is an important cause of bacterial meningitis causing death in ca. 25% of the cases and long-term neurological sequelae in up to one-third of the survivors (9
). Proinflammatory and directly cytotoxic pneumococcal products (such as pneumococcal cell wall products, pneumolysin, and bacterial DNA) contribute to neuronal injury in S. pneumoniae
Microglial cells are the major constituents of innate immunity within the CNS (20
). Parenchymal microglia, as well as meningeal and perivascular macrophages, which become activated by bacterial products are critically involved in protecting the brain from infection (30
). On the one hand, microglial cells can exert protective effects by phagocytosis of both pathogens and injured cells, and by mediating repair mechanisms (20
). When MyD88 bone marrow chimeric mice were studied after intracerebral injection of Staphylococcus aureus
, lack of MyD88 expression in the CNS compartment led to elevated intracerebral S. aureus
burdens despite the presence of immunocompetent bone marrow-derived cells (14
). On the other hand, activated microglial cells can be toxic to surrounding neurons by releasing, e.g., nitric oxide, glutamate, TNF-α, and IL-1β. The diminished inflammatory response decreased hearing loss in pneumococcal meningitis in MyD88-deficient mice, and neuronal injury caused by group B streptococci depended on the presence of TLR2 and MyD88 (18
). Thus, activation of microglia during infections seems to be a double-edged sword. The innate immune response can protect neurons by preventing the entry of pathogens into the brain, but its dysregulation can also be harmful for neuronal integrity and can cause neuronal injury (6
). Deeper understanding of the roles for TLRs in resident CNS glia and infiltrating immune cells will provide insights into how the immune response to bacterial infection can be tailored to achieve effective pathogen destruction without inducing excessive bystander damage of surrounding brain parenchyma (13
In this context, we focused our research on the phagocytosis of microglia activated by TLR stimulation. We hypothesized that the activation of the TLR system in microglial cells by agonist stimulation may enhance their phagocytic activity, thereby enabling them to protect the brain in pneumococcal CNS infections in patients with an impaired immune system.
The release of cytokines and chemokines in the CSF during pneumococcal meningitis has been analyzed. IL-6 is one of the major early response cytokines that can trigger an inflammatory cascade in pneumococcal meningitis (15
). In many resident cells, such as microglial cells and astrocytes, chemokine production is rapidly upregulated upon activation by stimuli such as bacteria or inflammatory mediators (24
). An upregulation of the expression of CCL2, CCL5, and CXCL2 chemokines was observed in lungs, blood, and brain tissue after intranasal inoculation of S. pneumoniae
strains (serotypes 2, 4, and 6A) in mice (25
). In the present study, when microglia were exposed to a TLR1/2, -4, or -9 ligand for 24 h, the release of IL-6 and CCL5 was strongly increased, confirming microglial activation.
Upon TLR stimulation, reactive microglia develop a phagocytic phenotype to engulf and kill microbes. In contrast to cytokine and chemokine induction, the phagocytic and bactericidal profiles of activated microglia have been explored less thoroughly. Our group has recently reported that TLR1/2, -4, and -9 agonists can increase the ability of murine microglial cells to phagocytose and kill intracellularly located Escherichia coli
). The present data demonstrate that microglia can also phagocytose and kill Gram-positive bacteria which have a thicker cell wall and that stimulation of TLRs can increase their phagocytic and bactericidal activity. This applies for both nonencapsulated apathogenic and encapsulated pathogenic pneumococci. Stimulation with either a TLR1/2, -4, or -9 agonist significantly increased the ability of microglia to phagocytose pneumococci. From our data, the effect of the stimulation through the TLR9 system was clearly greater than the effect caused via TLR1/2 or TLR4. Similarly, phagocytosis and killing of live S. pneumoniae
were found to be impaired in alveolar and bone marrow-derived macrophages from TLR9-deficient mice (1
) and in blood-derived polymorphonuclear leukocytes from TLR2-deficient mice (23
Once bacteria have been phagocytosed, they are incorporated into phagolysosomes and exposed to reactive oxygen species that eventually will result in bacterial lysis. The intracellular killing of S. pneumoniae
by microglial cells was more rapid than that of E. coli
studied in the same experimental setting (36
). For this reason, the number of viable intracellular bacteria determined after 90 min of phagocytosis was lower than the concentration of viable intracellular bacteria detected after 30 min.
The presence of the polysaccharide capsule is an important virulence factor of pneumococci because it decreases bacterial uptake into microglia by more than 10 times (Fig. ). In addition, we showed that the internalization of pneumococcal strains by murine microglia requires intact actin filaments since this process was blocked by >90% by cytochalasin D (Fig. ). Not only the phagocytic but also the bactericidal activities of reactive microglia depend on the stimulation of the TLR system. In our study, plotting the intracellular bacterial concentration versus time revealed higher absolute numbers of killed bacteria in TLR-stimulated than in unstimulated microglia, i.e., TLR stimulation clearly increased the efficacy of microglia in neutralizing the internalized S. pneumoniae (Fig. ).
An intact TLR signaling through the pathway organized by MyD88 appears to be necessary to protect the brain tissue against invading microorganisms. A poor outcome because of high bacterial counts in the CNS and severe bacteremia was observed in MyD88-deficient mice after intracisternal induction of pneumococcal meningitis (19
). Similarly, MyD88−/−
mice showed an increased susceptibility to pneumococcal colonization within the upper respiratory tract, an enhanced bacterial proliferation in infected lung tissue, precocious bacterial spread into the bloodstream, and increased mortality (2
). These findings illustrate the importance of an intact innate immune system to efficiently limit the spread of S. pneumoniae
Stimulation of the TLR system is a potential target for the development of new therapies in multiple diseases (45
). Several TLR agonists are currently at different stages of clinical trials (4
). The TLR7 agonist imiquimod has been successfully used and approved for the treatment of warts associated with human papillomavirus and is in a second phase trial as a therapeutic agent for herpes simplex virus (HSV) infections (43
). The TLR7/8 ligand resiquimod is also the subject of clinical investigations for the treatment of HSV infections (27
). CpG DNA has been tested as a vaccine adjuvant showing good results (8
). One of the most interesting clinical trials with CPG 7909 has been recently completed and aimed at comparing the immune responses after TLR9-boostered pneumococcal vaccination in human immunodeficiency virus-infected adults (www.clinicaltrials.gov/ct2/show/NCT00562939?term=TLR9&rank=3
Therefore, the agonists used in the present study or related compounds could be of value as adjuvants to improve the efficiency of the local immune system of the CNS against bacteria. In the pharmacological administration of TLR agonists as adjuvants, the dose, timing, and duration of the immunotherapy, as well as the route of administration, have to be selected not only to maximize the benefit of the enhancement of the immune response but also to restrict an excessive induced response that might lead to autoimmune diseases or increased neuronal injury (4
One clear advantage of using TLR agonists as adjuvants for the prophylaxis of bacterial meningitis is the low risk of development of resistance to the compound. For microglial activation, agonists with a low molecular mass would be preferable because of their higher penetration across the BBB (4
). The entry of LPS into the central nervous compartments is minimal (5
In conclusion, stimulation of TLRs increases phagocytosis of Gram-positive S. pneumoniae by microglia. Stimulation of the TLR system may be a therapeutic approach to protect the brain from invading pathogens. Further studies in immunocompromised mice are in progress in order to assess whether the resistance of the brain against infections can be increased by priming microglial cells with TLR agonists.