected in 89.7% of patients. No significant differences in SNP frequency were found between patients, family members, and the general population. CONCLUSIONS: The allele frequencies in the population studied are in accordance with the literature data. The study did not find an association between the analyzed SNPs and susceptibility to bacterial meningitis. The role of SNPs in genes coding toll-like receptors and the interactions between them in controlling inflammation in the central nervous system needs further evaluation.

Detection of Significant Pneumococcal Meningitis Biomarkers by Ego Network.

OBJECTIVE: To identify significant biomarkers for detection of pneumococcal meningitis based on ego network. METHODS: Based on the gene expression data of pneumococcal meningitis and global protein-protein interactions (PPIs) data recruited from open access databases, the authors constructed a differential co-expression network (DCN) to identify pneumococcal meningitis biomarkers in a network view. Here EgoNet algorithm was employed to screen the significant ego networks that could accurately distinguish pneumococcal meningitis from healthy controls, by sequentially seeking ego genes, searching candidate ego networks, refinement of candidate ego networks and significance analysis to identify ego networks. Finally, the functional inference of the ego networks was performed to identify significant pathways for pneumococcal meningitis. RESULTS: By differential co-expression analysis, the authors constructed the DCN that covered 1809 genes and 3689 interactions. From the DCN, a total of 90 ego genes were identified. Starting from these ego genes, three significant ego networks (Module 19, Module 70 and Module 71) that could predict clinical outcomes for pneumococcal meningitis were identified by EgoNet algorithm, and the corresponding ego genes were GMNN, MAD2L1 and TPX2, respectively. Pathway analysis showed that these three ego networks were related to CDT1 association with the CDC6:ORC:origin complex, inactivation of APC/C via direct inhibition of the APC/C complex pathway, and DNA strand elongation, respectively. CONCLUSIONS: The authors successfully screened three significant ego modules which could accurately predict the clinical outcomes for pneumococcal meningitis and might play important roles in host response to pathogen infection in pneumococcal meningitis.

Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis model.

BACKGROUND: Streptococcus pneumoniae is one of the most important causes of bacterial meningitis, an infection where unfavourable outcome is driven by bacterial and host-derived toxins. In this study, we developed and characterized a pneumococcal meningitis model in zebrafish embryos that allows for real-time investigation of early host-microbe interaction. METHODS: Zebrafish embryos were infected in the caudal vein or hindbrain ventricle with green fluorescent wild-type S. pneumoniae D39 or a pneumolysin-deficient mutant. The kdrl:mCherry transgenic zebrafish line was used to visualize the blood vessels, whereas phagocytic cells were visualized by staining with far red anti-L-plastin or in mpx:GFP/mpeg1:mCherry zebrafish, that have green fluorescent neutrophils and red fluorescent macrophages. Imaging was performed by fluorescence confocal and time-lapse microscopy. RESULTS: After infection by caudal vein, we saw focal clogging of the pneumococci in the blood vessels and migration of bacteria through the blood-brain barrier into the subarachnoid space and brain tissue. Infection with pneumolysin-deficient S. pneumoniae in the hindbrain ventricle showed attenuated growth and migration through the brain as compared to the wild-type strain. Time-lapse and confocal imaging revealed that the initial innate immune response to S. pneumoniae in the subarachnoid space mainly consisted of neutrophils and that pneumolysin-mediated cytolytic activity caused a marked reduction of phagocytes. CONCLUSIONS: This new meningitis model permits detailed analysis and visualization of host-microbe interaction in pneumococcal meningitis in real time and is a very promising tool to further our insights in the pathogenesis of pneumococcal meningitis.

Variation of 46 Innate Immune Genes Evaluated for their Contribution in Pneumococcal Meningitis Susceptibility and Outcome.

Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Early recognition of the pathogen and subsequent innate immune response play a vital role in disease susceptibility and outcome. Genetic variations in innate immune genes can alter the immune response and influence susceptibility and outcome of meningitis disease. Here we conducted a sequencing study of coding regions from 46 innate immune genes in 435 pneumococcal meningitis patients and 416 controls, to determine the role of genetic variation on pneumococcal meningitis susceptibility and disease outcome. Strongest signals for susceptibility were rs56078309 CXCL1 (p=4.8e-04) and rs2008521 in CARD8 (p=6.1e-04). For meningitis outcome the rs2067085 in NOD2 (p=5.1e-04) and rs4251552 of IRAK4 were the strongest associations with unfavorable outcome (p=6.7e-04). Haplotype analysis showed a haplotype block, determined by IRAK4 rs4251552, significantly associated with unfavorable outcome (p=0.004). Cytokine measurements from cerebrospinal fluid showed that with the IRAK4 rs4251552 G risk allele had higher levels of IL-6 compared to individuals with A/A genotype (p=0.04). We show that genetic variation within exons and flanking regions of 46 innate immunity genes does not yield significant association with pneumococcal meningitis. The strongest identified signal IRAK4 does imply a potential role of genetic variation in pneumococcal meningitis.

Exome Array Analysis of Susceptibility to Pneumococcal Meningitis.

Host genetic variability may contribute to susceptibility of bacterial meningitis, but which genes contribute to the susceptibility to this complex disease remains undefined. We performed a genetic association study in 469 community-acquired pneumococcal meningitis cases and 2072 population-based controls from the Utrecht Health Project in order to find genetic variants associated with pneumococcal meningitis susceptibility. A HumanExome BeadChip was used to genotype 102,097 SNPs in the collected DNA samples. Associations were tested with the Fisher exact test. None of the genetic variants tested reached Bonferroni corrected significance (p-value <5 × 10(-7)). Our strongest signals associated with susceptibility to pneumococcal meningitis were rs139064549 on chromosome 1 in the COL11A1 gene (p = 1.51 × 10(-6); G allele OR 3.21 [95% CI 2.05-5.02]) and rs9309464 in the EXOC6B gene on chromosome 2 (p = 6.01 × 10(-5); G allele OR 0.66 [95% CI 0.54-0.81]). The sequence kernel association test (SKAT) tests for associations between multiple variants in a gene region and pneumococcal meningitis susceptibility yielded one significant associated gene namely COL11A1 (p = 1.03 × 10(-7)). Replication studies are needed to validate these results. If replicated, the functionality of these genetic variations should be further studied to identify by which means they influence the pathophysiology of pneumococcal meningitis.

V-akt murine thymoma viral oncogene homolog 3 (AKT3) contributes to poor disease outcome in humans and mice with pneumococcal meningitis.

Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Fatality rates are substantial, and long-term sequelae develop in about half of survivors. Here, we have performed a prospective nationwide genetic association study using the Human Exome BeadChip and identified gene variants in encoding dynactin 4 (DCTN4), retinoic acid early transcript 1E (RAET1E), and V-akt murine thymoma viral oncogene homolog 3 (AKT3) to be associated with unfavourable outcome in patients with pneumococcal meningitis. No clinical replication cohort is available, so we validated the role of one of these targets, AKT3, in a pneumococcal meningitis mouse model. Akt3 deficient mice had worse survival and increased histopathology scores for parenchymal damage (infiltration) and vascular infiltration (large meningeal artery inflammation) but similar bacterial loads, cytokine responses, compared to wild-type mice. We found no differences in cerebrospinal fluid cytokine levels between patients with risk or non-risk alleles. Patients with the risk genotype (rs10157763, AA) presented with low scores on the Glasgow Coma Scale and high rate of epileptic seizures. Thus, our results show that AKT3 influences outcome of pneumococcal meningitis.

Functional polymorphisms of macrophage migration inhibitory factor as predictors of morbidity and mortality of pneumococcal meningitis.

Pneumococcal meningitis is the most frequent and critical type of bacterial meningitis. Because cytokines play an important role in the pathogenesis of bacterial meningitis, we examined whether functional polymorphisms of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) were associated with morbidity and mortality of pneumococcal meningitis. Two functional MIF promoter polymorphisms, a microsatellite (-794 CATT5-8; rs5844572) and a single-nucleotide polymorphism (-173 G/C; rs755622) were genotyped in a prospective, nationwide cohort of 405 patients with pneumococcal meningitis and in 329 controls matched for age, gender, and ethnicity. Carriages of the CATT7 and -173 C high-expression MIF alleles were associated with unfavorable outcome (P= 0.005 and 0.003) and death (P= 0.03 and 0.01). In a multivariate logistic regression model, shock [odds ratio (OR) 26.0, P= 0.02] and carriage of the CATT7 allele (OR 5.12,P= 0.04) were the main predictors of mortality. MIF levels in the cerebrospinal fluid were associated with systemic complications and death (P= 0.0002). Streptococcus pneumoniae strongly up-regulated MIF production in whole blood and transcription activity of high-expression MIF promoter Luciferase reporter constructs in THP-1 monocytes. Consistent with these findings, treatment with anti-MIF immunoglogulin G (IgG) antibodies reduced bacterial loads and improved survival in a mouse model of pneumococcal pneumonia and sepsis. The present study provides strong evidence that carriage of high-expression MIF alleles is a genetic marker of morbidity and mortality of pneumococcal meningitis and also suggests a potential role for MIF as a target of immune-modulating adjunctive therapy.

Genetic Variation in NFKBIE Is Associated With Increased Risk of Pneumococcal Meningitis in Children.

BACKGROUND: Streptococcus pneumoniae and Neisseria meningitidis are frequent pathogens in life-threatening infections. Genetic variation in the immune system may predispose to these infections. Nuclear factor-κB is a key component of the TLR-pathway, controlled by inhibitors, encoded by the genes NFKBIA, NFKBIE and NFKBIZ. We aimed to replicate previous findings of genetic variation associated with invasive pneumococcal disease (IPD), and to assess whether similar associations could be found in invasive meningococcal disease (IMD). METHODS: Cases with IPD and IMD and controls were identified by linking Danish national registries. DNA was obtained from the Danish Neonatal Screening Biobank. The association between SNPs and susceptibility to IPD and IMD, mortality and pneumococcal serotypes was investigated. RESULTS: 372 children with pneumococcal meningitis, 907 with pneumococcal bacteremia and 1273 controls were included. We included 406 cases with meningococcal meningitis, 272 with meningococcal bacteremia, and 672 controls. The NFKBIE SNP was associated with increased risk of pneumococcal meningitis (aOR 1.68; 95% CI: 1.20-2.36), but not bacteremia (aOR 1.08; 95% CI: 0.86-1.35). The remaining SNPs were not associated with susceptibility to invasive disease. None of the SNPs were associated with risk of IMD or mortality. CONCLUSIONS: A NFKBIE polymorphism was associated with increased risk of pneumococcal meningitis.

Interferon-γ-Induced Nitric Oxide Synthase-2 Contributes to Blood/Brain Barrier Dysfunction and Acute Mortality in Experimental Streptococcus pneumoniae Meningitis.

The proinflammatory cytokine interferon-gamma (IFNγ) recently was shown to play a crucial role in experimental pneumococcal meningitis (PM) pathogenesis, and we aimed in this study to investigate IFNγ-driven nitric oxide synthase-2 (NOS2)-mediated pathogenesis of murine PM. We demonstrate that costimulation of toll-like receptors and IFNγ receptors was synergistic for NOS2 expression in cultured murine microglia. Using an experimental PM model, wild-type mice treated with anti-IFNγ antibody, as well as IFNγ and NOS2 gene knockout (GKO) mice, were inoculated intracerebroventricularly with 10(3) colony-forming units of Streptococcus pneumoniae (WU2 strain). Mice were monitored daily during a 200-h disease course to assess survival rate and blood-brain barrier (BBB) permeability measured at 48 h. IFNγ deficiency was protective in PM, with an approximate 3-fold increase in survival rates in both antibody-treated and IFNγ GKO mice compared to controls (P < 0.01). At 48 h postinoculation, brain NOS2 mRNA expression was significantly increased in an IFNγ-dependent manner. Mortality was significantly delayed in NOS2 GKO mice compared to controls (P < 0.01), and BBB dysfunction was reduced by 54% in IFNγ GKO mice and abolished in NOS2 GKO. These data suggest that IFNγ-dependent expression of NOS2 in the brain contributes to BBB breakdown and early mortality in murine PM.