Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion.

The meninges are densely innervated by nociceptive sensory neurons that mediate pain and headache1,2. Bacterial meningitis causes life-threatening infections of the meninges and central nervous system, affecting more than 2.5 million people a year3-5. How pain and neuroimmune interactions impact meningeal antibacterial host defences are unclear. Here we show that Nav1.8+ nociceptors signal to immune cells in the meninges through the neuropeptide calcitonin gene-related peptide (CGRP) during infection. This neuroimmune axis inhibits host defences and exacerbates bacterial meningitis. Nociceptor neuron ablation reduced meningeal and brain invasion by two bacterial pathogens: Streptococcus pneumoniae and Streptococcus agalactiae. S. pneumoniae activated nociceptors through its pore-forming toxin pneumolysin to release CGRP from nerve terminals. CGRP acted through receptor activity modifying protein 1 (RAMP1) on meningeal macrophages to polarize their transcriptional responses, suppressing macrophage chemokine expression, neutrophil recruitment and dural antimicrobial defences. Macrophage-specific RAMP1 deficiency or pharmacological blockade of RAMP1 enhanced immune responses and bacterial clearance in the meninges and brain. Therefore, bacteria hijack CGRP-RAMP1 signalling in meningeal macrophages to facilitate brain invasion. Targeting this neuroimmune axis in the meninges can enhance host defences and potentially produce treatments for bacterial meningitis.

Case Report: Central Nervous System Immune Reconstitution Inflammatory Syndrome Related to Bacterial Meningitis.

Central nervous system immune reconstitution inflammatory syndrome (CNS-IRIS) describes clinical characteristics that may be observed in previously immunocompromised patients during rapid restoration of immunity function in the presence of a pathogen. There have been no reports about CNS-IRIS related to bacterial meningitis so far. Here, we report a 24-year-old pregnant female patient with bacterial meningitis. Her clinical and neuroradiological condition worsened after induced labor despite great effective anti-infective therapy. CNS-IRIS was considered. Corticosteroids were administered, and the patient gradually recovered. We present the first case of CNS-IRIS associated with bacterial meningitis.

Follow-up evaluation of the immunological status of children admitted for acute cerebral nervous system infections: a retrospective study.

BACKGROUND: Acute Cerebral Nervous System Infections (ACNS) may cause death or severe complications even to promptly treated children. The role of the immune system in influencing the course and the outcome of meningitis has been studied but it is not yet completely understood. The aim of the research is to ascertain whether children who experienced ACNS infection had a normal immune system. METHODS: Patients under 18 years of age admitted at Bambino Gesù Children from January 2006 till June 2016 for meningitis were asked to participate to the follow-up study. The immune status was evaluated both clinically and by laboratory investigations. RESULTS: Most of the patients over 3 years at follow up had at least one immunological alteration at follow-up evaluation (74%). Considering ACNS infection etiology, certain pathogens were almost exclusive of patients affected by some immunological alteration, regardless of their age. DISCUSSION: Our preliminary results indicate that sub-clinical immunological defects may be associated to ACNS pediatric infections. Moreover, to the best of our knowledges, this is the first study correlating pathogens to immune evaluation in ACNS infections. It is, however, important to underline the high frequency of persistent immunological alterations in the analyzed patients. Further studies are needed to confirm our conclusions. CONCLUSIONS: We recommend an immunological assessment at follow up evaluation in children who experienced an ACNS infection.

TRIM32 Drives Pathogenesis in Streptococcal Toxic Shock-Like Syndrome and Streptococcus suis Meningitis by Regulating Innate Immune Responses.

Streptococcus suis is an emerging zoonotic agent that causes streptococcal toxic shock-like syndrome (STSLS) and meningitis in humans, with high mortality and morbidity. The pathogenesis of both STSLS and central nervous system (CNS) infections caused by S. suis is not well understood. TRIM32, a member of the tripartite motif (TRIM) protein family, has been reported to regulate host inflammatory responses. In this study, we showed that TRIM32 deficiency significantly reduced the level of bacteremia and the production of proinflammatory cytokines following severe S. suis infection, protecting infected mice from STSLS. The influence of TRIM32 gene deletion on a range of processes known to be involved in S. suis meningitis was also examined. Both levels of bacterial loads and indications of brain hemorrhage were reduced in infected Trim32-/- mice compared with infected wild-type (WT) controls. We also found that TRIM32 deficiency increased the permeability of the blood-brain barrier (BBB) and the recruitment of inflammatory monocytes during the early course of S. suis infection, potentially limiting the development of S. suis meningitis. Our results suggest that TRIM32 sensitizes S. suis-induced infection via innate immune response regulation.

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.

Cytokine and immune cell profiling in the cerebrospinal fluid of patients with neuro-inflammatory diseases.

BACKGROUND: Cytokines play multiple roles during neuro-inflammatory processes and several cytokines have been studied in the context of specific diseases. This study provides a comprehensive picture of cerebrospinal fluid (CSF) changes during neuro-inflammation by analyzing multiple cytokines in combination with immune cell subsets and standard CSF parameters. METHODS: Using multiplex assays, we simultaneously measured 36 cytokines (CCL1-3, CCL7, CCL8, CCL11, CCL13, CCL19, CCL20, CCL22-27, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL9, CXCL11-13, CXCL16, CX3CL1, IL2, IL4, IL6, IL10, IL16, GM-CSF, IFNγ, MIF, TNFα, and MIB1ß) in the CSF and serum of 75 subjects. Diagnoses included clinically isolated syndrome and relapsing-remitting multiple sclerosis (MS, n = 18), secondary progressive MS (n = 8), neuro-syphilis (n = 6), Lyme neuro-borreliosis (n = 13), bacterial and viral meningitis (n = 20), and patients with non-inflammatory neurological diseases (NIND, n = 10). Cytokine concentrations were correlated with CSF standard parameters and CSF immune cell subsets (CD4 and CD8 T cells, B cells, plasmablasts, monocytes, and NK cells) quantified by flow cytometry. RESULTS: We observed increased levels of multiple cytokines (26/36) in patients with neuro-inflammatory diseases when compared to NIND that consistently correlated with CSF cell count and QAlbumin. Most CSF cytokine concentrations correlated with each other, but correlations between CSF and serum values were scarce (3/36). Within the CSF compartment, CXCL13 showed a strong association with B cells when analyzing all patients, as well as patients with an intact blood-brain barrier (BBB). NK cells positively correlated with CSF concentrations of multiple cytokines (22/36) when analyzing all patients. These correlations were maintained when looking at patients with a disrupted BBB but not detectable in patients with an intact BBB. CONCLUSIONS: Under conditions of neuro-inflammation, multiple CSF cytokines are regulated in parallel and most likely produced locally. A combined increase of CSF CXCL13 levels and B cells occurs under conditions of an intact BBB. Under conditions of a disrupted BBB, CSF NK cells show significantly increased values and seem to have a major contribution to overall inflammatory processes, reflected by a strong correlation with multiple cytokines. Future studies are necessary to address the exact kinetics of these cytokines during neuro-inflammation and their relation to specific diseases phenotypes.

Targeting the complement system in bacterial meningitis.

Bacterial meningitis is most commonly caused by Streptococcus pneumoniae and Neisseria meningitidis and continues to pose a major public health threat. Morbidity and mortality of meningitis are driven by an uncontrolled host inflammatory response. This comprehensive update evaluates the role of the complement system in upregulating and maintaining the inflammatory response in bacterial meningitis. Genetic variation studies, complement level measurements in blood and CSF, and experimental work have together led to the identification of anaphylatoxin C5a as a promising treatment target in bacterial meningitis. In animals and patients with pneumococcal meningitis, the accumulation of neutrophils in the CSF was mainly driven by C5-derived chemotactic activity and correlated positively with disease severity and outcome. In murine pneumococcal meningitis, adjunctive treatment with C5 antibodies prevented brain damage and death. Several recently developed therapeutics target C5 conversion, C5a, or its receptor C5aR. Caution is warranted because treatment with C5 antibodies such as eculizumab also inhibits the formation of the membrane attack complex, which may result in decreased meningococcal killing and increased meningococcal disease susceptibility. The use of C5a or C5aR antagonists to specifically target the harmful anaphylatoxins-induced effects, therefore, are most promising and present opportunities for a phase 2 clinical trial.

Bacterial Meningitis Caused by ß-lactamase Non-producing Ampicillin-resistant Haemophilus influenzae Type f in an Immunocompetent Woman.

We report the case of a 36-year-old previously healthy woman who presented with fever and headache. Blood and cerebrospinal cultures and a bacterial analysis revealed the presence of ß-lactamase non-producing ampicillin-resistant Haemophilus influenzae type f (Hif) with sequence type 124. Accordingly, the patient was diagnosed with bacterial meningitis with bacteremia caused by Hif. She had normal humoral immunity, and antibiotic therapy rapidly improved her condition. Our case indicates that serotype replacement can occur in Japan and suggests that a certain sequence type causes invasive Haemophilus influenzae disease, regardless of host immunity.

Bactericidal/Permeability-Increasing Protein Is an Enhancer of Bacterial Lipoprotein Recognition.

Adequate perception of immunologically important pathogen-associated molecular patterns like lipopolysaccharide and bacterial lipoproteins is essential for efficient innate and adaptive immune responses. In the context of Gram-negative infection, bactericidal/permeability-increasing protein (BPI) neutralizes endotoxic activity of lipopolysaccharides, and thus prohibits hyperactivation. So far, no immunological function of BPI has been described in Gram-positive infections. Here, we show a significant elevation of BPI in Gram-positive meningitis and, surprisingly, a positive correlation between BPI and pro-inflammatory markers like TNFα. To clarify the underlying mechanisms, we identify BPI ligands of Gram-positive origin, specifically bacterial lipopeptides and lipoteichoic acids, and determine essential structural motifs for this interaction. Importantly, the interaction of BPI with these newly defined ligands significantly enhances the immune response in peripheral blood mononuclear cells (PBMCs) mediated by Gram-positive bacteria, and thereby ensures their sensitive perception. In conclusion, we define BPI as an immune enhancing pattern recognition molecule in Gram-positive infections.

Baseline Circulating Activated TFH and Tissue-Like Exhausted B Cells Negatively Correlate With Meningococcal C Conjugate Vaccine Induced Antibodies in HIV-Infected Individuals.

Since 2006, meningococcal serogroup C (MenC) conjugate (MCC) vaccines have been supplied by the Brazilian government for HIV-infected children under 13 years old. For measuring protection against MenC, the serum bactericidal antibody (SBA) assay is the method of choice. The characterization of T follicular helper cells (TFH) cells has been an area of intensive study because of their significance in multiple human diseases and in vaccinology. The objective of this study was to characterize the phenotype of peripheral TFH cells and B cells and how they associated with each other and with SBA levels induced by vaccination as well as with serum cytokine levels of HIV-infected and non-infected children and adolescents. We found that CD27-IgD-CD21-CD38+ (exhausted B cells) as well as short-lived plasmablasts (CD27+IgD-CD21-CD38+) are increased in cART treated HIV patients and negatively associated with MCC vaccine induced SBA levels. Baseline frequency of activated peripheral TFH cells was a negative correlate for SBA response to MCC vaccine but positively correlated with circulating plasmablast frequency. Baseline IL4-levels positively associated with SBA response but showed a negative correlation with activated peripheral TFH cells frequency. The increased frequency of activated peripheral TFH cells found in non-responders to the vaccine implies that higher activation/differentiation of CD4 T cells within the lymph node is not necessarily associated with induction of vaccine responses.

Lack of chemokine (C-C motif) ligand 3 leads to decreased survival and reduced immune response after bacterial meningitis.

Pneumococcal meningitis, caused by Streptococcus pneumoniae, is the most common type of bacterial meningitis. The clinical management of this disease has been challenged by the emergence of multidrug-resistant Streptococcus pneumoniae, requiring the urgent development of new therapeutic alternatives. Over the course of bacterial meningitis, pathogen invasion is accompanied by a massive recruitment of peripheral immune cells, especially neutrophil granulocytes, which are recruited under the coordination of several cytokines and chemokines. Here, we used chemokine (C-C motif) ligand 3 (Ccl3)-deficient mice to investigate the functional role of CCL3 in a mouse model of pneumococcal meningitis. Following intrathecal infection with Streptococcus pneumoniae Ccl3-deficient mice presented a significantly shorter survival and higher bacterial load than wildtype mice, paralleled by an ameliorated infiltration of neutrophil granulocytes into the CNS. Blood sample analysis revealed that infected Ccl3-deficient mice showed a significant decrease in erythrocytes, hemoglobin and hematocrit as well as in the number of banded neutrophils. Moreover, infected Ccl3-deficient mice showed an altered cytokine expression profile. Glial cell activation remained unchanged in both genotypes. In summary, this study demonstrates that CCL3 is beneficial in Streptococcus pneumoniae-induced meningitis. Pharmacological modulation of the CCL3 pathways might, therefore, represent a future therapeutic option to manage Streptococcus pneumoniae meningitis.

How many single-nucleotide polymorphisms (SNPs) must be tested in order to prove susceptibility to bacterial meningitis in children? Analysis of 11 SNPs in seven genes involved in the immune response and their effect on the susceptibility to bacterial meningitis in children.

The aim of this study is to describe the prevalence of single single-nucleotide polymorphisms (SNPs) as well as their combinations in genes encoding proteins involved in the immune response in children with bacterial meningitis. The prospective study group consisted of 39 children with bacterial meningitis and 49 family members surveyed between 2012 and 2016. Eleven SNPs in seven genes involved in immune response were analysed. The mean number of minor frequency alleles (MAF) of studied SNPs was lowest in the control group and highest in patients with pneumococcal meningitis. We found that carrying ≥6 MAF of studied SNPs was associated with an increased risk of pneumococcal meningitis. The prevalence of risky variants was noted to be higher in patients with pneumococcal meningitis as compared to the control group. In conclusion, genetic factors are a relevant factor in determining the susceptibility to bacterial meningitis. A statistically significant cumulative effect of mutated variants on increasing the risk of bacterial meningitis was detected. Combining all three SNPs in MBL2 improves the prediction of susceptibility to pneumococcal meningitis. Analysis of risky alleles can help indicate people prone to the disease who are 'gene-immunocompromised'.

Cytotoxic Escherichia coli strains encoding colibactin isolated from immunocompromised mice with urosepsis and meningitis.

Immune-compromised mouse models allow for testing the preclinical efficacy of human cell transplantations and gene therapy strategies before moving forward to clinical trials. However, CRISPR/Cas9 gene editing of the Wsh/Wsh mouse strain to create an immune-compromised model lacking function of Rag2 and Il2rγ led to unexpected morbidity and mortality. This warranted an investigation to ascertain the cause and predisposing factors associated with the outbreak. Postmortem examination was performed on 15 moribund mice. The main lesions observed in these mice consisted of ascending urogenital tract infections, suppurative otitis media, pneumonia, myocarditis, and meningoencephalomyelitis. As Escherichia coli strains harboring polyketide synthase (pks) genomic island were recently isolated from laboratory mice, the tissue sections from the urogenital tract, heart, and middle ear were subjected to E. coli specific PNA-FISH assay that revealed discrete colonies of E. coli associated with the lesions. Microbiological examination and 16S rRNA sequencing confirmed E. coli-induced infection and septicemia in the affected mice. Further characterization by clb gene analysis and colibactin toxicity assays of the pks+ E. coli revealed colibactin-associated cytotoxicity. Rederivation of the transgenic mice using embryo transfer produced mice with an intestinal flora devoid of pks+ E. coli. Importantly, these barrier-maintained rederived mice have produced multiple litters without adverse health effects. This report is the first to describe acute morbidity and mortality associated with pks+ E. coli urosepsis and meningitis in immunocompromised mice, and highlights the importance of monitoring and exclusion of colibactin-producing pks+ E. coli.

The Thyroid Hormone Inactivating Type 3 Deiodinase Is Essential for Optimal Neutrophil Function: Observations From Three Species.

Neutrophils are essential effector cells of the innate immune system that have recently been recognized as thyroid hormone (TH) target cells. Cellular TH bioavailability is regulated by the deiodinase enzymes, which can activate or inactivate TH. We have previously shown that the TH inactivating enzyme type 3 deiodinase (D3) is present in neutrophils. Furthermore, D3 knockout (D3KO) mice show impaired bacterial killing upon infection. We hypothesized that D3 plays a role in neutrophil function during infection by actively regulating local TH availability. We measured TH concentrations in cerebrospinal fluid (CSF) from patients with bacterial meningitis and controls. Bacterial meningitis resulted in marked changes in CSF TH levels, characterized by a strong increase of thyroxine and reverse-triiodothyronine concentrations. This altered TH profile was consistent with elevated D3 activity in infiltrating neutrophils at the site of infection. D3 knockdown in zebrafish embryos with pneumococcal meningitis resulted in increased mortality and reduced neutrophil infiltration during infection. Finally, stimulated neutrophils from female D3KO mice exhibited impaired NADPH-oxidase activity, an important component of the neutrophil bacterial killing machinery. These consistent findings across experimental models strongly support a critical role for reduced intracellular TH concentrations in neutrophil function during infection, for which the TH inactivating enzyme D3 appears essential.

Human microglia and astrocytes constitutively express the neurokinin-1 receptor and functionally respond to substance P.

BACKGROUND: The tachykinin substance P (SP) is recognized to exacerbate inflammation at peripheral sites via its target receptor, neurokinin 1 receptor (NK-1R), expressed by leukocytes. More recently, SP/NK-1R interactions have been associated with severe neuroinflammation and neuronal damage. We have previously demonstrated that NK-1R antagonists can limit neuroinflammatory damage in a mouse model of bacterial meningitis. Furthermore, we have since shown that these agents can attenuate bacteria-induced neuronal and glial inflammatory mediator production in nonhuman primate (NHP) brain explants and isolated neuronal cells, and following in vivo infection. METHODS: In the present study, we have assessed the ability of NHP brain explants, primary human microglia and astrocytes, and immortalized human glial cell lines to express NK-1R isoforms. We have utilized RT-PCR, immunoblot analysis, immunofluorescent microscopy, and/or flow cytometric analysis, to quantify NK-1R expression in each, at rest, or following bacterial challenge. Furthermore, we have assessed the ability of human microglia to respond to SP by immunoblot analysis of NF-kB nuclear translocation and determined the ability of this neuropeptide to augment inflammatory cytokine release and neurotoxic mediator production by human astrocytes using an ELISA and a neuronal cell toxicity assay, respectively. RESULTS: We demonstrate that human microglial and astrocytic cells as well as NHP brain tissue constitutively express robust levels of the full-length NK-1R isoform. In addition, we demonstrate that the expression of NK-1R by human astrocytes can be further elevated following exposure to disparate bacterial pathogens or their components. Importantly, we have demonstrated that NK-1R is functional in both human microglia and astrocytes and show that SP can augment the inflammatory and/or neurotoxic immune responses of glial cells to disparate and clinically relevant bacterial pathogens. CONCLUSIONS: The robust constitutive and functional expression of the full-length NK-1R isoform by human microglia and astrocytes, and the ability of SP to augment inflammatory signaling pathways and mediator production by these cells, support the contention that SP/NK-1R interactions play a significant role in the damaging neuroinflammation associated with conditions such as bacterial meningitis.

Vaccination With a Latch Peptide Provides Serotype-Independent Protection Against Group B Streptococcus Infection in Mice.

Streptococcus agalactiae (group B streptococcus [GBS]) is a leading cause of invasive diseases in neonates and severe infections in elderly individuals. GBS serine-rich repeat glycoprotein 1 (Srr1) acts as a critical virulence factor by facilitating GBS invasion into the central nervous system through interaction with the fibrinogen Aα chain. This study revealed that srr1 is highly conserved, with 86.7% of GBS clinical isolates expressing the protein. Vaccination of mice with different Srr1 truncated peptides revealed that only Srr1 truncates containing the latch domain protected against GBS meningitis. Furthermore, the latch peptide alone was immunogenic and elicited protective antibodies, which efficiently enhanced antibody-mediated opsonophagocytic killing of GBS by HL60 cells and provided heterogeneous protection against 4 different GBS serogroups. Taken together, these findings indicated that the latch domain of Srr1 may constitute an effective peptide vaccine candidate for GBS.

Utility of the urine reagent strip leucocyte esterase assay for the diagnosis of meningitis in resource-limited settings: meta-analysis.

OBJECTIVE: Diagnosis of bacterial meningitis often requires cytometry, chemistry and/or microbiologic culture capabilities. Unfortunately, laboratory resources in low-resource settings (LRS) often lack the capacity to perform these studies. We sought to determine whether the presence of white blood cells in CSF detected by commercially available urine reagent strips could aid in the diagnosis of bacterial meningitis. METHODS: We searched PubMed for studies published between 1980 and 2016 that investigated the use of urine reagent strips to identify cerebrospinal fluid (CSF) pleocytosis. We assessed studies in any language that enrolled subjects who underwent lumbar puncture and had cerebrospinal fluid testing by both standard laboratory assays and urine reagent strips. We abstracted true-positive, false-negative, false-positive and true-negative counts for each study using a diagnostic threshold of ≥10 white blood cells per microlitre for suspected bacterial meningitis and performed mixed regression modelling with random effects to estimate pooled diagnostic accuracy across studies. RESULTS: Our search returned 13 studies including 2235 participants. Urine reagent strips detected CSF pleocytosis with a pooled sensitivity of 92% (95% CI: 84-96), a pooled specificity of 98% (95% CI: 94-99) and a negative predictive value of 99% when the bacterial meningitis prevalence is 10%. CONCLUSIONS: Urine reagent strips could provide a rapid and accurate tool to detect CSF pleocytosis, which, if negative, can be used to exclude diagnosis of bacterial meningitis in settings without laboratory infrastructure. Further investigation of the diagnostic value of using protein, glucose and bacteria components of these strips is warranted.

Pneumococcal conjugate vaccine in HIV-infected and HIV-exposed, uninfected children.

INTRODUCTION: Incidence of invasive pneumococcal disease (IPD) among HIV-infected children is 20-40 fold greater compared to HIV-uninfected children, including among HIV-infected children on antiretroviral therapy (ART). Also, HIV-exposed, uninfected children have 2.7-fold greater risk of IPD compared to HIV-unexposed children. Areas covered: We reviewed studies identified on Pubmed database with the terms 'PCV' and 'HIV'; studies involving adults only were excluded. Expert commentary: While ART and pneumococcal conjugate vaccines (PCV) have reduced IPD morbidity and mortality in HIV-infected children, ART-naïve and immunosuppressed children have inferior immunogenicity to most PCV serotypes; highlighting the need for concomitant use of ART with PCV. Furthermore, studies to determine optimal PCV dosing schedules, timing and number of doses, are urgently required to ensure sustained vaccine efficacy in HIV-infected children.