Mercedes Valls Serón

Genetic variation in inflammasome genes is associated with outcome in bacterial meningitis

Bacterial meningitis is a severe and deadly disease, most commonly caused by Streptococcus pneumoniae. Disease outcome has been related to severity of the inflammatory response in the subarachnoid space. Inflammasomes are intracellular signaling complexes contributing to this inflammatory response. The role of genetic variation in inflammasome genes in bacterial meningitis is largely unknown. In a prospective nationwide cohort of patients with pneumococcal meningitis, we performed a genetic association study and found that single-nucleotide polymorphisms in the inflammasome genes CARD8 (rs2043211) and NLRP1 (rs11621270) are associated with poor disease outcome. Levels of the inflammasome associated cytokines interleukin (IL)-1β and IL-18 in cerebrospinal fluid also correlated with clinical outcome, but were not associated with the CARD8 and NLRP1 polymorphisms. Our results implicate an important role of genetic variation in inflammasome genes in the regulation of inflammatory response and clinical outcome in patients with bacterial meningitis.

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

Significance Pneumococcal meningitis, the most frequent cause of bacterial meningitis in adults, is associated with substantial morbidity and mortality. In a prospective, nationwide cohort of patients with pneumococcal meningitis, macrophage migration inhibitory factor (MIF), a proinflammatory mediator, was identified as a previously unidentified genetic marker of patient’s outcome. High-expression MIF alleles were associated with disease severity and death, a finding consistent with the harmful consequences of robust proinflammatory cytokine responses on brain edema and neuronal damage in the course of bacterial meningitis. These results provide strong evidence that functional MIF polymorphisms are genetic predictors of morbidity and mortality of pneumococcal meningitis and suggest that MIF is a potential target for immune-modulating adjunctive therapies. 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.

Streptococcus pneumoniae Arginine Synthesis Genes Promote Growth and Virulence in Pneumococcal Meningitis

Streptococcus pneumoniae (pneumococcus) is a major human pathogen causing pneumonia, sepsis and bacterial meningitis. Using a clinical phenotype based approach with bacterial whole-genome sequencing we identified pneumococcal arginine biosynthesis genes to be associated with outcome in patients with pneumococcal meningitis. Pneumococci harboring these genes show increased growth in human blood and cerebrospinal fluid (CSF). Mouse models of meningitis and pneumonia showed that pneumococcal strains without arginine biosynthesis genes were attenuated in growth or cleared, from lung, blood and CSF. Thus, S. pneumoniae arginine synthesis genes promote growth and virulence in invasive pneumococcal disease.

Cerebrospinal fluid inflammatory markers in patients with Listeria monocytogenes meningitis

Background Listeria monocytogenes meningitis is the third most common cause of bacterial meningitis and is associated with high rates of mortality and unfavorable outcome. Methods We analyzed 101 cytokines, chemokines and complement factors in CSF of adult patients with Listeria meningitis included in a prospective cohort study and compared these biomarkers between Listeria meningitis patients and negative controls, and between Listeria meningitis patients with a favorable and an unfavorable outcome. Results CSF was available from 26 of 62 (42%) Listeria meningitis patients and 19 negative controls. Fifteen (58%) Listeria meningitis patients had an unfavorable outcome. In Listeria meningitis CSF levels of 51 biomarkers were significantly elevated compared to negative controls after Bonferroni correction. The 11 most significantly elevated (P < .01) biomarkers of unfavorable outcome in Listeria meningitis were markers of T-cell activation (sIL-2Rα, sCD40L and IL-1), interferon-related (IFN-α2, IL-18, CX3CL1, CCL20), markers of complement activation (C3a), and endothelial growth factor related (VEGF, CXCL7). Conclusions Our data suggest that T-cell activation, complement activation, interferon- and endothelial growth factor production are important in the immune response to Listeria meningitis, and thereby influence outcome. General significance Our study provides target pathways for further studies in the pathophysiology of Listeria meningitis.

Large scale genomic analysis shows no evidence for pathogen adaptation between the blood and cerebrospinal fluid niches during bacterial meningitis

Recent studies have provided evidence for rapid pathogen genome diversification, some of which could potentially affect the course of disease. We have previously described such variation seen between isolates infecting the blood and cerebrospinal fluid (CSF) of a single patient during a case of bacterial meningitis. Here, we performed whole-genome sequencing of paired isolates from the blood and CSF of 869 meningitis patients to determine whether such variation frequently occurs between these two niches in cases of bacterial meningitis. Using a combination of reference-free variant calling approaches, we show that no genetic adaptation occurs in either invaded niche during bacterial meningitis for two major pathogen species, Streptococcus pneumoniae and Neisseria meningitidis. This study therefore shows that the bacteria capable of causing meningitis are already able to do this upon entering the blood, and no further sequence change is necessary to cross the blood–brain barrier. Our findings place the focus back on bacterial evolution between nasopharyngeal carriage and invasion, or diversity of the host, as likely mechanisms for determining invasiveness.

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.

Mannose-binding lectin-associated serine protease 2 (MASP-2) contributes to poor disease outcome in humans and mice with pneumococcal meningitis

BackgroundPneumococcal 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. Disease outcome has been related to the severity of the pro-inflammatory response in the subarachnoid space. The complement system, which mediates key inflammatory processes, has been implicated as a modulator of pneumococcal meningitis disease severity in animal studies.MethodsWe investigated mannose-binding lectin-associated serine protease (MASP-2) levels in cerebrospinal fluid (CSF) samples derived from the diagnostic lumbar puncture, which was available for 307 of 792 pneumococcal meningitis episodes included in our prospective nationwide cohort study (39%), and the association between these levels and clinical outcome. Subsequently, we studied the role of MASP-2 in our experimental pneumococcal meningitis mouse model using Masp2−/− mice and evaluated the potential of adjuvant treatment with MASP-2-specific monoclonal antibodies in wild-type (WT) mice.ResultsMASP-2 levels in cerebrospinal fluid of patients with bacterial meningitis were correlated with poor functional outcome. Consistent with these human data, Masp2-deficient mice with pneumococcal meningitis had lower cytokine levels and increased survival compared to WT mice. Adjuvant treatment with MASP-2-specific monoclonal antibodies led to reduced complement activation and decreased disease severity.ConclusionsMASP-2 contributes to poor disease outcome in human and mice with pneumococcal meningitis. MASP-2-specific monoclonal antibodies can be used to attenuate the inflammatory response 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.

CCAAT/enhancer-binding protein δ (C/EBPδ) aggravates inflammation and bacterial dissemination during pneumococcal meningitis

BackgroundThe prognosis of bacterial meningitis largely depends on the severity of the inflammatory response. The transcription factor CAAT/enhancer-binding protein δ (C/EBPδ) plays a key role in the regulation of the inflammatory response during bacterial infections. Consequently, we assessed the role of C/EBPδ during experimental meningitis.MethodsWild-type and C/EBPδ-deficient mice (C/EBPδ−/−) were intracisternally infected with Streptococcus pneumoniae and sacrificed after 6 or 30 h, or followed in a survival study.ResultsIn comparison to wild-type mice, C/EBPδ−/− mice showed decreased bacterial loads at the primary site of infection and decreased bacterial dissemination to lung and spleen 30 h after inoculation. Expression levels of the inflammatory mediators IL-10 and KC were lower in C/EBPδ−/− brain homogenates, whereas IL-6, TNF-α, IL-1β, and MIP-2 levels were not significantly different between the two genotypes. Moreover, C/EBPδ−/− mice demonstrated an attenuated systemic response as reflected by lower IL-10, IL-6, KC, and MIP-2 plasma levels. No differences in clinical symptoms or in survival were observed between wild-type and C/EBPδ−/− mice.ConclusionC/EBPδ in the brain drives the inflammatory response and contributes to bacterial dissemination during pneumococcal meningitis. C/EBPδ does, however, not affect clinical parameters of the disease and does not confer a survival benefit.

Large scale genomic analysis shows no evidence for repeated pathogen adaptation during the invasive phase of bacterial meningitis in humans

Recent studies have provided evidence for rapid pathogen genome variation, some of which could potentially affect the course of disease. We have previously detected such variation by comparing isolates infecting the blood and cerebrospinal fluid (CSF) of a single patient during a case of bacterial meningitis. To determine whether the observed variation repeatedly occurs in cases of disease, we performed whole genome sequencing of paired isolates from blood and CSF of 938 meningitis patients. We also applied the same techniques to 54 paired isolates from the nasopharynx and CSF. Using a combination of reference-free variant calling approaches we show that no genetic adaptation occurs in the invasive phase of bacterial meningitis for four major pathogen species: Streptococcus pneumoniae, Neisseria meningitidis, Listeria monocytogenes and Haemophilus influenzae. From nasopharynx to CSF, no adaptation was seen in S. pneumoniae, but in N. meningitidis mutations potentially mediating adaptation to the invasive niche were occasionally observed in the dca gene. This study therefore shows that the bacteria capable of causing meningitis are already able to do this upon entering the blood, and no further sequence change is necessary to cross the blood-brain barrier. The variation discovered from nasopharyngeal isolates suggest that larger studies comparing carriage and invasion may help determine the likely mechanisms of invasiveness. Author Summary We have analysed the entire DNA sequence from bacterial pathogen isolates from cases of meningitis in 938 Dutch adults, focusing on comparing pairs of isolates from the patient’s blood and their cerebrospinal fluid. Previous research has been on only a single patient, but showed possible signs of adaptation to treatment within the host over the course of a single case of disease. By sequencing many more such paired samples, and including four different bacterial species, we were able to determine that adaptation of the pathogen does not occur after bloodstream invasion during bacterial meningitis. We also analysed 54 pairs of isolates from pre- and post-invasive niches from the same patient. In N. meningitidis we found variation in the sequence of one gene which appears to provide bacteria with an advantage after invasion of the bloodstream. Overall, our findings indicate that evolution after invasion in bacterial meningitis is not a major contribution to disease pathogenesis. Future studies should involve more extensive sampling between the carriage and disease niches, or on variation of the host.