Escherichia coli at the Blood-Brain Barrier Interface

E. coli is a gram-negative bacterium implicated in the majority of cases of neonatal meningitis. In vitro studies of infection of human brain microvascular endothelial cells (HBMECs) by E. coli K1, an encapsulated strain responsible for most cases of neonatal meningitis, have shown that E. coli K1 interacts with these cells in a unique manner involving both host- and pathogen-specific structures and signaling pathways. These interactions lead to alterations in the host actin cytoskeleton, membrane protrusion and ruffling around bacteria, and endocytosis of bacteria into membrane-bound vacuoles, where bacterial determinants act to prevent lysosome fusion and influence intracellular vacuole trafficking to achieve transcytotic passage.² Several bacterial determinants have been identified as part of the initial binding and invasion of HBMECs, including type 1 fimbriae, outer membrane protein A (OmpA), Ibe proteins, and cytotoxic necrotizing factor-1 (CNF-1).² Type 1 fimbriae are adhesins that bind to α-D-mannosides on the surface of host cells, thereby allowing binding and interaction of the bacterium with the host cell. Type 1 fimbriae have been shown to play an important role in the binding of E. coli K1 to HBMECs; deletion of fimH, the gene for the major adhesin protein of type 1 fimbriae, significantly decreases binding of E. coli K1 to HBMECs, a finding that is reversed by genetic complementation of fimH in deletion mutants.³ OmpA also facilitates binding of E. coli K1 to HBMECs via interaction with surface glycoproteins containing N-acetylglucosamine residues.⁴ In vivo studies using an experimental neonatal rat model of meningitis have shown that deletion mutants of ompA are impaired in their ability to enter the CNS in comparison to the parent K1 strain, and N-acetylglucosamine oligosaccharides are able to block penetration of the CNS by wild-type E. coli K1 in the same animal model.⁵

Once binding has occurred, the process of cellular invasion must take place for E. coli K1 to ultimately infect the CNS. Invasion of HBMECs is dependent on the host actin cytoskeleton; E. coli K1 invasion can be completely inhibited in vitro by using inhibitors of the actin cytoskeleton such as cytochalasin D.⁶ Entry of E. coli K1 into membrane-bound vacuoles in HBMECs involves the formation of membrane projections, described as zipper-like structures, and subsequent membrane ruffling before internalization (Fig. 39-1).⁷ These events are related to several important signal transduction pathways in the host cell known to be involved in the regulation of endocytosis, cell membrane interactions, and the actin cytoskeleton. In particular, E. coli K1 leads to activation of focal adhesion kinase (FAK) and phosphorylation of paxillin, a cytoskeletal protein that interacts with and regulates the actin cytoskeleton.⁸ The exact mechanisms underlying FAK activation by E. coli K1 are unknown but appear to play a role in HBMEC invasion because overexpression of a dominant-negative form of FAK significantly inhibits HBMEC invasion by E. coli K1.⁸

FIGURE 39-1 Transmission electron micrographs of Escherichia coli K1 strain RS 218 undergoing attachment (A) and internalization (B and C) into human brain microvascular endothelial cells. Scale bar, 1 µm.

(From Kim KS. Microbial translocation of the blood-brain barrier. Int J Parasitol. 2006;36:610.)

Another important signal pathway involved in E. coli K1 invasion of HBMECs is the family of phosphatidylinositol-3′-kinases (PI3Ks), which lie downstream of FAK activation. Pharmacologic inhibition of PI3Ks with LY-294002 significantly limits HBMEC invasion by E. coli K1.⁹ Akt/protein kinase B, a downstream effector of PI3K, is increased during E. coli K1 invasion of HBMECs.⁹ These observations indicate a role for the PI3K/Akt pathway in E. coli invasion of HBMECs, although the exact roles that these kinases play in the process have yet to be described.

Rho family guanosine triphosphatases (GTPases) have been shown to regulate a diverse array of processes that affect the actin cytoskeleton, cell motility, and cell-cell/cell-matrix interactions.¹⁰ E. coli strains associated with urinary tract infections and meningitis have been shown to produce CNF-1, an AB-type toxin with deaminase activity that targets this family of GTPases.¹¹ CNF-1 activates Rho GTPases and increases the in vitro uptake of bacteria by nonprofessional phagocytes such as epithelial and endothelial cells.¹¹ Deletion of the cnf1 gene from E. coli K1 significantly decreases the invasion of HBMECs, and this deletion is associated with decreased activation of RhoA and Cdc42, GTPases that are activated during E. coli K1 invasion of these cells.¹¹

Once E. coli K1 bacteria have been internalized in membrane-bound vacuoles within HBMECs, these organisms must survive the internal hostile milieu and avoid destruction in the lysosome to gain entry into the CNS. The K1 capsule appears to play a very important role in preventing the normal maturation of endosomes and fusion of vacuoles with the lysosome. K1 isogenic deletion mutants (K1⁻) have been shown to traffic through the endosomal system and colocalize with cathepsin D, thus confirming fusion of the lysosome with the vacuoles containing these bacteria.¹² The exact mechanisms underlying the K1 capsular effect on endosomal trafficking have yet to be elucidated, however.

In summary, a neuroinvasive strain of E. coli uses specific tools to gain entry into the CNS by binding/uptake into HBMECs and subsequent diversion of the normal protective trafficking of endosomal compartments to the lysosome (Fig. 39-2). These events allow E. coli K1 strains to cross the highly selective BBB and cause meningitis. The next section reviews the most recent information pertaining to crossing the BBB by GBS, gram-positive bacteria that remain a prominent cause of sepsis and meningitis in neonates and infants.

FIGURE 39-2 Summary diagram of Escherichia coli K1 binding and invasion of the blood-brain barrier. CNF-1, cytotoxic necrotizing factor-1; FAK, focal adhesion kinase; GTPase, guanosine triphosphatase; OmpA, outer membrane protein A; PI3K, phosphatidylinositol-3′-kinase.

Group B Streptococci: If You Can’t Beat Them, Join Them

GBS (i.e., Streptococcus agalactiae) are highly effective at colonizing the human female genitourinary tract and are responsible for significant morbidity and mortality in neonates and infants, predominantly in the form of pneumonia, sepsis, and meningitis. GBS use both similar and different strategies to gain entry into the CNS when compared with E. coli K1; these strategies are discussed here to highlight the diversity of virulence factors and pathogenic mechanisms used by neuroinvasive pathogens.

Like E. coli K1 strains, GBS express a polysaccharide capsule that serves many functions in the process of host invasion, including protection from opsonization, complement-mediated lysis, antibody-mediated clearance, and phagocytosis.¹³ Nine distinct GBS capsular serotypes have been identified, with serotype III strains dominating the clinical isolates associated with meningitis.¹³ Virulent strains of GBS have terminal sialic acid residues coating the surface of the polysaccharide capsule; GBS strains lacking these residues are less virulent than isogenic counterparts carrying these sialic acid groups.¹⁴ Many host glycoproteins have the same terminal sialic acid residues (α2 → α3 N-acetylneuraminic acid), so the presence of these residues on the GBS capsule may provide the bacterium with some protection from immune surveillance via molecular mimicry.¹³ These residues also inhibit activation of the alternative complement pathway, thus preventing opsonophagocytosis of GBS.¹³

Like E. coli K1, GBS must bind to the luminal surface of BMECs to initiate invasion of the CNS. Importantly, GBS are able to bind to several components of the extracellular matrix, including fibrinogen, laminin, and immobilized but insoluble fibronectin. ScpB, a C5 peptidase anchored in the GBS membrane, has been identified as a selective fibronectin-binding protein that differentially associates with bound fibronectin.¹³ FbsA is a surface-anchored protein that binds fibrinogen, and the gene fbsA is regulated by RogB, a transcriptional regulator that positively regulates several genes involved in binding to the extracellular matrix.¹³

Recently, GBS pili have been shown to play a role in GBS interactions with HBMECs, and targeted deletion of the gene for the pilus accessory surface protein PilA significantly reduces the ability of GBS to adhere to HBMECs, whereas deletion of pilB, the gene encoding the major pilus structural protein, does not affect adherence but significantly reduces HBMEC invasion by GBS.¹⁵

The dissociation of adherence and invasion observed with the pili mutants just described supports the hypothesis that these events, adherence and invasion, are mediated by distinct virulence factors expressed by GBS. Other invasion mediators that have been identified for GBS and HBMECs include a gene involved in modification of lipoteichoic acid (LTA), expression of a β-hemolysin/cytolysin, and factors that alter host cell signaling pathways. LTA is a major structural component of the surface of gram-positive bacteria and may mediate amphiphilic interactions with host cell membrane phospholipids. Allelic exchange of a glycosyltransferase homologue in GBS, iagA, reduces LTA anchoring in the GBS cell wall and significantly reduces HBMEC invasion in vitro and the development of meningitis in a murine in vivo model.¹⁶ β-Hemolysin/cytolysin is a pore-forming exotoxin expressed by GBS that is known to promote GBS invasion of HBMECs at sublytic concentrations and cause HBMEC cytolysis at higher concentrations.¹⁷ β-Hemolysin/cytolysin also induces HBMEC synthesis of interleukin-8 (IL-8), an extremely potent neutrophil chemoattractant, and promotes neutrophil transmigration across HBMEC monolayers.¹⁷ Mice hematogenously infected with a GBS mutant lacking this exotoxin had lower brain bacterial counts and lower mortality than did mice infected with the parent wild-type strain expressing this toxin.¹⁷

As with E. coli K1, GBS have been shown to modulate a number of host signaling pathways to facilitate passage across the BBB. In fact, the FAK/paxillin/PI3K pathway is a common target involved in both E. coli K1 and GBS invasion of HBMECs. Inhibition of FAK signaling via a dominant-negative form of FAK and pharmacologic inhibition of PI3K with LY-294002 both significantly inhibit GBS invasion of HBMECs.¹⁸ Another shared pathway involved in E. coli K1 and GBS invasion of HBMECs involves RhoA GTPase; RhoA levels are increased during GBS invasion of HBMECs, as are levels of Rac1.¹⁹ Inhibition of RhoA and Rac1 with a geranylgeranyl transferase I inhibitor, GGTI-288, and expression of dominant-negative forms of these GTPases both result in significantly reduced HBMEC invasion by GBS.¹⁹

In summary, GBS take advantage of cloaking, molecular mimicry, exotoxin synthesis, and usurpation of host signaling pathways to bypass the BBB and cause meningitis. The common theme of modulating FAK and Rho GTPase signaling pathways during HBMEC invasion by E. coli K1 or GBS may provide future therapeutic targets for the prevention or treatment of CNS infections caused by these pathogens.

Microglia: Ramón y Cajal’s “Third Element”

Santiago Ramón y Cajal (1852-1934) divided the histology of the brain into “elements,” including neurons (first element), astrocytes (second element), and a third category of non-neuronal, nonastrocytic cells that he termed the third element. This third element contained cells later identified as oligodendrocytes, as well as a group of small, highly branched cells that were morphologically distinct from other cells in the CNS. These cells were eventually termed microglia by Pio del Rio Hortega (1882-1945), who went on to further characterize these cells as a distinct entity in the brain parenchyma.²⁰ Like monocytes and macrophages, these cells are derived from the bone marrow and share many features of monocytes/macrophages with respect to immune modulation.²⁰

Microglia can exist in several different states, as defined by surface markers, morphology, migration status, and function. Resting microglia are small cells with few surface markers and prominent thin branches that are constantly reorganizing and sampling the microenvironment of the brain parenchyma.²¹ A large number of stimuli activate resting microglia, with the nature of the stimulus influencing the structural and functional changes that occur during activation. Cellular debris from CNS damage, particularly free adenosine triphosphate, activates microglia and induces a morphologic change from a small, ramified cell to an ameboid cell capable of phagocytosing such debris in a manner similar to macrophages.²¹ Microglia, like macrophages, express a large number of receptors associated with various mediators of the inflammatory response to tissue damage, pathogens, and immune stimuli. A major family of receptors expressed by microglia includes pathogen-associated molecular pattern (PAMP) receptors known as the Toll-like receptors (TLRs). Eleven TLRs (TLR1 to TLR11) have been identified to date in humans. Microglia express TLR1 to TLR9, which allows them to detect and respond to a huge array of PAMPs; LTA (TLR2), double-stranded RNA (TLR3), lipopolysaccharide (LPS; TLR4), flagellin (TLR5), single-stranded RNA (TLR7), and unmethylated CpG DNA (TLR9) are examples.²² Engagement of these receptors by cognate ligands triggers signal transduction of multiple intracellular pathways responsible for the inflammatory response. LPS-induced activation of TLR4 has been well characterized in microglia and leads to activation of nuclear factor κB (NF-κB), cytokine production (interferon-β [IFN-β]), tumor necrosis factor-α (TNF-α)], signal transducer and activator of transcription-1α (STAT-1α), production of reactive oxygen species (ROS), and production of nitric oxide (·NO, or NO).²³ Flagellin-mediated activation of TLR5 on microglia has been shown to upregulate expression of TLRs 1, 2, 4, and 5, as well as IL-6.²⁴ TLR3 activation in microglia has been demonstrated in response to HIV, and TLR9 activation in microglia leads to the production of multiple cytokines and chemokines (TNF-α, IL-1β, IL-6, IL-12, macrophage inflammatory protein-1α [MIP-1α], MIP-1β).^25,26

Activation of microglia by invading pathogens has many consequences, some beneficial to the host and some detrimental. Microglia, like other members of the monocyte lineage, are capable of NO synthesis and a respiratory burst, processes directed at producing oxidative damage to the offending pathogen. Microglia produce NO via inducible nitric oxide synthase (iNOS, NOS-2), which leads to the formation of peroxynitrite (ONOO⁻), a highly toxic product capable of damaging both host and pathogen.²⁷

Microglia also possess the metabolic machinery (reduced nicotinamide adenine dinucleotide phosphate oxidase) necessary for the generation of superoxide anion (·O₂⁻), an extremely reactive oxygen species that can damage nucleic acids, lipids, and proteins.²⁸ Pathogens expressing superoxide dismutase are able to neutralize this effective defense. Microglia also function as phagocytes in the CNS under both physiologic and pathophysiologic conditions. Microglia are the major scavengers of cell debris in the CNS and interact, in part, with apoptotic bodies expressing externalized phosphatidylserine.²⁹ The orphan receptor TREM-2 (triggering receptor expressed on myeloid cells-2) is important in transforming microglia into phagocytes, and activation of TREM-2 enhances phagocytosis while suppressing the production of proinflammatory cytokines, events that may be important for prevention of the autoimmune responses to the autoantigens present in apoptotic bodies.³⁰

Microglia also regulate the response of other CNS cells to injury or infection and recruit cells from outside the CNS via the production of a wide variety of cytokines, chemokines, and lipid mediators. Table 39-1 lists some of the cytokines and chemokines known to be generated by microglia in response to a number of activating stimuli.³¹ Microglia also produce factors that support glial and neuronal cells in their microenvironment, including nerve growth factor, NT-3, brain-derived neurotropic factor, glial-derived neurotropic factor, and basic fibroblast growth factor.³¹ Many potent lipid mediators are synthesized by microglia, including prostaglandins (D₂, E₂, F_2α, thromboxane B₂), leukotriene B₄, and platelet-activating factor.^32,33 Several of these lipid mediators serve an autocrine role; the EP2 receptor on microglia participates in the activation of microglia, and antagonism of this receptor may have a neuroprotective effect by preventing excessive microglial neurotoxicity.³⁴ The critical role of microglia in orchestrating the innate immune response has been established, and experimental evidence for this role is expanding rapidly; an extensive review of this evidence can be found elsewhere.^31,35,36

TABLE 39-1 Cytokines and Chemokines Produced by Microglial Cells

IL, interleukin; IP-10, interferon-γ–inducible protein-10; MCP-1, monocyte chemoattractant protein-1; M-CSF, macrophage colony-stimulating factor; MIP-1α, macrophage inflammatory protein-1α; RANTES, regulated on activation, normal T cell expressed and secreted; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α.

Data from Hanisch UK. Microglia as a source and target of cytokines. Glia. 2002;40:140-155.

In addition to initiating the innate immune response to a variety of pathogens in the CNS, microglia also function as antigen-presenting cells and are able to prime CD4⁺ T cells to initiate the adaptive immune response. Various TLR ligands (LPS, peptidoglycan, polyinosinic-polycytidylic acid [poly-I:C], CpG DNA) and infection with Theiler’s murine encephalomyelitis virus result in increased expression of major histocompatibility class II complexes and costimulatory molecules on microglia, events that favor efficient antigen presentation and development of an adaptive immune response.²⁶ Cytomegalovirus (CMV), a gamma herpes virus capable of invading the human CNS, elicits CXCL10 (IFN-γ–inducible protein-10 [IP-10]) production from primary microglia but not from astrocytes (see later).³⁷ CXCL10 is an important chemokine involved in IFN-γ–induced T-cell recruitment, a process critical for control of CMV infection.³⁷ Remarkably, astrocytes infected with CMV produce the viral homologue of IL-10, UL111a, which suppresses the production of CXCL10 from activated microglia.³⁷ Thus, CMV is able to suppress T-cell recruitment in the CNS by subverting the production of an important antiviral chemokine by microglia.

Microglia, as potent regulators of the proinflammatory response to CNS injury and infection, are also able to downregulate these proinflammatory responses. Microglia express anti-inflammatory cytokines (IL-4, IL-10, IL-13, transforming growth factor-β [TGF-β]) and actively phagocytose apoptotic T cells after stimulation by IFN-β and IFN-γ.^31,38,39 Production of IL-4 and IL-13 ultimately triggers microglial apoptosis via autocrine receptor engagement, thereby providing a means of balancing inflammation in response to a specific CNS insult.⁴⁰

Microglia are clearly multifunctional cells that serve as central regulators of the innate and adaptive immune response in the CNS. However, these cells do not act in a vacuum and interact with both immune and nonimmune cells to coordinate the events surrounding CNS injury or invasion. One important cell type included in this response is the astrocyte. The next section briefly addresses the importance of astrocytes in the CNS during various stages of infection.

Astrocytes: Stellar Actors in Central Nervous System Immunopathogenesis

Astrocytes are resident glial cells derived from neuroectoderm and are often thought of as “nurse” cells for neurons in the brain parenchyma. These cells compose a large portion of the brain parenchyma and can be identified by their star-shaped morphology and expression of glial fibrillary acidic protein (GFAP). In addition to maintaining the BBB, astrocytes also participate in the immunologic processes that are associated with CNS injury and infection. The overt histologic response of astrocytes to CNS damage is termed reactive astrogliosis and is manifested as an increase in the amount of GFAP expressed by resident astrocytes at the site of insult. However, these observations are a small reflection of the complex responses of astrocytes to CNS injury or infection.

Like microglia, astrocytes express several TLRs, but to a more limited degree, which allows them to participate in the initial innate immune response to CNS invasion. The close proximity of astrocyte foot processes to BBB tight junctions and the huge surface area presented by the brain endothelium also highly favor early interaction between astrocytes and invading pathogens. TLRs expressed by astrocytes include 1, 3, 4, 5, and 9, with little or no TLRs 2, 6, 7, 8, or 10.^22,41 Among the expressed TLRs, TLR3 levels are the most prominent by quantitative real-time polymerase chain reaction (PCR).⁴¹ TLR3 ligation in astrocytes (by poly-I:C) triggers the production and release of IFN-β, CXCL10, and IL-6, as well as an increase in the expression of TLRs 1 to 5 and 9 mRNA by quantitative real-time PCR.⁴¹ Astrocytes are the main source of IL-6 production in the CNS; IL-6 is a multifunctional cytokine with diverse biologic activities, including neurotropism, protection of neurons from glutamate toxicity and ischemic injury, astrocyte proliferation, inflammation, and modulation of Fas/FasL expression in astrocytes.⁴² This latter effect on Fas/FasL expression may affect the general immunologic state of the CNS in that astrocytes normally express both elements of the Fas apoptosis apparatus yet do not succumb to this autocrine signaling.⁴³ The lack of astrocyte apoptosis despite Fas/FasL expression has been attributed to low-level expression of procaspase-8, whereas FasL expression by astrocytes is thought to provide a molecular barrier to circulating lymphocytes entering the CNS, cells that are highly sensitive to Fas/FasL-induced apoptosis.^44,45

In addition to the production of important immunologically active cytokines and chemokines, astrocytes also respond to chemokines through expression of chemokine receptors. Multiple chemokine receptors have been identified in astrocytes, and ligation of these receptors has many downstream effects on astrocyte function, including regulation of chemokine production and receptor expression (Table 39-2).⁴⁶ Of note, astrocytes express CXCR4, the receptor for stromal cell–derived factor-1α (SDF-1α), a chemokine with autocrine activity leading to influx of Ca²⁺ and chemostasis of astrocytes. Both CCR5 and CXCR4 serve as CD4-associated coreceptors for HIV. Although astrocytes lack CD4 receptors, CXCR4 binds to the gp120 glycoprotein of HIV, an event that leads to activation of the mitogen-activated protein kinases (MAPKs) extracellular signal–regulated kinase-1 (ERK-1) and ERK-2.⁴⁶ HIV also infects astrocytes via a CD4-independent mechanism, which results in a restricted (i.e., nonlytic, nonproductive) infection with incorporation of the HIV provirion into genomic DNA. Nonetheless, this restrictive infection alters astrocyte functions: increased expression of immunomodulatory molecules such as monocyte chemotactic protein-1 (MCP-1), complement factor 3 (C3), and iNOS and decreased glutamate uptake (see later).⁴⁷ The decreased glutamate uptake and increased iNOS expression by HIV-infected astrocytes probably contribute to the neurotoxicity observed with HIV infection of the CNS.

TABLE 39-2 Select Astrocyte Chemokine Receptors and Ligands and Astrocyte Responses to Receptor Activation

cAMP, cyclic adenosine monophosphate; ERK, extracellular signal–regulated kinase.

Data from Dorf ME, Berman MA, Tanabe S, et al. Astrocytes express functional chemokine receptors. J Neuroimmunol. 2000;111:109-121.

One of the major functions of astrocytes is uptake of glutamate at synaptic junctions in the CNS. Glutamate is an excitatory neurotransmitter that is highly neurotoxic, mainly via two mechanisms: (1) hyperactivation of neurons and (2) inhibition of cysteine uptake leading to oxidative damage to neurons through glutathione depletion. Astrocytes “soak up” glutamate in the CNS via excitatory amino acid transporters and rapidly convert glutamate to nontoxic glutamine by expression of the enzyme glutamine synthase.⁴⁸ This glutamine is then exported from astrocytes, taken back into neurons, and converted back to glutamate via a mitochondrial-based glutaminase for use as a neurotransmitter.⁴⁸ Pathogen-associated factors or inflammatory mediators released in the CNS during infection, or both, can alter the ability of astrocytes to control the CNS glutamate concentration and result in glutamate-induced neurotoxicity as a by-product of infection. For example, HIV gp120 has been shown to reduce astrocyte expression of glutamine synthase, and patients with HIV-associated dementia have higher brain glutamate levels than do nonaffected controls.^49,50 Glucocorticoid-induced expression of glutamine synthase is inhibited by IL-1β and TNF-α.⁵¹

Astrocytes also contribute to the balance of CNS protection/destruction during infection by secretion of matrix metalloproteinases (MMPs). MMPs are a family of proteases produced in a wide range of tissues, including the CNS, and are discussed in the next section, with particular attention on their roles in bacterial meningitis.

Matrix Metalloproteinases, “You Can’t Have Your Cake and Eat It Too!”

MMPs are a subgroup of the metzincin family of proteases that share a common Zn²⁺ binding site in their catalytic domain (…HExxHxxGxxH…, where x is any amino acid) and an associated methionine within a β turn, biochemical elements important for proteolytic activity.⁵² There are 24 members of the MMP group in mammals, and they can be further subdivided according to their domain structure (additional details can be found in the review by Yong⁵²). Most MMPs are secreted from cells, although some may be associated with cell surface molecules such as integrins (e.g., pro-MMP-2) or the hyaluronan receptor (active MMP-9) or are linked to the cell membrane via a glycophosphatidylinositol link.⁵² MMPs degrade a wide variety of substrates, including extracellular matrix components, receptors, growth factors, and adhesion molecules.⁵² As a consequence, MMPs must be tightly regulated to prevent extensive tissue destruction or unintended biologic sequelae. The first level of control involves the transcriptional regulation of MMP genes based on specific activation signals. MMPs may also undergo posttranslational modification and are highly compartmentalized within cells, thereby allowing further intracellular control of MMP activity.⁵² The second level of MMP regulation is based on secretion of these enzymes as zymogens, or proenzymes, which require additional cleavage to become active proteases. Finally, specific inhibitors, known as tissue inhibitors of metalloproteinases (TIMP-1 to TIMP-4), are expressed in tissues to counteract MMPs.⁵²

MMPs have been implicated as playing a role, either beneficial or detrimental, in many different diseases affecting the CNS, including infections, ischemic or traumatic injury, and autoimmune disorders.⁵² The cellular sources of these MMPs vary depending on the specific pathologic condition but generally include cellular elements of the CNS (microglia, astrocytes, neurons), endothelial cells, and infiltrating leukocytes, especially neutrophils and macrophages.⁵² Neutrophils, in particular, represent an important early source of MMP-9 because these cells are among the “first responders” of the innate immune response to injury or infection and contain preformed stores of this MMP.⁵² In general, MMPs can have complex effects on the inflammatory state of the CNS. The BBB is rich in potential substrates for MMPs, including type IV collagen, fibronectin, and laminin; MMP degradation of the BBB may favor transmigration of leukocytes, as well as the movement of macromolecules and water, thus contributing to brain edema. Injection of heat-killed Neisseria meningitidis results in BBB disruption and increased intracranial pressure (ICP), phenomena that are inhibited by the administration of batimastat, an MMP inhibitor.⁵³ MMPs also modify or degrade cytokines and chemokines relevant to this inflammatory state. For example, MMP-9 cleaves six amino acids from the N-terminal of IL-8 to produce a truncated form of IL-8 that is more potent with respect to neutrophil chemoattraction.⁵⁴ In contrast, MMPs degrade IL-1β, thus demonstrating anti-inflammatory activity.⁵⁵

CNS infections in which MMPs have specifically been shown to play a role include pneumococcal, gram-negative bacterial, and tuberculous meningitis, as well as viral infections such as HIV, human T-cell lymphotropic virus type I, and mumps and parasitic infections such as cerebral malaria and Angiostrongylus-associated meningoencephalitis.^56–61 We focus here on the MMPs and pneumococcal meningitis as an example of the complex roles played by MMPs in CNS infection.

Streptococcus pneumoniae is a gram-positive bacterium responsible for a number of infections in humans, including pneumonia, otitis media, sinusitis, sepsis, and meningitis. A rabbit model of experimental pneumococcal meningitis demonstrated a positive correlation between MMP-9 levels and CSF leukocyte counts and total protein levels.⁶² MMP-9 activity, as measured by gelatin zymography, localized predominantly to the intrathecal compartment and not to brain parenchyma, thus supporting the conclusion that the increase in MMP-9 activity during meningitis is derived from infiltrating leukocytes.⁶² MMP-9 may become activated during pneumococcal infection by ROS, as shown by the inhibition of MMP-9 activity in but not release from rat brain slices or neutrophils exposed to heat-inactivated pneumococci in the presence of ROS inhibitors.⁶³ Pneumococci can directly activate MMP-9 via production of ZmpC, a pneumococcal zinc metalloproteinase.⁶⁴ Interestingly, MMP-9 knockout mice (MMP-9^−/−) do not develop different CNS pathology than wild-type controls when S. pneumoniae is injected directly into the brain parenchyma, but these mice are less able to clear systemic bacteremia, which suggests that MMP-9 expression may play a more important role in clearing systemic pneumococcal infection.⁶⁵ Infant rat pups with experimental pneumococcal meningitis demonstrated a peak in brain parenchymal MMP-2 and MMP-9 levels 20 hours after infection and a peak in TIMP-1 levels 24 hours after infection.⁶⁶ The MMP-9/TIMP-1 ratio was significantly elevated during the first 20 hours of infection, thus supporting an imbalance in proteinase and inhibitor levels.⁶⁶ The elevation in MMP-9 was associated with proteolysis of collagen type IV in the meninges, perivascular spaces, and brain parenchyma, and parenchymal gelatinolytic activity correlated well with the degree of cortical damage.⁶⁶ In a rat model of pneumococcal meningitis, brain levels of MMP-9 increased in infected rats treated with ceftriaxone but not in saline-injected rats, whereas treatment with ceftriaxone plus dexamethasone reduced MMP-9 levels in comparison to untreated controls or animals treated with just ceftriaxone.⁶⁷ Glucocorticoids are known to inhibit MMP expression, and these findings are consistent with a potential protective effect of dexamethasone during pneumococcal meningitis. Tetracyclines inhibit the proteolytic activity of many MMPs, as well as the activity of TNF-α–converting enzyme (TACE), another proteolytic enzyme implicated in propagating CNS damage during meningitis.⁶⁸ Infant rats with pneumococcal meningitis that were treated with ceftriaxone plus doxycycline had lower mortality, less cortical damage, and less BBB disruption than did rats treated with ceftriaxone alone.⁶⁸ Both groups had sterile CSF by 40 hours after infection, with no differences in the time-kill curves between these groups during this time frame, thus suggesting that the effect of doxycycline was not due to enhanced bacterial clearance from CSF.⁶⁸ These animal models provide evidence that MMPs, particularly MMP-9, play a pathophysiologic role in bacterial meningitis and that targeting MMPs with inhibitors may reduce mortality and cortical damage without significantly altering sterilization of the CNS.

Data regarding MMPs in human disease also exist and are consistent with the observations described in animal models. In 27 children with bacterial meningitis, 91% and 97% had elevated CSF levels of MMP-8 and MMP-9, respectively, when compared with uninfected control children.⁶⁹ The majority of these children were infected with Haemophilus influenzae (n = 14) or N. meningitidis (n = 11), with only 2 infected with S. pneumoniae. However, elevated MMP-9 levels in CSF were associated (P < .05) with an increased risk for neurologic sequelae, including hearing loss and postinfection seizures.⁶⁹ In 19 adults with bacterial meningitis (n = 7 with S. pneumoniae), all had elevated MMP-9 activity, as measured by gelatin zymography, in comparison to uninfected controls or patients with Guillain-Barré syndrome.⁵³ Patients with bacterial meningitis also demonstrated increases in CSF TIMP-1 levels, and the MMP-9/TIMP-1 ratio was significantly elevated when compared with noninfected controls.⁵³ MMP-9 levels in the infected patients correlated with CSF protein concentrations but not CSF leukocyte counts, whereas TIMP-1 levels correlated with CSF leukocyte counts but not protein levels.⁵³ An investigation of 111 paired CSF and serum samples from patients with a range of neurological disorders, including both aseptic and bacterial meningitis, found that a CSF leukocyte count greater than 5/µL correlated well with elevated CSF MMP-9 activity by zymography (Spearman r = .755, P < .0001).⁷⁰ A more recent study examined the correlation between serum MMP-2 levels and the α₂-macroglobulin (α2M) index as a marker of increased BBB permeability in patients with infectious meningitis. The α2M index was defined as the ratio of α2M (CSF/serum) to albumin (CSF/serum). This study found that serum MMP-2 levels, as measured with an enzyme immunoassay, correlated well (r = .64, P < .0001) with the α2M index and were higher in patients with bacterial meningitis than in those with viral or fungal meningitis.⁷⁰

In summary, MMPs contribute to the pathogenesis of bacterial meningitis by degrading components of the BBB, a process that favors the formation of brain edema and propagation of the inflammatory response via leukocyte transmigration across the impaired BBB. MMPs also have more complex actions on other components of the immune response to infection, including modulation of cytokines and chemokines involved in balancing a successful immune response to infection in the CNS. These proteinases are potential targets for therapeutic interventions aimed at minimizing the collateral damage that occurs during the immunologic assault on CNS pathogens. A major component of this collateral damage involves brain edema, the development of ischemia, and neurotoxicity from elements of the immune response and from the invading pathogens themselves. The next section addresses mechanisms in the development of brain edema, increased ICP, and neurotoxicity during CNS infections.