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.

Dysfunctional Glymphatic System with Disrupted Aquaporin 4 Expression Pattern on Astrocytes Causes Bacterial Product Accumulation in the CSF during Pneumococcal Meningitis.

Pneumococcal meningitis, inflammation of the meninges due to an infection of the Central Nervous System caused by Streptococcus pneumoniae (the pneumococcus), is the most common form of community-acquired bacterial meningitis globally. Aquaporin 4 (AQP4) water channels on astrocytic end feet regulate the solute transport of the glymphatic system, facilitating the exchange of compounds between the brain parenchyma and the cerebrospinal fluid (CSF), which is important for the clearance of waste away from the brain. Wistar rats, subjected to either pneumococcal meningitis or artificial CSF (sham control), received Evans blue-albumin (EBA) intracisternally. Overall, the meningitis group presented a significant impairment of the glymphatic system by retaining the EBA in the CSF compartments compared to the uninfected sham group. Our results clearly showed that during pneumococcal meningitis, the glymphatic system does not function because of a detachment of the astrocytic end feet from the blood-brain barrier (BBB) vascular endothelium, which leads to misplacement of AQP4 with the consequent loss of the AQP4 water channel's functionality. IMPORTANCE The lack of solute drainage due to a dysfunctional glymphatic system leads to an increase of the neurotoxic bacterial material in the CSF compartments of the brain, ultimately leading to brain-wide neuroinflammation and neuronal damage with consequent impairment of neurological functions. The loss of function of the glymphatic system can therefore be a leading cause of the neurological sequelae developing post-bacterial meningitis.

NOD2-RIP2 signaling alleviates microglial ROS damage and pyroptosis via ULK1-mediated autophagy during Streptococcus pneumonia infection.

Meningitis occurs when S. pneumonia invade the blood-brain barrier, provoking inflammatory host response and neurological injury. Nucleotide-binding oligomerization domain 2 (NOD2) has been identified to promote microglial activation and autophagy during pneumococcal meningitis, but the mechanism remains unclear. In the present study, we investigated the passway of NOD2-mediated autophagy activation and the role of autophagy in inflammatory damage of murine microglia and mouse meningitis model. We demonstrated that autophagy was activated during S. pneumonia infection, and NOD2-RIP2 signaling was involved in the process. Treatment of microglia with GSK583, the RIP2 kinase inhibitor resulted in reduced autophagy-related protein and p-ULK1, indicating that RIP2 regulated autophagy in a kinase-dependent manner by phosphorylating ULK1. In addition, microglia with ULK1 knockdown exhibited enhanced production of ROS, leading to IL-1ß and IL-18 release and cellular pyroptosis. Similar to the in vitro results, NOD2-RIP2 signaling induced autophagy in the brain in a mouse meningitis model. Moreover, ULK1 or RIP2 silencing significantly increased pyroptosis of brain and induced more inflammatory damage of pneumococcal meningitis mice. Taken together, our study demonstrate that NOD2-RIP2 signaling is involved in the activation of autophagy by promoting ULK1 phosphorylation, which alleviates microglial ROS damage and pyroptosis during S. pneumonia infection.

Streptococcus pneumoniae meningitis and the CNS barriers.

Streptococcus pneumoniae (SPN) is a globally significant cause of meningitis, the pathophysiology of which involves damage to the brain by both bacterial virulence factors and the host inflammatory response. In most cases of SPN meningitis bacteria translocate from the blood into the central nervous system (CNS). The principal site of SPN translocation into the CNS is not known, with possible portals of entry proposed to be the cerebral or meningeal blood vessels or the choroid plexus. All require SPN to bind to and translocate across the vascular endothelial barrier, and subsequently the basement membrane and perivascular structures, including an additional epithelial barrier in the case of the blood-CSF barrier. The presence of SPN in the CNS is highly inflammatory resulting in marked neutrophilic infiltration. The secretion of toxic inflammatory mediators by activated neutrophils within the CNS damages pathogen and host alike, including the non-replicative neurons which drives morbidity and mortality. As with the translocation of SPN, the recruitment of neutrophils into the CNS in SPN meningitis necessitates the translocation of neutrophils from the circulation across the vascular barrier, a process that is tightly regulated under basal conditions - a feature of the 'immune specialization' of the CNS. The brain barriers are therefore central to SPN meningitis, both through a failure to exclude bacteria and maintain CNS sterility, and subsequently through the active recruitment and/or failure to exclude circulating leukocytes. The interactions of SPN with these barriers, barrier inflammatory responses, along with their therapeutic implications, are explored in this review.

Regulation of the p75 neurotrophin receptor attenuates neuroinflammation and stimulates hippocampal neurogenesis in experimental Streptococcus pneumoniae meningitis.

BACKGROUND: Streptococcus pneumoniae meningitis is a destructive central nervous system (CNS) infection with acute and long-term neurological disorders. Previous studies suggest that p75NTR signaling influences cell survival, apoptosis, and proliferation in brain-injured conditions. However, the role of p75NTR signaling in regulating pneumococcal meningitis (PM)-induced neuroinflammation and altered neurogenesis remains largely to be elucidated. METHODS: p75NTR signaling activation in the pathological process of PM was assessed. During acute PM, a small-molecule p75NTR modulator LM11A-31 or vehicle was intranasally administered for 3 days prior to S. pneumoniae exposure. At 24 h post-infection, clinical severity, histopathology, astrocytes/microglia activation, neuronal apoptosis and necrosis, inflammation-related transcription factors and proinflammatory cytokines/mediators were evaluated. Additionally, p75NTR was knocked down by the adenovirus-mediated short-hairpin RNA (shRNA) to ascertain the role of p75NTR in PM. During long-term PM, the intranasal administration of LM11A-31 or vehicle was continued for 7 days after successfully establishing the PM model. Dynamic changes in inflammation and hippocampal neurogenesis were assessed. RESULTS: Our results revealed that both 24 h (acute) and 7, 14, 28 day (long-term) groups of infected rats showed increased p75NTR expression in the brain. During acute PM, modulation of p75NTR through pretreatment of PM model with LM11A-31 significantly alleviated S. pneumoniae-induced clinical severity, histopathological injury and the activation of astrocytes and microglia. LM11A-31 pretreatment also significantly ameliorated neuronal apoptosis and necrosis. Moreover, we found that blocking p75NTR with LM11A-31 decreased the expression of inflammation-related transcription factors (NF-κBp65, C/EBPß) and proinflammatory cytokines/mediators (IL-1ß, TNF-α, IL-6 and iNOS). Furthermore, p75NTR knockdown induced significant changes in histopathology and inflammation-related transcription factors expression. Importantly, long-term LM11A-31 treatment accelerated the resolution of PM-induced inflammation and significantly improved hippocampal neurogenesis. CONCLUSION: Our findings suggest that the p75NTR signaling plays an essential role in the pathogenesis of PM. Targeting p75NTR has beneficial effects on PM rats by alleviating neuroinflammation and promoting hippocampal neurogenesis. Thus, the p75NTR signaling may be a potential therapeutic target to improve the outcome of PM.

Neuronal death in pneumococcal meningitis is triggered by pneumolysin and RrgA interactions with ß-actin.

Neuronal damage is a major consequence of bacterial meningitis, but little is known about mechanisms of bacterial interaction with neurons leading to neuronal cell death. Streptococcus pneumoniae (pneumococcus) is a leading cause of bacterial meningitis and many survivors develop neurological sequelae after the acute infection has resolved, possibly due to neuronal damage. Here, we studied mechanisms for pneumococcal interactions with neurons. Using human primary neurons, pull-down experiments and mass spectrometry, we show that pneumococci interact with the cytoskeleton protein ß-actin through the pilus-1 adhesin RrgA and the cytotoxin pneumolysin (Ply), thereby promoting adhesion and invasion of neurons, and neuronal death. Using our bacteremia-derived meningitis mouse model, we observe that RrgA- and Ply-expressing pneumococci co-localize with neuronal ß-actin. Using purified proteins, we show that Ply, through its cholesterol-binding domain 4, interacts with the neuronal plasma membrane, thereby increasing the exposure on the outer surface of ß-actin filaments, leading to more ß-actin binding sites available for RrgA binding, and thus enhanced pneumococcal interactions with neurons. Pneumococcal infection promotes neuronal death possibly due to increased intracellular Ca2+ levels depending on presence of Ply, as well as on actin cytoskeleton disassembly. STED super-resolution microscopy showed disruption of ß-actin filaments in neurons infected with pneumococci expressing RrgA and Ply. Finally, neuronal death caused by pneumococcal infection could be inhibited using antibodies against ß-actin. The generated data potentially helps explaining mechanisms for why pneumococci frequently cause neurological sequelae.

Oligodeoxynucleotides containing unmethylated cytosine-guanine motifs are effective immunostimulants against pneumococcal meningitis in the immunocompetent and neutropenic host.

BACKGROUND: Bacterial meningitis is a fatal disease with a mortality up to 30% and neurological sequelae in one fourth of survivors. Available vaccines do not fully protect against this lethal disease. Here, we report the protective effect of synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG ODN) against the most frequent form of bacterial meningitis caused by Streptococcus pneumoniae. METHODS: Three days prior to the induction of meningitis by intracerebral injection of S. pneumoniae D39, wild-type and Toll-like receptor (TLR9)-/- mice received an intraperitoneal injection of 100 µg CpG ODN or vehicle. To render mice neutropenic, anti-Ly-6G monoclonal antibody was daily administrated starting 4 days before infection with a total of 7 injections. Kaplan-Meier survival analyses and bacteriological studies, in which mice were sacrificed 24 h and 36 h after infection, were performed. RESULTS: Pre-treatment with 100 µg CpG ODN prolonged survival of immunocompetent and neutropenic wild-type mice but not of TLR9-/- mice. There was a trend towards lower mortality in CpG ODN-treated immunocompetent and neutropenic wild-type mice. CpG ODN caused an increase of IL-12/IL-23p40 levels in the spleen and serum in uninfected animals. The effects of CpG ODN on bacterial concentrations and development of clinical symptoms were associated with an increased number of microglia in the CNS during the early phase of infection. Elevated concentrations of IL-12/IL-23p40 and MIP-1α correlated with lower bacterial concentrations in the blood and spleen during infection. CONCLUSIONS: Pre-conditioning with CpG ODN strengthened the resistance of neutropenic and immunocompetent mice against S. pneumoniae meningitis in the presence of TLR9. Administration of CpG ODN decreased bacterial burden in the cerebellum and reduced the degree of bacteremia. Systemic administration of CpG ODN may help to prevent or slow the progression to sepsis of bacterial CNS infections in healthy and immunocompromised individuals even after direct inoculation of bacteria into the intracranial compartments, which can occur after sinusitis, mastoiditis, open head trauma, and surgery, including placement of an external ventricular drain.

Individual responsiveness of macrophage migration inhibitory factor predicts long-term cognitive impairment after bacterial meningitis.

BACKGROUND: Patients with pneumococcal meningitis are at risk for death and neurological sequelae including cognitive impairment. Functional genetic polymorphisms of macrophage migration inhibitory factor (MIF) alleles have shown to predict mortality of pneumococcal meningitis. METHODS: We investigated whether MIF concentrations during the acute phase of disease were predictive for death in a nationwide prospective cohort study. Subsequently, we studied whether individual ex vivo MIF response years after meningitis was associated with the development of cognitive impairment. RESULTS: We found that in the acute illness of pneumococcal meningitis, higher plasma MIF concentrations were predictive for mortality (p = 0.009). Cognitive impairment, examined 1-5 years after meningitis, was present in 11 of 79 patients after pneumococcal meningitis (14%), as compared to 1 of 63 (2%) in controls, and was consistently associated with individual variability in MIF production by peripheral blood mononuclear cells after ex vivo stimulation with various infectious stimuli. CONCLUSIONS: Our study confirms the role of MIF in poor disease outcome of pneumococcal meningitis. Inter-individual differences in MIF production were associated with long-term cognitive impairment years after pneumococcal meningitis. The present study provides evidence that MIF mediates long-term cognitive impairment in bacterial meningitis survivors and suggests a potential role for MIF as a target of immune-modulating adjunctive therapy.

Vitamin B12 is neuroprotective in experimental pneumococcal meningitis through modulation of hippocampal DNA methylation.

BACKGROUND: Bacterial meningitis (BM) causes apoptotic damage to the hippocampus and homocysteine (Hcy) accumulation to neurotoxic levels in the cerebrospinal fluid of children. The Hcy pathway controls bioavailability of methyl, and its homeostasis can be modulated by vitamin B12, a cofactor of the methionine synthase enzyme. Herein, the neuroprotective potential and the underlying mode of action of vitamin B12 adjuvant therapy were assessed in an infant rat model of BM. METHODS: Eleven-day old rats were intracysternally infected with Streptococcus pneumoniae serotype 3, or saline, treated with B12 or placebo, and, 24 h after infection, their hippocampi were analyzed for apoptosis in the dentate gyrus, sulfur amino acids content, global DNA methylation, transcription, and proximal promoter methylation of candidate genes. Differences between groups were compared using 2-way ANOVA followed by Bonferroni post hoc test. Correlations were tested with Spearman's test. RESULTS: B12 attenuated BM-induced hippocampal apoptosis in a Hcy-dependent manner (r = 0.80, P < 0.05). BM caused global DNA hypomethylation; however, B12 restored this parameter. Accordingly, B12 increased the methylation capacity of hippocampal cells from infected animals, as inferred from the ratio S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) in infected animals. BM upregulated selected pro-inflammatory genes, and this effect was counteracted by B12, which also increased methylation of CpGs at the promoter of Ccl3 of infected animals. CONCLUSION: Hcy is likely to play a central role in hippocampal damage in the infant rat model of BM, and B12 shows an anti-inflammatory and neuroprotective action through methyl-dependent epigenetic mechanisms.

NOD2 regulates microglial inflammation through the TAK1-NF-κB pathway and autophagy activation in murine pneumococcal meningitis.

Streptococcus pneumoniae is responsible for pneumococcal meningitis, with significant mortality and morbidity worldwide. Microglial inflammation plays a vital role in meningitis. The peptidoglycan sensor NOD2 (nucleotide-binding oligomerization domain 2) has been identified to promote microglia activation, but the role in autophagy following pneumococcal meningitis remains unclear. In the present study, we investigated the role of NOD2 in microglial inflammation and autophagy, as well as related signaling pathways, during S. pneumonia infection. NOD2 expression was knocked down by the injection of lentivirus-mediated short-hairpin RNA (shRNA). Our results revealed that NOD2 promotes microglial inflammation by increasing inflammatory mediators. We also showed that the TAK1-NF-κB pathway is involved in this process. In addition, NOD2 increased the expression of autophagy-related proteins and induced autophagosome formation. Rapamycin and 3-MA were utilized to assess the role of autophagy in microglial inflammation induced by S. pneumonia. We demonstrated that autophagy serves as a cellular defense mechanism to reduce inflammatory mediators. Similar to the in vitro results, NOD2 induced inflammation and autophagy in the brain in a mouse meningitis model. Moreover, NOD2 silencing significantly reduced brain edema and improved the neurological function of pneumococcal meningitis mice. Taken together, these data demonstrate that NOD2 promotes microglial inflammation and autophagy in murine pneumococcal meningitis, and the TAK1-NF-κB pathway is involved in microglial activation.

Emergence of Ceftriaxone Resistance during a Case of Pneumococcal Meningitis with Fatal Evolution.

We report a case of a 62-year-old man treated for Streptococcus pneumoniae meningitis by ceftriaxone and dexamethasone. After neurological improvement, neurological degradation by vasculitis occurred, despite effective concentrations of ceftriaxone in the serum and cerebrospinal fluid (CSF). S. pneumoniae with increased MICs to third-generation-cephalosporins (3GC) was isolated from the ventricular fluid 10 days after the isolation of the first strain. Isolate analysis showed that a mutation in the penicillin-binding protein 2X (PBP2X) has occurred under treatment.

Notch1 Signaling Pathway Promotes Proliferation and Mediates Differentiation Direction in Hippocampus of Streptococcus pneumonia Meningitis Rats.

BACKGROUND: Streptococcus pneumonia meningitis (PM) is a major cause of childhood neurological deficits. Although the Notch1 signaling pathway regulates neurogenesis and neuroinflammation, we know little about its expression or influence on hippocampal neurogenesis and gliogenesis during PM. METHODS: We used immunofluorescence and Western blots to detect Notch1 signaling expression during experimental PM. Through double-labeling immunofluorescence, we investigated proliferation and differentiation in the dentate gyrus (DG) in PM before and after treatment with exogenous Notch1 activator (Jagged1) and inhibitor (IMR-1). RESULTS: Our results showed that Notch1 was activated after 24 hours in PM. Compared with the phosphate-buffered saline (PBS) control, Jagged1 increased the proliferation of neural stem cells and progenitor cells (NS/PCs) in DG. After 14 and 28 days of meningitis, astrocyte differentiation increased compared with control. Astrocyte differentiation was higher in the Jagged1 versus the PBS group. In contrast, IMR-1 increased neuronal differentiation but decreased astrocyte differentiation compared with dimethyl sulfoxide treatment. CONCLUSIONS: Under PM, Notch1 signaling promotes NS/PC proliferation and astrocyte differentiation in DG, while decreasing neuronal differentiation. Transient activation of the Notch1 signaling pathway explains the reactive gliogenesis and limited neuronal differentiation observed in PM.

Myeloid-Related Protein 8/14 Participates in the Progression of Experimental Pneumococcal Meningitis by Augmentation of Inflammation.

It has been reported that myeloid-related protein 8/14 (MRP8/14) participates in the progression of inflammation after release from neutrophils and monocytes. This study aimed to clarify the mechanism(s) of the MRP8/14-augmented inflammatory response in mice with pneumococcal meningitis. Streptococcus pneumoniae (SP) meningitis was established by intracerebral injection of SP suspension. Balb/c mice were randomly divided into four groups and received the following injections: phosphate-buffer saline (PBS), MRP8/14 alone, SP alone, and SP plus MRP8/14. At 6 h, 24 h and 48 h postinfection, the clinical disease status was measured by the modified neurological severity score test, body weight loss and degree of cerebral edema; mice were anaesthetized, blood samples and brain samples were collected and brain inflammation was detected by haematoxylin and eosin (HE) staining; tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP) and monocyte chemoattractant protein-1 (MCP-1) levels in serum and brain homogenates were assessed by an enzyme-linked immunosorbent assay (ELISA), and the mRNA levels of the above cytokines in brain homogenates were measured by polymerase chain reaction (PCR); and the expression of nuclear factor-kappa B (NF-κB) p65 in brain tissues was determined by immunohistochemical assay. In this study, we identified that MRP8/14 substantially augmented the SP-stimulated inflammatory response, aggravated clinical disease status and exacerbated SP-induced brain edema in a murine model of pneumococcal meningitis. Exogenous administration of MRP8/14 significantly enhanced mRNA and protein expression of the proinflammatory cytokines and chemokines TNF-α, CRP, IL-6 and MCP-1 in brain homogenates and serum from mice with pneumococcal meningitis, which may be related to the NF-κB signalling pathway. We further found that MRP8/14 strongly augmented SP-induced phosphorylation of NF-κB p65 in brain tissue slices from the same model. In conclusion, our results indicated that MRP8/14 augmented the inflammatory response in mice with pneumococcal meningitis and contributed to the development of disease, which was probably through NF-κB signalling pathway activation.

Upregulation of CBP by PLY can cause permeability of blood-brain barrier to increase meningitis.

BACKGROUND: Streptococcus pneumoniae causes many human diseases including bacterial meningitis. Previous study proposed that pneumolysin (PLY), a cytotoxin from pneumococcus, is related to the infection across blood-brain barrier (BBB). However, the mechanism of how PLY break through BBB remains elusive. The present study showed that PLY can increase the permeability of BBB both in vitro and in vivo in our experiments. RESULTS: Further we found out that PLY leads to the high expression of CERB-binding protein (CBP) which can lead to releasing of tumor necrosis factor α then enhance apoptosis of cells which is a significant factor leading to permeabilization of BBB. CONCLUSION: Our findings demonstrate that CBP plays an important role in the pneumococcus infection in the brain and could be a potential therapeutic target against pneumococcal meningitis.

Diosmetin Suppresses Neuronal Apoptosis and Inflammation by Modulating the Phosphoinositide 3-Kinase (PI3K)/AKT/Nuclear Factor-κB (NF-κB) Signaling Pathway in a Rat Model of Pneumococcal Meningitis.

Bacterial meningitis has a high mortality rate and can be challenging to diagnose and manage. This study aimed to evaluate the effect of diosmetin in a rat model of Streptococcus pneumoniae meningitis and to investigate the mechanism of action. Forty rats included a treatment group (n=30) that underwent intracisternal injection with S. pneumoniae, and a sham group (n=10) that underwent intracisternal injection with normal saline. In the treatment group, four days before the inoculation of the bacteria, rats were pre-treated with oral diosmetin 100 mg/kg (n=10) and 200 mg/kg (n=10), and the negative control was pre-treated with normal saline (n=10). Bacterial meningitis was confirmed one day after inoculation by cerebrospinal fluid (CSF) bacterial titer and neurological score. In rat brain tissue, levels of inflammatory mediators were determined by enzyme-linked immunosorbent assay (ELISA) and western blot for protein kinase B (Akt), phosphoinositide 3-kinase (PI3K), myeloid differentiation primary response 88 (MyD88), and nuclear factor-kappaB (NF-kappaB), and the TUNEL assay for apoptosis was performed. In the diosmetin-treated group compared with negative control group, the CSF bacterial titer and the level of pro-inflammatory mediators, and the neurological score, were significantly reduced (p<0.01). In the rat hippocampal tissue, levels of Akt, PI3K, MyD88 and NF-kappaB, and the number of TUNEL-positive apoptotic cells were significantly reduced in the diosmetin-treated group compared with negative control group (p<0.01). In a rat model of bacterial meningitis due to S. pneumoniae, diosmetin reduced neuroinflammation, and neuronal apoptosis by modulating the PI3K/AKT/NF-kappaB signaling pathway.

Patterns of cerebral tissue oxygen tension and cytoplasmic redox state in bacterial meningitis.

BACKGROUND: Compromised cerebral energy metabolism is common in patients with bacterial meningitis. In this study, simultaneous measurements of cerebral oxygen tension and lactate/pyruvate ratio were compared to explore whether disturbed energy metabolism was usually caused by insufficient tissue oxygenation or compromised oxidative metabolism of pyruvate indicating mitochondrial dysfunction. SUBJECT AND METHODS: Ten consecutive patients with severe streptococcus meningitis were included in this prospective cohort study. Intracranial pressure, brain tissue oxygen tension (PbtO2 ), and energy metabolism (intracerebral microdialysis) were continuously monitored in nine patients. A cerebral lactate/pyruvate (LP) ratio <30 was considered indicating normal oxidative metabolism, LP ratio >30 simultaneously with pyruvate below lower normal level (70 µmol/L) was interpreted as biochemical indication of ischemia, and LP ratio >30 simultaneously with a normal or increased level of pyruvate was interpreted as mitochondrial dysfunction. The biochemical variables were compared with PbtO2 simultaneously monitored within the same cerebral region. RESULTS: In two cases, the LP ratio was normal during the whole study period and the simultaneously monitored PbtO2 was 18 ± 6 mm Hg. In six cases, interpreted as mitochondrial dysfunction, the simultaneously monitored PbtO2 was 20 ± 6 mm Hg and without correlation with the LP ratio. In one patient, exhibiting a pattern interpreted as ischemia, PbtO2 decreased below 10 mm Hg and a correlation between LP and PbtO2 was observed. CONCLUSION: This study demonstrated that compromised cerebral energy metabolism, evidenced by increased LP ratio, was common in patients with severe bacterial meningitis while not related to insufficient tissue oxygenation.

Prevention of Memory Impairment and Neurotrophic Factors Increased by Lithium in Wistar Rats Submitted to Pneumococcal Meningitis Model.

The aim of this study was to investigate the effects of lithium on brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial cell line-derived neurotrophic factor (GDNF) expression in the hippocampus and on memory in experimental pneumococcal meningitis. The mood-stabilizer lithium is known as a neuroprotective agent with many effects on the brain. In this study, animals received either artificial cerebrospinal fluid or Streptococcus pneumoniae suspension at a concentration of 5 × 109 CFU/mL. Eighteen hours after induction, all animals received ceftriaxone. The animals received saline or lithium (47.5 mg/kg) or tamoxifen (1 mg/kg) as adjuvant treatment, and they were separated into six groups: control/saline, control/lithium, control/tamoxifen, meningitis/saline, meningitis/lithium, and meningitis/tamoxifen. Ten days after meningitis induction, animals were subjected to open-field habituation and the step-down inhibitory avoidance tasks. Immediately after these tasks, the animals were killed and their hippocampus was removed to evaluate the expression of BDNF, NGF, and GDNF. In the meningitis group, treatment with lithium and tamoxifen resulted in improvement in memory. Meningitis group showed decreased expression of BDNF and GDNF in the hippocampus while lithium reestablished the neurotrophin expression. Lithium was able to prevent memory impairment and reestablishes hippocampal neurotrophin expression in experimental pneumococcal meningitis.

DNA repair protein APE1 is involved in host response during pneumococcal meningitis and its expression can be modulated by vitamin B6.

BACKGROUND: The production of reactive oxygen species (ROS) during pneumococcal meningitis (PM) leads to severe DNA damage in the neurons and is the major cause of cell death during infection. Hence, the use of antioxidants as adjuvant therapy has been investigated. Previous studies have demonstrated the possible participation of apurinic/apyrimidinic endonuclease (APE1) during PM. The aims of this study were to investigate the APE1 expression in the cortical and hippocampal tissues of infant Wistar rats infected with Streptococcus pneumoniae and its association with cell death and understand the role of vitamin B6 (vitB6) as a protective factor against cell death. METHODS: APE1 expression and oxidative stress markers were analyzed at two-time points, 20 and 24 h post infection (p.i.), in the cortex (CX) and hippocampus (HC) of rats supplemented with vitB6. Statistical analyses were performed by the nonparametric Kruskal-Wallis test using Dunn's post test. RESULTS: Our results showed high protein levels of APE1 in CX and HC of infected rats. In the CX, at 20 h p.i., vitB6 supplementation led to the reduction of expression of APE1 and apoptosis-inducing factor, while no significant changes in the transcript levels of caspase-3 were observed. Furthermore, levels of carbonyl content and glutamate in the CX were reduced by vitB6 supplementation at the same time point of 20 h p.i.. Since our data showed a significant effect of vitB6 on the CX at 20 h p.i. rather than that at 24 h p.i., we evaluated the effect of administering a second dose of vitB6 at 18 h p.i. and sacrifice at 24 h p.i.. Reduction in the oxidative stress and APE1 levels were observed, although the latter was not significant. Although the levels of APE1 was not significantly changed in the HC with vitB6 adjuvant therapy, vitB6 supplementation prevented the formation of the truncated form of APE1 (34 kDa) that is associated with apoptosis. CONCLUSIONS: Our data suggest that PM affects APE1 expression, which can be modulated by vitB6. Additionally, vitB6 contributes to the reduction of glutamate and ROS levels. Besides the potential to reduce cell death and oxidative stress during neuroinflammation, vitB6 showed enhanced effect on the CX than on the HC during PM.

Inhibition of DAMP signaling as an effective adjunctive treatment strategy in pneumococcal meningitis.

BACKGROUND: Pneumococcal meningitis remains a potentially lethal and debilitating disease, mainly due to brain damage from sustained inflammation. The release of danger-associated molecular patterns (DAMPs), like myeloid-related protein 14 (MRP14) and high mobility group box 1 protein (HMGB1), plays a major role in persistence of inflammation. In this study, we evaluated if paquinimod, an MRP14-inhibitor, and an anti-HMGB1 antibody can improve clinical outcome as adjunctive therapeutics in pneumococcal meningitis. METHODS: We tested the adjuvant administration of paquinimod and the anti-HMGB1 antibody in our pneumococcal meningitis mouse model assessing clinical (clinical score, open-field-test, temperature) and pathophysiological parameters (intracranial pressure, white blood cell count in CSF, bleeding area) as well as bacterial titers in blood and brain 24 h after administration and 48 h after infection. Furthermore, we explored the interactions of these two agents with dexamethasone, the standard adjuvant treatment in pneumococcal meningitis (PM), and daptomycin, a non-bacteriolytic antibiotic preventing pathogen-associated molecular pattern (PAMP) release. RESULTS: Adjunctive inhibition of MRP14 or HMGB1 reduced mortality in mice with PM. This effect was lost when the two anti-DAMP agents were given simultaneously, possibly due to excessive immunosuppression. Combining anti-PAMP (daptomycin) and anti-DAMP treatments did not produce synergistic results; instead, the anti-DAMP treatment alone was sufficient and superior. The combination of anti-HMGB1 with dexamethasone did not diminish the effect of the former. CONCLUSIONS: DAMP inhibition possesses good potential as an adjuvant treatment approach in PM, as it improves clinical outcome and can be given together with the standard adjuvant dexamethasone without drug effect loss in experimental PM.

Role of purinergic signaling in experimental pneumococcal meningitis.

Excessive neutrophilic inflammation contributes to brain pathology and adverse outcome in pneumococcal meningitis (PM). Recently, we identified the NLRP3 inflammasome/interleukin (IL)-1ß pathway as a key driver of inflammation in PM. A critical membrane receptor for NLRP3 inflammasome activation is the ATP-activated P2 purinoceptor (P2R) P2X7. Thus, we hypothesized involvement of ATP and P2Rs in PM. The functional role of ATP was investigated in a mouse meningitis model using P2R antagonists. Brain expression of P2Rs was assessed by RT-PCR. ATP levels were determined in murine CSF and cell culture experiments. Treatment with the P2R antagonists suramin or brilliant blue G did not have any impact on disease course. This lack of effect might be attributed to meningitis-associated down-regulation of brain P2R expression and/or a drop of cerebrospinal fluid (CSF) ATP, as demonstrated by RT-PCR and ATP analyses. Supplemental cell culture experiments suggest that the reduction in CSF ATP is, at least partly, due to ATP hydrolysis by ectonucleotidases of neutrophils and macrophages. In conclusion, this study suggests that ATP-P2R signaling is only of minor or even no significance in PM. This may be explained by down-regulation of P2R expression and decreased CSF ATP levels.