Joerg R. Weber

Pneumococcal pneumolysin and H2O2 mediate brain cell apoptosis during meningitis

Pneumococcus is the most common and aggressive cause of bacterial meningitis and induces a novel apoptosis-inducing factor-dependent (AIF-dependent) form of brain cell apoptosis. Loss of production of two pneumococcal toxins, pneumolysin and H(2)O(2), eliminated mitochondrial damage and apoptosis. Purified pneumolysin or H(2)O(2) induced microglial and neuronal apoptosis in vitro. Both toxins induced increases of intracellular Ca(2+) and triggered the release of AIF from mitochondria. Chelating Ca(2+) effectively blocked AIF release and cell death. In experimental pneumococcal meningitis, pneumolysin colocalized with apoptotic neurons of the hippocampus, and infection with pneumococci unable to produce pneumolysin and H(2)O(2) significantly reduced damage. Two bacterial toxins, pneumolysin and, to a lesser extent, H(2)O(2), induce apoptosis by translocation of AIF, suggesting new neuroprotective strategies for pneumococcal meningitis.

A vicious cycle involving release of heat shock protein 60 from injured cells and activation of toll-like receptor 4 mediates neurodegeneration in the CNS.

Infection, ischemia, trauma, and neoplasia elicit a similar inflammatory response in the CNS characterized by activation of microglia, the resident CNS monocyte. The molecular events leading from CNS injury to the activation of innate immunity is not well understood. We show here that the intracellular chaperone heat shock protein 60 (HSP60) serves as a signal of CNS injury by activating microglia through a toll-like receptor 4 (TLR4)-dependent and myeloid differentiation factor 88 (MyD88)-dependent pathway. HSP60 is released from CNS cells undergoing necrotic or apoptotic cell death and specifically binds to microglia. HSP60-induced synthesis of neurotoxic nitric oxide by microglia is dependent on TLR4. HSP60 induces extensive axonal loss and neuronal death in CNS cultures from wild-type but not TLR4 or MyD88 loss-of-function mutant mice. This is the first evidence of an endogenous molecular pathway common to many forms of neuronal injury that bidirectionally links CNS inflammation with neurodegeneration.

A Mechanism for Neurodegeneration Induced by Group B Streptococci through Activation of the TLR2/MyD88 Pathway in Microglia

Group B Streptococcus (GBS) is a major cause of bacterial meningitis and neurological morbidity in newborn infants. The cellular and molecular mechanisms by which this common organism causes CNS injury are unknown. We show that both heat-inactivated whole GBS and a secreted proteinaceous factor from GBS (GBS-F) induce neuronal apoptosis via the activation of murine microglia through a TLR2-dependent and MyD88-dependent pathway in vitro. Microglia, astrocytes, and oligodendrocytes, but not neurons, express TLR2. GBS as well as GBS-F induce the synthesis of NO in microglia derived from wild-type but not TLR2−/− or MyD88−/− mice. Neuronal death in neuronal cultures complemented with wild-type microglia is NO-dependent. We show for the first time a TLR-mediated mechanism of neuronal injury induced by a clinically relevant bacterium. This study demonstrates a causal molecular relationship between infection with GBS, activation of the innate immune system in the CNS through TLR2, and neurodegeneration. We suggest that this process contributes substantially to the serious morbidity associated with neonatal GBS meningitis and may provide a potential therapeutic target.

Apoptosis-Inducing Factor Mediates Microglial and Neuronal Apoptosis Caused by Pneumococcus

Streptococcus pneumoniae is the major cause of bacterial meningitis and it damages the hippocampus by inducing neuronal apoptosis. The blocking of caspases provides only partial protection in experimental meningitis, which suggests that there is an additional apoptotic pathway. A trigger of this pathway is the bacterium itself, as exposure of microglia or neurons to live pneumococci induces rapid apoptosis. In this study, apoptosis was not associated with the activation of caspases-1-10 and was not inhibited by z-VAD-fmk, a broad-spectrum caspase inhibitor. Rather, apoptosis was attributed to damage to mitochondria, which was followed by the release of apoptosis-inducing factor (AIF) from the mitochondria, large-scale DNA fragmentation, and hypodiploidy. Furthermore, intracytoplasmatic microinjection of AIF-specific antiserum markedly impaired pneumococcus-induced apoptosis. These findings indicate that AIF may play a central role in brain cell apoptosis and bacterial pathogenesis.

TLR2 Mediates Neuroinflammation and Neuronal Damage

Innate immunity relies on pattern recognition receptors to detect the presence of infectious pathogens. In the case of Gram-positive bacteria, binding of bacterial lipopeptides to TLR2 is currently regarded as an important mechanism. In the present study, we used the synthetic bacterial lipopeptide Pam3CysSK4, a selective TLR2 agonist, to induce meningeal inflammation in rodents. In a 6-h rat model, intrathecal application of Pam3CysSK4 caused influx of leukocytes into the cerebrospinal fluid (CSF) and induced a marked increase of regional cerebral blood flow and intracranial pressure. In wild-type mice, we observed CSF pleocytosis and an increased number of apoptotic neurons in the dentate gyrus 24 h after intrathecal challenge. Inflammation and associated neuronal loss were absent in TLR2 knockout mice. In purified neurons, cytotoxicity of Pam3CysSK4 itself was not observed. Exposure of microglia to Pam3CysSK4 induced neurotoxic properties in the supernatant of wild-type, but not TLR2-deficient microglia. We conclude that TLR2-mediated signaling is sufficient to induce the host-dependent key features of acute bacterial meningitis. Therefore, synthetic lipopeptides are a highly specific tool to study mechanisms of TLR2-driven neurodegeneration in vivo.

Recognition of pneumococcal peptidoglycan: an expanded, pivotal role for LPS binding protein.

Lipopolysaccharide binding protein (LBP) has a well-established role in LPS-induced immune responses. Here, we report that LBP also plays an essential role in the innate immune response to Gram-positive pneumococci, specifically to their major inflammatory component, pneumococcal cell wall (PCW). LBP was present in the CSF of patients with meningitis, and LBP-deficient mice failed to develop meningeal inflammation. LBP enhanced PCW-induced cell signaling and TNF-alpha release. LBP bound specifically to PCW multimers, indicating novel lipid-independent binding capability for LBP. We propose the iterative anionic groups along the glycan backbone of the cell wall are a crucial structure for recognition by LBP. Such a function for LBP expands its role to Gram-positive infections.

Anti ICAM-1 (CD 54) monoclonal antibody reduces inflammatory changes in experimental bacterial meningitis

We investigated whether monoclonal antibodies directed against intracellular adhesion molecule 1 (ICAM-1 mAb) inhibit brain edema, increase of intracranial pressure (ICP), regional cerebral blood flow (rCBF) and recruitment of white blood cells (WBC) into the cerebrospinal fluid (CSF) in the rat model of the early phase of bacterial meningitis. Brain edema was assessed by brain water content determinations. rCBF measured by laser Doppler flowmetry and ICP were recorded continuously for 6 h after intracisternal challenge. Meningitis was induced with pneumococcal cell walls (PCW). Increase of ICP and brain water content were significantly inhibited (P <0.05) by intravenous treatment with ICAM-1 mAb (TM-8, 1 mg/kg). Furthermore, ICAM-1 mAb treatment profoundly attenuated (P <0.05) rCBF increase and WBC invasion into the CSF. These results suggest that the ICAM-1 pathway is critically involved in the early phase of bacterial meningitis.

Interferon‐γ differentially modulates the release of cytokines and chemokines in lipopolysaccharide‐ and pneumococcal cell wall‐stimulated mouse microglia and macrophages

During bacterial infections of the CNS, activated microglia could support leucocyte recruitment to the brain through the synthesis of cyto‐ and chemokines. In turn, invading leucocytes may feedback on microglial cells to influence their chemokine release pattern. Here, we analyzed the capacity of interferon‐γ (IFNγ) to serve as such a leucocyte‐to‐microglia signal. Production of cyto‐ and chemokines was stimulated in mouse microglia cultures by treatments with lipopolysaccharide (LPS) from Gram‐negative Escherichia coli or cell walls from Gram‐positive Streptococcus pneumoniae (PCW). IFNγ presence during the stimulation (0.1–100 ng/mL) modulated the patterns of LPS‐ and PCW‐induced cyto‐ and chemokine release in a dose‐dependent, potent and complex manner. While amounts of TNFα and IL‐6 remained nearly unchanged, IFNγ enhanced the production of IL‐12, MCP‐1 and RANTES, but attenuated that of KC, MIP‐1α and MIP‐2. Release modulation was obtained with IFNγ preincubation (treatment of cells before LPS or PCW administration), coincubation and even delayed addition to an ongoing LPS or PCW stimulation. Together the changes observed for the microglial chemokine release under IFNγ would shift the chemoattractive profile from favouring neutrophils to a preferential attraction of monocytes and T lymphocyte populations – as actually seen during the course of bacterial meningitis. The findings support the view of activated microglia as a major intrinsic source for an instant production of a variety of chemokines and suggest that leucocyte‐derived IFNγ could potentially regulate the microglial chemokine release pattern.

Microglial Activation by Components of Gram-Positive and -Negative Bacteria: Distinct and Common Routes to the Induction of Ion Channels and Cytokines

Gram-positive Streptococcus pneumoniae is the major pathogen causing lethal meningitis in adults. We used pneumococcal cell walls (PCW) to investigate microglial consequences of a bacterial challenge and to determine the role of serum in the activation process. PCW caused the characteristic induction of an outwardly rectifying K+ channel (IK+(OR)), together with a concomitant suppression of the constitutively expressed inward rectifier K+ current, and evoked the release of tumor necrosis factor-alpha (TNF alpha), interleukin-6 (IL-6), IL-12, KC, macrophage inflammatory protein (MIP) 1alpha and MIP-2. Serum presence strongly facilitated the PCW effects, similarly as observed for lipopolysaccharide (LPS) from gram-negative Escherichia coli. The inflammatory cytokine, interferon-gamma (IFNgamma) induced the same electrophysiological changes, but independent of serum. Recombinant LPS binding protein (LBP) could partially replace serum activity in LPS stimulations. In contrast, neither LBP nor an antibody-mediated blockade of the LPS receptor, CD14 had significant influences on PCW-inducible changes. Cell surface interactions and cofactor involvement in microglial activation by gram-positive bacteria are thus distinct from the mechanisms employed by LPS. Moreover, tyrphostin AG126, a protein kinase inhibitor that prevents activation of the mitogen-activated protein kinase, p42MAPK (ERK2), potently blocked the PCW-stimulated cytokine release while having only a limited effect on LPS-inducible cytokines. In contrast, AG126 did not influence IK+(OR) inductions. This indicates that PCW recruits more than 1 intracellular signaling pathway to trigger the various responses and that different bacterial agents signal through both common and individual routes during microglial activation.

Lipopolysaccharide and pneumococcal cell wall components activate the mitogen activated protein kinases (MAPK) erk-1, erk-2, and p38 in astrocytes

Cell wall compounds of gram‐positive bacteria are capable of inducing the biosynthesis of proinflammatory cytokines in CNS cells in a similar way as lipopolysaccharide (LPS) of gram‐negative bacteria does. Astrocytes, which lack the CD14 LPS receptor, have also been shown to respond to LPS‐stimulation by increased cytokine synthesis. However, almost nothing is known about signaling steps involved in this process. We have therefore examined signaling events in primary cultures of rat astrocytes and the human astrocytoma cell line U373MG, brought about by LPS and pneumococcal cell walls (PCW). Of particular interest to us was the tyrosine phosphorylation patterns and activation states of three members of the mitogen activated protein kinase (MAPK) family, i.e., extracellular signal‐regulated protein kinase (erk)‐1, erk‐2, and the recently identified p38. We show that LPS and PCW initiate tyrosine phosphorylation and activation of erk‐1, erk‐2, and p38 in a dose‐dependent fashion. Inhibitors of tyrosine phosphorylation were able to alleviate this effect and also blocked cytokine production of astrocytes. Both, LPS‐ and PCW‐induced responses of astrocytic cells required the presence of soluble CD14 (sCD14) present in serum. Unraveling the signaling steps induced by bacterial compounds in cells of the CNS may potentially help to elucidate the pathomechanisms of meningitis and central nervous complications of sepsis and may offer options for novel treatment strategies. GLIA 22:295–305, 1998.