Characterization of a pneumococcal meningitis mouse model

<p>Abstract</p> <p>Background</p> <p><it>S. pneumoniae </it>is the most common causative agent of meningitis, and is associated with high morbidity and mortality. We aimed to develop an integrated and representative pneumococcal meningitis mouse model resembling the human situation.</p> <p>Methods</p> <p>Adult mice (C57BL/6) were inoculated in the cisterna magna with increasing doses of <it>S. pneumoniae </it>serotype 3 colony forming units (CFU; n = 24, 10⁴, 10⁵, 10⁶and 10⁷CFU) and survival studies were performed. Cerebrospinal fluid (CSF), brain, blood, spleen, and lungs were collected. Subsequently, mice were inoculated with 10⁴CFU <it>S. pneumoniae </it>serotype 3 and sacrificed at 6 (n = 6) and 30 hours (n = 6). Outcome parameters were bacterial outgrowth, clinical score, and cytokine and chemokine levels (using Luminex^®) in CSF, blood and brain. Meningeal inflammation, neutrophil infiltration, parenchymal and subarachnoidal hemorrhages, microglial activation and hippocampal apoptosis were assessed in histopathological studies.</p> <p>Results</p> <p>Lower doses of bacteria delayed onset of illness and time of death (median survival CFU 10⁴, 56 hrs; 10⁵, 38 hrs, 10⁶, 28 hrs. 10⁷, 24 hrs). Bacterial titers in brain and CSF were similar in all mice at the end-stage of disease independent of inoculation dose, though bacterial outgrowth in the systemic compartment was less at lower inoculation doses. At 30 hours after inoculation with 10⁴CFU of <it>S. pneumoniae</it>, blood levels of KC, IL6, MIP-2 and IFN- γ were elevated, as were brain homogenate levels of KC, MIP-2, IL-6, IL-1β and RANTES. Brain histology uniformly showed meningeal inflammation at 6 hours, and, neutrophil infiltration, microglial activation, and hippocampal apoptosis at 30 hours. Parenchymal and subarachnoidal and cortical hemorrhages were seen in 5 of 6 and 3 of 6 mice at 6 and 30 hours, respectively.</p> <p>Conclusion</p> <p>We have developed and validated a murine model of pneumococcal meningitis.</p

Leukocyte Attraction by CCL20 and Its Receptor CCR6 in Humans and Mice with Pneumococcal Meningitis

We previously identified CCL20 as an early chemokine in the cerebrospinal fluid (CSF) of patients with pneumococcal meningitis but its functional relevance was unknown. Here we studied the role of CCL20 and its receptor CCR6 in pneumococcal meningitis. In a prospective nationwide study, CCL20 levels were significantly elevated in the CSF of patients with pneumococcal meningitis and correlated with CSF leukocyte counts. CCR6 deficient mice with pneumococcal meningitis and WT mice with pneumococcal meningitis treated with anti-CCL20 antibodies both had reduced CSF white blood cell counts. The reduction in CSF pleocytosis was also accompanied by an increase in brain bacterial titers. Additional in vitro experiments showed direct chemoattractant activity of CCL20 for granulocytes. In summary, our results identify the CCL20-CCR6 axis as an essential component of the innate immune defense against pneumococcal meningitis, controlling granulocyte recruitment

Pathophysiology of pneumococcal meningitis

Bacterial meningitis is a serious infectious disease, involving the membranes surrounding the brain and spinal cord, and the subarachnoid space. In the Netherlands most common causative agents are Streptococcus pneumoniae (72%) and Neisseria meningitidis (11%). The incidence of pneumococcal meningitis in the Netherlands is 0.7 per 100.000, and has rapidly declined over the last decade as a result of herd immunity established by the introduction of a pneumococcal conjugate vaccine in the national childhood immunization programme. However, mortality and morbidity of pneumococcal meningitis remain high. In chapter 2 the pathophysiology of pneumococcal meningitis is reviewed. We discuss the different routes of infection, the host innate and adaptive immune response, brain damage described in humans and different animal models and potential targets for adjunctive therapy. In chapter 3 we describe the development and validation of an experimental mouse model for pneumococcal meningitis. We describe the different features of the immune response and pathophysiology. This animal model is used to study the role of the inflammasome pathway in the pathogenesis of pneumococcal meningitis in chapter 4. In chapter 5 we describe the association of a single-nucleotide polymorphism in two genes that are involved in the inflammasome-signaling pathway with unfavorable outcome in patients with pneumococcal meningitis. In chapter 6 we show that pneumococcal virulence also plays a role in the outcome of pneumococcal meningitis in patients. The absence of a pneumococcal arginine synthetase system is associated with favorable outcome in patients. In chapter 7 we report an explorative study in which we measured a large set of inflammatory mediators in the CSF of patients with pneumococcal meningitis. In chapter 8 we discuss our results with regard to other studies, we address any methodological shortcomings and give directions for future research

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

Thrombin-activatable fibrinolysis inhibitor influences disease severity in humans and mice with pneumococcal meningitis

Background Mortality and morbidity in patients with bacterial meningitis result from the proinflammatory response and dysregulation of coagulation and fibrinolysis. Thrombin-activatable fibrinolysis inhibitor (TAFI) is activated by free thrombin or thrombin in complex with thrombomodulin, and plays an antifibrinolytic role during fibrin clot degradation, but also has an anti-inflammatory role by inactivating proinflammatory mediators, such as complement activation products. Objective To assess the role of TAFI in pneumococcal meningitis. Methods We performed a prospective nationwide genetic association study in patients with bacterial meningitis, determined TAFI and complement levels in cerebrospinal fluid (CSF), and assessed the function of TAFI in a pneumococcal meningitis mouse model by using Cpb2 (TAFI) knockout mice. Results Polymorphisms (reference sequences: rs1926447 and rs3742264) in the CPB2 gene, coding for TAFI, were related to the development of systemic complications in patients with pneumococcal meningitis. Higher protein levels of TAFI in CSF were significantly associated with CSF complement levels (C3a, iC3b, and C5b-9) and with more systemic complications in patients with bacterial meningitis. The risk allele of rs1926447 (TT) was associated with higher levels of TAFI in CSF. In the murine model, consistent with the human data, Cpb2-deficient mice had decreased disease severity, as reflected by lower mortality, and attenuated cytokine levels and bacterial outgrowth in the systemic compartment during disease, without differences in the brain compartment, as compared with wild-type mice. Conclusions These findings suggest that TAFI plays an important role during pneumococcal meningitis, which is likely to be mediated through inhibition of the complement system, and influences the occurrence of systemic complications and inflammation

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