Aging and Microglial Response following Systemic Stimulation with Escherichia coli in Mice.

Systemic infection is an important risk factor for the development cognitive impairment and neurodegeneration in older people. Animal experiments show that systemic challenges with live bacteria cause a neuro-inflammatory response, but the effect of age on this response in these models is unknown. Young (2 months) and middle-aged mice (13-14 months) were intraperitoneally challenged with live Escherichia coli (E. coli) or saline. The mice were sacrificed at 2, 3 and 7 days after inoculation; for all time points, the mice were treated with ceftriaxone (an antimicrobial drug) at 12 and 24 h after inoculation. Microglial response was monitored by immunohistochemical staining with an ionized calcium-binding adaptor molecule 1 (Iba-1) antibody and flow cytometry, and inflammatory response by mRNA expression of pro- and anti-inflammatory mediators. We observed an increased microglial cell number and moderate morphologically activated microglial cells in middle-aged mice, as compared to young mice, after intraperitoneal challenge with live E. coli. Flow cytometry of microglial cells showed higher CD45 and CD11b expressions in middle-aged infected mice compared to young infected mice. The brain expression levels of pro-inflammatory genes were higher in middle-aged than in young infected mice, while middle-aged infected mice had similar expression levels of these genes in the systemic compartment. We conclude that systemic challenge with live bacteria causes an age-dependent neuro-inflammatory and microglial response. Our data show signs of an age-dependent disconnection of the inflammatory transcriptional signature between the brain and the systemic compartment.

Complement factor H contributes to mortality in humans and mice with bacterial meningitis.

BACKGROUND: The complement system is a vital component of the inflammatory response occurring during bacterial meningitis. Blocking the complement system was shown to improve the outcome of experimental pneumococcal meningitis. Complement factor H (FH) is a complement regulatory protein inhibiting alternative pathway activation but is also exploited by the pneumococcus to prevent complement activation on its surface conferring serum resistance. METHODS: In a nationwide prospective cohort study of 1009 episodes with community-acquired bacterial meningitis, we analyzed whether genetic variations in CFH influenced FH cerebrospinal fluid levels and/or disease severity. Subsequently, we analyzed the role of FH in our pneumococcal meningitis mouse model using FH knock-out (Cfh-/-) mice and wild-type (wt) mice. Finally, we tested whether adjuvant treatment with human FH (hFH) improved outcome in a randomized investigator blinded trial in a pneumococcal meningitis mouse model. RESULTS: We found the major allele (G) of single nucleotide polymorphism in CFH (rs6677604) to be associated with low FH cerebrospinal fluid concentration and increased mortality. In patients and mice with bacterial meningitis, FH concentrations were elevated during disease and Cfh-/- mice with pneumococcal meningitis had increased mortality compared to wild-type mice due to C3 depletion. Adjuvant treatment of wild-type mice with purified human FH led to complement inhibition but also increased bacterial outgrowth which resulted in similar disease outcomes. CONCLUSION: Low FH levels contribute to mortality in pneumococcal meningitis but adjuvant treatment with FH at a clinically relevant time point is not beneficial.

Microglial Activation After Systemic Stimulation With Lipopolysaccharide and Escherichia coli.

Background: Microglial activation after systemic infection has been suggested to mediate sepsis-associated delirium. A systematic review of animal studies suggested distinct differences between microglial activation after systemic challenge with live bacteria and lipopolysaccharide (LPS). Here, we describe a mouse model of microglial activation after systemic challenge with live Escherichia coli (E. coli) and compare results with systemic challenge with LPS. Methods: Sixty mice were intraperitoneally injected with E. coli (1 × 104 colony-forming units) and sacrificed at 12, 20, 48, and 72 h after inoculation. For 48 and 72 h time points, mice were treated with ceftriaxone. Thirty mice were intraperitoneally injected with LPS (5 mg/kg) and sacrificed 3 and 48 h after inoculation; 48 control mice were intraperitoneally injected with isotonic saline. Microglial response was monitored by immunohistochemical staining with Iba-1 antibody and flow cytometry; and inflammatory response by mRNA expression of pro- and anti-inflammatory mediators. Results: Mice infected with live E. coli showed microglial activation 72 h post-inoculation, with increased cell number in cortex (p = 0.0002), hippocampus (p = 0.003), and thalamus (p = 0.0001), but not in the caudate nucleus/putamen (p = 0.33), as compared to controls. At 72 h, flow cytometry of microglia from E. coli infected mice showed increased cell size (p = 0.03) and CD45 expression (p = 0.03), but no increase in CD11b expression, and no differences in brain mRNA expression of inflammatory mediators as compared to controls. In mice with systemic LPS stimulation, microglial cells were morphologically activated at the 48 h time point with increased cell numbers in cortex (p = 0.002), hippocampus (p = 0.0003), thalamus (p = 0.007), and caudate nucleus/putamen (p < 0.0001), as compared to controls. At 48 h, flow cytometry of microglia from LPS stimulated mice showed increased cell size (p = 0.03), CD45 (p = 0.03), and CD11b (p = 0.04) expression. Brain mRNA expression of TNF-α (p = 0.02), IL-1ß (p = 0.02), and MCP-1 (p = 0.03) were increased as compared to controls. Interpretation: Systemic challenge with live E. coli causes a neuro-inflammatory response, but this response occurs at a later time point and is less vigorous as compared to LPS stimulation.The E. coli model mimics the clinical situation of infection associated delirium more closely than stimulation with supra-natural LPS.

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

BACKGROUND: 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. 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. METHODS: We 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. RESULTS: MASP-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. CONCLUSIONS: MASP-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.

Detrimental role for CCAAT/enhancer binding protein δ in blood-borne brain infection.

BACKGROUND: The most frequent pathogen that causes bacterial meningitis is the Gram-positive bacterium Streptococcus (S.) pneumoniae. CCAAT/enhancer binding protein δ is a transcription factor that has recently been hypothesized to play a detrimental role in outcome of meningitis caused by S. pneumoniae. Here, we studied the role of C/EBPδ prior to the development of pneumococcal meningitis. METHODS: Wild-type and C/EBPδ-deficient mice (C/EBPδ-/-) were intraveneously infected with S. pneumoniae and sacrificed after 24 or 48 h. cebpδ expression, bacterial loads, inflammatory response and pathology in the brain were assessed. RESULTS: S. pneumoniae induces cebpδ expression in the brain during blood-borne brain infection. In comparison to wild-type mice, C/EBPδ-/- animals showed decreased bacterial loads in blood and brain 48 h after inoculation. In the blood compartment, the host inflammatory response was significantly lower upon infection in C/EBPδ-/- mice as compared to wild-type mice. CONCLUSION: C/EBPδ facilitates bacterial dissemination to the brain and enhances the immune response in the blood compartment. Our study suggests that C/EBPδ plays a detrimental role during the initial development of blood-borne brain infection.

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.

Adjuvant treatment with dexamethasone plus anti-C5 antibodies improves outcome of experimental pneumococcal meningitis: a randomized controlled trial.

BACKGROUND: We compared adjunctive treatment with placebo, dexamethasone, anti-C5 antibodies, and the combination of dexamethasone plus anti-C5 antibodies in experimental pneumococcal meningitis. METHODS: In this prospective, investigator-blinded, randomized trial, 96 mice were infected intracisternally with 10(7) CFU/ml Streptococcus pneumoniae serotype 3, treated with intraperitoneal ceftriaxone at 20 h, and randomly assigned to intraperitoneal adjunctive treatment with placebo (saline), dexamethasone, anti-C5 antibodies, or dexamethasone plus anti-C5 antibodies. The primary outcome was survival during a 72-h observational period that was analyzed with the log-rank test. Secondary outcome was clinical severity, scored on a validated scale using a linear mixed model. RESULTS: Mortality rates were 16 of 16 mice (100%) in the placebo group, 12 of 15 mice (80%) in the dexamethasone group, 25 of 31 mice (80%) in the anti-C5 antibody group, and 18 of 30 mice (60%) in the dexamethasone plus anti-C5 antibody group (Fisher's exact test for overall difference, P = .012). Mortality of mice treated with dexamethasone plus anti-C5 antibodies was lower compared to the anti-C5 antibody-treated mice (log-rank P = .039) and dexamethasone-treated mice (log-rank P = .040). Clinical severity scores for the dexamethasone plus anti-C5 antibody-treated mice increased more slowly (0.199 points/h) as compared to the anti-C5 antibody-treated mice (0.243 points/h, P = .009) and dexamethasone-treated mice (0.249 points/h, P = .012). Modeling of severity data suggested an additive effect of dexamethasone and anti-C5 antibodies. CONCLUSION: Adjunctive treatment with dexamethasone plus anti-C5 antibodies improves survival in severe experimental meningitis caused by S. pneumoniae serotype 3, posing an important new treatment strategy for patients with pneumococcal meningitis.

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

BACKGROUND: The 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. METHODS: Wild-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. RESULTS: In 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-1ß, 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. CONCLUSION: C/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.

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.

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)-1ß 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.

Binding characteristics of thrombin-activatable fibrinolysis inhibitor to streptococcal surface collagen-like proteins A and B.

Streptococcus pyogenes is the causative agent in a wide range of diseases in humans. Thrombin-activatable fibrinolysis inhibitor (TAFI) binds to collagen-like proteins SclA and SclB at the surface of S. pyogenes. Activation of TAFI at this surface redirects inflammation from a transient to chronic state by modulation of the kallikrein/kinin system. We investigated TAFI binding characteristics to SclA/SclB. Thirty-four overlapping TAFI peptides of ~20 amino acids were generated. Two of these peptides (P18: residues G205-S221, and P19: R214-D232) specifically bound to SclA/SclB with high affinity, and competed in a dose-dependent manner with TAFI binding to SclA/SclB. In another series of experiments, the binding properties of activated TAFI (TAFIa) to SclA/SclB were studied with a quadruple TAFI mutant (TAFI-IIYQ) that after activation is a 70-fold more stable enzyme than wild-type TAFIa. TAFI and TAFI-IIYQ bound to the bacterial proteins with similar affinities. The rate of dissociation was different between the proenzyme (both TAFI and TAFI-IIYQ) and the stable enzyme TAFIa-IIYQ. TAFIa-IIYQ bound to SclA/SclB, but dissociated faster than TAFI-IIYQ. In conclusion, the bacterial proteins SclA and SclB bind to a TAFI fragment encompassing residues G205-D232. Binding of TAFI to the bacteria may allow activation of TAFI, whereafter the enzyme easily dissociates.

Recent developments in thrombin-activatable fibrinolysis inhibitor research.

Thrombin-activatable fibrinolysis inhibitor (TAFI) provides an important molecular link between the coagulation and fibrinolytic systems. In this review, recent major advances in TAFI research, including the elucidation of crystal structures, the development of small inhibitors and the role of TAFI in systems other than hemostasis, are described and discussed.

C-reactive protein and natural IgM antibodies are activators of complement in a rat model of intestinal ischemia and reperfusion.

BACKGROUND: The role of C-reactive protein (CRP), natural immunoglobulin M (IgM), and natural IgM against phosphorylcholine (anti-Pc IgM) was investigated in relation with complement activation in a rat model of intestinal ischemia and reperfusion (II/R). The effect of C1-esterase inhibitor (C1-Inh) on this complement activation along with other inflammatory mediators was also studied. METHODS: Rats were subjected to 1 h of superior mesenteric artery occlusion and 3 h of reperfusion. Intravenous administration of vehicle (human albumin) or C1-Inh (200 U/kg) was performed before (n = 8) or after ischemia (n = 8). II/R increased levels of C4b/c, CRP, IgM, anti-Pc IgM, and myeloperoxidase activity in the intestinal homogenates and induced vascular leakage. RESULTS: A good correlation was observed in the intestinal homogenates between C4b/c and CRP levels. Clear depositions of C3, CRP, and IgM in intestinal tissue were demonstrated after II/R, and a strong correlation of both CRP and IgM with complement was observed. C1-Inh administered before ischemia reduced the complement activation response after II/R, as reflected by decreased levels of C4b/c in conjunction with reduced anti-Pc IgM in the intestinal homogenates. C1-Inh also decreased leakage of albumin when administered before ischemia. C1-Inh after ischemia reduced C4b/c levels and myeloperoxidase activity in the homogenates. CONCLUSIONS: CRP and IgM depositions correlated well with local complement activation, which suggests a role of these molecules in complement activation. Furthermore, C1-Inh inhibited potentially II/R injury either administered before or after ischemia, by attenuating complement activation induced by CRP and/or natural IgM antibodies.